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Non-canonical roles of the nucleoporin Nup153 in influenza A virus infection, intracellular transport,… Acevedo, Maria 2016

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   NON-CANONICAL ROLES OF THE NUCLEOPORIN NUP153 IN INFLUENZA A VIRUS INFECTION, INTRACELLULAR TRANSPORT, AND CELLULAR ARCHITECTURE  by Maria Acevedo B.Sc (Hons.), Universidad Simón Bolívar, 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2016 © Maria Acevedo, 2016 ii  Abstract  The nuclear pore complex (NPC) mediates the bidirectional transport of macromolecules across the nuclear envelope. Nup153 is a nucleoporin localized in the nuclear side of the NPC that was identified as a host factor required for influenza A virus (IAV) infection in two genome-wide RNA interference (RNAi) screens (Hao et al., 2008, Nature 454:890-893; König et al., 2010, Nature 463:813-817). Since IAV uses the NPC at different steps of its infective cycle, the hypothesis that Nup153 could be required for the nuclear transport of IAV mRNAs, viral proteins, and viral ribonucleoproteins (vRNPs) was tested. The main objective of this thesis was to investigate the role of Nup153 during IAV infection. This was studied by following the progression of IAV infection in HeLa cells where Nup153 was knocked down (KD) with siRNA. These cells produced less infectious particles than control cells; however, no significant changes were detected in the nuclear import of vRNPs and the influenza nucleoprotein. To explain why Nup153 KD cells produced less infectious viral particles, the sub-cellular localization of the viral proteins nucleoprotein, matrix 1, and hemagglutinin were analyzed. The results indicate deficiency in the cytoplasmic trafficking of these proteins. To understand the observed defects in IAV infected Nup153 KD cells, the cellular changes associated with transient RNAi depletion of Nup153 were studied. Strikingly, defects in the intracellular traffic of other host cell proteins and in the distribution of endocytic organelles and the cytoskeleton were found. In addition, pronounced blebbing of the plasma membrane and alterations of the nuclear and cellular architecture were observed. This is the first time that a systematic characterization of the plethora of cellular defects resulting from Nup153 RNAi has been conducted. Collectively, the results suggest that iii  Nup153 depletion has an effect during the late stages of IAV infection and leads to defective vRNPs and/or the inadequate assembly or budding of progeny viral particles. This work adds to the cumulative evidence that nucleoporins have non-canonical roles, including roles during viral infection, and opens the possibility of considering nucleoporins as important factors for the development of future antiviral treatments.    iv  Preface I designed, performed, and analyzed the data from all experiments with the guidance of my supervisor Dr. Nelly Panté. Suggestions made by members of my doctoral advisory committee were incorporated for designing experiments and for writing the thesis. The first draft of this thesis was thoroughly revised and edited by Dr. Nelly Panté. From January to August, 2014, I supervised Jonathan Simkin, who was a UBC undergraduate student at the time. He assisted in repeating the plaque assays used to generate Figure 3-1 and establishing the knockdown conditions used to generate Figure A-1. Additionally, Julian Nguyen, a UBC undergraduate student working in our laboratory from January 2015 to May, 2016, also assisted in repeating the plaque assays used to generate Figures 3-1 and A-2. Dr. Lixin Zhou, a research associate in the Panté lab, constructed the 5GFP-NLS1 and 5GFP-NLS2 fusion plasmids used in the experiments shown in Figure 3-2. Dr. Zhou also assisted with the Western blots shown in Figures 4-4 and 4-9, and the cell flow cytometry analysis shown in Figures 5-4 and 5-7. Dr. Wei Wu, a postdoctoral fellow in the Panté lab, generated the 5GFP-NLS1 stable cell line used in the experiments shown in Figure 3-2. Dr. Shelly Au, a postdoctoral fellow in the Panté lab, performed the sample processing for electron microscopy (section 2.14) and obtained the images shown in Figures 4-15, 5-3, and 5-8. Astrid Arismendi created the base graphics used for Figures 1-1, 1-2, and 1-3, following my figure designs and indications.  The research presented in this thesis was approved by the UBC Bio-Safety Committee (Certificate B14-0137).   v  Table of Contents Abstract ..................................................................................................................................... ii Preface...................................................................................................................................... iv Table of Contents ...................................................................................................................... v List of Tables ............................................................................................................................ x List of Figures .......................................................................................................................... xi List of Abbreviations ............................................................................................................. xiv Acknowledgments................................................................................................................ xviii Dedication .............................................................................................................................. xxi Chapter 1 Introduction .............................................................................................................. 1 1.1. Influenza A virus ............................................................................................................ 2 1.1.1. Introduction to influenza A virus ............................................................................. 2 1.1.2. Overview of the IAV life cycle ............................................................................... 3 1.1.3. Internalization and endosomal traffic of IAV .......................................................... 8 1.1.4. Nuclear transport of influenza vRNPs and NP ...................................................... 10 1.1.5. Viral assembly and budding of IAV ...................................................................... 13 1.2. The NPC and nucleoporins .......................................................................................... 15 1.2.1. NPC structure and composition ............................................................................. 15 1.2.2. Nuclear transport ................................................................................................... 18 1.2.3. FG nucleoporins .................................................................................................... 21 1.2.4. Non-canonical roles of nucleoporins ..................................................................... 23 1.2.5. Nucleoporins and viral infections .......................................................................... 27 1.2.6. Nucleoporins and IAV ........................................................................................... 29 1.3. The nucleoporin Nup153 .............................................................................................. 31 1.3.1. Nup153 structure and cellular localization ............................................................ 31 1.3.2. Nup153 involvement in the cell cycle ................................................................... 35 1.3.3. Nup153 and nuclear trafficking of macromolecules ............................................. 36 1.3.4. Nup153 and its role during viral infections ........................................................... 39 1.4. The cytoskeleton and nucleoskeleton ........................................................................... 41 1.4.1. The cytoskeleton and nucleoskeleton .................................................................... 41 1.4.2. The LINC complex ................................................................................................ 43 vi  1.4.3. Nup153 and the nucleoskeleton ............................................................................. 44 1.5. Research aims ............................................................................................................... 46 1.5.1 Aim 1: To determine the role of Nup153 during the IAV infective cycle ............. 46 1.5.2. Aim 2: To determine the downstream effects of Nup153 depletion on endocytosis and intracellular traffic in HeLa cells .............................................................................. 47 1.5.3. Aim 3: To determine the effect of Nup153 RNAi depletion on nuclear and cell morphology ...................................................................................................................... 48 Chapter 2 Materials and Methods ........................................................................................... 49 2.1. Cell cultures.................................................................................................................. 49 2.2. Virus, reagents and antibodies ..................................................................................... 49 2.3. Small interference RNA (siRNA) and recombinant DNA transfection ....................... 51 2.4. Influenza A virus infection ........................................................................................... 52 2.5. Plaque assay ................................................................................................................. 55 2.6. Blocking nuclear export of NP with leptomycin B ...................................................... 56 2.7. Indirect immunofluorescence microscopy ................................................................... 56 2.8. Confocal microscopy.................................................................................................... 57 2.9. Ligand uptake experiments .......................................................................................... 57 2.9.1. Uptake of EGF ....................................................................................................... 57 2.9.2. Uptake of Tfn......................................................................................................... 58 2.9.3. Uptake of dextran .................................................................................................. 58 2.10. Plasma membrane and organelles labelling ............................................................... 59 2.10.1. Plasma membrane labelling ................................................................................. 59 2.10.2. LysoTracker and MitoTracker labelling .............................................................. 59 2.11. Image analysis–Quantification ................................................................................... 60 2.11.1. Quantification of nuclear import of NLS chimeric proteins and NP in transfected cells .................................................................................................................................. 60 2.11.2. Quantification of fluorescence intensity of NP, Nup153, EGF, and Tfn ............ 61 2.11.3. Gray intensity profile of WGA and HA transversal section ................................ 61 2.11.4. EGFR/TfR fluorescence intensity ....................................................................... 62 2.11.5. Quantification of pHrodo EGF particles ............................................................. 62 2.11.6. Dextran field index and vesicle size .................................................................... 63 2.11.7. Endosomal marker vesicle size and circularity ................................................... 64 vii  2.11.8. Spatial distribution analysis of endosomal markers ............................................ 64 2.11.9. Determination of the ER texture .......................................................................... 65 2.12. Statistical analysis ...................................................................................................... 65 2.13. Western blot (WB) ..................................................................................................... 66 2.14. Electron microscopy (EM) ......................................................................................... 67 2.15. Cell cycle analysis ...................................................................................................... 68 Chapter 3 Role of nucleoporin Nup153 during IAV infection ............................................... 70 3.1. Introduction .................................................................................................................. 70 3.2. Results .......................................................................................................................... 72 3.2.1. Infectious IAV particles production is significantly reduced in Nup153 depleted cells .................................................................................................................................. 72 3.2.2. Depletion of Nup153 results in decreased nuclear import of a chimeric protein containing the non-classical NLSs of NP ........................................................................ 77 3.2.3. Depletion of Nup153 does not prevent nuclear trafficking of influenza NP ......... 81 3.2.4. Cell entry and early traffic of IAV is affected in Nup153 KD HeLa cells ............ 85 3.2.5. Depletion of Nup153 does not prevent nuclear import of NP and vRNPs in IAV infected HeLa cells .......................................................................................................... 92 3.2.6. Nuclear/cytoplasmic distribution of newly made vRNP/NP is affected in Nup153 depleted cells infected with IAV ..................................................................................... 98 3.2.7. NP production, vRNP export, and overall IAV reinfection is hindered in Nup153 depleted cell after 24 h of IAV infection ....................................................................... 104 3.2.8. Hemagglutinin (HA) traffic is affected in Nup153 depleted cells infected with IAV after 24 h ................................................................................................................ 108 3.3. Discussion .................................................................................................................. 111 Chapter 4 Effects of Nup153 depletion on cargo uptake and endocytic organelle distribution............................................................................................................................................... 117 4.1. Introduction ................................................................................................................ 117 4.2. Results ........................................................................................................................ 119 4.2.1. Wheat germ agglutinin (WGA) labelling is altered in Nup153 depleted cells .... 119 4.2.2. The epidermal growth factor receptor (EGFR) cell distribution is altered and epidermal growth factor (EGF) uptake is markedly decreased in Nup153 depleted cells ....................................................................................................................................... 124 viii  4.2.3. The transferrin receptor (TfR) cell distribution is altered and transferrin (Tfn) uptake is markedly decreased in Nup153 depleted cells ............................................... 132 4.2.4. Dextran uptake decreases in Nup153 depleted cells ........................................... 138 4.2.5. Transient depletion of Nup153 by RNAi affects the distribution of the endocytic marker EEA1 ................................................................................................................. 142 4.2.6. Cellular distribution of the recycling endosome marker Rab11 is not affected by Nup153 depletion .......................................................................................................... 145 4.2.7. Transient depletion of Nup153 by RNAi induces changes in the distribution of the late endocytic marker CI-M6PR and late endosome/lysosome marker, LAMP1 ......... 147 4.2.8. Late endosomal/lysosomal compartments are redistributed in Nup153-depleted cells ................................................................................................................................ 152 4.2.9. Study of the secretory pathway elements in Nup153 depleted cells ................... 157 4.2.10. EM insights into Nup153-depleted cells ........................................................... 162 4.2.11. Nup153 depleted cells exhibit drastic cytoskeletal modifications ..................... 166 4.3. Discussion .................................................................................................................. 171 Chapter 5 Effect of Nup153 RNAi depletion on nuclear and cell morphology .................... 175 5.1. Introduction ................................................................................................................ 175 5.2. Results ........................................................................................................................ 177 5.2.1. Nup153-depleted cells exhibit plasma membrane blebbing and actin reorganization ................................................................................................................ 177 5.2.2. Nup153 depletion results in altered nuclear morphology and minor changes in the cell cycle ........................................................................................................................ 182 5.2.3. Nup153 depletion in HeLa LAP2β-GFP cells resulted in altered cytoskeleton and abnormal nuclear morphology ....................................................................................... 188 5.2.4. Depletion of Nup153 results in the formation of cytoplasmic vacuole-like structures and nuclear membrane herniations ............................................................... 197 5.2.5. Examining apoptotic cell death in Nup153 depleted cells .................................. 203 5.3. Discussion .................................................................................................................. 210 Chapter 6 General discussion and future perspectives ......................................................... 217 6.1. Steps of the IAV infection cycle affected in Nup153-depleted cells ......................... 218 6.2. Biology of the IAV: lessons learned in the absence of Nup153 ................................ 224 6.3. Nup153 and the intracellular domino effect ............................................................... 226 6.4. Future directions ......................................................................................................... 231 ix  6.5. Concluding remarks ................................................................................................... 232 References ............................................................................................................................. 234 Appendices ............................................................................................................................ 252 Appendix A – Supplementary results and figures from Chapter 3 ................................... 252 Appendix B – Supplementary results and figures from Chapter 4 ................................... 255 B.1. Quantification of changes in the distribution of EEA1, CI-M6PR, and LAMP-1 in Nup153 KD cells ........................................................................................................... 255 Appendix C – Supplementary results and figures from Chapter 5 ................................... 268    x  List of Tables   Table 1-1. Nup153 binding partners ....................................................................................... 34 Table 2-1. List of primary and secondary antibodies ............................................................. 53            xi  List of Figures Figure 1-1. Diagram of the influenza A virus and the vRNP ................................................... 5 Figure 1-2. Representation of the IAV life cycle. ..................................................................... 7 Figure 1-3. Structural components of the NPC. ...................................................................... 17 Figure 1-4. Diagram of the Nup153 organization. .................................................................. 33 Figure 1-5. Diagram of the LINC complex organization. ...................................................... 45 Figure 3-1. Production of infectious viral particles is reduced in Nup153 depleted cells. ..... 76 Figure 3-2. Nuclear import of a chimeric protein containing the main NLSs of NP is reduced in Nup153 depleted cells ......................................................................................................... 80 Figure 3-3. Nuclear import of NP still occurs in Nup153 KD cells ....................................... 84 Figure 3-4. Cellular uptake of IAV is reduced in Nup153 transiently depleted HeLa cells during early infection. ............................................................................................................. 87 Figure 3-5. The sub-cellular distribution of NP and M1 is altered in IAV infected Nup153 KD HeLa cells 60 min post-infection (p.i.). ........................................................................... 91 Figure 3-6. Depletion of Nup153 does not prevent nuclear import of vRNPs and nuclear import of NP in IAV infected HeLa cells ............................................................................... 95 Figure 3-7. Cellular distribution of M1 is altered in IAV infected Nup153 KD HeLa cells. . 97 Figure 3-8. Nuclear/cytoplasmic distribution of newly made vRNP/NP is affected in Nup153 depleted cells infected with IAV........................................................................................... 101 Figure 3-9. Newly synthesized M1 localization is affected in Nup153 depleted cells 8 h post infection with IAV. ............................................................................................................... 103 Figure 3-10. IAV reinfection appears hindered in Nup153 depleted cell after 24h of infection with IAV. .............................................................................................................................. 107 Figure 3-11. HA traffic is affected in Nup153 depleted cells infected with IAV after 24 h. 110 Figure 4-1. Extracellular binding of WGA is affected in live Nup153 depleted cells.......... 123 Figure 4-2. Transient depletion of Nup153 by RNAi induces defects on the cellular distribution of EGFR............................................................................................................. 127 Figure 4-3. Depletion of Nup153 reduces the cellular uptake and traffic of EGF ................ 131 Figure 4-4. Transient depletion of Nup153 by RNAi induces defects on the intracellular distribution of TfR. ............................................................................................................... 134 Figure 4-5. Tfn endocytosis is affected in Nup153 depleted cells. ....................................... 137 xii  Figure 4-6. Transient depletion of Nup153 by RNAi results in defects in dextran uptake and dextran-containing vesicle size ............................................................................................. 141 Figure 4-7. Transient depletion of Nup153 by RNAi affects the distribution of the early endocytic marker EEA1. ....................................................................................................... 144 Figure 4-8. Cellular distribution of the recycling endosome marker Rab11 is not affected by Nup153 depletion. ................................................................................................................. 146 Figure 4-9. Transient depletion of Nup153 by RNAi induces changes in the distribution of the late endocytic marker CI-M6PR and the late endocytic marker/lysosome LAMP-1. .... 150 Figure 4-10. The late endosomal/lysosomal marker Rab7 is redistributed in Nup153-depleted cells ....................................................................................................................................... 155 Figure 4-11. Acidic/lysosomal compartments identified by LysoTracker are redistributed in Nup153-depleted cells. ......................................................................................................... 156 Figure 4-12. The ER intraluminal marker ERp72 texture appears smoother in HeLa cells upon Nup153 depletion. ........................................................................................................ 159 Figure 4-13. Depletion of Nup153 in HeLa cells perturbs the distribution of the ER to Golgi vesicle marker, COPI. ........................................................................................................... 160 Figure 4-14. Cellular distribution of HA is altered in live Nup153 depleted cells. .............. 161 Figure 4-15. Nup153 depleted cells display a wide variety of vesicle bodies ...................... 165 Figure 4-16. Immunostaining of α-tubulin and F-actin are altered in Nup153-depleted cells............................................................................................................................................... 168 Figure 4-17. Immunostaining of vimentin and F-actin are altered in Nup153-depleted cells............................................................................................................................................... 170 Figure 5-1. Nup153-depleted cells exhibit plasma membrane blebbing and actin reorganization ....................................................................................................................... 180 Figure 5-2. F-actin changes upon Nup153 depletion are not related to Arp2/3 ................... 181 Figure 5-3. Nuclei of Nup153-depleted cells display invaginations and deformations ........ 186 Figure 5-4. Nup153-depleted cells exhibit minor cell cycle alterations ............................... 187 Figure 5-5. Nup153-depleted LAP2β-GFP cells exhibit changes in nuclear shape, cell morphology, and organization of the three cytoskeletal elements ........................................ 192 Figure 5-6. Nup153 depletion in LAP2β-GFP cells results in multi-lobed nuclei ............... 195 Figure 5-7. Nup153-depleted cells exhibit cell cycle alterations in LAP2β-GFP cells ........ 196 Figure 5-8. Nup153 depleted cells exhibit cytoplasmic vacuoles-like structures and outer nuclear membrane herniations .............................................................................................. 200 xiii  Figure 5-9. Nup153 depletion results in HA localization to the vacuoles-like structures in HA transfected and nuclear membrane in IAV infected cells. .................................................... 202 Figure 5-10. Depletion of Nup153 does not trigger caspase-3 mediated apoptosis. ............ 205 Figure 5-11. Nup153-depleted cells do not exhibit changes in mitochondria immunolabelling, size, interconnectivity, and elongation. ................................................................................. 208 Figure 5-12. Nup153 depletion does not alter the permeability of the NPC ........................ 209 Figure 6-1. Proposed model of alterations in the internalization and endosomal trafficking of IAV in Nup153 KD cells ...................................................................................................... 221 Figure 6-2. Proposed model of alterations in late steps of IAV infection in Nup153 KD cells............................................................................................................................................... 223 Figure 6-3. The domino effect: summary model of the major cellular defects observed in Nup153 KD cells................................................................................................................... 230 Figure A-1. Depletion of Nup358 and Nup214 in HeLa result in a decrease of IAV viral titer............................................................................................................................................... 252 Figure A-2. Subcellular localization of NP cannot be reliably quantified in NP-transfected Nup153 KD cells................................................................................................................... 253 Figure B-1. Quantification of changes observed in the distribution of the cellular markers EEA1 presented in Figure 4-7A ............................................................................................ 259 Figure B-2. Quantification of changes observed in the distribution of the cellular markers CI-M6PR presented in Figure 4-9A. .......................................................................................... 263 Figure B-3. Quantification of changes observed in the distribution of the late endocytic marker/lysosome LAMP-1 presented in Figure 4-9E ........................................................... 267 Figure C-1. Phosphorylation or accumulation of myosin light chain might be related to changes in the actin cytoskeleton observed in Nup153 depleted cells. ................................ 268   xiv  List of Abbreviations °C degree Celsius ANOVA analysis of variance BSA bovine serum albumin CDF cumulative distribution function  cDNA complementary deoxyribonucleic acid CI-M6PR cation independent mannose-6-phosphate receptor COP I coat protein complex I CRBPP completely random binomial point process  CRM1 chromosomal maintenance 1 cRNA complementary ribonucleic acid CSE1L chromosome segregation 1 like CTCF corrected total cell fluorescence Dapi 4',6-diamidino-2-phenylindole DIC differential interference contrast DMEM Dulbecco's modified Eagle medium DNA deoxyribonucleic acid EEA1 early endosome antigen 1 EGF epidermal growth factor EGFR epidermal growth factor receptor EM electron microscopy ER endoplasmic reticulum ERC endocytic recycling compartment  F phenylalanine F-actin filamentous actin FBS fetal bovine serum FG phenylalanine glycine FIJI FIJI is just ImageJ FITC fluorescein isothiocyanate G glycine xv  G2/M gap 2 phase/mitosis GFP green fluorescent protein GTP guanosine-5'-triphosphate h hour HA hemagglutinin HBV hepatitis B virus HCl hydrogen chloride HIV-1 human immunodeficiency virus type 1 HSV-1 herpes simplex virus type 1 IAV influenza A virus IBB importin-β binding domain INM inner nuclear membrane KD knockdown kDa kilodalton LAMP-1 lysosomal-associated membrane protein 1 LAP2β lamina-associated polypeptide 2, isoform beta LINC linker of nucleoskeleton and cytoskeleton LMB leptomycin B M1 matrix protein 1 MDCK Madin-Darby canine kidney mg milligram min minutes ml milliliter MLC2 myosin light chain 2 mM millimolar MOI multiplicity of infection mRNA messenger ribonucleic acid MTOC microtubule-organizing center MW molecular weight NA neuraminidase NE nuclear envelope xvi  NEP nuclear export protein NES nuclear export signal NLS nuclear localization signal nM nanomolar NP nucleoprotein NPC nuclear pore complex NS1 non-structural protein 1 NS2 non-structural protein 2 Nup  nucleoporin NXF1 nuclear RNA export factor 1 ONM outer nuclear membrane p.i. post-infection PA polymerase acidic PB1 polymerase basic 1 PB2 polymerase basic 2 PBS phosphate buffer saline PFU plaque forming unit PIC pre-integration complex PM plasma membrane RanGDP Ras-related nuclear protein guanine diphosphate RanGTP Ras-related nuclear protein guanine triphosphate RNA ribonucleic acid RNAi RNA interference ROI region of interest RTK receptor tyrosine kinase SAC spindle assembly checkpoint SDI spatial distribution index SDS sodium dodecyl sulfate siRNA small interfering RNA SV40 simian virus 40 Tfn transferrin xvii  TfR transferrin receptor TGN trans-Golgi network TPCK L-1-tosylamide-2-phenylethyl-chloromethyl ketone vRNA viral ribonucleic acid vRNP viral ribonucleoprotein WGA wheat germ agglutinin       xviii  Acknowledgments First, I would like to express my deepest thanks and appreciation to my supervisor, Dr. Nelly Panté, for her unconditional support and guidance during my PhD. She provided me with the opportunity to join her lab and allowed me the freedom to pursue my research interests. Thanks to her encouragement, dedication, patience, and excellent eye for detail, this manuscript has come to fruition.  I want to express my gratitude to the members of my advisory committee, Dr. Linda Matsuuchi, Dr. Ninan Abraham, and Dr. François Jean. They have come together to provide me with guidance, valuable ideas and scientific insights throughout the years; all while being incredible patient and encouraging.  I want to thank my research family, current and former members of the Panté lab. To our new members, Shuang Yuang, Shaghayegh Sadrekarimi, Julian Nguyen, thank you for your friendship and encouragement during the time I spent writing this manuscript, I wish you all success during your graduate studies. Special thanks to Dr. Lixin Zhou for helping me with my research and laying the foundations for the work I developed in this thesis. I would like to acknowledge the past members of the Panté Lab, Dr. Winco Wu, Dr. Sarah Cohen, Dr. Alexandra Marr, Dr. Nikta Fay, and Dr. Pierre Garcin. All of you have been instrumental to my success by helping me to further my scientific understanding, and teaching me important life lessons that will always stay with me.  Separately, I want to express my profound gratitude to former Panté Lab members Dr. Wei Wu and Dr. Shelly Au. Thank you for your love, friendship and guidance since I arrived to the lab. I consider myself the luckiest graduate student for working not only among xix  high caliber scientist but also great friends. You both have been my peer mentors in school and life. I am most thankful that we all traveled this path together. Without the two of you supporting this great academic journey, the culmination would not be possible. Thank you for always pushing me to grow as a person while showing me what compassion, tolerance, understanding, and hard work looked like. Additionally, since Wei and Bo come as a package deal, thank you Bo, for always taking care of me like a brother, and thanks too to little Wilson, for motivating me to finish this manuscript before his first steps. I want to recognize and be grateful to my mentor and friend, Dr. Kathryn Zeiler. You made teaching fun, challenging and (sometimes) exhausting, but always a place where I learned something new, despite the content (supposedly) remaining the same. You have seen me develop over the years while always there to listen to me and provide advice, whether it is life or school related. You have the soul of a true scientist and spending time with you is inspiring.  What is more, I am so grateful to have met wonderful people during my graduate studies that also became great friends and skiing buddies. Our skiing days inspired me to be bold and to believe in myself. Thank you for your support all these years. Dear Jenya Petoukhov, thank you for your constant love and support as a friend, even from the other side of the pond. Querida Ana Chavez, I am so happy I had the opportunity to meet you. You were the first person I befriended outside of the lab. I truly appreciate our scientific chats, girls skiing weekends, wine nights, among many other wonderful social events you have organized throughout the years.  xx  In addition, I want to thank my dearest friend and putative sister Dr. Ashley Sanders. I am so fortunate to have you in my life. With you, I have discussed science, ski across mountains and danced all night. You are a great source of inspiration, by showing me the definition of determination. You are not only one of the greatest emerging scientist I know but also a loving and supporting friend. Thanks to our friendship I have grown as a person and I have left many fears behind. From the bottom of my heart, thank you for always pushing me and believing I can.  I want to recognize and thank those who have been of great support during this journey, Mike Petersen, Guðrún Jónsdóttir, Farnaz Pournia, Marli Vlok, Astrid Arismendi, Carolina Novoa, Dr. Matt Taves, Dr. Sonja Christian, and Dr. Adam Plumb. Thanks for the good times whether we were discussing data, pondering about our future careers, playing board games or having a beer. Lastly, I want to thank my parents, without them, I would not be here. They have sacrificed themselves for my career and my success since before I was born. Everything I am, I owe it to them. They have taught me that I can achieve anything I set my mind to; all it takes is hard work and perseverance. They inculcated in me the knowledge thirst that pushed me to become a scientist. They have encouraged me to have deep, thought-provoking conversations since I was a little child. As an adult, I have understood I have the best parents in the world.    xxi    Dedication To God Because despite my attempts, you have never abandoned me  To my parents You are worth your weight in gold diamonds Your love and affection is the oil that keeps this engine going 1        Chapter 1 Introduction Introduction As the influenza A virus (IAV) produces a limited number of proteins, it requires cellular factors for its replication and viral production. During the last few years, IAV research has moved towards understanding not only the role of IAV proteins, but also the role of cellular proteins during IAV infection. Detailed studies of viruses such as IAV, which makes use of the cell cytoskeleton for trafficking and must access the nucleus of their host cells to replicate, will increase the knowledge about the biology of these processes. At the same time, this information will allow us to consider cytoplasmic and nuclear trafficking of viruses as a potential target in the development of antivirals. This thesis explores the requirements of the nucleoporin Nup153 during IAV infection. Nup153 was identified in two genome-wide RNA interference (RNAi) screens as a cellular protein required for IAV replication (Hao et al., 2008; König et al., 2010). The impact of silencing Nup153 by RNAi in HeLa cells was also studied; results revealed the importance of Nup153 for the maintenance of cellular processes related to the endocytic pathway and cellular architecture. This introduction begins with information on the biology of the IAV and discusses detailed aspects of its infective cycle that are relevant for this thesis. Subsequently, the nuclear pore complex (NPC) structure and composition is introduced, and the role of nucleoporins in nuclear transport and other cellular processes is discussed. Next, the focus is on the current knowledge regarding the function of Nup153. Since Nup153 is located in a key position to interact with components of the LINC (linker of the nucleoskeleton and cytoskeleton) complex and therefore may connect the nucleoskeleton and cytoskeleton, a short introduction 2  to the nucleoskeleton, its components, and its relevance to the biology of the cell is presented.  1.1. Influenza A virus 1.1.1. Introduction to influenza A virus IAV is the prototype for the Orthomyxoviridae family of viruses. The genome is fragmented and consists of eight negative-sense single-stranded RNA that must enter the nucleus of the host cell for replication (reviewed by Eisfeld et al. 2015). Each vRNA encodes a major viral protein; these are: hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), non-structural protein 1 (NS1), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and nucleoprotein (NP) (reviewed by Shaw and Palese, 2013). In addition, matrix protein 2 (M2) and non-structural protein 2/nuclear export protein (NS2/NEP) are produced as splicing variants of the M1 and NS1 genes (reviewed by Shaw and Palese, 2013). For a long time it was thought that the IAV genome encodes only these 10 viral proteins. However, research over the last 15 years has identified eight other IAV proteins present in some IAV strains that are expressed by splicing, alternative initiation, or ribosomal frameshifts. These are PB1-F2, PB1-N40, PB2-S1, PA-X, PA-N155, PA-N182, M42, and NS3 (Yamayoshi et al., 2015 and references therein). Although the function of some of these proteins remains to be established, the function of others, such as PB1-F2 and PA1-N40, have been associated with IAV pathogenicity (reviewed by Medina & García-Sastre, 2011). 3  The envelope of the IAV is a lipid bilayer membrane originating from the host cell once the virus has budded and contains projections formed by HA, NA, and M2 (Figure 1-1A) (reviewed by Noda, 2011). Inside the virion there is a matrix formed by M1 wherein the viral genome is anchored. There are eight RNA segments (only in influenza A and B), and each segment is helically wrapped around NP (Figure 1-1B). Each copy of NP (56 kDa) binds to approximately 24 nucleotides of viral RNA (vRNA) (Ortega et al., 2000; Ye et al., 2006). The trimeric polymerase complex (PA, PB1, and PB2) is also associated with the vRNA and binds to both ends of the vRNA, forming a non-covalent circular complex. NP is stoichiometrically bound to vRNA and together with the polymerase complex, forms the viral ribonucleoprotein complex (vRNP) (reviewed by Boulo et al. 2007). 1.1.2. Overview of the IAV life cycle This Section is an overview of the IAV infective cycle and precedes a more detailed description of several steps of the IAV infection, including: cellular uptake (Section 1.1.3), endosomal traffic (Section 1.1.3), nuclear transport (Section 1.1.4), assembly (Section 1.1.5), and budding (Section 1.1.5). These steps are explained in further detail because of their relevance to the interpretation and discussion of this thesis’s results.  The IAV life cycle starts when the virus enters the cell via receptor-mediated endocytosis. This step occurs through the interaction between the viral HA glycoprotein and the plasma membrane’s glycolipids and glycoproteins, which contain sialic acid (reviewed by Sun & Whittaker, 2013) (Figure 1-2, step 1). The virus is then internalized primarily through clathrin-dependent mechanisms (reviewed by Sun & Whittaker, 2013) (Figure 1-2, step 2). The viral disassembly occurs in late endosomes, wherein the low pH induces a 4  conformational change in HA that triggers the fusion of the endosomal and viral membrane, which in turn releases the vRNPs into the cytoplasm (Figure 1-2, step 3) to be translocated into the nucleus (Figure 1-2, step 4). The acidic environment of the endosome also triggers the dissociation of the matrix protein M1 from the vRNPs (reviewed by Edinger et al. 2014).  Once inside the nucleus, the vRNAs are transcribed by the viral RNA polymerase complex into mRNAs (Figure 1-2, step 5). Afterwards, the vRNA segments are copied into cRNA (complementary RNA) (Figure 1.2, step 9) to be used to generate more mRNA (secondary transcription). Thus, the viral transcripts are mRNA (transcription) (Figure 1-2, step 5), cRNA (first replication step) (Figure 1-2, step 9), and vRNA (second replication step) (Figure 1-2, step 10) (reviewed by Eisfeld et al. 2014). Later in infection the newly synthesized progeny vRNAs associate with NP, PB1, PB2, and PA, and then with M1, NS2/NEP, which lead to the formation of progeny vRNPs in the nucleus (Figure 1-2, step 11). The vRNPs are then exported through the NPC to the cytosol (Figure 1-2, step 12) (Martin & Helenius, 1991). New vRNPs traffic to the plasma membrane by “piggybacking” on Rab11 (Figure 1-2, step 13). The progeny virion starts its assembly (Figure 1-2, step 14) and buds from areas of the plasma membrane that are enriched in HA, NA, and M2 (Figure 1-2, step 15) (reviewed by Eisfeld et al. 2014). Post-translational modifications occur in NA and HA while they are transported through the secretory pathway (Figure 1-2, step 8) and targeted to lipid rafts in which virus particles are assembled. M2 also follows the secretory pathway. However, M2 is not targeted to lipid rafts, but is required for the scission of budded viruses (reviewed by Eisfeld et al. 2014). Other accessory proteins such as PB1-F2 and PA-X contribute to the modulation of the host response (reviewed by Eisfeld et al. 2014). 5    Figure 1-1. Diagram of the influenza A virus and the vRNP. Representation of key features of IAV. Depicted are the viral structural proteins forming the virion (A) and vRNPs (B). The virus contains eight vRNP fragments, which are constituted by the polymerase acid (PA), polymerase basic 1 (PB1), polymerase basic 2 (PB2), and negative sense single stranded (ss) vRNA wrapped around several copies of nucleoprotein (NP). Inside the virion the vRNPs interact with the matrix 1 protein (M1). The viral membrane contains the ion-channel protein M2 and the antigenic transmembrane proteins hemagglutinin (HA) and neuraminidase (NA). Small amounts of NS2/NEP can be found within the virion. Non-structural proteins NS1, PB-F1, and PA-X are not shown here.  6   7  Figure 1-2. Representation of the IAV life cycle. IAV HA binds to sialic acid moieties (step 1). Then, IAV is internalized mostly through endocytosis (step 2). The endosome containing the virus maturates into a “late endosome”, releasing M1 from the vRNPs and triggering a conformational change in HA that allows for endosomal and viral membrane fusion and release of vRNPs into the cytosol (step 3). The vRNPs are imported to the nucleus using the classical nuclear import pathway (step 4). In the nucleus, the vRNAs are transcribed to mRNA (step 5) and exported to the cytosol where they are translated either by free ribosomes (step 6A) or by endoplasmic reticulum (ER)-associated ribosomes to be transported along the secretory pathway (step 6B). The internal viral proteins assembled in the cytosol are imported back into the nucleus to assemble progeny vRNPs and aid in their nuclear export (step 7). Transmembrane viral proteins, such as HA and NA, traffic through the secretory pathway and are subjected to post-translational modifications, such as glycosylation (step 8). In the nucleus, the vRNAs are copied into cRNAs (step 9) and then again into vRNAs (step 10). The new vRNPs are assembled after vRNP transcription and the nuclear import of NP and the viral polymerase proteins (step 11). Binding of M1 and NS2/NEP to the vRNPs facilitates their nuclear export (step 12). The progeny vRNPs accumulate at the microtubule-organizing center (MTOC) and are recruited by Rab11-containing vesicles, and carried along microtubules to the plasma membrane and membrane periphery (step 13). The new virion assembles at the plasma membrane periphery (step 14) and buds off from the plasma membrane of the host cell containing HA, NA, and M2 (step 15). Proteolytic cleavage of HA (scissors = proteases) can take place in the secretory pathway (step 8), at the plasma membrane during budding of progeny virus (step 14), or in incoming viruses (step 1).    8  1.1.3. Internalization and endosomal traffic of IAV The attachment of IAV to the host cell determines viral tropism and restricts host range. The viral glycoprotein HA binds to specific sialic acid moieties, such as α-2,6-linked, which is found in the human lung and upper respiratory tract; galactose linked to α-2,3-linked sialic acid, which is found in the avian enteric tract, or; α-2,3 and α-2,6 sialic acid found together in swine trachea (reviewed by Shi et al. 2014). Amino acid residues found in the receptor-binding site of HA are the major determinants of HA specificity, and mutations in these residues confer the viral ability to engage with different cellular receptors (reviewed by Medina & García-Sastre, 2011; Sun & Whittaker, 2013). It is critical for viral infectivity that HA is cleaved from its precursor HA0 (75 kDa) to HA1 (55 kDa) and HA2 (25 kDa) (reviewed by Böttcher-Friebertshäuser et al. 2013). The cleavage of HA is necessary for its conformational change in the endosome, which is trigged by this organelle’s low pH and results in the fusion of the viral and endosomal membranes (reviewed by Böttcher-Friebertshäuser et al. 2013). In humans, low pathogenic strains of IAV predominantly contain monobasic HA cleavage sites, while the HA of highly pathogenic IAV contains a multibasic motif (reviewed by Böttcher-Friebertshäuser et al. 2013). In low pathogenic strains, HA can be cleaved at different times during the viral life cycle by transmembrane protease serine S1 member 2, human airway trypsin-like protease, or matriptase (in H9N2 viruses) (reviewed by Böttcher-Friebertshäuser et al. 2013). The cleavage of HA by the transmembrane protease serine S1 member 2 occurs within the cell, and evidence suggests that it takes place at the trans-Golgi network (TGN) (reviewed by Böttcher-Friebertshäuser et al. 2014). The human airway trypsin-like protease is found in the plasma membrane of human bronchial epithelial cells and cleaves HA at the cell surface, either during the 9  formation of the new virion or early during infection after viral attachment to the host cell (reviewed by Böttcher-Friebertshäuser et al. 2014). On the other hand, the cleavage of HA in the TGN of high pathogenic IAV strains such as H5 and H7 is mediated by intracellular proteases such as furin, which is ubiquitously expressed in multiple tissues (reviewed by Böttcher-Friebertshäuser et al. 2013).  After viral attachment, IAV’s uptake occurs through endocytosis or macropinocytosis. Entry through clathrin-mediated endocytosis and caveolae as well as through non-clathrin/non-caveolae-mediated endocytosis have also been reported for IAV (reviewed by Sun & Whittaker, 2013; Edinger et al. 2015). Viral entry through any of these pathways seems to be dependent upon cell polarization, serum presence in the media, and viral morphology (reviewed by Edinger et al. 2014). Actin filaments are essential for successful apical (but not basolateral) viral infection in polarized cells, while disruption of the actin cytoskeleton does not affect IAV entry in non-polarized cells (Gottlieb et al. 1993; Sun & Whittaker, 2007). Clathrin-mediated endocytosis is more frequent in the absence of serum, while viral entry still occurs through dynamin-dependent and independent routes in the presence of serum (de Vries et al., 2011). Lastly, filamentous IAV particles − which are observed primarily in clinical infection samples − favor macropinocytosis as their primary mechanism of entry (Rossman et al., 2012). There is also increasing evidence suggesting that receptor tyrosine kinases (RTKs) participate in the uptake of IAV. The binding of IAV to the cell surface results in the activation of the epidermal growth factor receptor (EGFR) and c-Met kinase to promote viral uptake and trigger RTK cascades (Eierhoff et al., 2010).  10  Once internalized, IAV viruses can be recruited to clathrin-coated or clathrin-non-coated pits. However, it has been observed that up to 65% of the bound IAV is internalized in clathrin-dependent endocytosis through the de novo formation of clathrin-coated pits at virus binding sites (reviewed by Edinger et al. 2014). The early endosomes containing the viral particles mature into late endosomes. However, these late endosomes have been deemed ‘intermediate’ endosomes because they are positive for both Rab5 and Rab7 GTPase (Sieczkarski & Whittaker, 2003). In the early endosome, a mildly acidic pH (6.0-5.0) increases the activity of the M2 ion-channel, which allows proton influx into the virion and initiates the dissociation of M1 from the vRNPs. Then, in the late endosomes, the low pH (4.5-5.5) induces a conformation change in HA that exposes the fusion peptide, allowing it to be inserted in the endosomal membrane. This in turn mediates the fusion of the viral envelope with the endosomal membrane, which then results in the release of the vRNPs. These must be dissociated from M1, because M1’s failure to dissociate from the vRNPs results in the inhibition of vRNP nuclear import (reviewed by Edinger et al. 2014; Sun & Whittaker, 2013). Additionally, components of the host aggresome processing machinery that mediate the accumulation of misfolded proteins aggregates such as histone deacetylase 6, dynein, dynactin, and myosin II have been implicated in successful IAV endosomal fusion and M1-vRNP uncoating (Banerjee et al. 2014). 1.1.4. Nuclear transport of influenza vRNPs and NP The nuclear import of individual viral proteins and vRNPs are crucial steps during the IAV life cycle. Due to its size, the nuclear import of IAV vRNPs is a receptor- and energy-dependent process that uses the importin-α/β nuclear import pathway (reviewed by Eisfeld et 11  al. 2014). All proteins forming the vRNP complex possess their own nuclear localization signals (NLSs) and must be transported into the nucleus to assemble progeny vRNPs (Whittaker et al., 1996). These proteins enter the nucleus individually (i.e. NP and PB2) or in a complex (i.e. PB1 and PA) (reviewed by Eisfeld et al. 2014). However, to date it is believed that only the NLSs of NP are necessary for vRNP nuclear import, which requires the presence of importin-α, importin-β, and Ran (O’Neill et al., 1995; Wang et al., 1997). Nevertheless, there is evidence indicating that another NLS motif in either NP or in the polymerases can also mediate the nuclear import of vRNPs (Ketha & Atreya, 2008; Wu et al. 2007a). Once the IAV vRNP-importin-α-importin-β complex is inside the nucleus, it is dissociated by means of the GTPase Ran and the importin-α export receptor CSE1L (chromosome segregation 1 like), allowing the importin-α and importin-β to be transported back to the cytoplasm (reviewed by Eisfeld et al. 2014). The nNLS (referred to here as NLS1) was the first NLS discovered in NP (Wang et al. 1997). It is considered to be a non-classical NLS because it is not rich in arginine or lysine. Wang et al. (1997) also discovered two NP interacting proteins (NPI-1 and NPI-3), which were later shown to be two importin-α isoforms that facilitate the nuclear import of NP. A second NLS was identified on NP (Weber et al. 1998). This sequence conforms to the description of a classical bipartite, NLS (cNLS, referred to here as NLS2), because it contains two basic clusters of amino acids (Weber et al. 1998). Studies suggest that the contribution of NLS1 to the nuclear import of vRNP is more important than the contribution of NLS2 (Cros et al., 2005) because the former seems to be more densely exposed on the vRNPs than the latter (Wu et al., 2007b). Additional experiments demonstrated that NLS1 on NP is the main contributor to the nuclear import of vRNPs (O’Neill et al. 1995; Wu et al. 12  2007a). Nevertheless, NLS2 is still able to contribute to the nuclear import of vRNPs if NLS1 is blocked by antibodies (Wu et al., 2007a). The assembly of new vRNPs takes place in the host cell nucleus and the nuclear export of vRNPs is largely dependent upon the chromosomal maintenance 1 (CRM1) nuclear export pathway (reviewed by Eisfeld et al. 2015). CRM1 (also called exportin-1 or Xpo1p) is a nuclear export receptor that recognizes motifs rich in leucine, known as nuclear export signals (NES) in proteins (Stade et al., 1997). CRM1 binds the NES-containing protein and forms a trimeric complex with Ran-GTP that is exported from the nucleus across the NPC (Stade et al., 1997). CRM1-mediated nuclear export is inhibited by leptomycin B (LMB), which binds covalently to a cysteine residue (cysteine 528 in humans) in the NES binding region of CRM1 (reviewed by Hutten & Kehlenbach, 2006). The addition of LMB to IAV infected cells results in the retention of vRNPs inside the nucleus at the nuclear periphery, which suggests not only a dependence on the CRM1 nuclear export pathway, but also an association of vRNPs with chromatin (reviewed by Eisfeld et al. 2015).  The binding of M1 and NS2/NEP viral proteins to vRNPs is required for nuclear export in a proposed model called the “daisy chain” complex (reviewed by Eisfeld et al. 2015). In this model, the C-terminus of M1 interacts with the vRNP. Then, the vRNP-M1 interacts with the viral protein NS2/NEP (which contains two NES motifs), which in turn binds CRM1-RanGTP and mediates the nuclear export of the complex (reviewed by Eisfeld et al. 2015). Interestingly, the nuclear export of vRNPs is also dependent upon the viral activation of the mitogen-activated protein kinases (MAPK) signaling cascade (reviewed by Zheng & Tao, 2013). The accumulation of HA in the plasma membrane can trigger the 13  activation of this pathway and induce the nuclear export of vRNPs (reviewed by Zheng & Tao, 2013). In summary, through conventional biochemical approaches, genome-wide siRNA screenings and protein interaction assays, a list of nuclear transport factors relevant for influenza A viral infection has been compiled (Watanabe et al., 2014). These host proteins include the following nuclear transport factors: CSE1L, transportin-3 (König et al., 2010), nuclear RNA export factor 3 (Karlas et al., 2010), heat shock protein 70, heat shock protein 90, NXF1, protein kinase B α, MDM2 proto-oncogene, inhibitor of kappa light polypeptide gene enhancer in B-cells kinase epsilon, CRM1, karyopherin subunit beta 1 (importin-β), importin-5, importin-α1/α2, importin-α1/α5 (reviewed by Watanabe et al., 2014). The role of some of these host factors in the nuclear import/export of viral components has been characterized and described above. However, the role of other host proteins remains to be validated. 1.1.5. Viral assembly and budding of IAV After nuclear export, the vRNPs must be transported to discrete viral assembly sites at the plasma membrane. Recent studies have shown that vRNPs transport is dependent on Rab11-positive recycling endosomes and the microtubules network (reviewed by Gerber et al. 2014; Eisfeld et al. 2015). Extensive colocalization between vRNA and vRNPs with Rab11-positive endosomes has been detected; initially the vRNPs and Rab11 are concentrated at the MTOC in order to later be transported along the microtubules to the plasma membrane (reviewed by Gerber et al. 2014; Eisfeld et al. 2015). The disruption of microtubules with depolymerizing agents or the knockdown (KD) of Rab11 result in a 14  decrease in viral titer (reviewed by Eisfeld et al. 2015). In addition, a reduction in Rab11 expression results in a decrease of M2 on the apical membrane in infected polarized cells (Rossman et al., 2010). It has been suggested that once the vRNPs have reached the plasma membrane, the interactions between vRNP-M1 and M1-M2 are responsible for recruiting the vRNPs to the budding site (reviewed by Eisfeld et al. 2015). The mechanism for packaging vRNP fragments into new virions was long elusive. However, the packaging of vRNPs was recently found to be a selective process that depends upon the terminal coding regions present in all vRNP segments and the internal coding regions unique to each segment (reviewed by Eisfeld et al. 2015). In addition, in the virion the vRNPs are packaged in a distinctive “7+1” pattern, which suggests specific vRNP-vRNP interactions (Noda et al., 2006). Finally, new IAV particles originate from lipid rafts in the plasma membrane. M1 is recruited by both HA and NA, which are viral transmembrane proteins that are targeted to lipid rafts through the secretory pathway (reviewed by Veit & Thaa, 2011). Mutations in the HA transmembrane domain responsible for the association of HA with lipid rafts result in the decrease of viral titer (Chen et al., 2005). The M2 protein mediates a cholesterol-dependent modification in membrane curvature during virus budding and mediates membrane scission independent of the endosomal sorting complex required for the transport (ESCRT) pathway (reviewed by Rossman & Lamb, 2011). Lastly, NA is responsible for cleaving sialic acids from glycoproteins and allows new viral particles to be released into the extracellular space (reviewed by Rossman & Lamb, 2011; Eisfeld et al. 2015).  15  1.2. The NPC and nucleoporins One of the main goals of this thesis is to determine the role of Nup153 during IAV infection. Thus, in this section the canonical and non-canonical roles of nucleoporins are discussed. First, the structure, composition, and function of the NPC are reviewed  1.2.1. NPC structure and composition The nucleus compartmentalization exists to maintain the integrity of the genome and regulate gene expression. The traffic of macromolecules between the nucleus and cytoplasm occurs through the NPC. The NPC has a tripartite architecture (Figure 1-3) comprised of a central framework or spoke ring located between a cytoplasmic ring and a nucleoplasmic ring. The main framework of the NPC forms a cylindrical structure composed of eight spokes. Each spoke consists of two roughly identical halves located between the nuclear and cytoplasmic ring structures that are embedded in the nuclear envelope (NE) (reviewed by Kabachinski & Schwartz, 2015). Eight knobs protruding into the cytoplasm are attached to the cytoplasmic face and these are believed to be attachment sites for the eight NPC cytoplasmic filaments (reviewed by Grossman et al. 2012). The nuclear ring is the anchor for eight filaments attached to a distal ring that forms a cage-like structure called the nuclear basket (reviewed by Grossman et al. 2012).  The NPC has a mass of approximately 125 MDa (Reichelt et al., 1990) and its overall structure is conserved among eukaryotes. There are ~30 different proteins called nucleoporins (Nups) building the NPC. These proteins are organized in discrete subcomplexes and are present in multiples copies that form the NPC’s eight-radial 16  characteristic symmetry (reviewed by Grossman et al. 2012). Nups have various dwell times at the NPC, with only a few of them being stably attached to the NPC at all times (Rabut et al., 2004). Nups have been grouped in different categories according to their location at the NPC (i.e. outer ring Nups, inner ring Nups, transmembrane Nups, cytoplasmic filament Nups, and nuclear basket Nups) (reviewed by Schwartz, 2016). The Nup107-160 complex forms the NPC scaffold as the outer ring Nups; the Nup93 complex corresponds to the inner ring complex; the cytoplasmic side of the NPC is constituted by cytoplasmic FG (Phenylalanine-Glycine) Nups such as Nup214, Nup88 and cytoplasmic filament Nups (Nup358); the central channel contains the central FG Nups (Nup68, Nup58, Nup54), and; the transmembrane Nups complex Ndc1 contains Nups such as Pom121, which anchor the NPC to the nuclear membrane. Lastly, the nuclear basket is built up by Nup153, Nup50, and Tpr (translocated promoter region) on the nucleoplasmic side (reviewed by Strambio-De-Castillia et al. 2010; Schwartz, 2016).  17   Figure 1-3. Structural components of the NPC. The NPC model represents the three major structures: the cytoplasmic filaments, the central framework, and the nuclear basket. The most notable nucleoporin complexes that form the major NPC structures are depicted and grouped according to their interactions and location. ONM: outer nuclear membrane. INM: inner nuclear membrane. PNS: perinuclear space. Tpr: translocated promoter region. Nups: nucleoporins.     18  The Nup107-160 scaffolding complex has been well characterized and is crucial for NPC assembly (Walther et al., 2003). It is symmetrically located in both sides of the NPC central framework. This complex is also called the Y complex due to the Y-shaped architecture it assumes when it is isolated from yeast and observed using electron microscopy (EM) (Lutzmann et al., 2002). The Nup93 complex is highly conserved across species and serves as a link between nucleoporin sub-complexes. For example, the complex links transmembrane Nups with the Nup107-160 complex at the NPC scaffold (reviewed by Kabachinski & Schwartz, 2015).  Although NPCs share a global architecture, there are variations in the size of the framework among species such as Xenopus and humans. The NPC framework provides its shape and the permeability barrier is formed by the FG repeats of the nucleoporins that are believed to extend to the center of the NPC channel in a permeable but selective manner (reviewed by Wente & Rout, 2010; Duheron and Fahrenkrog, 2014). 1.2.2. Nuclear transport Translocation through the NPC is limited to cargo with a maximum diameter of 39 nm (Panté & Kann, 2002) and occurs through two mechanisms: passive transport and facilitated or receptor-mediated transport. The passive transport of ions and metabolites occurs by the diffusion of small soluble macromolecules that are ~5 nm or >45 kDa (reviewed by Pouton et al. 2007; Kapon et al. 2010). Facilitated transport through the NPC is signal- and receptor-dependent, and is used by larger macromolecules and often viruses to enter the nucleus (Cohen et al., 2011; Pouton et al., 2007). The nuclear import of proteins is dependent upon the capacity of cytosolic receptors (called karyopherins or importins) to 19  recognize NLSs in the cargo. To date, six different classes of NLSs have been identified (Kosugi et al., 2009). The best characterized is the classical NLS (cNLS), which can be defined as either monopartite or bipartite (reviewed by Kosugi et al. 2009). The monopartite NLS contains a single cluster of basic amino acids residues with a minimum of three basic amino acids such as lysine or arginine, while the bipartite NLS contains two clusters of basic amino acids spaced by a 10-12 amino acids linker (reviewed by Kosugi et al. 2009). The first monopartite cNLS was discovered in the simian virus 40 (SV40) large T antigen, and the NLS of the Xenopus nuclear protein nucleoplasmin is the best example of a bipartite cNLS (reviewed by Lange et al. 2007).  The cNLS-mediated nuclear import of cargo requires importin-α to recognize the cNLS. Importin-α contains two distinctive NLS binding sites, a major grove and a minor grove, and acts as an adapter protein by linking cargos to importin-β1 (reviewed by Kosugi et al. 2009). Importin-α binds importin-β1 through its importin-β binding domain (IBB), and the IBB also functions as an autoinhibitor by binding to the major NLS binding pocket and reducing the affinity of importin-α for NLSs (reviewed by Pouton et al. 2007; Alvisi et al. 2008). Importin-β belongs to the karyopherin-β family, which contains at least 20 human proteins (reviewed by Soniat & Chook, 2015). Importin-β contain a set of 19 tandem helical HEAT (histidine, glutamate, alanine, and threonine) repeat motifs and is composed of two antiparallel helices A and B that are arranged in ring-like structures (reviewed by Soniat & Chook, 2015). Importin-β contains an importin-α binding domain, a GTP binding domain, and two sites that weakly bind directly to FG Nups, which facilitate cargo translocation across the NPC through transient interactions with FG Nups (reviewed by Pouton et al. 2007; Soniat & Chook, 2015). 20  Directionality of nuclear transport results from the compartmentalized distribution of RanGTP. Although Ran is found in both the nucleus and the cytoplasm, RanGDP is found predominantly in the cytoplasm, where the RanGTPase activating protein resides, while RanGTP is mostly found in the nucleus, where the Ran exchange factor converts RanGDP to RanGTP (Pouton et al. 2007; Soniat & Chook, 2015). The trimeric complex cargo-importin-α-importin-β forms in the presence of RanGDP in the cytoplasm and disassemble in the presence of RanGTP in the nucleus. Once the trimeric complex cargo-importin-α-importin-β is translocated to the nucleus, importin-α dissociates from the cargo and recycles back to the cytoplasm by binding to the nuclear export factor CAS (cellular apoptosis susceptibility) (Pouton et al. 2007; Soniat & Chook, 2015). Moreover, importin-β binds RanGTP with negative cooperativity and the complex is recycled back to the cytoplasm. In the cytoplasm, RanGTP is converted to RanGDP by the RanGTPase activating protein, which results in the release of importin-β in the cytoplasm for a new cycle (Pouton et al. 2007; Soniat & Chook, 2015). The nuclear export of proteins is mediated by other members of the karyopherin β family, which are better known as exportins. The export receptor CRM1/Xpo1p binds mainly to proteins that contain a leucine-rich signal (NES) in the presence of RanGTP (Wente and Rout, 2010). The NES was initially discovered in the cellular protein kinase inhibitor (PKI) and the human immunodeficiency virus type 1 (HIV-1) Rev protein (reviewed by Nguyen et al. 2012). CRM1 is also involved in the nuclear export of different classes of cellular RNAs or RNPs, such as U SnRNA, ribosomal RNAs, the signal recognition particle (SRP), and mRNP complexes (reviewed by Nguyen et al. 2012). In summary, the NPC allows bidirectional molecular trafficking by permitting the transport of fundamental proteins and 21  RNAs (mRNAs, ribosomal RNPs, spliceosomal small nuclear ribonucleic particles, and heterogeneous ribonucleoprotein particles) between the nucleus and the cytoplasm. The NPC is also used by viruses to deliver their genome into the nucleus (reviewed by Cohen et al. 2011), taking advantage of the cellular machinery as well as the nuclear import and export pathways.  1.2.3. FG nucleoporins As described above (Section 1.2.1) Nups have been grouped according to their location within the NPC, but they have also been categorized into three distinctive groups according to their predicted secondary structure. Nups in the first group have transmembrane α-helices and cadherin-like domains, and anchor the NPC to the NE. The second group consists of Nups containing α-solenoid and β-propeller folds, which form the NPC scaffold. The last group consists of the FG nucleoporins, which are characterized by repeated clusters of FG motifs and coiled-coil domains, and are involved in receptor-mediated nuclear transport across the NPC (Devos et al. 2006). Approximately 30% of all nucleoporins contain FG regions. The FG motifs are usually FXFG (where X is any amino acid), the FG motifs could also be separated by a small number of hydrophilic linker residue, which is usually 10-20 amino acids in length (Dölker et al. 2010; Milles & Lemke, 2011). These long FG rich regions provide low affinity but high-specificity interactions with the transport receptors involved in cargo translocation across the NPC. The FG regions are natively disordered and lack a secondary structure (Denning et al., 2003). Hence, they are expected to coat the surface of the NPC central channel, while the 22  coiled-coil domains are responsible for anchoring the FG Nups to the NPC scaffold (Devos et al., 2006).  It is well accepted that interactions between the FG repeats with each other constitute an essential part of the permeability barrier of the NPC (reviewed by Zahn et al. 2016). However, even though much research has been conducted in the field of nuclear traffic, researchers have not reach an overall consensus about how the FG nucleoporins facilitate nuclear transport through the NPC permeability barrier. Various cargo translocation models have been proposed to explain the NPC selectivity and the two major models in consideration at the moment are the “selective phase” (hydrogel) model and the “polymer brush” model (reviewed by Kabachinski & Schwartz, 2015). The hydrogel model proposes that FG repeats are extended into the central channel of the NPC, where they become crosslinked, resulting in the formation of a gel that has been shown to occur in vitro (reviewed by Kabachinski & Schwartz, 2015). The transport receptors would carry the cargo across the NPC by binding to the FG repeats and disrupting the FG repeat crosslinking, which would result in the “melting” of the hydrogel (reviewed by Kabachinski & Schwartz, 2015). In the hydrogel model, the charges of the amino acid sequences in the linker between the FG regions appear to be responsible for the strength in the aggregation of these motifs (Dölker et al., 2010). Consequently, there is variability in the strength of FG aggregates, which leads the FG regions to interact with different nuclear transport receptors (Dölker et al., 2010). This interaction provides further specificity to the receptor-mediated nuclear transport process, excluding larger macromolecules. 23  On the other hand, the “polymer brush” model proposes that FG regions are extended and do not interact with each other (reviewed by Musser & Grünwald, 2016). Hence, unfolded FG areas push away the macromolecules through “entropic exclusion”, which the cargo overcomes when transport receptors bind to the FG motifs (reviewed by Musser & Grünwald, 2016). The extended FG filaments collapse in the presence of importin-β, and it has been suggested that this is important for the cargo to cross the NPC (reviewed by Wӓlde & Kehlenbach, 2010). In this model, the FG filaments are non-cohesive, which has been proven to not be the case in vitro by the proponents of the hydrogel model (reviewed by Kabachinski & Schwartz, 2015). However, in vivo, the affinity of FG filaments for transport receptors is expected to differ from those under experimental conditions in which the hydrogel is formed, resulting in a weaker affinity between the transport receptors and the FG regions (reviewed by Musser & Grünwald, 2016).  1.2.4. Non-canonical roles of nucleoporins It has been over 60 years since the NPC was first discovered (Callan & Tomlin, 1950). It was proposed as early as 1985 that the nuclear organization of chromatin in combination with the presence of active transcription regions was related to the distribution of NPCs in the NE (Blobel, 1985). Moreover, the “gene gating” hypothesis proposed that certain transcriptionally active areas were associated to a particular NPC and that the nuclear export of the transcripts would be regulated by the NPC, thereby regulating gene expression (reviewed by Burns & Wente, 2014). Nucleoporins have been involved in gene expression by using the NPC as a scaffolding platform for the recruitment of transcription factors, thus affecting gene expression through “gene gating” (reviewed by Dieppois & Stutz, 2010; Burns 24  & Wente, 2014). Examples of “gene gating” have been observed for highly transcribed and inducible genes in the yeast Saccharomyces cerevisiae, and on occasion it has also been found in Caenorhabditis elegans, Plasmodium falciparum, and Drosophila melanogaster, and mammalian cells (reviewed by Dieppois & Stutz, 2010; Burns & Wente, 2014).  The important notion of “gene gating” would suggest that the defects observed when nucleoporins are mutated or silenced could be a consequence of the role of the NPC facilitating transcription, rather than a consequence of transporting cargo across to the cytoplasm. In yeast and Drosophila, NPC tethered genes can be transcribed and exported out of the nucleus in a coordinated fashion. A co-activator transcriptional complex, SAGA, interacts with the TREX2 complex, a multiprotein complex, forming SAGA-TREX2 (Spt–Ada–Gcn5–acetyltransferase– transcription export complex 2) and anchoring transcription sites to the NPC. Then, THO, a tetrameric protein complex (formed by Hpr1, Tho2, Mft1, Thp2) and a TREX complex known as THO-TREX, facilitate the transcription elongation and export of mRNA to the cytoplasm of the NPC tethered genes (reviewed by Dieppois & Stutz, 2010; Raices & D’Angelo, 2012). However, in human cells, it has been shown that TREX-2 associates to the NPC’s nuclear basket independently of the transcriptional state (Umlauf et al., 2013). The concept of nucleoporins regulating gene expression has been deeply explored in yeast, where Nups have been found bound to transcriptionally active genes (reviewed by Taddei, 2007). Some active genes are associated with NPCs through mRNA production, while others are tied to NPCs through gene recruitment sequences. In addition, the NPC has been involved in the modulation of gene expression through its association with gene loops 25  (reviewed by Raices & D’Angelo, 2012). In yeast, Nups were found to be post-translationally modified by ubiquitylation, which could have implications for the yeast NPC architecture and function (Hayakawa et al., 2012). For example, Nup159 − located on the cytoplasmic side of the NPC − is mono-ubiquitylated, which prevents it from targeting the dynein light chain to the NPC, resulting in a nuclear segregation defect at the onset of mitosis (Hayakawa et al., 2012) Research has also shown that the NPC is important for genome architecture in cases when the chromatin is not condensed in areas around the NPC (reviewed by Raices & D’Angelo, 2012). Further research has shown that the nuclear basket protein nucleoporin Tpr is involved in the maintenance of areas free of heterochromatin (Krull et al., 2010). In Drosophila melanogaster, Megator (Mtor, orthologue of Tpr), and Nup153 have been found to be associated with up to 25% of the genome in areas dominated by nucleoporin-associated regions, which are markers for active transcription (Vaquerizas et al., 2010). Other FG Nups, such as Nup62, Nup50, and Nup88, have also been found interacting with chromatin (reviewed by Hou and Corces, 2010), particularly those involved in regulation of developmental processes and cell cycle. These findings suggest that the NPC’s nuclear basket can play an important role in gene expression and regulation due to its key positioning.  More recent findings have indicated that the NPC is important for cell cycle regulation, gene expression, chromosome positioning in mitosis, and cell differentiation (reviewed by Raices & D’Angelo, 2012). Moreover, new views are starting to emerge suggesting that nucleoporins play important roles in gene expression and regulation, which 26  extends beyond Blobel’s (1985) proposed “gene gating” (reviewed by Raices & D’Angelo, 2012). The fact that the NPC disassembles at the onset of mitosis facilitates nucleoporins’ placement in different areas of the cell to carry out specific roles. An example of these roles is found when the Nup107-160 complex positioning is examined during the early stages of mitosis (Mossaid & Fahrenkrog, 2015). The Nup358 and the Nup107-160 complex localize to the kinetochore, which ensures adequate progression through the cell cycle and proper spindle assembly (Mossaid & Fahrenkrog, 2015). The importance of the Nup107-160 complex is further explained by its involvements in microtubule nucleation at the kinetochores during mitosis, which occurs through cooperation with the gamma-tubulin ring complex (Mishra et al., 2010). Additionally, Tpr is an anchoring site for the spindle assembly checkpoint (SAC) proteins (Schweizer et al., 2013), which has implications for chromosome segregation and cell division. Nup98 also greatly affects cell division and genome integrity due to the interaction of Nup98 with Rae1, which is required for the anaphase-promoting complex/cyclosome (APC/C) (reviewed by Xu & Powers, 2009). Finally, expression levels of nucleoporins such as Nup96, are tightly regulated and dependent on the cell cycle progression, which in turns controls the expression of other cell-cycle proteins that play a role in cell-cycle regulation and progression (Chakraborty et al., 2008).   In higher eukaryotes, the expression of Nup133 (a member of the Nup107-160 complex) is dependent on the cell type and the developmental stage of a developing embryo, resulting essential during neural differentiation (Lupu et al. 2008). Similarly, Nup153 was shown to repress developmental genes and maintain cell pluripotency (Jacinto et al., 2015). 27  Furthermore, a change in NPC composition by the small hairpin RNA depletion or the overexpression of the transmembrane Nup210 was shown to play a role in myogenesis/muscle generation (especially during embryonic development) and neuronal differentiation (D’Angelo et al., 2012). Nup358 has also been implicated in myogenesis through increased Nup358 expression, which is required for myoblast differentiation (Asally et al., 2011).  Nup155, a member of the scaffolding Nups complex, has been involved in mutation that causes atrial fibrillation and early sudden cardiac death in mice (Zhang et al., 2008). Similarly, mutations of Nup154 in Drosophila (a homolog of Nup155) showed tissue-specific phenotypes, and affects male and female fertility (Gigliotti et al., 1998). In mammalian cells, Nup98 has been involved in transcriptional regulation through transcriptional memory, altering expression of an interferon-γ induced gene (reviewed by Ptak et al., 2014). Altogether, mutations or alterations in nucleoporin expression can result in tissue-specific defects by means of gene expression, cell cycle regulation, and cell proliferation. Nucleoporins can thus serve multiple functions beyond nuclear-cytoplasmic transport, although nuclear traffic remains a very important one. 1.2.5. Nucleoporins and viral infections In the previous section the roles of nucleoporins that extend beyond nucleocytoplasmic traffic were discussed. In this section, roles of nucleoporins during 28  various viral infections and how viruses modify nucleoporins upon infection is reviewed. The involvement of Nup153 during various viral infections is further discussed in Section 1.3.4.  The vesicular stomatitis virus matrix protein targets the mRNA export factor Rae1/Gle2, which interacts with Nup98, resulting in inhibition of the host cell mRNA export during the interphase and mitosis (reviewed by Yarbrough et al. 2014). In addition, once the host immune response is activated, expression of Nup98, Nup96, and Rae1/Gle2 increases, resulting in the restoration of the nuclear export of mRNA (reviewed by Xylourgidis & Fornerod, 2009; Yarbrough et al. 2014). Nuclear access for HIV-1 and successful integration of the genome of HIV-1 into the host cell DNA depends on many factors, including the nucleoporins Nup98, Nup85, Nup133, Nup107, Nup160, Nup153, Nup214, Nup358, Nup155, Nup50, and Nup62 (reviewed by Le Sage & Mouland, 2013). The HIV-1 pre-integration complex (PIC) docks to the cytoplasmic filaments of the NPC, specifically binding to Nup358, and is imported into the nucleus and integrated into chromosomes with the aid of Nup98, Nup62, and Nup153 (reviewed by Cohen et al., 2011). Once Nup98 has been depleted, accumulation of HIV-1 complimentary DNA (cDNA) in the nucleus is reduced (Ebina et al., 2004), which suggests participation of Nup98 in the nuclear import of the PIC (reviewed by Cohen et al. 2011). Additionally, localization of Nup62 has been found to be altered during late stages of HIV-1 replication, implicating this nucleoporin in processes other than the nuclear import of the PIC (reviewed by Cohen et al. 2011). Viruses from the Picornaviridae family such as enteroviruses and cardioviruses (both positive-stranded RNA that replicate in the cell cytoplasm) alter the NPC by different 29  mechanisms (reviewed by Yarbrough et al. 2014). Enterovirus infections alter the NPC composition due to the degradation of Nup62, Nup98, and Nup153 at different times post-infection (p.i.), while cardiovirus infections promote the hyperphosphorylation of Nup62, Nup98, Nup153, and Nup214 (reviewed by Cohen et al. 2012; Yarbrough et al. 2014).  During herpes simplex virus-1 (HSV-1) infection, the depletion of Nup214 delays the nuclear import of viral DNA but does not prevent it, while Nup358 KD results in reduced capsid attachment to the NPC (Copeland et al., 2009). Since both Nup214 and Nup358 are part of the NPC cytoplasmic filaments, it has been proposed that the HSV-1 capsid interacts with the NPC cytoplasmic filaments and docks at the NPC to trigger viral DNA release from the capsid through the interactions of the capsid protein UL25 with Nup214 (reviewed by Cohen et al. 2011). Similarly to HSV-1, adenovirus interactions with nucleoporins are related to the capsid docking step at the NPC (reviewed by Fay & Panté, 2015). However, unlike HSV-1, the adenovirus capsid disassembles at the cytoplasmic side of the NPC, which is triggered by the interaction of the capsid with activated kinesin-1 once it has bound to Nup358 (Strunze et al., 2011). Additionally, evidence suggests that during adenovirus infection Nup358, Nup214, and Nup62 are displaced from the NPC to the cytoplasm in order to increase the permeability of the NE (Strunze et al., 2011). 1.2.6. Nucleoporins and IAV Some nucleoporins have been identified as proteins relevant for influenza A viral infection.  In a genome-wide RNAi screen carried on with modified IAV particles to infect 30  Drosophila cells that expressed a influenza-derived reporter gene, it was found that Nup43, Nup153, and Nup98 were important for IAV replication (Hao et al., 2008). Subsequently, another genome-wide siRNA screen performed in human lung epithelial cell line (A549), found Nup214 and Nup153 expression to be required for influenza A viral replication (König et al., 2010). In these two genome-wide RNAi screens a modified IAV expresses a reporter gene from Renilla luciferase instead of containing one of the viral proteins. In both studies, the nucleoporins were initially detected when the cells were infected with the engineered IAV and the expression of the reporter gene was reduced. Due to this methodology, the host factors identified by Konig et al. (2010) only concerned earlier steps of IAV infection, since HA was not expressed and the modified viral particles could proceed to viral assembly, budding, or release. However, the authors (König et al., 2010) further validated the proposed host factors (Nup214 and Nup153) relevant to IAV infection by confirming their requirement to grow wild IAV.  Concurrently, Karlas et al. (2010) also performed a genome-wide siRNA screening using a modified IAV, and found that Nup205 and Nup98 were important to reduce IAV infection by at least 35%.  More recently, Watanabe et al. (2014) through initial means of coimmunoprecipitation of viral and host proteins and subsequent RNAi analysis of host proteins, found that reduced expression of Nup205 and Nup160, affects influenza A viral replication. Some of the common nucleporins found in the genome-wide RNAi screens mentioned above are Nup98, Nup205, and Nup153. The IAV downregulates the levels of Nup98 during infection to negatively affect host mRNA export and increasing cell 31  permissibility to infection (Satterly et al., 2007). This is most likely a viral strategy, as Nup98 and Rae1 expression are induced by interferon (Satterly et al., 2007). Additionally, Nup62 KD inhibits the nuclear export of virus mRNA and vRNA and results in lower viral titers (Morita et al., 2013). Due to the consistent findings that Nup153 expression is important for IAV replication, the role of Nup153 in IAV infection needs to be fully elucidated.  1.3. The nucleoporin Nup153 1.3.1. Nup153 structure and cellular localization Nup153 is a 1,475 amino acid protein localized in the nuclear side of the NPC and is an integral component of the NPC nuclear basket (Pante et al., 1994). This nucleoporin can be divided into three regions based on the sequence of its amino acids: the N-terminus region, the zinc finger region, and the C-terminus region (Figure 1-4).  The N-terminus located at the nuclear ring of the NPC contains an NPC targeting domain, an RNA binding domain, and a NE targeting cassette (NETC) (reviewed by (Duheron and Fahrenkrog, 2014). The zinc finger region at the distal ring of the NPC binds DNA in vitro (Sukegawa and Blobel, 1993). The zinc finger motifs also facilitates Nup153 recruitment of COPI to the NPC, which allows COPI to participate in remodeling the nuclear membrane during NE breakdown (Liu et al., 2003; Prunuske et al., 2006). The C-terminal region contains ~30 irregularly spaced FXFG repeats (reviewed by Ball & Ullman, 2005). 32  The FG repeats appears to be flexible in their location within the NPC, reaching as far as the cytoplasmic side (reviewed by Lim et al. 2006). Table 1-1 provides a complete list of Nup153 binding partners according to the protein region. Studies have shown that the assembly and integrity of the NPC nuclear basket are not affected when Nup153 is depleted (Duheron et al., 2014). However, studies have also shown that the incorporation of Nup153 to the NPC has been deemed necessary for the association of Nup50 to the nuclear basket, while there is controversy as to whether Nup153 recruitment of Tpr to the nuclear basket is required (reviewed by Duheron and Fahrenkrog, 2014).  On the other hand, Nup153 overexpression results in an increase in the formation of annulate lamellae in the endoplasmic reticulum (ER) (Daigle et al. 2001). Annulate lamellae are dense stacks of membranes derived from the ER membrane that contain a high density of NPCs (Daigle et al., 2001). Additionally, the overexpression of Nup153 has been shown to induce changes in nuclear organization, specifically due to the excess of Nup153’s zinc finger motifs (Duheron et al., 2014). The association of Nup153 to the NPC is highly dynamic (Daigle et al., 2001). Nup153 is a highly mobile nucleoporin that shuttles between a nucleoplasmic associated pool and an NPC associated pool. Its mobility appears to be dependent on active transcription, which suggests that Nup153 is involved in mRNA cargo recruitment to the NPC (reviewed by Duheron and Fahrenkrog, 2014).  33   Figure 1-4. Diagram of the Nup153 organization. According to its amino acid sequence, Nup153 is divided into three regions. The N-terminal region is shown as a box containing the NPAR, NETC, and RBD regions. The zinc finger motifs are depicted with folds. The C-terminal region contains the FG-rich region commonly associated with nuclear transport factors. NPAR: nuclear pore association region, amino acids 39-339. NETC: nuclear envelope targeting cassette, amino acids 2-144. RBD: RNA binding domain, amino acids 250-400. Figure modified from Ball & Ullman, 2005.   34  Table 1-1. Nup153 binding partners Protein region N-terminus Zinc finger region C-terminus/ FG rich region Not specified/all regions Mad1 Nup107-160 complex Tpr lamin A/C lamin B Nup50 SENP1/2 transportin 1 RanBP7 RNA COPI Ran GDP/GTP DNA  Nup50 lamin B lamin A/C NXF1/TAP Stat 1 importin-β importin-α HBV HIV-1 integrase SENP1/2 Sun1 NTF2  RanBP5 CRM1 EIF5A Smad2 PU.1 Table adapted from the following reviews: Ball & Ullman, 2005; Duheron & Fahrenkrog, 2014. New information from Woodward et al. 2009; Schmitz et al. 2010; Lussi et al. 2010; Ogawa et al. 2012; Li & Noegel, 2015 was incorporated.   . 35  1.3.2. Nup153 involvement in the cell cycle During interphase, Nup153 recruits the NPC Y complex (Nup107-160 complex) to the inner nuclear membrane (INM) (Vollmer et al., 2015). Using in vitro NE and assembling NPCs from Xenopus egg extracts, it was found that Nup153 interaction with the NE through the Nup153 N-terminal amphipathic helix was required for interphasic NPC assembly (Vollmer et al., 2015). Additionally, it has been proposed that Nup153 is a major player in the spacing of NPCs in the NE through its interactions with the nuclear lamina (Hutchison, 2002; Walther et al., 2001). Lamin B3 was found to bind the C-terminus of Nup153 in cell-free extracts of Xenopus eggs, and when the NE was reconstituted using a dominant-negative lamin B, Nup153 was not found at the NE (Smythe et al., 2000). Furthermore, nuclear aggregates of Nup153 dependent on its N-terminus interactions with mutated lamin A were found in laminopathies (degenerative set of diseases cause by lamin A gene mutations) studies (Hübner et al., 2006). This suggested alterations in Nup153 distribution in laminopathies that could contribute to the pathology of the disease.  Cell cycle progression is also closely linked to the DNA damage response. Due to chromosome accessibility, DNA breakages are repaired in different ways depending on the cell cycle stage. Cyclin-dependent kinases are responsible for regulating cell-cycle transitions by degrading checkpoint proteins (reviewed by Branzei & Foiani, 2008). In this way, the C-terminus of Nup153 is necessary for the nuclear import of 53BP1 (a DNA damage response factor) through the 53BP1-Nup153/importin-β pathway (Moudry et al., 2012). Through its involvement with 53BP1, Nup153 becomes essential for the activation of the DNA damage 36  checkpoints and is responsible for choosing homology-based repair or non-homologous end-joining in DNA double-strand breaks (Lemaître et al., 2012). In a genome-wide siRNA screen, Nup153 was identified as a protein that plays a role in early mitosis (particularly in cells arrested in metaphase) by eliciting defects in the mitotic spindle (Rines et al., 2008). In addition, the reduction in Nup153 levels results in a delay in the progression of mitosis that depends on Nup153 levels of expression (Mackay et al., 2009). Interestingly, only FG motifs of Nup153 are required for the cell to exit from mitosis when Nup153 expression is reduced (Mackay et al., 2009). Further research has linked Nup153 depletion with an increase in unresolved midbodies, failed cytokinesis, and multilobed nuclei (Mackay et al., 2009). On the other hand, the overexpression of Nup153 results in multinucleated cells and the formation of multipolar spindles (Lussi et al. 2010). More importantly, Nup153 expression is associated with cell cycle defects through the inactivation of the SAC, due to the hyperphoshorylation of Mad1, a SAC protein (Lussi et al. 2010). Lastly, active Aurora B, a kinase that prevents cells from dividing aberrantly and is essential for chromosome orientation, is mislocalized in cells expressing reduced levels of Nup153 or a dominant-negative form of this protein (Mackay et al., 2010). These results highlight the importance of Nup153 for cell cycle progression through the direct and indirect effects on the SAC regulators and the Aurora B-dependent abscission checkpoint.  1.3.3. Nup153 and nuclear trafficking of macromolecules The nuclear-cytoplasmic traffic of macromolecules is regulated by the NPC, where FG nucleoporins have been of particular interest due to their interaction with transport receptors. The regulation of nuclear traffic can have consequences for a wide range of 37  cellular events. For example, FG domains in Nup153 are involved in facilitating the nuclear import and nuclear export of a wide variety of cargo (reviewed by Ball & Ullman, 2005). Nup153 interacts with the transport adaptor protein importin-α (Ogawa et al., 2012) and the transport receptors importin-β, transportin, importin-5, and importin-7 (reviewed by Duheron & Fahrenkrog, 2014). The interaction between Nup153 and importin-β is dependent upon the FG domain, while a fragment containing more of the Nup153’s N-terminal region is important for the relationship between Nup153 and transportin-1. These interactions demonstrate that importin-β- and transportin-1-mediated-nuclear transport are two Nup153-related import pathways (Shah & Forbes, 1998). Additionally, Nup153 binds nuclear export receptors such as CRM1, exportin-5, exportin-t, and NXF1, an mRNA export receptor (reviewed by Ball & Ullman, 2005; Duheron & Fahrenkrog, 2014). Lastly, Nup153 binds NTF2, the import receptor for Ran (Cushman et al., 2004). Nup153 interacts with import and export receptors in a RanGTP-dependent manner through the zinc finger region of Nup153 (Nakielny et al., 1999). Furthermore, it has been proposed that the positioning of the Nup153 FG repeats could be relevant to the movement of the cargo-importin-α/β import complex through the NPC. This is because the positioning aids in cargo translocation from the cytoplasm to the nucleus through importin-α/β interactions with the FG rich region of Nup153 (reviewed by Ball & Ullman, 2005). Further structural biology studies have characterized the conformational plasticity of Nup153’s PXFG rich domain (Milles et al., 2015). The individual FG motifs in Nup153 provide fast but specific transport since FG interactions with importins are of low-affinity, but when the FG motifs are combined there is efficient binding of these motifs with the transport receptors (Milles et al., 2015).  38  With respect to nuclear import, it has been reported that the interaction between the N-terminus of Nup153 and Nup50 is not only critical to the NPC targeting of Nup50, but also facilitates binding of Nup50 and importin-α, in addition to other transport factors (Makise et al., 2012). Likewise, disruption of the Nup153-Nup50 relationship has a detrimental effect on nuclear import (Makise et al. 2012). Similarly, multiple reports suggest that in vitro Nup153 facilitates classical NLS mediated nuclear import (Ogawa et al., 2012; Zhou & Panté, 2010). Studies in Xenopus oocytes with an antibody that blocks Nup153 found no changes in nuclear import, but the nuclear export of snRNA, mRNA and 5S rRNA was blocked, as were the NES-dependent protein export and Rev-dependent RNA export (Ullman et al., 1999). However, other studies have found no changes to mRNA export after Nup153 depletion, or few changes (Jacinto et al., 2015; Mackay et al., 2009; Wickramasinghe et al., 2010). This suggests that the effects observed when Nup153 was blocked with an antibody could have been more widespread in the NPC or disrupting the interaction of Nup153 with export factors. In contrast, the overexpression of Nup153 leads to a dramatic nuclear accumulation of poly(A)+ mRNA that is dependent on the C-terminus of the protein (Bastos et al., 1996). In sum, it appears that Nup153 is of key importance to the cell due to its localization in the nuclear basket and its wide range of protein interactions. However, further investigation is required to determine the biological relevance of some of the afore-mentioned interactions and distinguish the ones that are a product of chance due to Nup153’s location from those that are necessary for cell functioning.  39  1.3.4. Nup153 and its role during viral infections As mentioned above, various viruses of the Picornaviridae family trigger the proteolytic degradation of several nucleoporin, including Nup153 (reviewed by Yarbrough et al. 2014). Poliovirus infection results in the degradation of Nup153 by the viral protease 2A, while human rhinovirus (from the genus Enterovirus) infection degrades Nup153 through viral protease 3C pro and its precursor form, 3CD (reviewed by Yarbrough et al. 2014). The degradation of Nup153 among other nucleoporins due to enterovirus infection results in NPC permeabilization, disruption of nuclear traffic, and the mislocalization of nuclear proteins such as Sam 68, nucleolin, and La (reviewed by Yarbrough et al. 2014; Le Sage & Mouland, 2013). Additionally, the encephalomyocarditis virus, also a member of the Picornaviridae family, utilizes the host cell kinases by means of its L protein to hyperphosphorylate multiple nucleoporins, including Nup153. The L protein also binds to Ran and disrupts the cytoplasmic traffic of new mRNAs (Porter et al., 2006). HSV-1 infection results in a decrease of Nup153 expression and the displacement of Nup153 from the NPC into the cytoplasm during early infection (reviewed by Le Sage & Mouland, 2013). Additionally, the gammaherpesvirus Epstein-Barr virus encodes the viral protein kinase BGLF4, which is located predominantly in the nucleus (reviewed by Chang et al. 2012). The nuclear import of BGLF4 appears to occur independently of NLSs and relies on interaction with Nup62 and Nup153 to facilitate its translocation into the host cell nucleus (Chang et al., 2012). Hepatitis B virus (HBV) is a DNA virus that requires access to the cell nucleus to replicate its genome. The HBV capsid is imported through the NPC since they contain NLSs 40  in the capsid protein that are exposed upon phosphorylation and interact with the importin-α/ β complex to facilitate nuclear import (reviewed by Le Sage & Mouland, 2013; Fay & Panté, 2015). Interestingly, HBV capsids bind the FxFG domains of Nup153 in the nuclear basket and only mature capsids are released into the nucleoplasm, while immature HBV capsids remained arrested in the nuclear basket (Schmitz et al., 2010). Moreover, the interaction of HBV capsids with Nup153 results in a reduction of nuclear import of NLS-tagged bovine serum albumin (BSA), which Schmitz et al. (2010) suggest is due to Nup153’s obstruction of the importin-β interaction site (Schmitz et al., 2010). Nup153 was identified in two genome-wide RNAi screens for host factors involved in HIV-1 infection (Brass et al., 2008; König et al., 2008). The HIV-1 is a retrovirus and the PIC requires access to the host cell nucleus in order to replicate and to incorporate its pro-viral cDNA into the host genome. The depletion of Nup153 (and Nup358) decreased HIV-1 integration into the host cell genome and has been deemed important for the nuclear import of the PIC (reviewed by Cohen et al. 2011; Matreyek & Engelman, 2013). The HIV-1 capsid’s interaction with Nup153 is the dominant determinant in Nup153 dependency, since missense mutations in HIV-1 viral capsid rendered the virus insensitive to Nup153 depletion (reviewed by Le Sage & Mouland, 2013). Additionally, it has been proposed that Nup153 directs the integration of the PIC to sites in the host cell chromosome (along with Nup62 and Nup98) (reviewed by Le Sage & Mouland, 2013). Finally, there is data proposing that Nup153, along with Nup214, Nup98, and Nup62, interact indirectly with the HIV-1 vRNP transport complex to facilitate nuclear export (reviewed by Cohen et al. 2011; Le Sage & Mouland, 2013). 41  1.4. The cytoskeleton and nucleoskeleton 1.4.1. The cytoskeleton and nucleoskeleton The complex network of the cell cytoskeleton is formed primarily by microtubules, filamentous actin (F-actin), and intermediate filaments. Together, the different cytoskeletal elements provide the cell with shape, intracellular organization, functional specialization, movement, and a connection with the extracellular environment (reviewed by Fletcher & Mullins, 2010; Huber et al. 2013). Microtubules are polar protofilaments formed by the polymerization of α-tubulin and β-tubulin in the presence of GTP (reviewed by Wickstead & Gull, 2011). Microtubules are dynamic and facilitate intracellular transport, organelle positioning, and chromosomal segregation during cell division (reviewed by Alfaro-Aco & Petry, 2015). Actin monomers (G-actin) polymerize into dynamic helical filaments called F-actin (reviewed by Huber et al. 2013). The assembly and disassembly of actin filaments are highly responsive to signaling pathways (reviewed by Lee & Dominguez, 2010). Through the different organization of actin filaments (branched or bundled), the actin network facilitates cell motility (for example during cell migration and chemotaxis) and shapes cells in response to stress or signals (reviewed by Fletcher & Mullins, 2010). Finally, intermediate filaments are composed of overlapping polypeptide dimers resulting in the formation of cables without polarity, and aid in organelle positioning and the mechanical stabilization of the cell to guarantee mechanical integrity (reviewed by Wickstead & Gull, 2011). The intermediate filaments in vertebrates are an entire family of proteins composed primarily by five major types (I-V) which vary in expression level according to cell type (reviewed Lowery et al. 2015). The intermediate filaments that are of importance for this thesis are type III, vimentin, 42  which is expressed in fibroblast and endothelial cells, and type V, nuclear lamins (reviewed Lowery et al. 2015). The latter include lamin A/C, lamin B1, and lamin B2, all of which are key members of the nucleoskeleton and contribute to the integrity of the nucleus (reviewed by Fletcher & Mullins, 2010). The actin filaments, microtubules, and intermediate filaments are anchored to the NE through the LINC complex (linker of the nucleoskeleton and cytoskeleton) (Figure 1-5). This connection allows the transmission of force that occurs between the cytoskeleton and the nucleoskeleton (reviewed by Isermann & Lammerding, 2013). The nucleoskeleton is a term that groups all the skeletal elements from the cell nucleus. Similarly to the cytoskeleton, but with a higher degree of stiffness, the nucleoskeleton provides the cell nucleus shape and mechanical responses to stimuli (reviewed by Simo & Wilson, 2011). More importantly, the nucleoskeleton is implicated in gene expression through the organization of chromatin and nuclear architecture (reviewed by Simon & Wilson, 2011; Chow et al. 2012). The nucleoskeleton components include nuclear lamins, nuclear mitotic apparatus networks, spectrin, titin, nuclear actin, and various myosin and kinesins (reviewed by Simon & Wilson, 2011). Interestingly, defects in nucleoskeleton elements such as the lamins result in diseases known as laminopathies, which include Hutchinson-Gilford progeria syndrome and Emery-Dreifuss muscular distrophy (reviewed by Simon & Wilson, 2011). More specifically, cells in which lamin A/C or emerin are absent display a supression of mechanoresponsive genes (reviewed by Isermann & Lammerding, 2013).   43  1.4.2. The LINC complex The LINC complex couples both the nucleoskeleton and the cytoskeleton by bridging the inner nuclear membrane (INM) with the outer nuclear membrane (ONM) through the perinuclear space (Figure 1-5) (reviewed by Horn, 2014). The LINC complex is formed by two families of proteins: SUN domain and KASH domain proteins. The SUN (Sad1 and UNC-84) domain proteins are located in the INM and interact with chromatin, the nuclear lamina, and nucleoporins (reviewed by Isermann & Lammerding, 2013). There are at least five SUN domain proteins in mammals, with Sun1 and Sun2 being expressed in many tissues, while Sun3, Sun4, and Sun5 are mostly associated with testes (reviewed by Horn, 2014). The localization of SUN domain proteins to the INM is essential for the formation of the LINC complex, since KASH domain proteins which reside in the ONM are connected with the nucleoskeleton by binding to SUN domain proteins (reviewed by Horn, 2014). The KASH (Klarsicht, ANC-1 and SYNE homology) domain proteins interact with various elements of the cytoskeleton. For example, nesprin-1 and nesprin-2 interact with F-actin and regulate fibroblast migration (reviewed by Horn, 2014). Nesprin-1 and nesprin-2 also interact with microtubules through kinesin-1 and dynein, while nesprin-3 interacts with intermediate filaments through plectin and is also expressed in a variety of tissues (reviewed by Horn, 2014).  Disruptions of the LINC complex result in an interruption of force transmission between the cytoskeleton and the nucleus, which results in defective cellular mechanosensing and mechanotransduction, as well as cell migration in processes such as wound healing or cancer (reviewed by Isermann & Lammerding, 2013). Moreover, in humans, nesprin-4 44  mutations that result in the mislocalization of the protein because it is missing the KASH domain are associated with hearing loss (reviewed by Horn, 2014). 1.4.3. Nup153 and the nucleoskeleton  The NE delimits the nucleus from the rest of the cell. On the nucleoplasmic side of the NE, the INM contains embedded proteins such as SUN domain proteins, LAP2, LBR, and emerin. These proteins are in direct contact with the nuclear lamina, chromatin, and NPCs (reviewed by Wilson & Berk, 2010). Because of the NPC’s positioning, elements of its nuclear basket can also interact with INM embedded proteins and nucleoskeleton elements. To that extent, Nup153 binds the INM through its N-terminus during interphase (Vollmer et al., 2015) and interacts with lamina and Sun1 (reviewed by Duheron & Fahrenkrog, 2014). Accordingly, a reduction in Nup153 expression results in alteration of lamin A/C and Sun1 immunolabelling (Zhou & Panté, 2010). Moreover, when lamina assembly is disrupted, Nup153 is not recruited to the nuclear basket – while other nucleoporins are (Smythe et al. 2000). Finally, it has been proposed that Nup153 anchors the NPCs to the nuclear lamina, while Sun1 is necessary to assemble and space NPCs (reviewed by Simon & Wilson, 2011). Combining these findings underscores the importance of Nup153 positioning in the nuclear basket of the NPC and its tight interrelationship with major nucleoskeleton elements such as lamins and LINC complex proteins.  45   Figure 1-5. Diagram of the LINC complex organization. The LINC complex interconnects the nucleoskeleton and the cytoskeleton through the NE. The LINC complex is comprised of SUN domain proteins and KASH domain proteins. The INM and ONM are bridged in high curvature areas through the insertion of the NPC. The ONM is continuous with the ER membrane. The INM hosts lamina-associated peptide (LAP) proteins, emerin, and SUN domain proteins that interact with chromatin, the nuclear lamina, and with KASH domain proteins, nesprins, in the perinuclear space. KASH domain proteins (nesprin-1 to nesprin-4) are found traversing the ONM and tethering cytoskeletal components directly, through adaptor proteins (plectin) or cytoskeletal motors (kinesin). ONM: outer nuclear membrane. INM: inner nuclear membrane. NPC: nuclear pore complex. ER: endoplasmic reticulum.    46  1.5. Research aims Several genome-wide RNAi screens identified multiple nucleoporins as host factors that negatively impacted IAV replication (Hao et al., 2008; Karlas et al., 2010; König et al., 2010). Nup153 was one of the few repeatedly proteins found in these screens that was important for IAV infection (Hao et al., 2008; König et al., 2010). IAV requires translocation through the NPC for the nuclear import of incoming vRNPs and viral proteins that assemble progeny vRNPs in the nucleus, as well as for the nuclear export of progeny vRNPs. Hence, we the hypothesis that Nup153 was required for the nuclear transport of IAV viral proteins and vRNPs was formulated. This hypothesis was tested by following the progression of IAV infection in cells depleted of Nup153 by RNAi and by studying the effect of Nup153 KD in the nuclear import of NP and chimeric proteins containing NP’s NLSs. Given that nucleoporins have roles in cellular processes other than nucleocytoplasmic transport, such as involvement in gene expression and cell cycle regulation, it is important to study not only the specific role of Nup153 in the nuclear transport of IAV vRNPs and/or viral proteins, but also during other steps of the IAV life cycle. Thus, whether other steps of the IAV infective cycle were defective in Nup153 KD cells was also studied. In addition, it is important to define the effects that Nup153 RNAi depletion exerts on the cell, which may explain defects on the IAV life cycle. Our studies of the role of Nup153 in IAV infection open the possibility of considering nucleoporins as important factors for the development of future antiviral treatments. The specific objectives of my thesis are as follows. 1.5.1 Aim 1: To determine the role of Nup153 during the IAV infective cycle 47  Multiple studies have associated Nup153 depletion with a reduction of IAV viral titers (Hao et al. 2008; König et al. 2010; York et al. 2014; Watanabe et al. 2014; Benitez et al. 2015). However, the step(s) of the IAV infective cycle that is/are affected by Nup153 KD have not yet been determined. We hypothesized that the nuclear transport of IAV vRNPs, vRNAs, or viral proteins might be disrupted in Nup153 KD cells due to the importance of Nup153 in mediating nuclear-cytoplasmic transport. The experiments in Chapter 3 were designed to study the progression of IAV infection in Nup153 depleted HeLa cells by monitoring the localization of NP and M1 (two of the main viral proteins) using immunofluorescence microscopy. Since NP is responsible for the nuclear import of influenza vRNPs, the nuclear import of both NP and chimeric proteins containing NP’s NLSs in Nup153 depleted cells was also studied.  1.5.2. Aim 2: To determine the downstream effects of Nup153 depletion on endocytosis and intracellular traffic in HeLa cells In pursuit of Aim 1, defects in the intracellular traffic of IAV were found. To determine whether this was specific to IAV infection or whether other cellular pathways were also affected, the cellular uptake and early trafficking of the epidermal growth factor (EGF) and transferrin (Tfn) in uninfected Nup153 KD cells were examined. In addition,  the cellular localization of the EGF and Tfn receptors (EGFR and TfR) in ininfected cells were determined. Given that the intracellular traffic of these two ligands was affected and they, as well as IAV, use the endocytic pathway, next the cellular localization of endocytic organelles were studied. The results showed defects on the distribution of these organelles. Since during an IAV infection HA uses these organelles to be targeted to the plasma membrane, HA 48  trafficking in Nup153 KD cells was also studied. The results of these experiments are presented in Chapter 4.  1.5.3. Aim 3: To determine the effect of Nup153 RNAi depletion on nuclear and cell morphology Analysis of the data in Chapter 4 allowed us to begin building a comprehensive view of the major changes arising in HeLa cells as a consequence of Nup153 depletion. One of the major alterations was the distribution of endocytic organelles. Since endosome position and distribution is impacted by the cytoskeleton (Granger et al., 2014), whether the three cytoskeletal elements were affected in Nup153 KD cells was studied in Chapter 5. In addition, because the cytoskeleton plays an important role in the establishment of cellular and nuclear morphology whether Nup153 depletion disturbed these was also determined. The results showed that Nup153 KD depletion resulted in a plethora of cellular defects, including alteration of the immunolabelling of F-actin, microtubules, and the vimentin network, as well as blebbing of the plasma membrane and alterations of the nuclear and cellular architecture. This is the first time that changes to all three major cytoskeletal elements related to nuclear deformations and transmembrane protein mistargeting have been described in Nup153 depleted cells. Thus, my findings established for the first time that Nup153 is an important player in the maintenance of cellular homeostasis.  49        Chapter 2 Materials and Methods Materials and Methods 2.1. Cell cultures  HeLa cells, Madin-Darby canine kidney (MDCK) cells, and HeLa cells stably expressing a fusion of GFP to lamina-associated polypeptide 2β (GFP-LAP2β, courtesy of Dr. U. Kutay, ETH Zurich) were cultured at 37°C and 5% CO2 in Dulbecco's modified Eagle medium (DMEM, Sigma-Aldrich, catalog number: D5671) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich; catalog number: F1051), 1% penicillin-streptomycin (Cellgro, catalog number: CO-02-CI), 2 mM L-glutamine (Cellgro, catalog number: 25-005-CI), and 1 mM sodium pyruvate (Thermo Fisher Scientific, catalog number: 11360-070).  2.2. Virus, reagents and antibodies  IAV strain A/X-31, A/Aichi/68 (H3N2), was purchased directly from Charles River (Charles River, Catalog number: 10100374). Leptomycin B (Sigma-Aldrich, catalog number: L2913) was used to block nuclear export of NP during transfection experiments. Because the viral infectivity of IAV (H3N2) is dependent upon cleavage of HA by exogenous proteases, endogenous trypsin was used to cleave HA during the infection experiments. The trypsin used was L-1-tosylamide-2-phenylethyl-chloromethyl ketone (TPCK)-treated mycoplasma- and extraneous virus-free trypsin (Worthington Biochemical Company, catalog number: LS003740). Rhodamine-phalloidin was used to detect F-actin during immunofluorescence experiments (Sigma-Aldrich, catalog number: P5282). Cell nuclei were visualized in fluorescence microscopy by using ProLong Gold Antifade Mountant with 4',6-diamidino-2-50  phenylindole (Dapi) (Thermo Fisher Scientific, catalog number: P36935) in fixed cells experiments and NucBlue Live ReadyProbes Reagent (Thermo Fisher Scientific, catalog number: R37605) during live cells experiments. The lectin wheat germ agglutinin (WGA) conjugated to Alexa Fluor 594 (Alexa Fluor 594 WGA; Thermo Fisher Scientific, catalog number: I34406) that binds to N-acetylglucosamine and sialic acid residues in the plasma membrane was used to detect the distribution of these glycosylated residues in live cells. The experiments to detect uptake of EGF were performed using Alexa Fluor 647 EGF complex (Thermo Fisher Scientific, catalog number: E35351) and pHrodoTM EGF conjugate (Thermo Fisher Scientific, Catalog number: P35374). The experiments to detect Tfn uptake were performed using Tfn from human serum, fluorescein conjugate (Thermo Fisher Scientific, catalog number: T2871). Live cell dextran uptake experiments were performed using Alexa Fluor 488 Dextran (10,000 Da MW; Thermo Fisher Scientific, catalog number: D-22910) as a ligand to stimulate macropinocytosis. LysoTracker Green DND-26 (Thermo Fisher Scientific, Catalog number: L7526) was used during live cell experiments to visualize acidic endocytic compartments. Staurosporine (Sigma-Aldrich, catalog number S5921) was used to induce apoptotic cell death. MitoTracker Red CMXRos (Thermo Fisher Scientific, catalog number: M7512) was used for mitochondria identification and visualization through live cell microscopy. To detect Nup153, two anti-Nup153 antibodies were used as needed, mouse monoclonal anti-Nup153 antibody derived from SA1 hybridoma clone (kindly provided by Dr. Brian Burke, Institute of Molecular and Cell Biology, Singapore) was used for immunofluorescence (dilution 1:250) and immunoblotting (dilution 1:10), and commercial 51  rabbit polyclonal anti-Nup153 antibody (dilution 1:500; Abcam, catalog number: Ab84872) was used for immunofluorescence. All other antibodies used are listed in Table 2-1. The SV40 cNLS-DS red plasmid was a kind gift from Dr. Robert Nabi’s lab (UBC). The pEGFP-5GFP construct used in this project was a kind gift of Dr. Gergely L. Lukacs (McGill University) and it was used by Dr. Wei Wu to generate the 5GFP-NLS1 and 5GFP-NLS2 constructs (Wu, 2014). The NP construct (A/WSN/1933/H1N1) used was kindly provided by Dr. Gary Whittaker (Cornell University). The HA (IAV A/PR8/1934/H1N1) construct was a kind gift from Dr. Honglin Chen (University of Hong Kong) and Dr. Robert Webster (St Jude Children Research Hospital).  2.3. Small interference RNA (siRNA) and recombinant DNA transfection For siRNA, HeLa cells were seeded at a confluency of approximately 75,000 cells per milliliter on round glass coverslips (Thermo Fisher Scientific, catalog number: 12-545-81) and transfected following manufacturer’s instructions with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, catalog number: 13778075) to a final concentration of 10 nM of Nup153 siRNA (Zhou & Panté, 2010) (Dharmacon) or 10 nM of ON-TARGET plus Non-targeting Control siRNA (Dharmacon, catalog number: D-001810-10-05) hereafter referred to as Control siRNA. The Nup153 siRNA targeting the nucleotide regions 2593-2615 of human Nup153 (AAGGCAGACUCUACCAAAUGUTT) previously reported to KD Nup153 (Zhou & Panté, 2010) was used. Expression of Nup153 was assessed by immunofluorescence microscopy or immunoblotting 48-72 h after initial transfection. 52  Cell transfection of recombinant DNA was performed 48 h after cells had been transfected with RNAi sequences. Transfection of 5GFP, 5GFP-NLS1, 5GFP-NLS2, cNLS SV40-DS red, NP (A/WSN/1933/H1N1), and HA (IAV A/PR8/1934/H1N1) plasmids were carried using Lipofectamine 2000 (Thermo Fisher Scientific, Catalog number: 11668027) following manufacturer’s instructions. After transfection, cells were then either prepared for immunofluorescence microscopy or live cell microscopy. 2.4. Influenza A virus infection HeLa cells were seeded on glass coverslips and treated with the siRNA as indicated above; 48 h after siRNA treatments, cells were mock infected or infected with purified IAV (strain A/X-31, A/Aichi/68 (H3N2)) at a multiplicity of infection (MOI) of 4 plaque unit formation per cell (PFU/cell). Cells were first incubated with the virus in infection media (DMEM supplemented with 0.2% FBS, 1% penicillin-streptomycin, 1mM sodium pyruvate, 2 mM L-glutamine, and 0.1% TPCK-trypsin) for 15 min at 4°C to allow for viral binding to the cell receptors at the plasma membrane. After this period, the virus-containing media was removed from the cells, fresh infection media was added, and cells were incubated for 1 h at 37°C (or less if the infection time in the experimental procedure was under 1 h) to promote internalization of the virions attached to the plasma membrane. After this period, a mild acidic wash with acidic phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 2.9 mM Na2HPO4, 1.8 mM KH2PO4, and HCl to pH to 5.5) was performed to remove non-internalized viral particles attached to the cell exterior. Then, cells were incubated with infection maintenance media (DMEM supplemented with 2% FBS, 1% penicillin-streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, and 0.1% TPCK-trypsin) at 37 °C 53  for 5 min to 24 h, as indicated in the figure legends, before being prepared for the next step of the experimental procedure. Table 2-1. List of primary and secondary antibodies  Primary antibodies Antibody name Host species Company Catalog number Dilution used Technique anti-Arp2 rabbit polyclonal Abcam Ab47654 1:250 IF anti-M6PR rabbit monoclonal Abcam Ab134153 1:100/ 1:1000 IF/ WB anti-cleaved caspase-3 rabbit polyclonal Cell Signaling Technology 9661 1:400 IF anti-EEA1 rabbit monoclonal Cell Signaling Technology 3288 1:100 IF anti-EGFR rabbit monoclonal Cell Signaling Technology 4267 1:50/ 1:1000 IF/ WB anti-ERp72 rabbit monoclonal Cell Signaling Technology 5033 1:100 IF anti-human influenza A (anti-H1) mouse monoclonal Takara Clontech M146 1:500 IF anti-human influenza A (anti-H3) mouse monoclonal Takara Clontech M145 1:500 IF anti-LAMP-1 rabbit polyclonal Abcam Ab24170 1:250/ 1:700 IF/ WB anti-M1 mouse monoclonal Acris SM1748P 1:1000 IF anti-NP mouse monoclonal Acris AM01375PU 1:1000 IF anti-Rab11 rabbit monoclonal Cell Signaling Technology 5589 1:100 IF anti-Rab7 rabbit monoclonal Cell Signaling Technology 9367 1:100/ 1:100 IF/ WB anti-TfR mouse monoclonal Thermo Fisher Scientific 136800 1:500/ 1:500 IF/ WB anti-tubulin rabbit serum Sigma Aldrich T3526 1:10000 IF Antibody name Host species Company Catalog number Dilution used Technique anti-vimentin mouse monoclonal Sigma Aldrich V6389 1:400 IF 54  anti-β actin mouse monoclonal Abcam Ab6276 1:5000 WB anti-β COP rabbit polyclonal Abcam Ab2899 1:2000 IF  Secondary antibodies Antibody name Conjugate Host species Company Catalog number Dilution used Technique anti-Rabbit IgG (H+L), Alexa Fluor 488 goat Thermo Fisher Scientific A-11008 1:1500 IF anti-Mouse IgG (H+L) Alexa Fluor 488 A-11001 anti-Mouse IgG (H+L) FITC F-2761 anti-Mouse IgG (H+L) Alexa Fluor 405 A-31553 anti-Rabbit IgG (H+L) Alexa Fluor 405 A-31556 anti-Rabbit IgG (H+L) Alexa Fluor 594 A-11037 anti-Mouse IgG (H+L) Alexa Fluor 647 A-21236 anti-Rabbit IgG (H+L) Alexa Fluor 647 A-21244 anti-Mouse IgG (H+L) Alexa Fluor 568 A-11004 anti-Mouse IgG (H+L) Rhodamine Red R-6393 anti-mouse IgG (whole molecule) Peroxidase Sigma-Aldrich A4416 1:2000 WB anti-rabbit IgG (whole molecule) Peroxidase A0545 Abbreviations: immunofluorescence (IF), Western blot (WB), immunoglobulin G (IgG), heavy + light chains (H+L). 55  2.5. Plaque assay Plaque assays were performed to estimate the amount of infectious progeny virions produced during infection of HeLa cells with IAV. To obtain the cell culture supernatants used for plaque assays, HeLa cells were infected as indicated above for a period of 24 h. Next, the cell culture medium was collected and filter through a 0.22 or 0.45 µm filter (Sarstedt, catalog number: 83.1826) to remove cell debris. To perform the plaque assay, MDCK cells were seeded at a high confluency 2-3 days prior to the plaque assay in 6-well plates and deemed appropriate to use in plaque assay when a clear monolayer was established. The infectious supernatants were serially diluted in PBS containing 0.1% TPCK-trypsin and added to the monolayers of MDCK cells. Cells were then incubated for 1 h at room temperature in an orbital shaker and shake at 60 revolutions per minute. Next, the solutions were removed and cells were rinsed twice with PBS. Afterwards, cells were covered with a layer of nutrient agar overlay (1% agarose, 0.1% TPCK-trypsin and 1% penicillin-streptomycin in minimum eagle medium). Plates were then incubated at 37 °C and 5% CO2 for 72 h. Subsequently, the virus was inactivated with Carnoy’s reagent (60% ethanol, 30% chloroform, and 10% glacial acetic acid) and cells were fixed with 4% formaldehyde for 20 min. To visualize plaques, cells monolayers were stained with 1% crystal violet (in 20% methanol) for 1 h, rinsed with water and allowed to air dry. Non-stained circular spaces were identified as plaques. Plaques were counted and averaged from three separate wells. Viral titers were expressed as PFU/ml = [(numbers of plaques per well) X dilution]/(volume of inoculum).   56  2.6. Blocking nuclear export of NP with leptomycin B  HeLa cells were seeded on round 1.5 mm glass coverslips and transfected with either siRNA for 48 h as described in Section 2.3. Cells were then transfected with NP construct (as described in Section 2.3) for 6 h, cell culture media was then changed, leptomycin B added to a final concentration of 11 nM (diluted in cell culture media) and cells were incubated for 18 h as per Cross et al. (2005). Cells were then prepared for immunofluorescence and image acquisition (as described in Section 2.7.1). 2.7. Indirect immunofluorescence microscopy  Cells on round 1.5 mm glass coverslips treated as indicated above were fixed with 4% paraformaldehyde in PBS for 15 min, gently washed with PBS, followed by 5 min of permeabilization with 0.2% Triton X-100 (Sigma-Aldrich, Catalog number: T8787) or 0.2% Tween20 (used only for anti-HA1 antibody). Coverslips were incubated in blocking buffer (PBS containing 2.5% bovine albumin serum (BSA) (Sigma-Aldrich, catalog number: T8787) and 10% goat serum (Sigma-Aldrich, catalog number: G9023) at room temperature for 1 h or 4°C overnight. After blocking, cells were incubated with primary antibodies diluted in blocking buffer for 1 h at room temperature or 4°C overnight. Next, cells were washed gently three times at 10 min intervals with PBS and then incubated with the appropriate fluorophore conjugated secondary antibody, diluted in blocking buffer, for 1 h at room temperature. When F-actin was labelled with rhodamine phalloidin, the reagent was added to the mix of secondary antibodies. Coverslips were then washed three times at 5 min interval with PBS and mounted with ProLong gold antifade reagent containing Dapi.  57  For immunolabelling using antibodies purchased from Cell Signaling Technology, no separate permeabilization step was performed. Instead, blocking and cell permeabilization were performed in one step incubating the cell with the blocking-permeabilization buffer (PBS containing 5% goat serum and 0.3% Triton X-100) for 1 h. The buffer used to dilute the antibodies was PBS containing 1% BSA and 0.3% Triton X-100, and the incubation of samples with primary antibodies was always performed overnight at 4 °C. 2.8. Confocal microscopy For both indirect immunolabelling and live cell microscopy images were acquired using a confocal laser scanning microscope (Olympus Fluoview FV1000). Live cell image acquisition was performed at 37 °C for the experimental time points indicated in the figure legends. All the fluorescence images shown are representative of at least three independent experiments. 2.9. Ligand uptake experiments 2.9.1. Uptake of EGF HeLa cells were seeded on 35 mm glass bottom dishes (MatTek, catalog number P35G-0.170-14-C) and treated with control siRNA or Nup153 siRNA for 72 h. After 48 h of RNAi transfection, media was changed and substituted for DMEM without FBS; cells were serum starved for 24 h prior to the assay. Cells were incubated with NucBlue and Alexa Fluor 647 EGF complex (10 μg/ml) or pHrodo red EGF conjugate (3 μg/ml) (both EGF ligands were diluted in serum free cell culture media) for 30 min at 4 °C. After this incubation time, cells were rinsed with PBS and then L-15 medium (Leibovitz; Sigma-58  Aldrich, catalog number: L5520) imaging media was added. Cells were warmed up to 37 °C for 5 min prior to the beginning of imaging.  2.9.2. Uptake of Tfn HeLa cells were seeded on 1.5 mm glass coverslips and transfected with control siRNA or Nup153 siRNA for 72 h as indicated above. After 48 h of RNAi transfection, media was changed and substituted for cell media without FBS. Cells were serum starved for 24 h prior to the assay and then incubated with 20 µg/ml of fluorescein isothiocyanate (FITC)-conjugated Tfn (diluted in serum free cell culture media) at 4 °C for 15 min. Then, cells were warmed up to 37 °C for the indicated time in the figure legend before fixation and sample processing for immunofluorescence as described in Section 2.7. 2.9.3. Uptake of dextran HeLa cells were seeded on glass bottom 35 mm dishes (MatTek, catalog number P35G-0.170-14-C) and treated with control siRNA or Nup153 siRNA for 72 h. The cell media was replaced with fresh cell culture media. Cells were incubated with NucBlue and Alexa Fluor 488 Dextran (0.5 mg/ml) diluted in serum free cell culture media, for 30 min at 37 °C to allow for cell uptake. Cells were then rinsed with PBS and L-15 medium (Leibovitz; Sigma-Aldrich, catalog number: L5520) imaging media was added. Live cell image acquisition was performed at 37 °C one hour after the initially incubating cells with dextran and 30 min after dextran was removed.    59  2.10. Plasma membrane and organelles labelling 2.10.1. Plasma membrane labelling  HeLa cells were seeded on glass bottom 5 mm dishes (MatTek, catalog number P35G-0.170-14-C) for live cell microscopy and treated with control or Nup153 siRNA for 72 h as indicated above. The media was removed and cells were then rinsed twice with PBS, then Alexa Fluor 594 WGA was added to cells to a final concentration of 5 μg/mL in Hank's balanced salt solution (Thermo Fisher Scientific, catalog number: 14175079) and incubated for 10 min at 37 °C. Cells were then rinsed twice with PBS and L-15 medium (Leibovitz; Sigma-Aldrich, catalog number: L5520) imaging media was added. Live cells were kept in a heated chamber at 37 °C during image acquisition.  2.10.2. LysoTracker and MitoTracker labelling HeLa cells were seeded on 8-well glass bottom slide (Ibidi, catalog number: 80827) and treated with control or Nup153 siRNA for 72 h as indicated above. LysoTracker Green DND-26 was added to a final concentration of 50 nM (diluted in cell culture media), and cells were incubated at 37 °C for 30 min. For mitochondria labelling, MitoTracker Red CMXRos was added to the samples to a final concentration of 100 nM (diluted in cell culture media) and cells were incubated at 37 °C for 30 min. After staining with either reagent, the media was replaced with fresh L-15 medium (Leibovitz; Sigma-Aldrich, catalog number: L5520) imaging media and cells were kept in a heated chamber at 37 °C for image acquisition.    60  2.11. Image analysis–Quantification  2.11.1. Quantification of nuclear import of NLS chimeric proteins and NP in transfected cells Quantification followed the corrected total cell fluorescence (CTCF) method by Burgess et al. (2010) using FIJI (Schindelin et al., 2012). To determine the fluorescence intensity in the nucleus, cytoplasm, and the background, the nucleus and the entire cell periphery were delineated as regions of interest (ROI) and their fluorescence intensity and areas were recorded. The background intensity measurements were obtained by defining three ROI where no cells were present. To obtain the cytoplasmic fluorescence, the nuclear fluorescence was subtracted from the entire cell fluorescence. The CTCF formula (CTCF = integrated density – (area of selected cell X mean fluorescence of background readings)) was then applied to obtain the final intensity levels for the nucleus and the cytoplasm. The nuclear/cytoplasmic (Fn/c) fluorescence ratio was calculated dividing nuclear CTCF values by cytoplasmic CTCF values. The selection of cells and delimitation of areas to be measured were done manually and because of it, data were obtained from an n >45 cell per condition from each experiment. Quantification of the Fn/c fluorescence ratio for the chimeric proteins containing NLSs (Figure 3-2B) was from one experiment, but represented the analysis obtained from three independent experiments. However, quantification of the Fn/c fluorescence ratio of NP (Figure 3-3C) was performed from a total of ~300 cells from three independent experiments.  61  The quantification for experiments performed with transfection of the DS-Red cNLS constructs was excluded because of scale purposes (CTCF values for cells expressing DS-Red cNLS were over ten times larger than for cells expressing 5GFP chimeric proteins).  2.11.2. Quantification of fluorescence intensity of NP, Nup153, EGF, and Tfn  Quantification of the total amount of NP in IAV infected cells (Chapter 3) and Nup153, EGF, and Tfn (Chapter 4) was carried out using the same CTCF methodology mentioned above (Burgess et al., 2010) using FIJI (Schindelin et al., 2012). Briefly, the whole cell was delineated (the F-actin label was used to determine the cell boundary) and its fluorescence intensity and total area was recorded. The total intensity was then corrected by the background intensity. The selection of the cell area was done manually; hence, fluorescence intensity measures were obtained from 75-100 cells per condition from one experiment. The relative fluorescence intensity was obtained by using the largest fluorescence intensity value (arbitrary units) from one of the control experimental values and converting it to 100%. Then, all other experimental values were adjusted in relation to it. Quantification reported corresponds to the mean and S.E.M from three independent experiments.  2.11.3. Gray intensity profile of WGA and HA transversal section In order to show the fluorescence intensity of WGA and HA related to their distribution inside/outside of the cell, a gray intensity profile was generated by selecting an image and converting it to an 8-bit (black, white, and gray values) in FIJI (Schindelin et al., 2012). Cell of interests were identified and a plot profile was generated by placing a defined 62  ROI in the cell. The result was a two-dimensional (X, Y) graph representing the pixel intensity along the horizontal distance of a particular ROI. 2.11.4. EGFR/TfR fluorescence intensity Quantification of the total fluorescence intensity of EGFR or TfR was performed using a pipeline built in Cell Profiler (Carpenter et al., 2006). Briefly, the program was set up (through implementation of filter and thresholds) to automatically recognize individual cells, the cell nucleus, and the cell cytoplasm (by subtracting the cell nucleus from the total cell area). The program was then used to measure fluorescence intensity of only the cytoplasmic region. The selection of cells and areas to be measured was automatized. Therefore, fluorescence intensity measures were obtained from a minimum of 150 cells per condition from one experiment. The relative fluorescence intensity was obtained by using the largest fluorescence intensity value (arbitrary units) from one of the control experimental values and converting it to 100%. Then, all other experimental values were adjusted in relation to it. Quantification reported corresponds to the mean and S.E.M from three independent experiments. 2.11.5. Quantification of pHrodo EGF particles Quantification of the total amount of pHrodo-EGF per cell calculated using the same CTCF methodology used by Burgess et al. (2010) through FIJI (Schindelin et al., 2012). The image was opened with FIJI and converted to an 8-bit image. The whole cell was delineated using the differential interference contrast (DIC) image channel from each figure and total particle density and abundance was registered through the use of analyze particle option, 63  where the minimum threshold size established was 2 pixels, to discard noise signal. Total number of particles and area was registered for each time point in each condition. The selection of cells to be measured was done manually and the data was obtained from a minimum of 10 cells per condition per time point. Due to the acquisition of the data within 140 minutes, only a limited amount of cells could be examined during the time frame of the experiment. Quantification reported is from one representative experiment from a total of three independent experiments due to slide variation in time points between different experiments. 2.11.6. Dextran field index and vesicle size To determine dextran uptake, the field index was calculated according to Commisso et al. (2014). The field index is determined by the total particle area (area occupied by dextran positive structures) in relation to the total cell area for each field. The computation for field index includes dividing the total area covered by a particle by the total cell area, and multiplying the result by 100. The data was acquired as per Commisso et al. (2014) instructions using FIJI (Schindelin et al., 2012). Likewise, while acquiring the data for the particle number we also collected data regarding particle size (dextran-containing vesicle size) using the analyze particle option in the program. A minimum of 50 cells for each condition was included in the analysis. The number of cells was limited by the acquisition methodology during the live cell experiment within the proposed time course. Quantification was performed from data from three different experiments.   64  2.11.7. Endosomal marker vesicle size and circularity With the purpose of examining changes to endosomal markers, we evaluated the fluorescence intensity and circularity (assuming signal belong to vesicles) of the immunolabelling of early endosome antigen 1 (EEA1), cation independent mannose-6-phosphate receptors (CI-M6PR), and lysosomal-associated membrane protein 1 (LAMP-1). Quantification of the total EEA1, CI-M6PR and LAMP-1 immunostained structures per cell was performed by thresholding the image (single confocal slices), selecting the cell, restricting measured structures to larger than 2 pixels, and employing the “analyze particle” option in FIJI (Schindelin et al., 2012). At least 20 cells from each condition were analyzed. The number of cells analyzed was restricted to 20 cells due to the manual selection of each cell and thresholding, which was performed prior to applying the 2D/3D spatial analysis plugin. Shown are the results corresponding to three independent experiments.  2.11.8. Spatial distribution analysis of endosomal markers  In order to quantify the changes observed in the distribution of the cellular markers EEA1, CI-M6PR, and LAMP1, we used the 2D/3D spatial analysis plugin in FIJI (Schindelin et al., 2012) as described by Andrey et al. (2010) and Ollion et al. (2013). A single confocal slice was selected; the cell area was identified through the labelling of F-actin and thresholded to create a cell mask. Then, the same cell area from the appropriate channel was thresholded to identify the endosomal/lysosomal marker (fluorescent dot). The functions evaluated were F, G, and H; where F-function is the cumulative distribution function (CDF) of the distance between fluorescent dots and the surrounding space, G-function is the CDF of the distance between fluorescent dots and its nearest neighbor, and H-function is the CDF of 65  the distance between a typical fluorescent dot and any other fluorescent dot within the cell. The default setup, with number of points (F function) at 10,000, samples 100, and 5% error margin (95% confidence), was used. The number of cells analyzed was restricted to 20 cells as above (Section 2.11.8). The results shown correspond to one representative experiment out of three independent experiments.  2.11.9. Determination of the ER texture As a way to study the distribution of the luminal ER protein, the ER texture (granularity) was quantified using a pipeline built in Cell Profiler (Carpenter et al., 2006). The program was set up (through implementation of filter and thresholds) to automatically recognize individual cells, cell nucleus, and the cell cytoplasm, and to identify the ER label signal in the cytoplasm of each cell. Then the texture (variation in grayscale intensity in images) was examined according to Haralick features (Haralick, 1979) at a scale of 10. The automatization of the cell analysis permitted the acquisition of data for a minimum of 100 cells per condition. The results shown correspond to three independent experiments.  2.12. Statistical analysis Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, Inc). When required, data was analyzed with a one-way analysis of variance (ANOVA) with Tukey’s post-test comparison. A p-value of less than 0.05 was established as the minimum required for considering the treatments statistically significant. When comparing only two treatments, a two-tailed paired Student’s t-test was performed and statistical significance was determined by a p-value of less than 0.05. 66  2.13. Western blot (WB) RNAi efficiently silence expression of Nup153, as well as the cellular expression of the EGF receptor and Rab7 was determined by WB analysis using β actin expression as loading control. After siRNA transfection as indicated above, cells were rinsed with PBS three times and collected through mechanical detachment with a scraper from 6-well tissue culture plates (Corning Inc., catalog number: 353046). Cells were centrifuged at 15,000 x g for 10 minutes at 4°C. Cell pellets were then treated with RIPA buffer (150 mM NaCl, 50 mM Tris–HCl pH 8.0, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.5% sodium deoxycholate, 0.1% SDS, 0.5% NP-40, 10 mM phenylmethylsulfonyl fluoride (PMSF), 1μM pepstatin, 10% μg/ml aprotinin, and 2 mg/ml leupeptin) for 1 h on ice. Lysates were cleared by centrifugation at 15,000 x g for 10 minutes at 4 °C and the supernatant was assessed for protein concentration using Pierce™ BCA protein assay (Thermo Fisher Scientific, catalog number: 23225). Supernatants were mixed with 2X Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS and 0.01% bromophenol blue, and 5% β mercaptoethanol) and boiled at 96 °C for 5 min in a thermomixer (Eppendorf). Equal amounts of protein were then loaded onto a 10% SDS-PAGE. Proteins were transferred to nitrocellulose membrane using a trans-blot semi-dry electrophoretic transfer cell (Biorad). Unspecific interactions were prevented by incubating the membrane with blocking solution (TBST: 5% skim milk in Tris-buffered saline and 0.1% Tween 20) for 1 h at room. Next, membranes were incubated with the appropriate primary antibody overnight at 4 °C. The antibody was removed and membranes were then washed four times, each for 25 min, with TBST. Membranes were then incubated with the appropriate secondary antibody for 1 h at room temperature, washed again with TBST four times, each for 25 min. After washes, the 67  membranes were developed using a chemiluminescence development kit (GE Healthcare, catalog number 45002401 or Millipore, catalog number: WBKLS0100) following the manufacturer’s instructions.  Quantifications of Nup153 expression (Figure 3-1A) was performed using FIJI to detect band intensities from WB according to Miller (2010). The image was first converted to gray-scale, a rectangular tool was used to identify specific bands and the band intensities of Nup153 and loading control, β actin, were measured. The relative expression of Nup153 was calculated by dividing the band intensity of Nup153 by the band intensity of β actin for each condition. This procedure was carried out for three independent experiments. 2.14. Electron microscopy (EM) HeLa cells were grown in 6-well tissue culture plates (Corning Inc., catalog number: 353046). Four experimental conditions were used: non-treated cells, cells treated with transfection reagent only, cells transfected with control siRNA, and cell transfected with Nup153 siRNA. Mock and siRNA transfection was for 72 h as indicated in Section 2.3. Transfected cells were washed with PBS three times and then fixed with 2% glutaraldehyde (Ted Pella) in PBS for 1 h. Cells were then rinsed with PBS three times for 5 min each time, scrapped off the plate and centrifuged for 15 sec at to obtain a cell pellet. The pellet was then embedded in 2% low-melting agarose (Sigma-Aldrich, catalog number: A9414). Once the agarose solidified, the agarose-embedded cell pellet was washed three times with PBS for 5 min; samples were constantly mixed using a rotary mixer during washing. Samples were then post-fixed with 1% osmium tetroxide in PBS for 1 h and washed again with PBS three times, each for 5 min. Samples were sequentially dehydrated in 50%, 70%, and 90% ethanol for 20 68  min each, followed by dehydration in 100% ethanol for 15 min two times followed by 100% acetone for another 15 min. Finally, fixed and dehydrated samples were infiltrated by incubation in a mixture of epoxi embedding medium (Epon; Sigma-Aldrich, catalog number: 45345) and acetone (1:1 ratio) for 1 h, then in a mixture of Epon and acetone at a 2:1 ratio for 2 h, and lastly, pure Epon for at least 8 h. Epon-infiltrated samples were then placed into flat embedding molds (Ted Pella), filled with pure Epon and allowed to polymerize for 48 h at 60 °C. Once samples were polymerized, a Leica ultracut ultramicrotome (Leica Microsystems) equipped with a diamond knife (Diatome) was used to obtain 50-nm-thin sections of the Epon-embedded cell pellets. The sections were then collected on parlodion and carbon-coated EM copper grids (Ted Pella), stained with 2% uranyl acetate for 30 min, followed by four washes with distilled water, stained again with 2% lead citrate for 5 minutes, and finally washed four times with distilled water.  Samples were visualized using a FEI Tecnai G2 spirit transmission electron microscope operated at an acceleration voltage of 120 kV. Micrographs were digitally recorded using an Eagle 4k CCD camera (FEI). 2.15. Cell cycle analysis Flow cytometry was used to determine the phase of the cell cycle in a cell population through its DNA content. For this, HeLa cells were grown in 6-well tissue culture plates (Corning Inc., catalog number: 353046). Three experimental conditions were used: non-treated cells, cells transfected with control siRNA, and cell transfected with Nup153 siRNA. Mock and siRNA transfection took place over a 72 h period. Afterwards, cells were rinsed with PBS and incubated with 10 mM EDTA for 10 min at 37 °C to detach them from the cell 69  culture plate. Detached cells were then collected by centrifugation for 4 min at 380 x g at 4°C to obtain a pellet, the EDTA was removed and cells were washed with PBS twice; the supernatant was removed after centrifugation at 380 x g for 4 mins. Cells were resuspended in 70% ice-cold ethanol and fixed overnight at 4 °C. Samples were then washed twice with PBS. Then, the cells were treated with RNAse in a concentration of 1 Unit/ml at 37 °C for 30 mins. Propidium iodide was then added to cells in a concentration of 50 μg/ml and the cells were incubated for at least 1 h at room temperature. Samples were analyzed using a LSRII flow cytometer (BD Biosciences). 70                                             Chapter 3 Role of nucleoporin Nup153 during IAV infection Role of nucleoporin Nup153 during IAV infection 3.1. Introduction As is characteristic of viruses, IAV requires host cellular factors for its replication and viral production. Given the constant risk of an influenza global pandemic and the threat of antiviral resistant strains, the scientific community has focused its efforts on understanding the role of cellular proteins during IAV infection, in addition to the role of viral proteins. Identifying fundamental interactions between viral proteins/genome and host cellular proteins would provide valuable information that could be used for the development of new drug targets that would not easily generate resistant viral strains. In an effort to further refine the cellular pathways required for IAV infection, Hao et al. (2008) and König et al. (2010) performed a separate genome-wide RNA interference screening and identified Nup153 as one of the host proteins important for viral replication. A review of all the identified genes and cellular pathways involved in multiple screenings further suggested that Nup153 could play a role during vRNP nuclear import and export and viral mRNA export in the IAV infection cycle (Watanabe et al. 2010). Furthermore, York et al. (2014) identified Nup153 as a protein that interacts with the PB2 subunit of the viral RNA-dependent RNA polymerase purified from infected cells employing a recombinant virus that expresses PB2 fused to a polypeptide tag. In addition, Watanabe et al. (2014) also recognized Nup153 as a binding partner of PB2 and M2 while studying the co-immunoprecipitation of transiently transfected IAV proteins. York et al. (2014) and Watanabe et al. (2014), in addition to Benitez et al. (2015), identified a decrease in IAV viral titer upon the infection of cells treated with 71  Nup153 siRNA. Together, these studies confirm the relevance of Nup153 during IAV infection. However, the specific role of Nup153 during the IAV infective cycle remains to be determined. Although IAV is a negative-stranded RNA virus, the import of its genomic unit (the vRNP) into the host cell nucleus is required to successfully establish an infection. In addition, IAV uses both the nuclear import and export machinery at other steps of its infective cycle. For example, progeny vRNPs are assembled in the nucleus of the host cells and exported to the cytosol to later be sorted and transported to the plasma membrane, where it forms new budding virions; viral mRNAs have to be nuclear exported to express progeny viral proteins; after synthesis in the cytoplasm NP and the viral polymerase proteins must enter the nucleus to assemble progeny vRNPs; and NS2/NEP, which assists in the nuclear export of newly made vRNPs, must enter the nucleus. Given the role of Nup153 in nuclear transport, Nup153 may be involved in the nuclear transport of IAV vRNPs and/or viral proteins. However, the location of Nup153 at the NPC’s nuclear basket and its relatively high mobility, position this protein to participate in a multitude of cellular processes associated with gene expression.  The main goal of this chapter was to determine the role of Nup153 during IAV infection. We first confirmed a reduction in viral titer in Nup153 depleted HeLa cells infected with IAV. Given the importance of Nup153 in nuclear transport and the role of NP in the nuclear import of vRNPs, we then studied the nuclear import of NP and chimeric proteins containing NP’s NLSs in HeLa cells where Nup153 was KD with siRNA. Our findings show that nuclear import of NP still took place despite the absence of Nup153 in the cells. Hence, we set out to investigate which steps of the IAV infective cycle, such as viral 72  entry, early traffic, and/or nuclear import/export of vRNPs, were affected in Nup153 depleted HeLa cells. Surprisingly, the results indicated defects in the cellular uptake and cytoplasmic trafficking of viral components in Nup153 depleted HeLa cells infected with IAV. Our findings also suggested possible alterations in IAV assembly and/or budding.  3.2. Results 3.2.1. Infectious IAV particles production is significantly reduced in Nup153 depleted cells  It has been previously observed that when Nup153 depleted cells are infected with IAV, the production of infectious viral particles decreased (König et al., 2010; Watanabe et al., 2014). In order to determine the role of Nup153 during IAV, we used siRNA to deplete Nup153 in HeLa cells and then infected them with IAV (H3N2). HeLa cells were used because they are a commonly employed cell line in the study of nuclear transport and there are documented effects of RNAi mediated depletion of Nup153 (Duheron et al., 2014; Jacinto et al., 2015; Lowe et al., 2015; Mackay et al., 2009; Matreyek and Engelman, 2011; Matreyek and Engelman, 2013; Moudry et al., 2012; Shain et al., 2013; Umlauf et al., 2013; Vaquerizas et al., 2010; Zhou and Panté, 2010). Although Nup153 KD HeLa cells are not apoptotic, several defects are present: altered nuclear morphology, cell cycle-related delays and a reduction in NLS-dependent nuclear import (Mackay et al., 2009; Zhou and Panté, 2010). Previous studies of Nup153 KD cells in HeLa cells have established a basic understanding of the effects of Nup153 depletion in such cell system. In addition, these cells have been extensively used for studies of the biology of IAV, thus they are also a valid system to study IAV infection (Chu & Whittaker, 2004; Sieczkarski & Whittaker, 2002).  73  The experimental conditions used in this study included a different siRNA sequence and cell line (HeLa) (Materials and Methods, Section 2.3) than those used in previously IAV-published studies (König et al., 2010; Watanabe et al., 2014). Hence, we first established the optimal conditions to deplete Nup153 through RNAi by both WB (Figure 3-1A) and immunofluorescence microscopy (Figure 3-1B). Nup153 detection through WB shows approximately a five-time reduction in the expression of Nup153 in Nup153 KD cells when compared with control conditions (Figure 3-1A). In contrast, quantification of the fluorescence intensity of Nup153 as detected by immunofluorescence microscopy shows an approximate three-fold reduction in Nup153 siRNA treated cells when compared to Control siRNA cells (Figure 3-1B). Once the Nup153 KD was confirmed, monolayers of Nup153-depleted HeLa cells were infected with IAV for a period of 24 h, and the supernatant was collected and used to perform plaque assays on MDCK cells monolayers (Materials and Methods, Section 2.5). As a control, the same infection procedure was repeated in HeLa cells untreated or treated with control siRNA or transfection reagent only. As documented in Figure 1-1C, there was a significant reduction (~3.5 fold) in the production of infectious viral particles between Nup153 siRNA treated cells and control siRNA cells. Similar differences were observed between Nup153 siRNA treated cells and cells treated with the transfection reagent only, and between Nup153 siRNA treated cells and non-treated cells. We consistently observed an increase in viral titer in cells treated with control siRNA. This was probably because of off-target effects from the pool of control siRNA sequences used during our experiments, which somehow facilitates viral replication. These results confirmed that Nup153 depleted cells produced significantly less infectious IAV particles than control treated cells, as found by 74  previously published studies (König et al., 2010; Watanabe et al., 2014). In addition, we verified that the effects of Nup153 KD were specific to the disruption of Nup153 instead of a consequence targeting nucleoporins in general. In order to do this, two other nucleoporins, Nup214 and Nup358 were tested. Previously, only Nup214 was recognized in a genome-wide RNAi screening as a nucleoporin important for IAV infection (Karlas et al., 2010). HeLa cells were treated with Nup214 siRNA or Nup358 siRNA and infected with IAV (as previously done in Nup153 KD cells), in both cases we observed a decrease in viral production, almost a five-fold reduction in IAV in infected Nup214KD cells and approximately a two-fold reduction in Nup358 KD HeLa cells (Figure A-1). These results demonstrate that nucleoporin depletion affects IAV infection to different degrees, depending on the targeted nucleoporin.  75   76   Figure 3-1. Production of infectious viral particles is reduced in Nup153 depleted cells. A) WB analysis of Nup153 and β actin expression in HeLa cell treated with Nup153 siRNA, Control siRNA or untreated after 72 h of transfection. In the left of (A) is the quantification of the expression label of Nup153 normalized to the amount of beta actin (the band intensity of Nup153 divided by the band intensity of β actin); this quantification is representative of at least three different WB (Y axis are arbitrary units, error bars represent the S.E.M.; ANOVA was used to determine statistical differences between conditions, *p<0.05). B) HeLa cells treated with Nup153 siRNA or Control siRNA for 72 h were fixed and prepared for immunofluorescence microscopy with an antibody against Nup153 (right panel). DNA was detected by staining with Dapi (left panel). Scale bar, 10 µm. Lack of Nup153 signal indicates the RNAi mediated KD was successfully. Quantification of the relative fluorescence intensity (%) of Nup153 (n>75 cells per condition) from of at least three independent RNAi KD experiments is shown in the left of this panel. Student’s t-test was used to determine statistical differences between conditions, ***p<0.001. C) Viral titer of supernatants collected from HeLa cells that had been treated with Nup153 siRNA, Control siRNA, transfection agent, or untreated for 72 h. The cells were then incubated with IAV for 15 min at 4°C, virus was removed, and infection was allowed to progress for 24 h at 37°C. Supernatants were then collected and subjected to plaque assay. PFU= plaque forming unit. Mean values are shown (bars) and error bars represent the S.E.M. Plaque assays shows significant (ANOVA, ***p<0.05) difference of the viral titer between Nup153 siRNA and the rest of the conditions. Results shown are representative of the plaque assay, which was repeated 3 times independently.  77  3.2.2. Depletion of Nup153 results in decreased nuclear import of a chimeric protein containing the non-classical NLSs of NP It has been suggested that Nup153 plays a role during the nuclear/cytoplasmic traffic of IAV vRNPs (König et al., 2010; Watanabe et al., 2010) because the IAV vRNPs are imported into the host cell nucleus through the classical nuclear import pathway (reviewed by Eisfeld et al. 2015). Previous research has found a decrease of classical NLS-dependent nuclear import in Nup153 depleted cells (Zhou & Panté, 2010). Hence, we hypothesized that nuclear import of IAV vRNPs might be disrupted in Nup153 depleted cells. In order to test this hypothesis, we first determined whether nuclear import of chimeric proteins containing either one of the two NLSs of NP (which are known to be important for vRNP nuclear import) (Wang et al., 1997; Weber et al., 1998) was affected in Nup153 depleted cells. For this purpose, 5GFP-NLS1 and 5GFP-NLS2 chimeric proteins were transiently transfected in HeLa cells treated with Nup153 siRNA (or control siRNA) and observed live with a scanning confocal laser microscope 48 h after transfection. As illustrated in Figure 3-2A and quantified in Figure 3-2B, nuclear import of the chimeric protein containing the NP primary NLS (5GFP-NLS1) was drastically reduced in Nup153 depleted HeLa cells. The chimeric protein with the secondary NP’s NLS (5GFP-NLS2) was also significantly reduced, but to a lesser degree than its 5GFP-NLS1 counterpart. In addition, Ds-Red coupled with three classical NLSs (cNLS sequences from SV40 large T antigen) was used as a control to determine whether the classical nuclear import was affected. There were no observed changes in the nuclear accumulation of Ds-Red cNLS between both siRNA treatments (Figure 3-2A, right panel). 78  To further test the hypothesis that Nup153 depletion would affect cNLS dependent nuclear import, a HeLa 5GFP-NLS1 stable cell line was treated with Nup153 siRNA or control siRNA and later transfected with DS-Red cNLS. The results are displayed in Figure 3-2C and show that while 5GFP-NLS1 was present in both cell cytoplasm and cell nucleus of control siRNA treated cells, it accumulated in the nucleus along with the DS-Red cNLS. In contrast, 5GFP-NLS1 was also present in the nucleus and cytoplasm of Nup153 depleted cells, yet the accumulation in the nucleus was minor and the DS-Red cNLS nuclear accumulation was not affected.  Overall, the results in Figure 3-2 generated through the use of chimeric proteins show that nuclear import of IAV NP’s NLSs is reduced in Nup153 depleted cells. This is the first study to demonstrate that nuclear import dependent on a non-classical NLS (5GFP-NLS1) is selectively affected during Nup153 depletion.    79     80  Figure 3-2. Nuclear import of a chimeric protein containing the main NLSs of NP is reduced in Nup153 depleted cells. A) Live cell imaging of HeLa cells treated with either Control siRNA or Nup153 siRNA and transfected with the following cDNA plasmids: 5GFP, 5GFP-NLS1, 5GFP-NLS2, or DS-Red cNLS (cNLS sequence of SV40). HeLa cells treated with either Control siRNA or Nup153 siRNA for 72 h were transfected for 48 h with cDNA constructs 24 h after beginning of siRNA treatment. Cells were imaged 72 h after the start of the siRNA transfection (48 h after start of cDNA transfection). Nuclear import of 5GFP-NLS1 was drastically reduced, while 5GFP-NLS2 was also reduced in Nup153 depleted cells. However, DS-Red cNLS accumulated in the nucleus in both siRNA treatments. B) Quantification of the nuclear/cytoplasmic fluorescence (Fn/c) from confocal images of live cells from the experiments shown in (A) for the GFP plasmids. Quantification of DS-Red SV40cNLS is not shown because fluorescence intensity values were several orders of magnitude higher than those shown for 5GFP constructs. Additionally, all fluorescence was found in the nucleus. Shown are the mean values and S.E.M (n>45 for each condition). Student’s t-test was used to determine statistical differences between conditions, ***p<0.05, n.s: not significant. C) Live cell imaging of 5GFP-NLS1 HeLa stable cell lines transiently expressing SV40 cNLS treated with Control siRNA or Nup153 siRNA and observed 72 h after the beginning of the treatment. Decreased GFP nuclear fluorescence was observed only in Nup153 depleted cells, while nuclear fluorescence of DS-Red was not altered. Scale bars, 10 µm.  81  3.2.3. Depletion of Nup153 does not prevent nuclear trafficking of influenza NP Given the results obtained in Figure 3-2, the hypothesis that nuclear import of vRNPs would be reduced in Nup153 depleted cells was further tested. First, the nuclear import of IAV NP was studied, since this protein is the most abundant in the vRNP complex and its nuclear import is essential during the IAV infection cycle. For this purpose, HeLa cells were treated with Nup153 siRNA for 48 h (or Control siRNA) and transfected with the NP cDNA from IAV H1N1 (A/WSN/33) for 24 h. As illustrated in Figure 3-3A, NP was present primarily in the nucleus for both conditions. Although Nup153 depleted cells displayed NP cytoplasmic staining, these results could at most suggest a decrease in the efficiency in the nuclear import of NP.  In order to determine whether the cytoplasmic staining of NP corresponded to NP exported from the nucleus or NP not able to be imported into the nucleus, we blocked NP nuclear export with leptomycin B (LMB). This drug inhibits CRM1- (exportin 1)-dependent nuclear export (Nishi et al., 1994), which is the pathway required for the nuclear export of NP (Ma et al., 2001). When LMB was added 8 h after NP transfection, it efficiently retained NP in the nucleus of both Nup153 KD cells and control cells (Figure 3-3B). In addition, NP immunostaining appeared stronger around the inner rim of the nucleus; this staining pattern is characteristic of NP localization (Bui et al., 2002). There was a strong NP fluorescence signal in the nucleus of Nup153 KD cells, which suggested that nuclear import of NP was not impeded in Nup153 depleted cells (Figure 3-3B). This is in contrast to the results obtained with chimeric proteins containing either one of the NP’s NLSs (Figure 3-2). Thus, it is highly likely that containing both NLS1 and NLS2 does not significantly disrupt the nuclear import 82  of NP. These findings suggest the possibility that nuclear import of IAV vRNPs might not be affected in Nup153 depleted cells. Quantifying the nuclear/cytoplasmic fluorescence ratio to determine the nuclear import efficiency of NP after transfection of its plasmid was performed from three independent experiments (Figure 3-3C). However, as NP intracellular localization (nuclear or cytoplasmic) was markedly affected by cell confluency, the quantification of nuclear/cytoplasmic fluorescence ratio was different for each experiment (Figure A-2). This was due to the effect of the treatment with Nup153 siRNA or control siRNA on cell proliferation which can be markedly different between different experiments That the import efficiency of NP depends on cell confluent has previously been documented (Bui et al., 2002).    83   84  Figure 3-3. Nuclear import of NP still occurs in Nup153 KD cells. A) HeLa cells treated with either Control siRNA or Nup153 siRNA for 72 h were transfected for 24 h with NP construct 48 h after beginning of siRNA treatment. B) Leptomycin B was added 8 h post NP transfection to a final concentration of 11 nM. Samples were prepared for indirect immunofluorescence microscopy using antibodies against NP (red) and Nup153 (white). Nuclei were stained with Dapi. Nuclear import of NP in (A) was not disrupted by depletion of Nup153. Due to possible nuclear-cytoplasmic shuttling of NP, in (B) leptomycin B was added to inhibit nuclear export of NP. Results suggest that NP is imported into the nucleus of Nup153 depleted cells. Scale bars, 10 µm. C) Quantification of the subcellular localization of NP in NP-transfected cells. The bars represent the mean values of the nuclear/cytoplasmic (Fn/c) fluorescence ratio from three independent experiments (n>150 cells per condition). Errors bars represent the S.E.M. Statistical significance was assayed through ANOVA and Tukey's Multiple Comparison Test.   85  3.2.4. Cell entry and early traffic of IAV is affected in Nup153 KD HeLa cells Since our results showed that Nup153 depletion did not affect nuclear import of NP, we hypothesized that other steps in the viral infective cycle could be affected prior to the nuclear import of vRNPs. To examine this hypothesis, we set up an IAV infection in Nup153 depleted cells to detect differences exclusively during the initial viral entry and cellular traffic steps. These steps are necessary for the successful trafficking of the IAV genome into the host cell nucleus. To investigate early viral traffic, HeLa cells were treated with Nup153 siRNA or control siRNA for 72 h and incubated with IAV for 15 min at 4 °C to allow viral attachment. The non-attached virus was then removed and the media substituted for fresh media (see Section 2.4 of Materials and Methods). The infection was allowed to continue for 5 min or 15 min at 37 °C. Cells were then fixed and prepared for immunofluorescence. To identify incoming vRNPs and follow the early cellular traffic of vRNPs, NP was immunolabelled and visualized by confocal microscopy (Figure 3-4A). In these experiments (and others below), F-actin was labelled with fluorescently-labelled phalloidin to help delineate the cell periphery. After 5 or 15 min of infection, NP was more readily detected in control siRNA cells than in Nup153 depleted cells (this is depicted well in the enlargement panels of Figure 3-4A). Quantification of the relative fluorescence intensity of NP showed significantly less NP signal after 5 min post-infection (Figure 3-4B) in Nup153 KD cells compared with control siRNA treated cells. Interestingly, at 15 min after infection the relative fluorescence intensity of NP increased in Nup153 KD cells (Figure 3-4C). Through the fluorescence intensity quantification of NP (relative to control siRNA cells), our results showed that the early uptake of IAV was significantly reduced in Nup153 depleted cells. 86  Thus, NP immunostaining at early stages of infection suggest that IAV uptake is reduced in Nup153 depleted cells.   87    Figure 3-4. Cellular uptake of IAV is reduced in Nup153 transiently depleted HeLa cells during early infection. Confocal images of HeLa cells treated with either Control siRNA or Nup153 siRNA and infected with IAV (H3N2 X:31) for 5 or 15 min. Infected cells were prepared for indirect immunofluorescence microscopy using fluorescently-labelled phalloidin to detect F-actin (white) and an antibody against NP (red). Nuclei were stained with Dapi. NP immunolabelling was used to identify incoming vRNPs. The results suggest that initial viral uptake was slower in Nup153 depleted cells than in control cells. Immunofluorescence results of Nup153 showed that Nup153 was depleted (data not shown). Dashed lines in left and middle panels delineate the cell boundary. Arrows point to NP accumulations near the edge of the cell. Scale bar, 10 µm. B) Quantification of relative NP fluorescence intensity (%) from experiments shown in (A, 5 min). C) Quantification of NP fluorescence intensity from experiments shown in (A, 15 min). For each condition, the relative fluorescence intensity (%) was quantified in n>80 cells from three independent experiments and the mean values and S.E.M are shown. Student’s t-test was used to determine statistical differences between conditions, ***p<0.0001.     88  With the purpose of examining whether the NP immunostaining difference persisted with an increasing time of infection, Nup153 depleted HeLa cells (and corresponding controls) were infected for 60 min and prepared for immunofluorescence microscopy. As illustrated in Figure 3-5A, in control siRNA treated cells, the NP immunostaining was easily distinguished near the nucleus, while in Nup153 KD cells NP appeared throughout the cytoplasm. This is a possible indication of defects on the trafficking of virion-containing endosomes towards the nucleus of Nup153 KD cells. Quantification of the NP fluorescence intensity per infected cell indicated a significant decrease in NP immunostaining in Nup153 KD cells (Figure 3-5C). Although this decreased was significant, it was only a ~20% reduction compared to the ~50% reduction after 5 min of infection (Figure 3-4B). Thus, suggesting that IAV entry into Nup153 KD cells is delayed when compared to control conditions. In separate experiments, M1, which forms a coat inside the viral envelope to which the vRNPs anchor, was immunolabelled after 60 min of infection. As documented in Figure 3-5B, the M1 distribution was drastically different between the Nup153 KD cells and the control cells. While M1 staining appeared dotty and dispersed throughout the cytoplasm in control siRNA treated cells, the Nup153 depleted cells displayed patches of M1 staining along the edges of the cells (Figure 3-5B). Again, this is an indication of defects on trafficking of the endosome-containing virions towards the nucleus of Nup153 KD cells. Similar to the NP immunostaining (Figure 3-5C), there was also a decreased of the fluorescence intensity of M1 per infected cell (Figure 4-5D), indicating reduced IAV uptake in Nup153 KD cells compared to control cells. 89  The contrasting distribution of NP and M1 in both Nup153 KD and control cells one hour after infection indicates that some vRNPs had been released from the late endosomes. While the M1 immunostaining corresponded to intact virions, which were in endosomes, the NP immunostaining corresponded to vRNPs that could have either escaped from the late endosomes or remained in the late endosomes. In summary, comparison of the sub-cellular localization of NP and HA at 5 min and 60 min p.i. in Nup153 KD cells and control cells suggests that IAV entry into the cells is reduced or defective in Nup153 KD cells and trafficking of virion-containing endosomes towards the nucleus appears to be delayed. The later could result in the released of the vRNPs far away from the nucleus, which may result in a reduction of viral infection as determined by plaque assay (Figure 3-1C).   90    91  Figure 3-5. The sub-cellular distribution of NP and M1 is altered in IAV infected Nup153 KD HeLa cells 60 min post-infection (p.i.). Confocal images of HeLa cells treated with either Control siRNA or Nup153 siRNA and infected with IAV (H3N2 X: 31) for 60 min. Infected cells were prepared for indirect immunofluorescence microscopy using fluorescently-labelled phalloidin to detect F-actin (white) and antibodies against NP (red) (A) and M1 (green) (B). Nuclei were stained with Dapi. NP immunolabelling was used to identify incoming vRNPs and M1 was used as a marker for total incoming viral particles. A) NP localization was found dispersed in the cytoplasm of Nup153 depleted cells in contrast to control siRNA treated cells, which showed NP predominantly around the nucleus. B) M1 was distributed through the cytoplasm in control siRNA treated cells, but was rather clustered adjacent to the plasma membrane in Nup153 depleted cells (arrows). Immunofluorescence results of Nup153 showed that Nup153 was depleted (data not shown). Dashed lines in left and middle panels of (A) and (B) delineate the cell boundary. Scale bars, 10 µm. C) Quantification of NP relative fluorescence intensity (%) from the experimental conditions shown in (A). D) Quantification of M1 relative fluorescence intensity (%) from the experimental conditions shown in (B). For each condition, the relative fluorescence intensity (%) was quantified in n>80 cells from three independent experiments and the mean values and S.E.M are shown. Student’s t-test was used to determine statistical differences between conditions, ***p<0.05.   92  3.2.5. Depletion of Nup153 does not prevent nuclear import of NP and vRNPs in IAV infected HeLa cells During IAV infection, the vRNP must enter the nucleus. To determine whether Nup153 depletion could affect the nuclear import of vRNPs within the context of a viral infection, HeLa cells were treated with Nup153 siRNA or control siRNA for 72 h, infected with IAV for 3 h or 5 h, and immunolabelled using an anti-NP antibody (details in Materials and Methods, Section 2.4). NP is a crucial component of the vRNPs; thus, depending of the time of infection, its immunolabelling during an IAV infection corresponds to vRNPs (either incoming or progeny vRNPs) and/or newly made NP. After 3 h of infection, NP immunostaining was both in the cytoplasm and nucleus for both siRNA treatment conditions (Figure 3-6). For this time point, NP immunolabelling corresponded to either incoming vRNPs or newly generated NP. Thus, the results suggested that the nuclear import of newly synthesized NP has occurred. After 5 h of infection, NP immunolabelling was found accumulated in the nucleus in both control siRNA treated cells and Nup153 depleted cells (Figure 3-6). These results indicate that nuclear import of both incoming vRNPs (which must have been transcribed to generate the mRNA of NP) and progeny NP was not affected in Nup153 KD cells. These results are in agreement with previously obtained results in Nup153 KD cells transiently expressing NP (Figure 3-3).  In contrast to NP, M1 is a late expressing protein during viral infection (Shapiro et al., 1987), and after it is synthesized enters the nucleus in preparation for the nuclear export of progeny vRNPs (Bui et al., 2000). Additionally, M1 interactions with HA, NA, and M2 at the cell membrane are required for the formation of new viral particles (reviewed by Rossman & 93  Lamb, 2011). For these reasons, the examination of M1 distribution during infection in conjunction with NP can be used to determine whether nuclear export of vRNP has occurred. Thus, we also performed immunolabelling of M1 in Nup153 KD cells and control cells at 3 h and 5 h after infection. Figure 3-7 illustrates that 3 h after infection, M1 immunostaining was largely dispersed in the cytoplasm in infected cells from both conditions. However, 5 h after infection, M1 immunolabelling was found at the cell periphery of Nup153 depleted cells (Figure 3-7, arrows). In contrast, M1 immunostaining in control siRNA treated cells was equally dispersed throughout the cell (Figure 3-7). Since at 5 h p.i M1 immunolabelling corresponds to newly synthesized M1 and was found accumulated at the plasma membrane, the results indicate early M1 oligomerization and accumulation at the plasma membrane due to the interaction of M1 with other viral proteins such as M2 and HA. Thus, it seems that in the infected Nup153 KD cells there are possible defects on assembly or budding of progeny viral particles. Overall, our findings regarding NP and M1 immunolocalization indicate that although there was a difference on the sub-cellular distribution of NP in Nup153 KD cells compared with control cells at 3 h p.i, IAV vRNPs were imported into nucleus of Nup153 KD cells and progeny NP and M1 viral proteins were synthesized in these cells. NP was also successfully imported into the nucleus to form progeny vRNPs. The major difference we found in Nup153 KD cells was the distribution of M1 at 5 h p.i, which was accumulated at the plasma membrane in these cells (white arrows, Figure 3-7) but not in control cells.     94     95  Figure 3-6. Depletion of Nup153 does not prevent nuclear import of vRNPs and nuclear import of NP in IAV infected HeLa cells. Confocal images of HeLa cells treated with either Control siRNA or Nup153 siRNA and infected with IAV (H3N2 X: 31) for 3 h or 5 h. Infected cells were prepared for indirect immunofluorescence microscopy using fluorescently-labelled phalloidin to detect F-actin (white) and an antibody against NP (red). Nuclei were stained with Dapi. Immunolabelled NP at 3 h p.i. can be attributed to either incoming vRNPs or newly generated NP, while nuclear NP detected at 5 h p.i. corresponds to newly translated NP, which has been imported into the nucleus. At 5 h p.i. NP localization in the cytoplasm can be attributed to newly made vRNPs, which have been exported out of the cell nucleus. Immunofluorescence results of Nup153 showed that Nup153 was depleted (data not shown). Dashed lines in left and middle panels delineate the cell boundary. Scale bar, 10 µm.    96      97  Figure 3-7. Cellular distribution of M1 is altered in IAV infected Nup153 KD HeLa cells. Confocal images of HeLa cells treated with either Control siRNA or Nup153 siRNA and infected with IAV (H3N2 X: 31) for 3 h or 5 h. Infected cells were prepared for indirect immunofluorescence microscopy using fluorescently-labelled phalloidin to detect F-actin (white) and an antibody against M1 (green). Nuclei were stained with Dapi. Observed M1 at 3 h can be attributed to incoming viral M1 or newly made M1 (although less likely), while M1 detected at 5 h corresponds to newly synthesized M1, which due to its small size (25 kDa) can diffuse into the nucleus. Immunofluorescence results of Nup153 showed that Nup153 was depleted (data not shown). Dashed lines in left and middle panel delineate the cell boundary. Arrow heads indicate accumulation of M1 at the cell periphery. Scale bar, 10 µm.    98  3.2.6. Nuclear/cytoplasmic distribution of newly made vRNP/NP is affected in Nup153 depleted cells infected with IAV It is possible that although the nuclear import of NP and vRNPs were not affected, Nup153 depletion could have an effect on the nuclear export of vRNPs in infected cells. We hypothesized that because Nup153 interacts with CRM1 (Nakielny et al., 1999), which mediates nuclear export of vRNPs (Ma et al., 2001), the nuclear export of vRNPs in Nup153 depleted cells will be disrupted. To test this hypothesis, HeLa cells were treated with Nup153 siRNA or control siRNA for 72 h, infected for either 8 h or 12 h (details in Section 2-4 of Materials and Methods), and prepared for immunofluorescence using antibodies to detect NP. After 8 h of infection, NP immunostaining was found primarily in the nucleus of infected cells treated with control siRNA (Figure 3-8). In contrast, NP staining was both nuclear and cytoplasmic in the majority of infected Nup153 KD cells (Figure 3-8). Because NP staining occurred predominantly in the nucleus of Nup153 depleted cells at 5 h (Figure 3-6), the 8 h results indicated that newly synthesized vRNPs had been exported from the nucleus and accumulated in the cytoplasm of the Nup153 KD cells at this time. These results suggest that the nuclear export of vRNPs occurs at an earlier time point in Nup153 depleted cells than in control cells. An alternative explanation is that newly made vRNPs were not being assembled into new virions and budded off from Nup153 depleted cells. Instead, the newly made vRNPs remained trapped in the cytoplasm after they were exported out of the nucleus.  After 12 h of infection control siRNA treated cells displayed NP immunofluorescence predominantly in the nucleus with some additional cytoplasmic staining (Figure 3-8, lower 99  panel), which likely corresponded to an increase in vRNP export. In contrast, the NP immunofluorescence in Nup153 depleted cells was present in both the cytoplasm and in the nucleus. It is possible that the nuclear export of vRNP and newly made NP/vRNP had reached equilibrium in Nup153 depleted cells. Alternatively, IAV could have affected NPC permeability in Nup153 depleted cells.  Experiments were also performed detecting M1 instead of NP, so as to use the combined knowledge of both proteins’ distribution in order to determine the progression of the IAV infective cycle in HeLa cells. After 8 h of infection, M1 accumulated in the nucleus of Nup153 depleted cells (Figure 3-9). This is in contrast to the control siRNA treated cells, in which M1 was preferentially located in the cytoplasm (Figure 3-9, upper panel). Nuclear accumulation of M1 occurs when the vRNPs have been assembled and M1 binds to them (prior to their nuclear egress) (Bui et al., 2000). Thus, although there was a considerable amount of NP in the cytoplasm (Figure 3-8), the nuclear accumulation of M1 in Nup153 depleted cells is a contradictory result if the cytoplasmic NP immunofluorescence is considered to be vRNPs. However, if the cytoplasmic immunolabelling of NP in Nup153 depleted cells at 8 h post infection (Figure 3-8) corresponds to newly synthesized NP, instead of the nuclear exported (or accumulated in the cytoplasm) vRNPs, then an accumulation of M1 in the nucleus at 8 h post infection would be indicative of M1 binding vRNPs in the nucleus in preparation for nuclear export.    100     101  Figure 3-8. Nuclear/cytoplasmic distribution of newly made vRNP/NP is affected in Nup153 depleted cells infected with IAV. Confocal images of HeLa cells treated with either Control siRNA or Nup153 siRNA, infected with IAV (H3N2 X: 31) for 8 h or 12 h and prepared for indirect immunofluorescence microscopy using fluorescently-labelled phalloidin to detect F-actin (white) and an antibody against NP (red). Nuclei were stained with Dapi. Immunostaining of NP at 8 h can be attributed to newly synthesized NP imported into the nucleus and possibly NP forming progeny vRNPs. At 12 h the NP detected in the nucleus could corresponds to newly synthesized NP and vRNPs, while the NP found in the cytoplasm can be attributed to newly made vRNPs that have been exported out of the nucleus. Immunofluorescence results of Nup153 showed that Nup153 was depleted (data not shown). Dashed lines in left and middle panels delineate the cell boundary. Scale bar, 10 µm.     102       103  Figure 3-9. Newly synthesized M1 localization is affected in Nup153 depleted cells 8 h post infection with IAV. Confocal images of HeLa cells treated with either Control siRNA or Nup153 siRNA, infected with IAV (H3N2 X: 31) for 8 h or 12 h, and prepared for indirect immunofluorescence microscopy using fluorescently-labelled phalloidin to detect F-actin (white) and an antibody against M1 (green). Nuclei were stained with Dapi. Observed M1 protein at 8 h can be attributed to newly synthesized M1. M1 is a late expressing protein during IAV infective cycle. Although only 25 kDa, M1 accumulates in the nucleus of the cell to form the complex of proteins required for the nuclear export of vRNPs. In contrast to control cells, M1 accumulated in the nucleus of Nup153 depleted cells at 8 h post infection. At 12 h post infection, M1 was primarily in the cytoplasm in both conditions. The arrows indicate dotty pattern of M1 found in Nup153 depleted cells at 12 h p.i. Immunofluorescence results of Nup153 showed that Nup153 was depleted (data not shown). Dashed lines in left and middle panels delineate the cell boundary. Scale bar, 10 µm.   104  At 12h after infection there was a larger accumulation of M1 in the cytoplasm in both control and Nup153 depleted cells (Figure 3-9, bottom panel). In the Nup153 siRNA treated condition, one particular cell (Figure 3-9, bottom panel, white arrow) displayed strong M1 dotty fluorescence at the edges of the plasma membrane. An accumulation of M1 at the plasma membrane would suggest clustered HA, NA and/or M2 viral proteins and a possible viral assembly site.  In summary, the immunolocalization of NP and M1 was altered at 8 h and 12 h p.i. in Nup153 depleted cells. If cytoplasmic NP staining represents newly made NP instead of assembled vRNPs at 8 h, then our data would be consistent with delayed nuclear export of vRNPs in Nup153 depleted cells. Alternatively, due to possible accumulation of newly made vRNPs in the cytoplasm and the clustering of M1 in the plasma membrane, there might be defects in the assembly/budding of new viral particles.  3.2.7. NP production, vRNP export, and overall IAV reinfection is hindered in Nup153 depleted cell after 24 h of IAV infection The initial results obtained from this study (Figure 3-1C) showed a decrease in infectious progeny viral particles when Nup153 depleted cells were infected with IAV. Even though we have identified differences between Nup153 siRNA treated cells and control siRNA treated cells during the early stage and mid-stage of IAV infection, it is necessary to detect additional changes in the life cycle of IAV in order to explain the decrease in the production of infectious virions. Thus, HeLa cells treated with either control siRNA or Nup153 infected for 24 h were examined. The expected findings were a mixed population of infected cells: cells recently infected from newly produced virus, mid-cycle infected cells (at 105  about 12 h), and infected cells actively producing vRNPs, but in the final stages of infection (before signs of apoptosis such as DNA condensation). We found that NP was localized in the nucleus and the cytoplasm in both control and Nup153 depleted cells (Figure 3-10, upper panel). In addition, patches of NP staining (most likely progeny vRNP) located at the cell periphery (Figure 3-10, arrows) were observed in infected Nup153 depleted cells. These results support the hypothesis that the budding of new viral particles might be faulty due to defects in the assembly of all viral components.  Besides NP, we also examined the localization of M1 after 24 h of infection in each siRNA treatment (Figure 3-10, lower panel). The results showed that the control siRNA treated cells exhibited M1 primarily in the cytoplasm, with observed accumulations of M1 staining throughout the cytoplasm. In contrast, M1 was located in the nucleus as well as the cytoplasm of IAV infected Nup153 depleted cells (Figure 3-10). These results could indicate that while control siRNA treated cells appeared to be in late stages of infection and actively producing new viral particles, Nup153 siRNA treated cells still contained M1 inside the nucleus. This suggests the presence of vRNPs inside the nucleus and defects in the viral cycle regarding the full nuclear export of vRNPs and the assembly of new virus.   106     107  Figure 3-10. IAV reinfection appears hindered in Nup153 depleted cell after 24h of infection with IAV. Confocal images of HeLa cells treated with either Control siRNA or Nup153 siRNA, infected with IAV (H3N2 X: 31) 24 h, prepared for indirect immunofluorescence microscopy using fluorescently-labelled phalloidin to detect F-actin (white) and antibodies against NP (red) or M1 (green). Nuclei were stained with Dapi. Observed NP protein at 24 h in control siRNA treated cells can be attributed to newly generated NP, which accumulated in the cell nucleus and was actively incorporated into vRNPs. The NP immunolabelling observed in the cytoplasm of control treated cells could correspond to newly synthesized NP or exported vRNPs. Nup153 depleted cells exhibited diminished NP nuclear fluorescence, indicative of reduced nuclear import of NP, and hence, reduced assembly of new vRNPs. Arrows point to the accumulations of NP near the cell periphery. In contrast, M1, in control treated cells was predominantly in the cytoplasm, which indicates that a large amount of progeny vRNP has been exported out of the nucleus. Alternatively, dotty M1 immunostaining could corresponds to M1 labelling found in endosomes in newly infected cells. In Nup153 depleted cells, M1 is seen primarily in the cytoplasm but is also present in the cell nucleus. Additionally, if dotty M1 is not present it could suggest lower reinfection rates. Immunofluorescence results of Nup153 showed that Nup153 was depleted (data not shown). Dashed lines in left and middle panel delineate the cell boundary. Scale bar, 10 µm.     108  3.2.8. Hemagglutinin (HA) traffic is affected in Nup153 depleted cells infected with IAV after 24 h In order to produce infectious progeny virions, IAV must pack all eight appropriate vRNPs in the budding virion for viral budding to take place on areas in the host cell plasma membrane where the transmembrane proteins HA, NA, and M2 are localized (reviewed by Noda and Kawaoka, 2012). In previous sections it was hypothesized that viral egress from Nup153 depleted cells might be deficient, which would result in the decrease of viral titer observed in Figure 1-1C. In order to further test this hypothesis, we evaluated the localization of HA in Nup153 depleted cells and control siRNA treated cells after 24 h IAV infection. The results showed that while HA distribution in control cells was primarily at the edge of the cell (Figure 3-11A, upper panel), HA predominantly formed patches at the plasma membrane of Nup153 depleted cells and in perinuclear areas of these cells (Figure 3-11A, mid and lower panel). Figure 3-11A depicts both a full field of view in which HA distribution can be observed in multiple cells and an enlargement panel that depicts the two main alterations in HA distribution found in Nup153 depleted cells. To better illustrate the modifications in HA distribution in Nup153 KD cells, we have included a cross-section of a three dimensional XYZ stack in Figure 3-11B. This figure shows that the fluorescence vesicle-like structures (white arrows) are located in the area adjacent to the nucleus in Nup153 KD cells.     109    110  Figure 3-11. HA traffic is affected in Nup153 depleted cells infected with IAV after 24 h. Confocal images of HeLa cells treated with either Control siRNA or Nup153 siRNA, infected with IAV (H3N2 X: 31) for 24 h, and prepared for indirect immunofluorescence microscopy using an antibody against H3 (red). Nuclei were stained with Dapi. Observed HA at 24 h can be attributed to newly produced HA, which follows the secretory pathway from ER to Golgi to plasma membrane. Control siRNA treated cells displayed HA primarily at the periphery of the cell, while Nup153 depleted cells displayed HA in clusters at the perinuclear area and/or at the cell periphery (second Nup153 panel). Bottom panel shows an X-Y composite from a XYZ acquired image showing the circular “vesicles” labelled by HA immunostaining in the perinuclear area (arrows). Immunofluorescence results of Nup153 showed that Nup153 was depleted (data not shown). Scale bar, 10 µm (A, left panel; B), 5 µm (A, enlargement).   111  Taken together, the results obtained in the two previous sections along with the observation regarding the accumulation of HA in the plasma membrane and other indeterminate intracellular structures indicate that Nup153 depleted cells support the nuclear import and export of the vRNPs, but the assembly or budding of newly infectious IAV particles is compromised.  3.3. Discussion In this chapter, we used Nup153RNAi in order to determine the role of Nup153 during the IAV infection cycle. Our findings showed a significant decrease in the production of infectious viral particles in Nup153 depleted cells infected with IAV (Figure 3-1C). Although we use a different cell line and a different IAV stain, our results are in agreement with previous published findings (Benitez et al., 2015; König et al., 2010; Watanabe et al., 2014; York et al., 2014). Our next objective was to determine at which stage of the IAV viral infective cycle Nup153 was required. Experiments with chimeric proteins containing the NLSs of NP indicated a decrease in NP NLSs-mediated nuclear import in Nup153 cells, although the nuclear import of vRNPs during infection did not appear to be largely affected. We further examined the IAV life cycle through the detection of NP and M1 distribution in a 24 h infection period, and found alterations in the nuclear and cytoplasmic distribution of NP and M1 throughout most of the infective cycle, which indicate defects in the early uptake and traffic of IAV. Finally, we visualized changes in the distribution of HA in IAV infected Nup153 depleted cells, which indicates possible defects in IAV assembly or budding in Nup153 KD cells.  112  We found a decrease in the nuclear import of 5GFP containing NLS1 or NLS2 of NP (Figure 3-2) in Nup153 depleted cells. Surprisingly, the nuclear import of NP or the nuclear import of vRNPs was not hindered. It is possible that the nuclear import of NP was not affected even though each NLS was affected independently. This is because both signals in the protein would be enough for the efficient import of NP into the cell nucleus. The nuclear import of vRNPs could be due to the large number of NLSs they have. The IAV vRNPs vary in sizes and each of them contains between 37 and 97 copies of NP (reviewed by Wu et al. 2007b). Given that each NP contains at least two NLSs, NLS1 and NLS2, each vRNP contains between 74 and 194 NLSs in total. It is likely that the presence of both NLSs in such large numbers resulted in the successful nuclear import of vRNPs. Overall, the combined data conclusively demonstrated that Nup153 depletion does not disrupt the nuclear import of IAV vRNPs within the context of viral infection. During early infection we detected a significant reduction in the NP immunostaining in Nup153 depleted cells (Figures 3-4B and 3-5C). This reduction indicates that although IAV entered Nup153 KD, its uptake was defective in Nup153 KD cells. Interestingly, NP immunolabelling at 1 h p.i. (Figure 3-5) indicates that incoming vRNPs localization was different that M1 localization, which was clustered along the edges of the cells. Since M1 dissociates from vRNPs in ‘late endosomes’ (Section 1.1.3), it is possible that endosomal-viral fusion in Nup153 depleted cells occurs nearby the plasma membrane instead of the nuclear periphery; thus, vRNPs were released far away from the nucleus. This would explain the lack of NP clusters (Figure 3-5) at the area adjacent to the cell membrane. These results further suggest a defect in the entry and/or early traffic of IAV in Nup153 depleted cells.  113  The nuclear export of IAV HA, M2, and M1 mRNA is mediated by the nuclear export receptor NXF1/TAP (Read & Digard, 2010). Moreover, IAV RNA can bind to NXF1-p15, which facilitates viral mRNA export and viral replication through interactions with the FG-nucleoporin Nup62 (Morita et al., 2013). Although Nup153 has been shown to bind NXF1 (reviewed by Ball & Ullman, 2005) and facilitate the nuclear export of mRNA, it appears that Nup153’s role in mRNA nuclear export is dispensable for IAV. It is possible that interactions of the IAV mRNA with other FG-repeats nucleoporins are the primary requirement. Although Nup153’s specific requirement for the nuclear export of IAV mRNA needs to be further investigated, our data allow us to speculate that Nup153 depletion does not alter the nuclear export of IAV mRNA.  The nuclear export of vRNPs is required for the assembly of newly produced IAV viruses. Interestingly, multiple steps are required for this to occur, such as the vRNP association with M1, the phosphorylation of a viral or cellular factor, the regulation of chromosome condensation 1 (RCC1) and CRM1-mediated nuclear export (Ma et al., 2001). Nup153 interacts with CRM1 (Nakielny et al., 1999) and there is evidence that suggests blocking Nup153 with antibodies in Xenopus oocytes results in obstruction of the NES export pathway, particularly with respect to the HIV-1 Rev protein (Ullman et al. 1999). However, our data shows no evidence that vRNPs/NP remains arrested in the nucleus of the Nup153 KD cells. On the contrary, we observed plenty of cytoplasmic staining of vRNPs/NP (Figure 3-8, lower panel) in Nup153 KD cells during late infection. Thus, we proposed that the nuclear export of vRNPs/NP is not affected in Nup153 KD cells 114  Despite our finding that the IAV cellular uptake was reduced and the trafficking of incoming virions/vRNPs was delayed in Nup153 KD cells, viral protein production and vRNP assembly (as judged by the immunolabelling of both NP and M1) appeared to occur in a timely manner in these cells. However, the nuclear/cytoplasmic distribution of both NP and M1 was dramatically altered at later time of infection in Nup153 KD cells (Figures 3-6 to 3-10). For example, at 12 p.i. NP was accumulated in the cytoplasm of Nup153 KD cells, but not in the control cells (Figure 3-8). A possible explanation for this cytoplasmic accumulation of NP immunolabelling, which corresponds to accumulation of progeny vRNPs in the cytoplasm, is that the nuclear export of vRNPs occurs at an earlier time point in Nup153 depleted cells than in control cells. It is possible that in IAV infected Nup153 KD cells, caspase-3 mediated alterations of the NPC and its permeability take place earlier during the infective cycle than in control siRNA cells. As consequence there could be a dysregulation in the tight control of the nuclear import/export of viral components. Interestingly, caspase-3 activation is required for viral propagation; the inhibition of caspase-3 was found to also result in the retention of vRNPs inside the nucleus of infected cells (Wurzer et al., 2003). Further studies suggested that Nup153 degradation and redistribution were involved in the caspase-mediated enlargement of the NPC during late infection, which was required for the nuclear export of vRNPs in MDCK cells (Mühlbauer et al., 2015). However, previous reports showed a downregulation of Nup98 during IAV infection, but no changes in the expression of Nup153 after 24 h infection in 293T cells (Satterly et al., 2007). During apoptosis, caspase-3 enters the nucleus of apoptotic cells and cleaves nucleoporins such as POM121 and later, Nup153 and Nup62 (Ferrando-May et al., 2001; Kihlmark et al., 2004). However, nuclear permeabilisation during apoptosis occurs 115  ahead of the cleavage of nucleoporins (Ferrando-May et al., 2001). Although Nup153 is indeed a substrate for degradation by caspase-3, Nup153 depletion does not affect nuclear permeability (Figure 5-10). Hence, degradation of this protein alone would not result in the widening of the NPC. It is therefore possible that the effects observed by Mühlbauer et al. (2015) apply not only to Nup153, but also to Nup62 and Nup214, which are two other nucleoporins recognized by the QE5 antibody (Pante et al., 1994) used in their study.  An alternative explanation for the accumulation of progeny vRNPs in the cytoplasm of Nup153 KD cells at 12 h p.i. is that the assembly/budding of new viral particles is defective. In Nup153 KD cells vRNPs accumulated in the cytosol after their nuclear export because they could not be assembled into progeny virion. In contrast, in control cells after progeny vRNPs were exported from the nucleus, they were immediately delivered to the plasma membrane to assemble progeny virions, which successfully budded from the cell; thus, vRNPs do not accumulate in the cytoplasm of control cells at later time of infection. In support of this explanation, M1 accumulates in the plasma membrane of Nup153 KD cells (Figure 3-9). Timely assembly of progeny virion in control cells does not allow for such accumulation of M1. Furthermore, our findings showed that HA distribution was severely affected in Nup153 depleted cells (Figure 3-11) due to viral protein accumulation in distinctive areas of the cell periphery as HA-filled vesicles or HA-delineated vesicles in the perinuclear area. These results support defects in the assembly/budding of progeny virion, and could indicate that there are substantial disturbances in the secretory pathway that affect the cellular distribution of HA, which could also affect other viral transmembrane proteins (i.e. M2 and NA) that traffic through the secretory pathway.  116  In summary, the results show that IAV appears to successfully enter Nup153 depleted cells and the vRNPs are imported into the nucleus. However, the cytoplasmic traffic of NP/vRNPs towards the nucleus was delayed in the early steps of the IAV life cycle in the Nup153 depleted cells, perhaps due to altered endosomal dynamics in these cells. In light of the findings regarding the localization of NP, M1, and HA in Nup153 KD cells during late infection, we propose that the observed reduction in viral titer could be caused by an overall decrease in viral egress from the cell. The reason for this decrease could be deficient assembly or reduced viral infectivity due to the altered composition of viral proteins in the membrane of the newly formed virion. Further studies are required to identify the reason for a ~3.5-fold decrease in infectious viral production in Nup153 depleted cells, as compared to control conditions (Figure 3-1C). Pleiotropic effects resulting from Nup153 KD could be the overall reason behind a decrease in viral titer, which will be discussed in the next two chapters.    117            Chapter 4 Effects of Nup153 depletion on cargo uptake  and endocytic organelle distribution Effects of Nup153 depletion on cargo uptake and endocytic organelle distribution 4.1. Introduction It is now well established that nucleoporins have beyond nuclear-cytoplasmic transport, nucleoporins have a diversity of functions within the cell. For example, the RNAi depletion of Nup153 results in growth arrest (Harborth et al., 2001) and altered gene expression (Vaquerizas et al., 2010). More recently, Nup153 has been implicated in epigenetic silencing that controls stem cell differentiation (Jacinto et al., 2015). In Chapter 3, we found a decrease in the production of infective viral particles in Nup153 depleted cells infected with IAV. After thoroughly following NP and M1 during a 24-hour infection period, we detected a deficiency in the viral uptake and early virus trafficking in Nup153 depleted cells. In addition, HA displayed changes in distribution between control and Nup153 siRNA treated cells 24 hours after infection; which suggested a disturbance in the protein targeting to the plasma membrane in Nup153 depleted cells. Such changes may be a consequence of Nup153 indirectly affecting both retrograde and anterograde intracellular trafficking. Thus, it was hypothesized that there may be changes to the endosomal/secretory pathway in Nup153 depleted cells. In this chapter this hypothesis was tested by studying the endocytic and exocytic pathways in non-infected Nup153 depleted cells in order to determine the changes occurring in the cell as a consequence of the Nup153 depletion. This study also aimed to further clarify whether the deficiencies observed during an infection with IAV were occurring only due to the virus or were a generalized consequence of Nup153 depletion. 118  The endocytic machinery is a major trafficking pathway in mammalian cells and defines the interactions between the cell and its environment. The endo-lysosomal pathway is essential for nutrient uptake, protein recycling, intracellular communication, pathogen entry, and degradation (reviewed by Repnik et al. 2013). It was hypothesized that intracellular traffic is perturbed in Nup153 depleted cells and as a consequence, processes such as the uptake of extracellular cargo would be affected. To test this hypothesis, the presence and distribution of sialic acids in the plasma membrane of live cell was first analyzed. This allowed inferring whether IAV binding would be affected in Nup153 depleted cells. Then immunofluorescence was used to determine changes in the localization and distribution of the epidermal growth factor receptor (EGFR) since it has been implicated in IAV uptake (Eierhoff et al., 2010). In addition, the transferrin receptor (TfR) was examined, which is unlike the EGFR in that it is recycled back to the plasma membrane after its cargo has been internalized and delivered. The uptake of two classical, well-characterized and distinctive receptor-mediated ligands was studied: EGF and Tfn. To further understand the endocytic and exocytic pathways, the localization (and in some cases abundance) of common endosomal and lysosomal markers was also evaluated. To gather data on the exocytic pathway, we detected the distribution of a luminal ER marker and used cells transfected with a HA plasmid to examine whether the ER-Golgi-plasma membrane exocytic pathway was disturbed in Nup153 KD cells. Finally, we examined the cell cytoskeleton in Nup153 depleted cells due to the close relationship between vesicular traffic and the cytoskeleton. The findings presented in this chapter showed a deficient uptake of ligands that was associated with receptor and non-receptor mediated endocytosis in Nup153 depleted cells. Additionally, it was found that endosomal markers corresponding to early endosomes, late 119  endosomes, and lysosomes were redistributed throughout the cell. The research presented here also showed possible alterations in the exocytic pathway, suggesting that there could be defects in the intracellular vesicle traffic. Furthermore, an altered cytoskeleton in Nup153 depleted cells was shown. The proposition is that the changes in the cytoskeleton are responsible for the alterations observed in vesicle distribution throughout the cell. Overall, we showed that the depletion of a nucleoporin can have serious consequences in processes extending beyond nuclear transport. This is the first time that Nup153 depletion has been associated with changes in the intracellular traffic. However, it is likely that the effects described in this chapter are downstream consequences resulting from alterations in gene expression or in the nuclear transport of any cellular factors that are negatively affecting the cell’s internal communication systems and the cytoskeleton.  4.2. Results 4.2.1. Wheat germ agglutinin (WGA) labelling is altered in Nup153 depleted cells In Chapter 3, we observed apparent changes in virus uptake/traffic during the first 15 min after the IAV infection of Nup153 depleted HeLa cells (Figure 3-4). Because of these changes, we hypothesized that the binding of IAV to Nup153 depleted cells was altered. The IAV HA protein binds sialic acids present at the cell’s plasma membrane as a first step for viral infection. The sialic acids and N-acetylglucosaminyl residues in the exterior of the cell also bind to the lectin WGA (Bhavanandan and Katlic, 1979; Brown and Hunt, 1978; Gallagher et al., 1985). To determine whether sialic acid distribution was altered in Nup153 depleted cells, we treated HeLa cells with either control siRNA or Nup153 siRNA and incubated them with WGA-Alexa fluor 594. Further, the live cells were visualized using 120  confocal microscopy. RNAi conditions were the same as previously shown in Chapter 3 (Figure 3-1B). We found that control siRNA treated cells displayed WGA-Alexa fluor 594 staining around the cell’s plasma membrane and in the center of the cell (the latter with a dotty pattern) (Figure 4-1A). In contrast, the WGA-Alexa fluor 594 label was patchy around the plasma membrane of Nup153 depleted cells (Figure 4-1A, white arrows) and the cell labelling was found throughout the plasma membrane and did not accumulate in the middle of the cells (Figure 4-1A).  Figure 4-1B graphically illustrates the differences in WGA fluorescence signal distribution observed in Figure 4-1A. The grey intensity profile across the arrow through two cells of the control sample first displays a well-defined peak labelled as “PM”, which corresponds to the plasma membrane of the first cell. Then, a series of higher peaks labelled with asterisks “*” represent the signal from the dotty accumulations within the two cells. This is followed by another peak representing the PM labelling in the boundary between both cells, which is in turn followed by a wide peak representing the dotty accumulations in the second cell (labelled with *). Finally, one last high peak indicates the PM signal of the second cell. In contrast to the WGA profile showed in control siRNA cell, the PM peaks in Nup153 KD cells are followed by several peaks of almost equal or larger intensity, indicating the patchy immunolabelling of WGA at the PM of Nup153 KD cells.  The differences in the WGA labelling of control and Nup153 KD cells is further illustrated in Figure 4-1C, which shows a high magnification of the cells in the boxed areas in Figure 4-1A. Figure 4-1C clearly shows that in control siRNA cells, the labelled WGA distribution in the plasma membrane is uniform and the dotty internal label is accumulated in 121  an area closer to the nucleus. This pattern is not observed in Nup153 depleted cells. Further enlargement of the images in Figure 4-1C documents the “patchy” accumulation of WGA at the edge of the cell in Nup153 depleted cells (Figure 1C, right panels). Altogether, the patchy distribution of the lectin WGA showed a defect in the distribution of sialic acid and N-acetylglucosaminyl sugar residues at the plasma membrane of Nup153 KD live cells. These findings could be indicative of further alterations in the location of plasma membrane receptors.   122      123  Figure 4-1. Extracellular binding of WGA is affected in live Nup153 depleted cells. HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h, and then incubated with 5 µg/ml of WGA-Alexa fluor 594 (red) at 37°C for 10 min. NucBlue was added to visualize the nucleus (blue). Live cells were imaged using confocal microscopy. A) Live HeLa cells from both conditions labelled with WGA-Alexa fluor 594. Arrow heads denote WGA label accumulated at the plasma membrane of Nup153 siRNA treated cells. B) Gray intensity profile of a transversal section (white long arrows) from two cells in (A). The white long arrows in A are the specific area where the histogram displays relative fluorescence intensity of gray values. Peaks labelled (PM) correspond to the plasma membrane and WGA dotty accumulations are labelled as (*). C) Magnification of boxed areas in (A), colored as observed during live cell microscopy (red, WGA-Alexa fluor 594; blue, DNA). Live cells were also observed by DIC imaging (merge). In (C), boxed areas are magnified (right panel) to show the accumulation of WGA label at the cell edges. Scale bars, 10 μm.   124  4.2.2. The epidermal growth factor receptor (EGFR) cell distribution is altered and epidermal growth factor (EGF) uptake is markedly decreased in Nup153 depleted cells The EGFR, as a representative of the RTKs, is engaged in signal transmission upon IAV binding to the cell that facilitates viral uptake (Eierhoff et al., 2010). Additionally, like IAV, the EGFR-EGF complex is internalized by the cell through clathrin-mediated endocytosis and follows the endocytic pathway (reviewed by Madshus and Stang, 2009). However, unlike IAV, a large population of the EGF-EGFR complex reaches the lysosomes, where it is degraded (reviewed by Madshus & Stang 2009). Because the activation of RTKs is important for IAV viral uptake, we examined the localization and distribution of EGFR in the plasma membrane of Nup153 depleted cells. Traditionally, fetal bovine serum (FBS) is added to cell cultures because it is rich in nutrients and contains growth factors (reviewed by Gstraunthaler, 2003). However, the presence of FBS in the culture media negatively interferes with IAV binding and uptake (Hartley et al., 1992; Iki et al., 2005). For this reason, IAV infections are performed under the condition of reduced FBS content or no FBS at all. Following this logic, we studied the cellular distribution of EGFR in either the presence or absence of FBS. To determine EGFR localization, control siRNA and Nup153 siRNA treated cells were kept in full growth media with FBS or serum starved (-FBS) overnight and then endogenous EGFR was immunolabelled and detected by confocal immunofluorescence microscopy. EGFR distribution in control siRNA treated cells in growth media (+FBS) was found mainly in the plasma membrane, with a small population of EGFR near the nuclear area (Figure 4-2A, white arrows). F-actin staining was used to identify the cell boundary and 125  EGFR was present at the cell boundary in the control cells, as shown by the merge image (Figure 4-2A). In contrast, Nup153 KD cells in growth media (+FBS) displayed irregular patchy staining of EGFR at the cell’s plasma membrane (Figure 4-2A). In addition, the intracellular staining of EGFR was visually distributed throughout the whole cell, and there were no easily identified populations near the cell nucleus. After the FBS was removed and the EGFR staining in serum-starved cells was examined, control siRNA treated cells showed EGFR staining primarily at the plasma membrane and dispersed throughout the cell. However, the EGFR label was accumulated asymmetrically (Figure 4-2A, small white arrows) at the cell edge in Nup153 depleted cells, presumably near the plasma membrane.  The quantification of whole cell EGFR fluorescence intensity from a large number of cells (n>200) shows significantly more EGFR signal in Nup153 depleted cells than in control cells, independently of whether the cells were serum starved (Figure 4-2B). However, when EGFR expression was assayed by WB (Figure 4-1C), there was no difference in the EGFR protein levels between non-treated cells, control siRNA treated cells, and Nup153 depleted cells under regular growth media conditions (+FBS). In combination, these results suggest that EGFR expression was not modified, although its cellular distribution was highly altered in Nup153 depleted cells.    126   127   Figure 4-2. Transient depletion of Nup153 by RNAi induces defects on the cellular distribution of EGFR. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h, cells were incubated in media with FBS (+FBS) or serum starved (-FBS) for 24 h and immunolabelled with antibodies against EGFR (green) and Nup153 (white). Cells were also labelled with Dapi (blue) to observe the nucleus and rhodamine-phalloidin to visualize F-actin (red). Boxed areas are shown in the small panels on the right of the merge images depicting EGFR, F-actin and merge channels. Scale bars, 10 μm. B) Quantitative analysis of EGF fluorescence intensity per cell for the experiments shown in (A). For each condition, the relative fluorescence intensity (%) was quantified in n>200 cells from three independent experiments and the average mean values are shown. Error bars are standard error of the media (S.E.M). Statistical analysis was determined using ANOVA with Tukey’s multiple comparison test; *** p < 0.0001. C) Abundance of EGFR was detected through WB analysis of Nup153 siRNA treated, control siRNA treated, and untreated HeLa cells in regular growth media (+FBS). Lysates were analyzed by immunoblotting with antibodies against Nup153 (SA1), EGFR, and β actin. β actin expression was used as a loading quality control.     128  Finally, these results prompted us to hypothesize that an irregular distribution of EGFR would have an effect on the uptake of its ligand, EGF. To determine EGF uptake, control siRNA and Nup153 siRNA treated cells were serum starved overnight and incubated with EGF coupled to Alexa fluor 647 (500 µg/ml) at 4°C for 15 min to allow binding to EGFR. The EGF-Alexa fluor 647 was then removed and the cells were prepared for confocal immunofluorescence microscopy after 15, 30, and 60 min. After uptake had occurred for 15 min, less EGF signal was found in Nup153 depleted cells than in control siRNA treated cells (Figure 4-3A). The quantification of EGF fluorescence intensity for both conditions at different times shows a significantly smaller initial amount of EGF being detected in comparison to control siRNA cells (Figure 4-3B). The EGF-Alexa fluor 647 fluorescence signal continued to decrease in both conditions as time increased (Figure 4-3B), most likely due to EGF-EGFR complex degradation.  The small amount of EGF that entered the Nup153 depleted cells at the initial time point posed a caveat for the experiment as it did not allow determining if traffic of EGF was affected. To address this caveat, pHrodo-EGF was used. The pHrodo is not fluorescent, or is weakly fluorescent at a neutral pH (external cell environment). Once the cell takes up the pHrodo-EGF, a slow acidification occurs when endosomal compartments shift from early endosomes to late endosomes and finally lysosomes. Then, the pHrodo-EGF becomes brightly fluorescent. For this experiment, serum starved cells treated with either control siRNA or Nup153 siRNA were incubated with pHrodo EGF at 37°C and imaged over a period of 140 minutes. The results show that the number of EGF particles detected (by fluorescence microscopy) increased with time under both conditions (Figure 4-3C) and 129  similar to the experiment with EGF-Alexa Fluor 647 (Figure 4-3A), more EGF was detected in control treated cells than in Nup153 treated cells (Figure 4-3C).  Taken together, these results suggest that Nup153 depleted cells exhibit defects in the distribution of EGFR, which results in the decreased binding and uptake of EGF. However, once the EGF-EGFR complex is endocytosed, there is no observable difference between control and Nup153 KD cells. This suggests that progressive traffic through the endocytic pathway and acidification is not affected in Nup153 KD cells, as shown by the experiment with the pHrodo dye. 130      131  Figure 4-3. Depletion of Nup153 reduces the cellular uptake and traffic of EGF. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h. Cells were serum starved for 24 h and then incubated with 500 µg/ml of EGF-Alexa Fluor 647 (A-B) or pHrodo-EGF (C) at 4°C for 15 min. A) Cells were immunolabelled with an anti-Nup153 antibody (white). Rhodamine-phalloidin was used to visualize F-actin (red) and Dapi (blue) to observe the nucleus. Scale bar, 10 μm. B) Quantification of relative fluorescence intensity (%) of EGF-Alexa Fluor 647 for experiments shown in A. For each condition, the relative fluorescence intensity (%) was quantified in n>100 cells from three independent experiments and the mean values are shown. Error bars represent S.E.M. Statistical significance between different conditions were determined by Student’s t-test (*** p < 0.05). C) Quantification of the number of vesicles (signal present >2 pixels) containing pHrodo-EGF through time for experiments performed as indicated in A but with pHrodo-EGF instead of EGF-Alexa Fluor 647. Quantifications were performed with n>20 cells. Data shown represents mean value of number of particles per cell.   132  4.2.3. The transferrin receptor (TfR) cell distribution is altered and transferrin (Tfn) uptake is markedly decreased in Nup153 depleted cells To examine whether the changes observed in EGFR distribution and EGF uptake were specific to the EGF receptor, we determined the distribution of TfR and the uptake of Tfn in Nup153 depleted cells. The TfR is a glycoprotein and is internalized independently of the EGFR (Leonard et al., 2008), but both EGF and Tfn receptors are internalized via clathrin-mediated endocytosis. However, unlike EGFR, TfR follows a different route in the endocytic pathway. The TfR is recycled back to the plasma membrane following the release of bound iron in the early endosome. Hence, the TfR is first found in a population of early endosomes, then in sorting endosomes and recycling endosomes, and then it traffics back to the plasma membrane. Alternatively it could bypass the recycling endosomes and traffic directly to the plasma membrane.  The cellular distribution of endogenous TfR in control and Nup153 siRNA treated cells was determined by immunofluorescence confocal microscopy. Cells were maintained in full growth media (+FBS) or serum starved overnight (-FBS) previous to cell fixation. We found that in full growth media, control siRNA treated cells showed a clear accumulation of TfR (Figure 4-4A, white arrows) in the area adjacent to the nucleus. This particular distribution was lost upon Nup153 depletion. Moreover, in Nup153 depleted cells there was TfR staining throughout the whole cell (Figure 4-4A). When the cells were serum starved the TfR was found to be dispersed through the whole cells in control cells, very much as it was found to be in full growth conditions in Nup153 depleted cells (Figure 4-4A).   133      134  Figure 4-4. Transient depletion of Nup153 by RNAi induces defects on the intracellular distribution of TfR. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h, after 48 h cells were incubated in media with FBS (+FBS) or serum starved (-FBS) for 24 h. Cells were immunolabelled with antibodies against TfR (green) and Nup153 (white). Samples were also labelled with rhodamine-phalloidin to visualize F-actin (red) and Dapi (blue) to observe the nucleus. Arrows indicate areas where the TfR is accumulated in the cytoplasmic area adjacent to the nucleus of controls cells cultured in growth media (+FBS). Arrows in Nup153-depleted cells (-FBS) indicate distinct cytoplasmic areas where TfR is accumulated. Scale bar, 10 μm. B) Quantitative analysis of TfR relative fluorescence intensity for experiments shown in (A). For each condition, the relative fluorescence intensity (%) was quantified in n>150 cells from three independent experiments and the mean values are shown. The error bars correspond to S.E.M. Statistical analysis for (B) were performed using ANOVA with Tukey’s multiple comparison test; *** p < 0.0001. C) Abundance of TfR was detected through WB analysis of Nup153 siRNA treated, control siRNA treated, and untreated HeLa cells in regular growth media (+FBS). Lysates were analyzed by immunoblotting with antibodies against Nup153 (SA1), TfR, and β actin. β actin expression was used as a loading quality control.    135  Finally, the results showed that when Nup153 depleted cells were serum starved, the TfR receptor was non-uniformly distributed and accumulated in visible patches (Figure 4-4A, white arrows), unlike its control siRNA treated cells counterpart (Figure 4-4A). The quantification of the fluorescence intensity showed more TfR in Nup153 depleted cells than in control siRNA treated cells under regular growth media conditions (Figure 4-4B). Likewise, fluorescence decreased in both conditions once the cells were under serum starvation, but there was still more fluorescence intensity in the Nup153 KD cells than in control cells (Figure 4-4B). Interestingly, more TfR fluorescence intensity (Figure 4-4B) in Nup153 KD cells did not correlate with an increase in TfR expression in regular growth media (Figure 4-4C).  As was the case for the EGFR-EGF experiments, we set up to determine whether the distribution of TfR would affect the uptake of Tfn. To determine this effect, control siRNA and Nup153 siRNA treated cells were serum starved overnight and incubated with 20 µg/ml of FITC-Tfn at 4°C for 15 min to allow Tfn to bind to TfR. The cells were then prepared for immunofluorescence microcopy at different times during the 1 hour period. As documented in Figures 4-5A and 4-5B, initially (0 minutes) less Tfn was found attached to Nup153 KD cells than to control cells. In the case of control siRNA cells, the Tfn fluorescence decreased with time. However, in Nup153 depleted cells, Tfn fluorescence remained low through all the time points (Figures 4-5A and 4-5B). Interestingly, as shown in Figure 4-5A, Nup153 depleted cells at 30 min show an accumulation of Tfn similar to that observed at the edge of the cells for TfR in Figure 4-4A (Nup153 depleted cells –FBS). The accumulation of Tfn could occur due to its binding to a cluster of TfR. However, Tfn was not taken up by the cell, which could indicate possible defects in the endocytosis of the TfR-Tfn complex.  136  On the whole, our results showed that the TfR distribution is altered in Nup153 depleted cells, which might result in the decreased binding and uptake of Tfn. The TfR accumulation at the plasma membrane also matches Tfn accumulation after 30 min, which could suggest defects in the uptake of the TfR-Tfn complex. The increase in Tfn vesicle density in Nup153 depleted cells could indicate defects in the endocytosis and traffic of the Tfn-TfR complex.   137   Figure 4-5. Tfn endocytosis is affected in Nup153 depleted cells. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h, and then incubated with 20 µg/ml of FITC-Tfn at 4°C for 15 min. Cells were then warmed up to 37°C for the indicated time before fixation. Early time point showed accumulation of FITC-Tfn at the plasma membrane in Nup153-depleted cells. Scale bar, 10 μm. B) Quantification of FITC-Tfn fluorescence intensity per cell of each time point as shown in (A). For each condition, the relative fluorescence intensity (%) was quantified in n>100 cells from three independent experiments and the mean values are shown. The error bars correspond to S.E.M. Statistical analysis for (B) were determined by Student’s t-test, ***p<0.05.  138  4.2.4. Dextran uptake decreases in Nup153 depleted cells Our findings in Sections 4.2.2 and 4.2.3 showed a defect in the uptake of EGF and Tfn. Once both cargoes are bound to their receptors, they are taken up by the cell through clathrin-mediated endocytosis. To further investigate whether other cellular uptake are defective in Nup153 KD cells, we examined macropinocytosis, which is an alternative cellular entry pathway. Macropinocytosis is among the multiple pathways through which IAV enters the cell (reviewed by Zhang & Whittaker, 2014). We hypothesized that since we detected low NP fluorescence after 5 min of infection and IAV entry into Nup153 KD cells was diminished (Figures 3-4A and 3-4B), the process of macropinocytosis could be affected in Nup153 KD cells. Dextran was chosen as the cargo to study macropinocytosis. This complex glucan lacks cell-specific membrane receptors and upon uptake, it follows the endocytic pathway and accumulates at lysosomes (Ohkuma & Poole, 1978). Nup153 depleted cells and control siRNA treated cells were incubated with dextran conjugated to Alexa fluor 594 for 15 min at 37 °C to allow cellular uptake. Dextran was later removed and the cells were incubated for an additional 15 min at 37 °C. Live imaging acquisition was performed using confocal microscopy. Our images showed a decrease in dextran-containing vesicles (red dots) uptake and size in Nup153 depleted cells (Figure 4-6). Moreover, the spatial distribution of dextran-containing vesicles was not observably different between Nup153 depleted cells and control siRNA treated cell (Figure 4-6A).  In order to quantify dextran uptake, we implemented the field index (Commisso et al., 2014). In brief, this index takes the sum of the measures of every particle’s area and divides 139  it by the total cell area, then multiplies the resulting number by 100. The average of the ‘field index’ for all the cells in each condition constitutes the ‘macropinocytic index’. The macropinocytic index shows that control siRNA cells took up twice more dextran than Nup153 depleted cells (Figure 4-6B). Additionally, the size of dextran-containing vesicles remained fairly consistent in control siRNA treated cells, while Nup153 depleted cells showed a wider variety of sizes of vesicles containing dextran (with an overall average of almost half the size of its control cell counterparts) (Figure 4-6C).   When combined, these results suggest that dextran uptake is negatively affected in Nup153 depleted cells. Thus, the previously observed deficiencies in uptake of EGF-EGFR and Tfn-TfR are not exclusive to cargoes that use receptor-mediated endocytosis. Furthermore, the data obtained from the size of dextran-containing vesicles (Figure 4-6C) in Nup153 depleted cells suggests that when cargo does successfully enter the cells, it accumulates in vesicles that are smaller than the vesicles in control siRNA treated cells.  140     141  Figure 4-6. Transient depletion of Nup153 by RNAi results in defects in dextran uptake and dextran-containing vesicle size. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h and incubated with fluorescent dextran (MW 3,000 Da) for 15 min at 37 °C, then dextran was removed and cells were incubated for 15 min at 37 °C. The dextran was conjugated with Alexa fluor 594 (red). NucBlue (blue) was added to detect the cell nucleus. Samples were observed live by confocal microscopy. Right panels show live cells though DIC imaging merged with fluorescence channels. Scale bar, 10 μm. B) Quantifications of dextran uptake through field index. Field index is calculated by quantifying the total number of fluorescence vesicles per cell (each dot) observed in each condition and divided by the total cell area. Line represents mean field index value, known as macropynocitic index. Field index values were obtained from three independent experiments, n≥18 cells per experiment. The error bars correspond to S.E.M. Statistical analysis for (B) were determined by Student’s t-test, **p<0.05. C) Quantification of dextran-Alexa fluor 594 vesicle size. Field index was quantified in n>12 cells from each condition. Line represents mean value. The error bars correspond to S.E.M. Statistical analysis for (C) were determined by Student’s t-test, **p<0.05.    142  4.2.5. Transient depletion of Nup153 by RNAi affects the distribution of the endocytic marker EEA1 The above results demonstrated significant changes in the binding and uptake of ligands in Nup153 depleted cells. All the cargoes studied in the previous sections are known to traffic through early endosomes. Thus, we hypothesize that the cellular localization and distribution of endocytic organelles might be affected in Nup153 KD cells. To test this hypothesis, we next used EEA1 as a marker for early endosomes to detect changes of these organelles in the Nup153 KD cells. HeLa cells were treated with either Nup153 siRNA or control siRNA for 72 h and prepared for immunolabelling with an anti-EEA1 antibody. The localization of EEA1 in control siRNA treated cells was predominantly at one side of the cell, while in Nup153 depleted cells, the EEA1 staining vesicles appeared to be randomly dispersed throughout the cytoplasm (Figure 4-7A). In order to further corroborate these findings, we used a spatial distribution analysis approach developed by Andrey et al. (2010) for a three-dimensional analysis of centromeres and chromocenters in plant and animal nuclei and found that indeed the EEA1 staining in Nup153 depleted cells follows a spatial distribution pattern closely related to a random distribution pattern (Appendix B, Figure B-1). In contrast, the spatial distribution pattern of control siRNA treated cells indicates that the EEA1 staining was not randomly distributed but instead was aggregated (Appendix B, Figure B-1B). In addition, calculations of the cumulative distribution function (CDF) of the distance between fluorescent dots and its nearest neighbor showed that in Nup153 depleted cells, the EEA1 stain was significantly further from its nearest neighbor (Appendix B, Figure B-1C). Thus, confirming that the EEA1 spatial distribution was more spread-out in Nup153 depleted cells than in control cells.  143  Further quantitative analysis indicated that EEA1 positive endosomes nearest-neighbor were distributed more randomly in Nup153 depleted cells than in control cells (Appendix B, Figure B-1F). Additionally, in control cells, the EEA1 staining had a non-random regularly space pattern, while the distribution observed in Nup153 depleted cells suggests that in these cells the EEA1 staining was random and unevenly distributed (Appendix B, Figure B-1H). Finally, to further characterize the EEA1 positive vesicles, we measured their sizes and circularity. Nup153 depleted cells resulted in having smaller EEA1 positive vesicles compared to control cells (Figure 4-7B). This is consistent with the results previously obtained from endocytosed cargo where we showed smaller dextran-containing vesicles (Figure 4-6C), which traffics through EEA1 positive early endosomes. In addition, the EEA1 immunostaining was regularly more circular in Nup153 depleted cells (Figure 4-7C).   144   Figure 4-7. Transient depletion of Nup153 by RNAi affects the distribution of the early endocytic marker EEA1. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h. Control and Nup153 siRNA treated cells were immunolabelled with antibodies against EEA1 (green) and Nup153 (white). Cells were also labelled with Dapi (blue) to observe the nucleus and fluorescently-labelled phalloidin to visualize F-actin (not shown); the latter was used to delineate cell boundaries. Scale bar, 10 μm. B) Quantification of EEA1 dots (or EEA1 positive vesicles). C) Quantification of EEA1 positive vesicles circularity shape, where a value closer to 1 resembles a perfect circle. The values shown in (B) and (C) are the mean values obtained from three independent experiments, n≥20 cells per experiment. The error bars represent the S.E.M and the significance in (B) and (C) were tested using a Student’s t-test, **p<0.05, ***p<0.001.  145  4.2.6. Cellular distribution of the recycling endosome marker Rab11 is not affected by Nup153 depletion There are other types of endosomes in addition to early endosome that directly interact with the plasma membrane. These are recycling endosomes, which are characterized for the presence of Rab11 positive vesicles (Grant & Donaldson, 2009). Recycling endosomes are of great importance in the recycling of the TfR and the targeting and recruiting of the influenza’s vRNPs towards the plasma membrane (reviewed by Eisfeld et al. 2015) Therefore, we investigated the localization and distribution of Rab11. Given that the Rab11 protein often serves as a marker for the recycling endosomes, the spatial changes observed when studying TfR and EEA1 led us to hypothesize that Rab11 distribution might be negatively affected in the Nup153 KD cells. To test this hypothesis, HeLa cells were treated with either Nup153 siRNA or control siRNA for 72 h, prepared for immunolabelling with an anti-Rab11 antibody, and visualized by confocal microscopy. As illustrated in Figure 4-8, the Rab11 marker was found dispersed throughout the cell in control siRNA treated cells and no visual changes regarding its localization/distribution were detected in Nup153 depleted cells. Therefore, despite the changes observed in the distribution of early endosomes and the deficiencies in Tfn uptake, the intracellular distribution of Rab11 appears to be not affected in the Nup153 KD cells. 146   Figure 4-8. Cellular distribution of the recycling endosome marker Rab11 is not affected by Nup153 depletion. HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h, and immunolabelled with an anti-Rab11 antibody (green), an anti-Nup153 antibody (white), and fluorescently-labelled phalloidin to visualize F-actin (not shown). The latter was used to delineate the cell boundary. Dapi (blue) was used to observe the nucleus. Distribution of Rab11 was not affected by the depletion of Nup153. Scale bar, 10 μm.   147  4.2.7. Transient depletion of Nup153 by RNAi induces changes in the distribution of the late endocytic marker CI-M6PR and late endosome/lysosome marker, LAMP1 The CI-M6PR is present in the late endosomal population and plays an essential role in the lysosomal degradation pathway by delivering over 60 different lysosomal enzymes (tagged with mannose-6-phosphate) from the TGN to the endo-lysosomal system (Bohnsack et al., 2009). The CI-M6PR is present not only on the endocytic pathway, but also on the secretory pathway (through its involvement with the TGN). Unlike CI-M6PR, LAMP-1 is a glycoprotein and transmembrane protein which resides primarily in lysosomal membranes, it traffics from the TGN to late endosomes/lysosomes in a different population set than CI-M6PR (Pols et al., 2013). We hypothesized that given the changes observed in the distribution of markers and ligands associated with the endocytic or the secretory pathway, distribution of CI-M6PR and LAMP-1 would be affected. To test this hypothesis, HeLa cells were treated with either Nup153 siRNA or control siRNA for 72 h, prepared for immunolabelling using an anti-CI-M6PR or anti-LAMP-1 antibody, and visualized by confocal microscopy. The results showed that the CI-M6PR immunostaining in control cells was punctate and located in an area closer to the nucleus (Figure 4-9A). In contrast, the distribution of CI-M6PR varied vastly among Nup153 depleted cells, with oddly shaped clusters of fluorescence signal in the vicinity of the cell nucleus or a fluorescence signal all around the nucleus (Figure 4-9A). Because of its non-uniform distribution tendencies, we performed the spatial distribution analysis (Andrey et al., 2010) as was previously performed with EEA1 (Appendix B, Figure B-1). Our findings showed that the CI-M6PR signal (presumably vesicles) was located closer to its nearest neighbor in Nup153 depleted cells (Appendix B, Figure B-2). Generally, each CI-M6PR signal in Nup153 depleted cells was 148  closer to every other CI-M6PR signal (not just its closest neighbor) and was more clustered in comparison to control siRNA treated cells. These results are consistent with the images shown in the enlargement panel in Figure 4-9A, in which clusters of CI-M6PR are seen in Nup153 depleted cells (second and third row).  Overall, the spatial distribution analysis (Appendix B, Figure B-2) corroborates visually observed changes shown in Figure 4-9A regarding the clustered and disorganized distribution of CI-M6PR in Nup153 depleted cells at a cell population level. To further analyze the CI-M6PR particle data, we evaluated the marker’s signal size (which presumably extrapolates into vesicle size) and its circularity. The results show a larger CI-M6PR vesicle size in Nup153 KD cells than in control cells (Figure 4-9B) but no significant changes in signal circularity in Nup153 depleted cells (Figure 4-9C), most likely as a result of vesicle aggregation and the consequent lack of signal segmentation when acquiring/processing the image. Interestingly, WB indicated a progressive reduction of CI-M6PR expression from untreated cells to control siRNA treated cells and Nup153 depleted cells, while LAMP-1 expression remained consistent (Figure 4-9D).   149    150  Figure 4-9. Transient depletion of Nup153 by RNAi induces changes in the distribution of the late endocytic marker CI-M6PR and the late endocytic marker/lysosome LAMP-1. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h. Control and Nup153 siRNA treated cells were immunolabelled with antibodies against CI-M6PR (green) and Nup153 (white). Cells were also labelled with Dapi (blue) to observe the nucleus and fluorescently-labelled phalloidin to visualize F-actin (not shown), which was used to delineate the cell periphery. Right panels in (A) are enlargements of boxed areas in merge panels; it compares the distribution pattern observed in Nup153 depleted cells with control siRNA treated cells. Scale bar, 10 μm, 5 μm (enlargement). B) Quantification of CI-M6PR signal (or CI-M6PR positive vesicles). C) Quantification of CI-M6PR signal circularity shape. The values shown in (B) and (C) are the mean values measured from three independent experiments, n≥20 cells per experiment. The error bars represent the S.E.M and the significance in (B) and (C) were tested using a Student’s t-test, *p<0.05. D) Abundance of CI-M6PR and LAMP-1 was detected through WB analysis of Nup153 siRNA treated, control siRNA treated, and untreated HeLa cells in regular growth media. Lysates were analyzed by immunoblotting with antibodies against Nup153 (SA1), CI-M6PR, LAMP-1, and β actin. β actin expression was used as a loading quality control. E) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h. Control and Nup153 siRNA treated cells were immunolabelled with antibodies against LAMP1 (green) and Nup153 (white). Cells were also labelled with Dapi (blue) to observe the nucleus. Right panels in (E) are enlargements of boxed areas in merge panels; through the enlargement panels we compare the LAMP-1 distribution pattern observed in Nup153 depleted cells with control siRNA treated cells. Scale bar, 10 μm.       151  Unlike what was observed for CI-M6PR where agglomeration of the signal was apparent, LAMP-1 was found dispersed all over the cell and the area where LAMP-1 was concentrated near the nucleus was no longer obvious in Nup153 depleted cells. In contrast, LAMP-1 was found to be dispersed throughout the cell, with a population concentrated in an area adjacent to the nucleus in control cell (Figure 4-9E).  Once again, we made use of the spatial distribution analysis (previously used for EEA1 and CI-M6PR) to determine whether there were major differences in LAMP-1 distribution between both experimental conditions. The spatial distribution pattern analysis in Nup153 KD cells showed that LAMP-1 signal appeared randomly distributed in relationship to each other (Appendix B, Figure B-3). In addition, calculations of the CDF of the accumulation of fluorescent dots showed that in Nup153 depleted cells, the LAMP-1 signal was equidistantly spaced out, more than seeing in control treated cells and even more so than more than expected in a random distribution (Appendix B, Figure B-3D). Put together, LAMP-1 in Nup153 KD cells showed a pattern that is more likely to be observed when there is no signal clustered or when there is a spread out-random like distribution of the feature at the cell population level. Our analysis is consistent with the results illustrated in the enlargement panel in Figure 4-9E, which show that the LAMP-1 expression has not changed (Figure 4-9D) but its signal is widely dispersed throughout Nup153 depleted cells. This suggests a possible intracellular widespread distribution of lysosomes.    152  4.2.8. Late endosomal/lysosomal compartments are redistributed in Nup153-depleted cells In order to further examine the distribution of late endosomes and lysosomes, we then studied Rab7 and LysoTracker positive compartments. Rab7 has been implicated in the downstream endocytic traffic of late endosomes and plays a key role in the aggregation and fusion of late endosomes in the perinuclear region and the biogenesis of lysosomal compartments (Bucci et al., 2000). On the other hand, LysoTracker is a weak base probe with high selectivity for acidic organelles that is traditionally used to label lysosomes. These two ways of detecting late endosomal/lysosomal compartments are not directly related to traffic from the TGN, like CI-M6PR and LAMP-1 are. Hence, the study of Rab7 and LysoTracker is another step towards isolating the intracellular traffic area/pathway that is disturbed in Nup153 depleted cells. Figure 4-10A depicts Rab7 distribution in control treated cells and Nup153 depleted cells. It is apparent that the perinuclear gathering characteristic of Rab7 in control siRNA treated cells was lost in Nup153 depleted cells. The cellular distribution of Rab7 was classified in two ways: clustered (as seen in control treated cells) or dispersed (as seen in Nup153 depleted cells). Such phenotypes were scored for both experimental groups and the mean results are shown in Figure 4-10B. The quantification indicates that although both phenotypes were present in the population of control and Nup153 siRNA treated cells, the clustered staining of Rab7 was the predominant phenotype in the control siRNA treated cells, while the Rab7 dispersed signal was the predominant phenotype in the Nup153 depleted cells.  153  In addition, it is apparent from the images in Figure 4-10A not only that the distribution of Rab7 has changed in Nup153 depleted cells, but also that there appears to be more staining. To determine if Rab7 levels were altered in Nup153 depleted cells, Nup153 depleted cells, control siRNA treated cells, and non-treated cells were examined for Rab7 levels of expression by WB and no difference was found (Figure 4-10C). These results lead us to confirm the findings that Rab7 distribution has changed and has become even more prominent.  Likewise, due to the close connection between Rab7 positive vesicles and lysosomes, we hypothesized that LysoTracker (labelling lysosomes) would follow a distribution pattern similar to that observed in Rab7 in control and Nup153 siRNA treated cells. The distribution of Rab7 in Figure 4-10A is indeed very similar to that of LysoTracker in the live HeLa cells observed in Figure 4-11A. The near-nuclear agglomeration of the LysoTracker observed in control siRNA treated cells was lost in Nup153 depleted cells, in which the signal was present all over the cell’s cytoplasm. These phenotypes were quantified in the same manner as the Rab7 phenotypes and the results indicated that both phenotypes (clustered and dispersed) were present. However, the clustered distribution of the LysoTracker in control siRNA treated cells was the predominant distribution, while the dispersed distribution was significantly predominant in Nup153 depleted cells (Figure 4-11B).  Overall, these results indicate that late endosome/lysosomal markers appear redistributed in Nup153 depleted cells, similar to the distribution observed in LAMP-1 (Figure 4-9E).    154     155  Figure 4-10. The late endosomal/lysosomal marker Rab7 is redistributed in Nup153-depleted cells. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h. Control and Nup153 siRNA treated cells were immunolabelled with antibodies against Rab7 (green) and Nup153 (white). Cells were also labelled with Dapi (blue) to observe the nucleus and fluorescently-labelled phalloidin to visualize F-actin (not shown), which was used to delineate the cell periphery. Scale bar, 10 μm. B) Rab7 dispersion phenotype was scored as clustered or dispersed as observed in (A). The graph represents the mean values of clustered/dispersed cells obtained from three independent experiments, n≥100 cells per experiment. Error bars represent S.E.M. Statistical analysis determined using Student’s t-test; ***p < 0.001. C) Abundance of Rab7 was detected through immunoblot analysis of Nup153 siRNA treated, control siRNA treated, and untreated HeLa cells (no siRNA). Lysates were analyzed by immunoblotting with antibodies against Nup153 (SA1), Rab7, and β actin as a loading quality control.  156   Figure 4-11. Acidic/lysosomal compartments identified by LysoTracker are redistributed in Nup153-depleted cells. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h. Live cells were labelled with LysoTracker green DND-26 (green) and NucBlue (blue) and observed by confocal microscopy. Cell boundaries were drawn from DIC images, right and middle panels. Scale bar, 10 μm. In (B) LysoTracker dispersion phenotype was scored as clustered or dispersed as observed in (A). The graph represents the mean values of clustered/dispersed cells obtained from three independent experiments, n>50 cells per experiment. Error bars represent S.E.M. Statistical analysis determined using Student’s t-test; *** p < 0.001. 157  4.2.9. Study of the secretory pathway elements in Nup153 depleted cells Our previous results showed changes to the distribution of CI-M6PR and LAMP-1, late endosomal/lysosomal markers, whose traffic is associated with the Golgi apparatus. Consequently, we hypothesized that the alterations observed in the endosomal pathway were widespread and affected the overall vesicular traffic. To test this hypothesis, we first determined the ER distribution throughout the cell. HeLa cells treated with either control siRNA or Nup153 siRNA were labelled with an anti-ERp72 antibody, an abundant protein found in the luminal portion of the ER (Mazzarella et al., 1990). The distribution of ERp72 in control siRNA treated cells and Nup153 depleted cells was very similar (Figure 4-12A). However, when the texture of the images was measured (showing a wide variety of pixel intensities), there was a small, albeit significant, difference between the signals of ERp72 in control siRNA treated cells and Nup153 depleted cells. That is, the ER staining in Nup153 KD cells appeared smoother than in control siRNA treated cells (Figure 4-12B).  We also set up to investigate the distribution of the beta subunit of the COPI complex. This subunit is found in the COPI complex, which is involved in the retrograde transport from the Golgi apparatus to the ER. The distribution of β COPI in control siRNA treated cells was found predominantly in an area adjacent to the nucleus (Figure 4-13). In contrast, β COPI fluorescence signal appeared more dispersed over the cytoplasm in Nup153 depleted cells (Figure 4-13).  The results obtained from examining the ER distribution and the beta subunit of the COPI complex suggest that possible changes are also occurring in the secretory pathway. To further investigate this hypothesis, we examined the immunostaining of influenza HA protein 158  in control and Nup153 KD cells that transiently express HA after transfection. Transfection (instead of infection) was used to remove the effects that influenza infection has on the cells. The results of HA transfection in control treated cells and Nup153 depleted cells showed that HA staining was clearly located in the perinuclear area (presumably the Golgi apparatus) and the plasma membrane (Figure 4-14A). This is better documented in the gray intensity profile in Figure 4-13B. However, the HA immunostaining was found all throughout the cell in Nup153 depleted cells and was also present in random patches, which would mean that it was not present exclusively in the plasma membrane. Additionally, the HA immunostaining only increased slightly in the cell edge areas, indicating that HA was dispersed throughout the cell and pointing to possible defects in the targeting of this glycoprotein to the cell’s plasma membrane when Nup153 is KD.    159    Figure 4-12. The ER intraluminal marker ERp72 texture appears smoother in HeLa cells upon Nup153 depletion. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h, and immunolabelled with an anti-ERp72 antibody (green), an anti-Nup153 antibody (white), and Dapi (blue) to observe the nucleus. Scale bar, 10 μm. B) Comparative analysis of texture of single confocal plane images from (A). Values closer to 0 indicate finer patterns of texture (more localized, granular) while larger values indicate larger patterns (smoother). Shown are the individual values and the mean three independent experiments, n>120 cells. Errors bars represent S.E.M. Statistical analysis in (B) was performed using Student’s t-test. *** p < 0.05.   160   Figure 4-13. Depletion of Nup153 in HeLa cells perturbs the distribution of the ER to Golgi vesicle marker, COPI. HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h. Control and Nup153 siRNA treated cells were immunolabelled with an antibody against β COPI (green) and Nup153 (white). Cells were also labelled with Dapi (blue) to observe the nucleus and fluorescently-labelled phalloidin to visualize F-actin (not shown) used to delineate the cell periphery. Right panels are high magnification images of cells indicated with white boxes in the merge images. Scale bar, 10 µm.    161    Figure 4-14. Cellular distribution of HA is altered in live Nup153 depleted cells. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 48h, then transfected with a influenza’s HA (H1N1 PR8 strain) plasmid for 24 h. Cells were then immunolabelled with an antibody against HA (red) and the nuclei were labelled with Dapi (blue). Scale bar, 10 µm. B-C) Gray intensity profile of a transversal section (white arrows) from cells depicted in (A) for either control siRNA cells (B) or Nup153 siRNA (C). The arrows in A show the specific area where the histograms display relative fluorescence intensity of gray values. Peaks in control siRNA treated cells correspond to edges of the plasma membrane (p.m) where the intensity of HA staining is increased. p.m: plasma membrane.  162  4.2.10. EM insights into Nup153-depleted cells Due to the intracellular changes observed in Nup153 KD cells by fluorescence microscopy in the endosomal and lysosomal markers, and to the changes in proteins that follow the secretory pathway, it was important to use electron microscopy to look at these cells and compare them with the morphology of cells treated with control siRNA. Electron micrographs of whole cells are shown in Figure 4-15A to generally compare control siRNA treated cells and Figure 4-15B for Nup153 depleted cells. The major changes observed in Nup153 depleted cells were an abnormal cell shape, a deformed nucleus, and the appearance of large empty vesicle-like structures in the cytoplasm (Figure 4-15B). Large magnification images show multiple vesicular structures with one or multiple membranes (Figure 4-15C, red arrows), other smaller empty-like vesicles with a single membrane, and a larger vesicle that appears to contain multiple organelles or membranes (Figure 4-15D, red arrows). We also observed putative vimentin filaments surrounding the cell nucleus (Figures 4-15C and 4-15D, cyan arrows,). The vesicles observed in Figures 4-15C and 4-15D, compared to vesicles shown in the literature, appear to be lysosomes, autophagosome, or autophagic structures (Vergarajauregui et al., 2008).  Additionally, Figures 4-15E and 4-15F depict an enlargement of the intracellular contents of another Nup153 depleted cell. In Nup153 depleted cells, we can observe other types of vesicles and features of interest (Figures 4-15E and 4-15F), such as double membrane vesicles (red arrows). Interestingly, in Figure 4-15E, the round empty-like vesicles with single membranes appear similar to the TGN, while in Figure 4-15F, we observe a vesicle resembling a multivesicular body (red arrow, upper right corner). 163  Despite all our observations, it is impossible to certify that any of the vesicles or structures observed is the proposed cellular structure without immunogold labelling or other labelling methods. However, the defects detected in the Nup153 KD cells at the level of EM are consistent with alterations in vesicular traffic in Nup153 depleted cells.   164   165  Figure 4-15. Nup153 depleted cells display a wide variety of vesicle bodies. Electron micrographs of cross sections from HeLa cells transfected with control siRNA or Nup153 siRNA for 72 h and then prepared for embedding thin section EM. A control siRNA treated cell is shown in (A) and Nup153 depleted cells are shown in (B). The two boxes in (B) are magnified in (C) and (D). High magnification images of other Nup153 depleted cells (whole cell not shown) are depicted in (E) and (F). Red arrows point to a variety of vesicles observed in Nup153 depleted cells, some resembling putative Golgi related vesicles, lysosomes, multivesicular bodies and possibly autophagosomes. In (C) and (D), cyan arrow also shows what appear to be vimentin filaments around the nuclear membrane. n, nucleus; c, cytoplasm. Scale bars are 2 μm (A), 5 μm (B), and 500 nm (C, D, E, and F).  166  4.2.11. Nup153 depleted cells exhibit drastic cytoskeletal modifications The cell cytoskeleton provides a framework that facilitates intracellular vesicular traffic and organelle’s positioning. It is well known that intracellular vesicles traffic along microtubules with the aid of motors. Due to the defects in the cargo uptake of endosomal markers and the modification in the spatial distribution of these markers, we hypothesized that Nup153 depletion must be having an effect on the cell cytoskeleton. In order to test this hypothesis, HeLa cells were transfected with either control siRNA or Nup153 siRNA for 72 h and then analyzed by immunofluorescence microscopy to detect α-tubulin (Figure 4-16) or vimentin (Figure 4-17). Fluorescently-labelled phalloidin was also used to detect F-actin. Our results showed that Nup153 depleted cells exhibited drastic changes in terms of the organization of microtubules and also displayed a rearrangement of actin filaments (Figure 4-16A). A montage of the confocal z-stack shows a concentration of α-tubulin and F-actin around the cell nucleus in Nup153 depleted cells (Figure 4-16B). We also observed alterations on the vimentin filament network in Nup153 KD cells (Figure 4-17). The vimentin filaments reorganized around the nucleus in some cells and primarily towards one side of the cell in others (Figure 4-16B). A montage of z-stack shows the distribution of vimentin and F-actin around the cell nucleus of Nup153 depleted cells and at the cell periphery of these cells (Figure 4-17B).  In summary, our results indicate that when levels of Nup153 depletion are high, there are drastic changes in all three major cytoskeletal elements, such as an accumulation of F-actin at the cell periphery, a disorderly distribution of microtubules, and a concentration of vimentin around the cell nucleus.  167     168  Figure 4-16. Immunostaining of α-tubulin and F-actin are altered in Nup153-depleted cells. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h, and immunolabelled with antibodies against Nup153 and α-tubulin. F-actin was detected with rhodamine-phalloidin, and the nucleus with Dapi. In Nup153-depleted cells, α-tubulin (A) exhibited a prominent labelling around the cell nucleus compared to control cells, while F-actin appeared concentrated in the cell boundaries. B) Z stacks montage progression (from left to right, from top to bottom) of control siRNA treated cells (left) and Nup153 depleted cells (right) showing the changes in microtubules and F-actin at different focal planes in the cell. Scale bars, 10 μm (A), 5 μm (B).    169      170  Figure 4-17. Immunostaining of vimentin and F-actin are altered in Nup153-depleted cells. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h, and immunolabelled with antibodies against Nup153 and vimentin. F-actin was detected with rhodamine-phalloidin, and the nucleus with Dapi. In Nup153-depleted cells, vimentin (A) exhibited a prominent labelling around the cell nucleus compared to control cells, while F-actin appeared concentrated in the cell boundaries. B) Z stacks montage progression (from left to right, from top to bottom) of control siRNA treated cells (left) and Nup153 depleted cells (right) showing the changes in vimentin and F-actin at different focal planes in the cell. Scale bars, 10 μm (A), 5 μm (B).    171  4.3. Discussion The results obtained in Chapter 3 indicated that there was a difference in the traffic of vRNPs during the early IAV infection of Nup153 KD cells in comparison to control siRNA treated cells, which helped us to focus our research on the endocytic pathway. In this chapter, we identified a defect in the uptake of cellular ligands such as EGF and Tfn in Nup153 depleted cells. We also observed changes in the spatial distribution of endosomal and vesicle markers and a reorganization of major cytoskeletal elements in Nup153 KD cells.  Our initial findings showed an uneven distribution of the lectin WGA (Figure 4-1) in Nup153 depleted cells. The presence of WGA patches at the plasma membrane of Nup153 KD cells could have implications for the distribution of the sialic-acid rich glycoproteins present in the plasma membrane. Accordingly, the transmembrane glycoprotein receptors EGFR and TfR displayed altered distribution in Nup153 depleted cells, particularly under conditions of serum starvation. The EGFR and TfR immunostaining aggregation observed in the Nup153 KD cells did not follow the same pattern, which suggests that the distribution of each receptor has a different response to Nup153 depletion, but results in a decrease binding and uptake of ligands in both cases. Thus, it appears that receptor targeting to the plasma membrane and/or endocytosis are defective in Nup153 depleted cells. Defects in EGF signaling have consequences for cell proliferation and migration. Interestingly, Zhou & Panté (2010) found impaired cell migration in Nup153 depleted human breast carcinoma cells. Lastly, Tfn uptake is essential for the regulation of iron homeostasis inside the cell. Iron deficiencies would therefore have deleterious effects on oxygen transport, DNA biosynthesis, and oxidative phosphorylation (Hentze et al., 2004).  172  Interestingly, EGFR and TfR have distinct and different distributions in the plasma membrane (Leonard et al., 2008). In addition, the uptake of their cargoes− EGF and Tfn respectively − are endocytosed into separate endocytic routes (Leonard et al., 2008). Because uptake of EGF was reduced, but the acidification of EGF containing vesicles (as a proxy for cargo progression through the endo-lysosomal pathway) was not affected, we propose that defects in the EGF/EGFR uptake occur prior to cargo endocytosis. Likewise, our findings suggest that the binding of Tfn to TfR is reduced (Figure 4-5B) and that there are defects in the uptake of Tfn/TfR complexes (Figure 4-5A). On the whole, the problems encountered in receptor localization and the binding of ligands were found to be not exclusive to RTKs because they also affected the localization of TfR. The defects we observed on the endocytic pathway in Nup153 depleted cells included reduced vesicle size in dextran uptake experiments and endocytic makers (interpreted from reduced signal size). These observations might suggest that Nup153 KD cells present defects in vesicle biogenesis. Moreover, the redistribution of EEA1 could affect the formation of the sorting endosome and the fusion of endocytic vesicles. Interestingly, the expression of the late endosomal marker CI-M6PR was slightly reduced while it was visually concentrated in the perinuclear area of Nup153 depleted cells (Figure 4-9). This location is commonly associated with the endocytic recycling compartment (ERC) or the TGN. According to Lin et al. (2004), the CI-M6PR follows a complex route to the late endosomes through the sorting endosome, the ERC, the plasma membrane, and then the TGN/late endosomes. The extensive traffic through compartments of this transmembrane glycoprotein could suggest a defect in CI-M6PR leaving the ERC or the TGN.  173  Unlike CI-M6PR, we found that LAMP-1 expression remained constant while its distribution became scattered all over the cell, losing the population that was concentrated in the perinuclear area. Interestingly, Rab7 controls aggregation and fusion of late endocytic compartments and lysosomes, and is crucial for the maintenance of the perinuclear lysosome compartment in HeLa cells (Bucci et al., 2000). Accordingly, in Nup153 depleted cells we found Rab7 and LAMP-1 dispersed throughout the cell, along with an extensive cellular labelling of acidic compartments by LysoTracker.  The endo-lysosomal pathway has a close relationship with the secretory pathway. After examining some components of the secretory pathway, we found slight changes in the distribution of an ER luminal protein and the β subunit of the coat protein complex I (COP I), and also found defects in the targeting of HA to the plasma membrane. These results combined with our previously discussed results, indicate that distribution of vesicular markers is highly modified in Nup153 depleted cells, which could possibly alter vesicle formation, cytoplasmic traffic and communication between cellular compartments. Finally, we detected dramatic changes in the organization and distribution of microtubules, F-actin, and intermediate filaments in Nup153 depleted cells. Although previous research has shown changes in the cytoskeleton of Nup153 depleted cells (Mackay et al. 2009; Lussi et al. 2010; Zhou & Panté, 2010), we detected simultaneous changes in microtubules and F-actin or vimentin and F-actin. Due to the importance of F-actin in endocytosis and microtubules in vesicular transport and organelle positioning, we propose that the defects observed in the spatial distribution of endosomal markers are a consequence of the alteration of the cellular cytoskeleton in Nup153 depleted cells. However, the changes 174  in the cytoskeleton and vesicular transport do not account for the increase in Rab7 staining in conjunction with the extensive LysoTracker labelling of intracellular acidic compartments. In addition, ultrastructural studies of Nup153 depleted cells showed a wide variety of vesicles that closely resembled undigested content in lysosomes or autophagosomes. Our initial investigations with the autophagic markers LC3 and p62 suggested possible alterations in the autophagic pathway (data not shown). We speculate defects in cell signaling and the intracellular traffic of important receptors such as CI-M6PR in Nup153 depleted cells resulted in a blockage of lysosomal or autophagic activity. Further experiments testing autophagic flux are required to prove/disprove this hypothesis.  In summary, we have shown in this chapter that Nup153 depletion has serious downstream effects on intracellular traffic and cytoskeletal organization within the cell, which arguably, could induce remodeling of the membrane transport apparatus. We are the first research group to show uptake defects and changes in the spatial distribution of endosomal markers in Nup153 depleted cells as well as the reorganization of the three major elements of the cellular cytoskeleton. We believe that the defects observed in Nup153 KD cells are downstream consequences of Nup153 depletion and are not taking place because of direct interaction between Nup153 and any of the cargoes or markers studied above. More details on the possible mechanisms of how Nup153 depletion exerts alterations in intracellular traffic and cell cytoskeleton are examined in the following chapter.    175             Chapter 5 Effect of Nup153 RNAi depletion on nuclear and cell morphology Effect of Nup153 RNAi depletion on nuclear and cell morphology 5.1. Introduction Complex roles of nucleoporins in gene regulation, cell development, and disease have emerged as a result of their physical interaction with chromatin (reviewed by Capelson & Hetzer, 2009) as well as perturbed nuclear transport. Furthermore, nuclear structural elements such as lamins have been associated with DNA replication, transcription, and chromatin organization (reviewed by Dechat et al. 2008). On the whole, the study of nucleoporins and their relationship with nuclear structural elements will promote an understanding of the relationships between nuclear morphology and gene expression.  The NPC nuclear basket protein Nup153 plays a role during mitosis and cytokinesis, and affects the cell cytoskeleton in slightly different ways depending on the level of protein expression (Mackay et al. 2009; Lussi et al. 2010; Zhou & Panté, 2010). For example, after 48 h of Nup153 RNAi depletion, HeLa cells show cytoskeleton and nuclear lamina alterations, as well as defects on cellular processes that depend on the cytoskeleton (i.e. deficient wound healing, cell migration, and polarized MTOC) (Zhou & Panté, 2010). Other defective processes observed in Nup153 depleted cells are incomplete cytokinesis and unresolved midbodies (Mackay et al., 2009), which are both related to cytoskeletal elements.  In the previous chapter it was observed that Nup153 depleted cells presented defects in the intracellular trafficking system in both the endocytic and the secretory pathway, and presented alterations of their cytoskeletal elements that included actin filaments, vimentin 176  filaments, and microtubules. Because the cytoskeleton plays an important role in the establishment of cellular and nuclear morphology, the phenotype of Nup153 depleted cells was further analyzed in this chapter. Moreover, nuclear shapes and sizes in Nup153 depleted cells were examined while relating them to deformations in the NE.  The findings in this chapter showed that Nup153 depleted cells exhibit dramatic cellular changes with a variety of cell phenotypes, including rounded cells and cells with lamellipodia and plasma membrane blebbing. At the same time, it was confirmed that changes in the nuclear morphology as previously established in the literature, such as deformed nucleus and/or the presence of multiple nuclei and multi-lobed nuclei (Mackay et al. 2009; Lussi et al. 2010). In addition, the observation of cytoplasmic vacuolar-like or vesicle-like structures was linked to a defect in the outer nuclear membrane/ER membrane. Finally, apoptosis was evaluated as a possible culprit for the observed nuclear and cytoplasmic alterations, but no evidence was found that cell death by apoptosis was a mechanism at play in Nup153 depleted cells. The proposed model suggests that changes in the levels of Nup153 indirectly affect the cell cytoskeleton by disturbing the nucleoskeleton through its interaction with the nuclear lamina and proteins of the LINC complex (see Chapter 1, Table 1-1) while simultaneously altering the endomembrane system through its effect on cell cycle progression. Defects of the nuclear membrane morphology and their possible relation to the cell cycle in Nup153 KD cells are also discussed.   177  5.2. Results 5.2.1. Nup153-depleted cells exhibit plasma membrane blebbing and actin reorganization In Chapter 4, we showed changes in the distribution of cytoskeletal elements in Nup153 depleted cells (Chapter 4, Figures 4-16 and 4-17). Hence, we further explored the morphological traits in Nup153 depleted cells in accordance with the hypothesis that cytoskeleton changes would result in distinguishable external morphological changes. In order to test this hypothesis, HeLa cells were transfected with either control siRNA or Nup153 siRNA for 72 h and cell morphology changes were visualized by DIC imaging. We detected prominent cell blebbing in Nup153 RNAi KD cells (Figures 5-1A and 5-1B). In addition to blebbing, 50% of the cells had extensive lamellipodia and there were some round-up and amorphous cells phenotypes (Figure 5-1A and 5-1C). Due to the association of plasma membrane blebbing with actin dynamics, we detected F-actin by labelling with rhodamine-phalloidin and found the label to be accumulated on the cell edges, at the cellular blebs, or in bright thick bundles of actin filaments (arrows in Figure 5-1D). In order to understand the mechanism behind the actin remodeling that was observed in Figure 5-1D, we studied the actin-related protein (Arp) 2/3 complex, which is a fundamental player in the process of actin dynamics and organization. The Arp2/3 complex plays a key role in nucleation of actin filaments, which produce branched filaments, and is heavily involved in cell motility, endocytosis, and pathogen invasion (reviewed by Goley & Welch, 2006). First, HeLa cells were transfected with either control siRNA or Nup153 siRNA for 72 h and Arp 2/3 was detected by immunofluorescence microscopy. As 178  documented in Figure 5-2, Arp 2/3 staining did not have any apparent changes in intensity or distribution in Nup153 depleted cells, despite the presence of thick bundles of actin filaments. Thus, the cellular actin changes subsequent to Nup153 depletion are not related to Arp2/3. Preliminary results with another protein involved in actin dynamics are presented in Appendix C, Figure C-1. Immunolabelling of the phosphorylated version of the regulatory light chain of myosin II (P-MLC2), which is associated with the formation of plasma membrane blebs (Torgerson & McNiven, 1998), was detected alongside bright condensed F-actin staining close to the plasma membrane in Nup153 depleted cells (Appendix C, Figure C-1). Cells were treated with the ROCK inhibitor Y27632, a Rho-GTPase effector that is as an important participant in the regulation of myosin light chain phosphorylation (Leung et al., 1995). As a result of the treatment, the close localization of P-MLC2 with the condensed filamentous actin at the plasma membrane of Nup153 KD cells was not present (Appendix C, Figure C-1) and active cell blebbing was abolished (data not shown). Hence, the changes in F-actin upon Nup153 depletion may possibly be related to the phosphorylation of MLC2. Taken together, our results show that Nup153 depleted cells exhibit not only internal changes in the distribution of cytoskeletal elements, but also external alterations such as modifications to the cell shape and plasma membrane dynamics. However, further research is required to establish the mechanisms that drive these changes.  179     180  Figure 5-1. Nup153-depleted cells exhibit plasma membrane blebbing and actin reorganization. HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h and examined by (A) confocal microscopy DIC or (D) immunofluorescence microscopy. A) DIC images of live cells showing that Nup153-depleted cells exhibit changes in the cell morphology compared to control siRNA; the 4 main phenotypes observed were: lamellipodia, round up cells, blebbing cells, and amorphous cells (blebbing cells that have completely lost a defined cell shape). B) Quantification of the number of cells (%) displaying plasma membrane blebbing for cells treated with control siRNA or Nup153 siRNA. C) Quantification of the number of cells (%) showing different phenotypes observed as described in (A). For (B) and (C) bars shown represent the mean values in % from four different experiments, n>700 examined cells in total; error bars indicate the S.E.M. Significant differences between different conditions were determined by Student’s t-test (B, ***p<0.001) or one-way ANOVA (C, ***p<0.05). D) Immunofluorescence of cells labelled with Nup153 antibody (white), rhodamine-phalloidin to visualize F-actin (red), and Dapi (blue) to observe the nucleus. Images in (D) show three different F-actin staining patterns observed in Nup153 depleted cells and one image of control cells. From left to right, white arrow points at the thickened actin at the cell periphery, at a cell bleb or at the thick bundle of filamentous actin. Scale bar, 10 μm.   181   Figure 5-2. F-actin changes upon Nup153 depletion are not related to Arp2/3. HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h and prepared for immunofluorescence microscopy. Immunofluorescence of cells labelled with antibodies against Arp2/3 (green) and Nup153 antibody (white). Rhodamine-phalloidin was used to visualize F-actin (red) and Dapi (blue) to observe the nucleus. Single confocal slices were examined for differences in the distribution of Arp2/3 but no changes were detected. Scale bar, 10 μm.   182  5.2.2. Nup153 depletion results in altered nuclear morphology and minor changes in the cell cycle In addition to the alterations to cellular shape and plasma membrane morphology, we observed alterations to the nuclear morphology of Nup153 KD cells. This is better documented in confocal image Z stacks through the nucleus of control and Nup153 siRNA treated cells: while control siRNA treated cells exhibited a predominantly round nucleus with no visible alterations to shape (Figure 5-3A), Nup153 depleted cells had amorphous nuclei, cell blebbing, and thick actin bundles (Figure 5-3B). For example, the nucleus in the upper cell in Figure 5-3B has an oval shape that progressively displays a dent as the images are taken through a sample and ends up looking like two separate nuclei in the last image. Nup153 KD cells also showed invaginations of the nuclear membrane that were filled with actin filaments. For example, in the lower cell in Figure 5-3B, actin filaments are going across a nuclear invagination (indicated by green arrow) as if they were dividing the nucleus. Additional examination of Nup153 KD cells using thin section EM showed multiple nuclear fragments that would most likely be connected in another section plane (Figure 5-3C), as well as invaginations in the nuclear membranes (Figure 5-3D, black arrow). Nuclear alterations during mitosis have been widely documented in Nup153 depleted cells (Mackay et al., 2009; Zhou & Panté, 2010), and have been related to alterations in the cell cycle. Thus, we next determine the cell cycle stage for a population of Nup153 depleted cells under our specific experimental conditions (80-90% reduction in Nup153 expression 72 h after transfection) through the use of flow cytometry. In Figure 5-4 we observe that Nup153 depleted cells showed approximately a significant 10% increase (when 100% is the 183  total cell population) of cells in the G1 phase, while decreasing to almost half in Gap 2 phase/mitosis (G2/M) (~10 percentage points decrease) and S phases (~3 percentage points decrease) when compared to control siRNA treated cells (and mock/untreated cells). Overall, the increase in the number of cells in the G1 cell cycle phase and the decrease in G2/M and S phase were found to be small but statistically significant when Nup153 expression was reduced by RNAi. Put together, the large percentage in cells displaying altered morphology (~50%) do not match the percentage difference observed at different stages of the cell cycle between control cells and Nup153 KD cells. Thus, we propose that there might be other mechanisms at play beyond mitotic defects that are responsible for the cytoskeletal and nuclear deformations observed in Nup153 KD cells.   184    185   186  Figure 5-3. Nuclei of Nup153-depleted cells display invaginations and deformations. HeLa cells were transfected with control siRNA (A) or Nup153 siRNA (B) for 72 h, prepared for immunofluorescence, and examined by confocal microscopy. Cells were labelled with rhodamine-phalloidin to visualize F-actin (red) and Dapi (blue) to observe the nucleus. Montage of series Z stacks of control siRNA treated cell (A) or Nup153 depleted cell (B). Arrows in (B) indicate areas where deep nuclear invaginations are present (white arrows) and actin filaments appear embedded in the nuclear indentations (green arrows). C-D) Electron micrographs of cross sections from HeLa cells transfected with Nup153 siRNA for 72 h and prepared for embedding thin section electron microscopy. In (C) a micrograph of a whole cell shows four nuclear compartments, which are more likely connected with each other in the 3-D shape of the cell as indicated by the confocal Z stacks in (B). D) High magnification micrograph of a Nup153 depleted cell displaying nuclear membrane invaginations (black arrow). n: nucleus, c: cytoplasm. Scale bars, 10 μm (A, B), 2 μm (C), and 500 nm (D).   187   Figure 5-4. Nup153-depleted cells exhibit minor cell cycle alterations. A) HeLa cells were transfected with Nup153 siRNA (left panel), control siRNA (middle panel) or mock transfected (right panel) for 72 h, fixed, and prepared for flow cytometry to analyze relative DNA content. Individual diagrams depict the percentage of cells from the correspondent condition in G1 or G2/M phase as measured by propidium iodide intensity as a proxy for estimation of DNA content in the cell. B) Quantification of the results of experiments shown in (A). Bars shown represent the mean values; error bars indicate the S.E.M of 3 different samples per condition (total 3 samples per condition, n= 5,000 cells per sample). Significant differences between different conditions were determined by two-way ANOVA (***, p<0.001). These results are representative from 3 different experiments repeated under the same conditions.   188  5.2.3. Nup153 depletion in HeLa LAP2β-GFP cells resulted in altered cytoskeleton and abnormal nuclear morphology  The presence of multi-lobed nuclei (Figure 5-3) in Nup153 KD cells could be considered a consequence of the role of Nup153 during mitosis (Mackay et al., 2009). However, our finding suggests that alterations in cell cycle stage in a population of Nup153 KD cells do not fully account for the drastic cytoskeletal changes observed. Because of its localization at the nucleus basket, Nup153 associates with nuclear lamin A/C and other nuclear membrane proteins (reviewed by Fahrenkrog et al. 2002). Previous research has shown that once Nup153 is depleted, both lamin A/C and Sun1 are redistributed in HeLa cells (Zhou & Panté, 2010). Hence, we hypothesized that the changes observed in the nuclear shape of Nup153 KD cells are a consequence of deformations that occur at the NE. To test this hypothesis, we examined the NE upon Nup153 depletion of a HeLa cell line that stably expresses a GFP NE maker, namely the GFP-LAP2β (lamina-associated polypeptide 2, beta isoform) expressing HeLa cells (Mühlhäusser and Kutay, 2007). The LAP2β protein is a membrane protein located in the inner nuclear membrane, where it interacts with chromatin and the nuclear lamina to aid in the organization of the NE and the maintenance of the NE’s structure (reviewed by Nili et al. 2001). Because LAP2β is inserted in the membrane of the ER during synthesis, it can also be found in the ER, albeit in a lower amount.  First, we examined the immunolabelling of the three cytoskeleton components subsequent to the depletion of Nup153 in the HeLa LAP2β-GFP stable cell line. Our findings showed that the organization of all three cytoskeletal components was altered and the cell 189  shape was drastically changed in this cell line (Figure 5-5). Strikingly, these cells showed an impressive impact (when compared to “regular” HeLa cells) on nuclear size and morphology, and a large number of cells appeared to have multiple nuclei. This is better documented in live cell imaging of Nup153 KD HeLa LAP2β-GFP cells labelled with NucBlue (a live cell nuclear labelling agent) (Figure 5-6). However, confocal image Z stacks seen through the cell nucleus revealed that most of the multiple nuclei that appeared to be independent in a single confocal slice were actually connected to each other (Figure 5-6F). Thus, the nuclei of these cells are primarily multi-lobed. The nuclear structures were as large as 30 μm or as small as 2 μm. Overall, over 60% of the HeLa LAP2β-GFP treated with Nup153 siRNA exhibited multiple nuclei/multi-lobed nuclei (Figure 5-6B), as determined by the appearance of multiple LAP2β-GFP and Dapi positive circular structures within the cell. There was also a wide variability in the number of nuclei/multi-lobed nuclei within each cell (Figure 5-6C).  The presence of nuclei with multiple lobules in the Nup153 KD LAP2β-GFP cells could be due to the deformation of a large nucleus or failure in cell division during the cell cycle. Although cell cycle analysis in HeLa cells (Figure 5-4) did not show cells arrested during G2/M, the multi-lobed phenotype was found to be more pronounced in HeLa LAP2β-GFP. To further investigate these differences, a cell cycle analysis through flow cytometry was performed (Figure 5-7) and showed a larger proportion of Nup153 KD HeLa LAP2β-GFP cells were arrested in the G2/M phase of the cell cycle. This suggests that the observed multi-lobed nuclei correspond to a single nucleus in which the replication of the DNA occurs, but the cell does not proceed to mitosis. Additionally, in Figure 5-7 an increase in dead cells for the Nup153 KD condition was observed in HeLa LAP2β-GFP cells (DNA 190  content found under 50K). However, this was not found in Nup153 KD HeLa cells (Figure 5-4).  Put together, we have found that Nup153 depletion in LAP2β-GFP cells alters nuclear shape, size, and morphology, all of which can be possibly associated with failure in the cell cycle. In contrast to the results found for “regular” HeLa cells where shifts in cell cycle stage, although significant, were not large when comparing Nup153 KD and control populations. These findings suggest that expression of LAP2β-GFP in HeLa cells modifies the cell response to Nup153 depletion.    191   192   Figure 5-5. Nup153-depleted LAP2β-GFP cells exhibit changes in nuclear shape, cell morphology, and organization of the three cytoskeletal elements. HeLa LAP2β-GFP cells were transfected with control siRNA or Nup153 siRNA for 72 h and prepared for immunofluorescence microscopy. A-C) Immunofluorescence of HeLa cell line stably expressing LAP2β-GFP (green) labelled with an anti-Nup153 antibody (white) and (A) rhodamine-phalloidin to visualize F-actin (red), (B) an antibody against α-tubulin (red), or (C) an anti-vimentin antibody (red). Dapi (blue) was used to observe the nucleus. Scale bars, 10 μm.   193   194   195  Figure 5-6. Nup153 depletion in LAP2β-GFP cells results in multi-lobed nuclei. HeLa LAP2β-GFP cells were transfected with Nup153 siRNA for 72 h and prepared for live cell microscopy. A) Live confocal imaging of Nup153 depleted HeLa LAP2β-GFP and stained with NucBlue to visualize the cell nucleus. Right panel shows merge with DIC image. The middle (1) and bottom rows (2) show higher magnification of cells in the white boxes (1 and 2) shown in the upper row. Scale bars, 10 μm for the upper panels and 5 μm for the middle and bottom panels. B) Quantification of the percentage of multinucleated HeLa-LAP2β cells. Bars correspond to mean values from three independent experiments. Error bars correspond to S.E.M. n> 100 cells. C) Number of nuclear lobes/micronuclei observed in multinucleated cells in HeLa LAP2β-GFP depleted of Nup153 by RNAi. Values shown are the average from three experiments, n>100 cells for each experiment. Significant differences in (B) and (C) between different conditions were determined by Student’s t-test (**p = 0.003 and p*** <0.0001). D) Two dimensional rendering of XYZ planes composite of the nuclear area positive for LAP2β-GFP (green) of Nup153 depleted cell (2) shown in box (A). Scale bar, 5 μm. E-F) Montage of series Z stacks of the nuclei of LAP2β-GFP cells treated with control siRNA (E) or Nup153 depleted cell (F). Scale bars, 5 μm.    196   Figure 5-7. Nup153-depleted cells exhibit cell cycle alterations in LAP2β-GFP cells. A) HeLa LAP2β-GFP cells were transfected with Nup153 siRNA, control siRNA or mock transfected for 72 h, fixed, and prepared for flow cytometry to analyze relative DNA content. Individual diagrams depict the percentage of cells from the correspondent condition in G1 or G2/M phase as measured by cell DNA content. B) Flow cytometry results from all conditions shown in (A) aggregated in one figure for the purpose of comparison.   197  5.2.4. Depletion of Nup153 results in the formation of cytoplasmic vacuole-like structures and nuclear membrane herniations Through DIC observation of the effects of Nup153 depletion in the cellular morphology of HeLa cells, we were able to identify a phenotype that occurred approximately in 13% of the cells. Nup153 depleted cells exhibited large circular structures that were normally located next to the nucleus (Figure 5-8A). These vacuole-like structures have a different appearance than the rest of the cell’s cytoplasm or nucleus (Figure 5-8A). A graphic representation of the observed phenomenon is shown in the bottom left panel of Figure 5-8A. These findings prompt us to hypothesize that Nup153 depletion could lead to the formation of nuclei-like structures that lack DNA. To test this hypothesis, we examined Nup153 depleted HeLa cells using thin section EM and found large alterations to the NE (Figure 5-8B, 5-8C). As depicted in the graphic representation of Figure 5-8B, we observed herniations of the outer nuclear membrane, which resulted in large expansions of the perinuclear space in some areas of the nucleus. In addition, we observed the presence of small vesicle-like electron dense material in the perinuclear space (Figure 5-8B, bottom panels). Lastly, an examination of other cells showed smaller nuclear membrane herniations and deformations of the nuclear membrane, resulting in accumulation of electron dense material (vesicle-like structures) (Figure 5-8C, white arrows).  The round vacuole-like structures described in Figure 5-8A are predominantly located adjacent to the nucleus. Additionally, we observed that nuclear membrane herniations (Figure 5-8A and 5-8B) occurred as a consequence of the separation of the outer nuclear membrane from the inner nuclear membrane. Hence, we hypothesized that the structures 198  observed in Figure 5-8A were vesicles originating from the outer nuclear membrane or the ER membrane, given that they are physically connected and continuous. We therefore studied the distribution of the influenza’s HA protein in a transfection system and then observed its distribution in Nup153 depleted infected cells. The influenza’s HA protein is a transmembrane protein that is inserted in the ER membrane and follows the secretory pathway to be targeted to the cell’s plasma membrane. After transfection of a HA plasmid, HA was found around large vesicles next to the nucleus (Figure 5-9A, left panel), but not inside these vesicles (Figure 5-9B, white arrows). Interestingly, HA in Nup153 KD cells infected with IAV for 24 h was found at the nuclear rim (Figure 5-9A, right panel) and even traversing the nucleus in what appears to be invaginations of the NE (Figure 5-9C, top cell), most likely localized to the outer nuclear membrane.  On the whole, it appears that Nup153 depletion induces nuclear membrane herniations and disturbances in the connection between the ONM and INM. The defects observed in the ONM could be leading to the formation of outer nuclear membrane or ER origin vesicles that alter the balance in the intracellular endomembrane system and possibly disturb the correct targeting of transmembrane proteins.  199    200   Figure 5-8. Nup153 depleted cells exhibit cytoplasmic vacuoles-like structures and outer nuclear membrane herniations. A) HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h and examined by DIC confocal microscopy. Arrows point at the cytoplasmic vacuoles-like structures located next to the cell nucleus. Bottom left panel of (A) shows a graphic representation of the observed phenomenon. Scale bar, 10 μm. B) Electron micrographs of cross sections from HeLa cells transfected with Nup153 siRNA for 72 h and prepared for embedding thin section electron microscopy. In (B) several electron micrographs depict herniations in the NE where the ONM and the INM separate (white arrows), creating an abnormal extended perinuclear space, which sometimes contained smaller vesicles (bottom two micrographs). Left in (B) there is graphic representation of the deformations observed in the nuclear membrane of Nup153 depleted cells. C) Electron micrographs of cross sections from HeLa cells transfected with Nup153 siRNA for 72 h and prepared for embedding thin section electron microscopy. In (C) micrographs depict herniations in the NE where electron dense material is found in the intermembrane space between the ONM and the INM; creating vesicle like intrusions (white arrow). n: nucleus, c: cytoplasm. ONM: outer nuclear membrane. INM: inner nuclear membrane.   201    202  Figure 5-9. Nup153 depletion results in HA localization to the vacuoles-like structures in HA transfected and nuclear membrane in IAV infected cells. HeLa cells were transfected with Nup153 siRNA (A) for 72 h, cells were transfected with a HA plasmid (A left, B) or infected with IAV (H3N2 X: 31) for 24 h (A right, C), prepared for immunofluorescence, and examined by confocal microscopy. Cells were labelled with antibodies against HA1/2 (A, left) or HA3 (A, right) and Dapi (blue) to observe the nucleus. C-D) Montage of series Z stacks of Nup153 depleted cell transfected with HA1 (C) or infected with IAV (D). Arrows in (B) indicate the progression in appearance of the round vacuole-like structures and confirm the presence of HA only in the membrane of such structures. In (D), an upper arrow points to an accumulation of HA in the nuclear adjacent area and lower arrow indicates HA labelling at the nuclear rim. Scale bars, 10 μm (A) and 5 μm (B-C).    203  5.2.5. Examining apoptotic cell death in Nup153 depleted cells  As shown in Figure 5-1, Nup153 depleted cells prominently exhibited cell blebbing and cell rounding. Since plasma membrane blebbing is considered one of the hallmarks of cell death in both apoptosis and necrosis, we hypothesized that apoptotic cellular death could be occurring in Nup153 depleted cells. This could be possible given that these cells have been shown to be defective in the uptake of important cellular factors such as Tfn (Chapter 4, Figure 4-5). To determine whether Nup153 depleted HeLa cells were apoptotic, we examined caspase-3 activation and mitochondrial morphology. Caspase-3 activation was studied by immunofluorescence using an anti-cleaved caspase 3 (Asp175) antibody. This approach is more sensitive than others assays, because it allows for examination of each cell and, therefore, would detect possible variations in caspase 3 activation while also detecting variations in Nup153 expression within the Nup153 KD cell population. The activation of caspase-3, which is designated as an effector caspase, is associated with the initiation of the apoptotic signaling cascade, which occurs very early during the apoptotic cell’s death (reviewed by Wolf & Green, 1999). Caspase activation is unlike the terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labelling (TUNEL) assay, which detects DNA strand breaks occurring in the late stages of apoptosis (Gavrieli et al., 1992) and has been previously tested and reported negative in Nup153 depleted cells (Zhou & Panté, 2010). For detection of caspase-3, both Nup153 siRNA or control siRNA cells were treated with staurosporine (a drug known to trigger caspase-3 dependent apoptosis) as a positive control (Segal-Bendirdjian and Jacquemin-Sablon, 1995). No active caspase-3 was detected in Nup153 depleted cells or control siRNA treated cells (Figure 5-10 left panel). In contrast, control experiments in which cells were treated with staurosporine showed activated caspase-204  3 in addition to other hallmarks of apoptosis such as nuclear fragmentation, chromatin condensation, and pronounced cell blebbing (Figure 5-10 right panel). Both during apoptosis and necrosis, mitochondria become permeable and later, dysfunctional. For this reason we examined the mitochondria of Nup153 depleted HeLa cells and compared them to control siRNA treated cells. As documented in Figure 5-11 we did not observe major changes to the mitochondrial labelling (Figure 5-11A) or fluorescence intensity (Figure 5-11B). In addition, the mitochondrial size (Figure 5-11C), interconnectivity (Figure 5-11D), and elongation (Figure 5-11E) showed no significant difference between both siRNA treatments, indicating that there were no changes to mitochondria morphology upon Nup153 depletion.  To conclude our examination of apoptosis as a possibility in Nup153 depleted cells, we monitored nuclear permeability. A degradation of nucleoporins takes place during apoptosis (Fahrenkrog, 2006) and NE permeability has been shown to increase even before other apoptotic hallmarks became evident (Strasser et al., 2012). It has been previously established that GFP oligomers with four or less tandem GFP copies freely diffuse into the nucleus, while 5GFP is excluded from the nucleus (Wang & Brattain, 2007). Hence, HeLa cells were treated with Nup153 siRNA and control siRNA for 48 h, transfected with a 5GFP construct, and prepared for immunofluorescence microscopy. We observed that 5GFP was excluded from the nucleus in both treatments (Figure 5-12), indicating that the depletion of Nup153 by RNAi did not permeabilize the NPC.  205   Figure 5-10. Depletion of Nup153 does not trigger caspase-3 mediated apoptosis. HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h, prepared for immunofluorescence, and examined by confocal microscopy. Immunofluorescence of cells labelled with anti-cleaved caspase 3 (Asp175) antibody (green) and anti-Nup153 antibody (white). Rhodamine-phalloidin was used to visualize F-actin (red) and Dapi (blue) to observe the nucleus. Control siRNA and Nup153 siRNA treated cells were treated with Staurosporin (1 nM) for 4 h to induce apoptosis (as positive control) and re-examined for cleaved-caspase 3 staining (right panel). Scale bar, 10 μm.     206  Overall, although we did not study other effector caspases such as caspase-6 and caspase-7, the study of caspase-3 activation, mitochondrial morphology, and nuclear permeability allows us to further conclude that Nup153 depleted cells are most likely not apoptotic, in agreement with previous report that failed to detect caspase-3 activation (Mackay et al., 2009) or DNA breaks with a TUNEL assay (Zhou & Panté, 2010). Moreover, while we did not examine necrosis in Nup153 depleted cells directly, signs such as mitochondrial and cell swelling, cell lysis instead of vesicle formation, DNA fragmentation, and a loss of membrane integrity were not observed.   207      208  Figure 5-11. Nup153-depleted cells do not exhibit changes in mitochondria immunolabelling, size, interconnectivity, and elongation. HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h and prepared for live confocal fluorescence microscopy. A) Mitochondria were identified using MitoTracker (red), nuclear area was identified using NucBlue (blue), and cell morphology was observed through DIC imaging (shown in merged image). Scale bar, 5 μm. B) Quantification of MitoTracker fluorescence intensity, adjusted to relative percentage to normalize samples in all experimental repeats. C) Quantification of mitochondrial size. D) Quantification of mitochondrial interconnectivity index, defined as the mean area/perimeter ratio. E) Quantification of mitochondrial elongation index, defined as inversed circularity. (D) and (E) were used as estimators of mitochondrial fission and fusion. Results in B-E are shown as compiled means (dots) from n>100 cells from four independent experiments. Error bars represent the S.E.M. Statistical analyses were performed using Student’s t-test and no significant differences (n.s) between conditions were found.   209   Figure 5-12. Nup153 depletion does not alter the permeability of the NPC. HeLa cells were treated with either control siRNA or Nup153 siRNA for 72 h. Cells were transfected 48 h after the siRNA treatment with 5GFP cDNA constructs for 24 h. Samples were prepared for indirect immunofluorescence microscopy using an antibody against Nup153 (white). Nuclei were stained with Dapi. Nuclear membrane permeability was intact because 5GFP was excluded from the nucleus of Nup153 depleted cells. Scale bar, 10 μm.   210  5.3. Discussion Our data showed that a reduction in Nup153 expression results in drastic changes to the cell cytoskeleton in HeLa and HeLa LAP2β-GFP, the two cell lines used. F-actin and microtubules were reorganized subsequent to Nup153 depletion (Chapter 4, Figure 4-16; Figure 5-5B). This is in agreement with the results published by Zhou and Panté (2010), who reported that Nup153 depletion by RNAi resulted in a rearrangement of the microtubules and F-actin. However, we also detected changes in the immunostaining of the intermediate filament protein vimentin. That is, vimentin filaments appeared collapsed to one side of the cell or wrapped around the cell nucleus (Chapter 4, Figure 4-17; Figure 5-5C). This is not surprising, because it is very well known that there is crosstalk between intermediate filaments, actin filaments, and microtubules (Chang & Goldman, 2004). Thus, changes in one of the cytoskeleton elements may modify one of the other cytoskeleton systems. Blebbing of the plasma membrane was a distinct phenotype in our Nup153 KD cells (Figure 5-1). Mackay et al. (2009) also reported an increase in non-oriented cortical blebbing in HeLa cells depleted of Nup153 by RNAi. According to Mackay et al. (2009), these changes in cell morphology were associated with profound defects in early mitosis;, their live cell imaging nicely showed that Nup153 KD cells that did not undergo cell division, had a rounded morphology (similar to our images in Figure 5-1A), and yielded waves of non-oriented cortical blebbing. In addition, the number of cells with this phenotype (24%) that were reported by Mackay et al. (2009) is similar to our finding of an average of approximately 26% of the Nup153 KD cells with the blebbing phenotype (Figure 5-1B).  211  Zhou and Panté (2010) found cell cycle progression defects in Nup153 KD cells, similar to ours, albeit with a comparatively larger population of cells arrested in G1 than what we found (Figure 5-4B). These results are on par with those of Mackay et al., (2009) and Lussi et al. (2010), who found that low Nup153 levels were associated with defects in mitotic timing and mitotic exit. A relationship between advancement in the cell cycle and the cellular cytoskeleton has been well established. For example, a disruption of the F-actin architecture through the use of drugs has led to arrest in G1 in multiple cellular types (reviewed by Moes et al. 2011). Finally, plasma membrane blebbing and cell rounding has been observed in cells during the early G1 phase (Porter et al., 1973) or during anaphase/early telophase (Boss, 1955). Although, the specific role of plasma membrane blebbing during cytokinesis is unknown, the presence of blebbing during different stages of the cell cycle suggest a delicate regulation mechanism in the cell and it is possible that Nup153 depletion has affected gene expression in a manner that ‘turns on’ the actin dynamics but does not ‘turn them off’. Moreover, we examined if Nup153 KD cells showed signs of apoptosis, and found that this phenotype does not appear to be due to apoptosis. Thus, more studies are required to explain how Nup153 KD leads to plasma membrane blebbing. Other striking phenotypes we observed upon depletion of Nup153 by RNAi were nuclear deformations (Figure 5-3) and cells with multiple nuclei and multi-lobed nuclei (Figures 5-5 and 5-6). The latter was more prominent in HeLa LAP2β-GFP cells (Figure 5-6). These results are in agreement with those of Mackay et al. (2009), who also observed multi-lobed nuclei in Nup153 KD cells and reported that the number of cells with multi-lobed nuclei increased with the increased depletion of Nup153. Thus, it is not surprising that 212  we observed this phenotype when Nup153 was completely depleted, as indicated by immunofluorescence microscopy using an anti-Nup153 antibody (Figure 5-6A). Interestingly, although both cell lines studied in this chapter are HeLa cells, the presence of LAP2β-GFP as a stable protein expressed in HeLa seems to have an effect on the formation of multi-lobed nuclei in Nup153 KD cells. In contrast to the cell cycle proportions observed in Nup153 KD HeLa cell population (G2/M ~13%) (Figure 5-4), flow cytometry results in the HeLa LAP2β-GFP stable cell line showed that the majority (~50%) of the cells were in G2/M phase of the cell cycle (Figure 5-7). It is possible that the differences in cell cycle stage and extreme multi-lobed/multiple nuclei phenotype might be due to overexpression of LAP2β. It has been shown (Anderson et al., 2009) that overexpression of LAP2β resulted in accelerated nuclear formation through acceleration of NE formation due to its interaction with chromatin. Moreover, in HeLa cells overexpression of LAP2β results in the formation of multilamellar structures in the cytoplasm originated from the ER membrane (Volkova et al., 2012). It appears that in HeLa LAP2β-GFP, cell cycle progression from G1 to G2/M might be due to overexpression of this protein, which allowed DNA replication perhaps due to the availability of NE.  As an interesting coincidence, Nup153 overexpression has been associated with the formation of membranous structures in the cytoplasm (Duheron et al., 2014) and inside the nucleus (Bastos et al., 1996; Duheron et al., 2014). In Nup153 KD cells, Mackay et al. (2009) were able to rescue the multi-lobed nuclei phenotype in their research by the transfection of a plasmid encoding the N-terminal domain or the zinc finger motifs of Nup153. The Nup153 N-terminal and C-terminal domains interact with lamin A/C, lamin B, and Sun1 (Al-Haboubi et al., 2011; Li and Noegel, 2015). The absence of Nup153 interaction with these proteins 213  may explain the multi-lobed nuclei phenotype by indirectly altering the regulatory mechanisms involved in NE formation. In fact, not only LAP2β immunostaining was affected in Nup153 KD cells. Zhou & Panté (2010) found that lamin A/C and the LINC complex protein Sun1 were redistributed in the nuclei of Nup153 depleted cells. Moreover, a lack of Nup153 in reconstituted Xenopus nuclei induces NPC clustering (Walther et al., 2001), while RNAi depletion of Sun1 (Liu et al., 2007) results in NPC clustering in mammalian cells. However, during our ultrastructural studies of Nup153depleted cells we were not able to identify NPC clusters, in agreement with other ultrastructural studies performed with Nup153 depleted cells (Duheron et al., 2014). The difference might be due to the experimental system used. Both Walther et al. (2001) and Vollmer et al. (2015) observed NPC clustering in reconstituted membranes from Xenopus oocytes lacking Nup153, while Duheron et al.’s (2014) and our work were performed with Nup153 KD mammalian cells. This suggests that NPCs might not cluster upon Nup153 depletion by RNAi in vivo in mammalian cells. Regardless, because of the role of the nuclear lamina in maintaining nuclear structure and shape, a disruption of the physiological abundance of either Nup153 or Sun1 would cause deformations of the nucleoskeleton and possibly explain the presence of multi-lobed nuclei in Nup153 KD cells. The disruption in the nucleoskeleton or the LINC complex can perturb nuclear-cytoskeletal associations, inducing changes in mechanotransduction, nuclear positioning, and cell differentiation (Chambliss et al., 2013; Wilson and Berk, 2010). Interestingly, Mackay et al. (2009) were able to partially recover the multi-lobed nuclei phenotype by expressing the exogenous N-terminus of Nup153, which targets Nup153 to the nuclear ring of the NPC where it interacts with lamin B (Smythe et al., 2000). This suggests that Nup153’s interaction 214  with the nuclear lamina is an important factor at play in nuclear shape, although not the only one. Moreover, the nucleoskeleton is responsible for nuclear shape, mechanical properties, and spatial-related gene regulation (reviewed by Dahl et al. 2008). The stiffness of the nucleoskeleton is approximately five or ten times higher than stiffness of the cytoskeleton (reviewed by Wang et al. 2009). Hence, drastic changes to the nucleoskeleton through the depletion of Nup153 could induce the changes observed in the cytoskeleton of the Nup153 KD cells.  The physical connections between the nucleus and the rest of the cell are further extended by the relationship between the ONM and the ER. Through a thin section EM, we observed herniation in the ONM and the presence of electron dense material in the perinuclear space or in the nucleus adjacent to the nuclear membrane (Figure 5-8B and 5-8C). The continuity of ONM with the ER could be the cause of the ONM herniation that we propose gives origin to the vacuole-like/vesicular structures observed by DIC in the cytoplasm of Nup153-depleted cells (Figure 5-8). In agreement with this, Nup153 KD cells expressing HA either after transfection with a HA plasmid or infection with IAV yielded HA immunolabelling at these vacuole-like/vesicular structures and sometimes at the NE (Figure 5-9). Interestingly, electron micrographs of nup116, nup145, or nup1 yeast null mutants exhibit a plethora of alterations in the NE structure. Nuclear membrane herniations and the presence of vesicle-like structures in the perinuclear space were among these alterations (Bailer et al., 1998; Bogerd et al., 1994; Emtage et al., 1997; Wente and Blobel, 1993). Yeast nup1 mutant cells (Nup1p gene product) are multinucleated and show a random orientation 215  of the mitotic spindle, in addition to the thin, ‘finger-like’ NE projections extending into the cytoplasm (Bogerd et al., 1994). Although Nup1p and Nup153 are not homologous proteins, Nup1p is also located in the nuclear basket of the yeast NPC, in a similar location to that of Nup153 (Davis and Fink, 1990; Pante et al., 1994). The similar location of Nup1p and Nup153 at the NPC alludes to the importance of the location of these nucleoporins at the base of the nuclear basket, which might be involved in the maintenance of the NE structure.  Overall, several nucleoporins in addition to Nup153 have been proposed as regulators of the cell cycle and gene expression (Nakano et al., 2011). Even yeast null nup116, nup145 ∆N, and null nup1 mutants, which yield similar defects on the NE morphology as those of Nup153 KD cells, display defects in cell growth (Bogerd et al., 1994; Wente and Blobel, 1993; Wente and Blobel, 1994). We propose that for Nup153 depleted cells, altered levels of ONM/ER membrane are being produced in preparation for cell division that does not fully occurs due to dysregulation in the cell cycle. We also propose that the ONM/ER presents the morphological defects we observed because absence of Nup153 weakens the interaction between the INM and the ONM. These alterations may in turn affect the endomembrane system within the cells and give rise to defects in organelles of the endocytic and secretory pathway, as was reported in Chapter 4. In support of this claim, we observed plasma membrane blebbing, defects in the cellular localization and distribution of endocytic vesicles, the intracellular traffic of several substrates, and the targeting the transmembrane protein HA to the plasma membrane (Chapter 3 and Chapter 4). In summary, in this chapter we examined consequences of Nup153 depletion in the cellular cytoskeleton and the nuclear membrane. Our findings indicate that the presence of 216  Nup153 is important for appropriate cell shape, nuclear morphology, and cell cycle regulation. In addition, we concluded that changes analyzed in this chapter are most likely not due to cell death by caspase-3 induced apoptotic mechanisms.    217            Chapter 6 General discuss ion and f uture perspectives General discussion and future perspectives  This thesis has contributed to the field of cell biology and virology by identifying new roles of nucleoporins in viral infections. It was found that Nup153 silencing by RNAi induces unexpected defects in several cellular processes and reduces infectivity of IAV. By examining the sub-cellular localization of several IAV proteins at different times of infection in Nup153 KD cells, a deficiency in several steps of the IAV infective cycle was established. Defects in the cellular uptake and intracellular traffic of IAV and other cellular cargos (e.g. EGF and Tfn) indicate that Nup153 is a versatile nucleoporin that is indirectly involved in these cellular processes, which have never before been observed in relation to Nup153 or any other nucleoporin. To explain these defects as well as defects on nuclear architecture and the arrangement of the three cytoskeletal elements upon Nup153 depletion, we propose that Nup153 is essential for adequate nuclear membrane organization through its interaction with nucleoskeletal elements. Thus, the absence of Nup153 results in tensions in the nucleoskeleton-cytoskeleton as a consequence of changes in nuclear morphology. This in turn negatively affects cargo uptake and intracellular communication pathways.   Overall the results presented in this thesis indicate that Nup153 have non-canonical roles, including roles during IAV infection. The results would lead to consider Nup153 as an important factor in the development of therapeutics against IAV. Below, the steps of the IAV infective cycle affected in Nup153-depleted cells are first discussed. Then, the model of how Nup153’s association to the nucleoskeleton and the cytoskeleton can affect cell morphology and gene expression is further discussed. 218  6.1. Steps of the IAV infection cycle affected in Nup153-depleted cells Our findings of the alterations that occurred in various cellular processes upon Nup153 depletion (Chapters 4 and 5) can explain the defects of the IAV infective cycle we observed in Nup153 cells (Chapter 3). As illustrated in Figure 6-1, although we did not directly detect the distribution of sialic acid, our results of the altered distribution of WGA label (Figure 4-1) and plasma membrane proteins (EGFR and TfR; Figures 4-2, and 4-4) indicates an uneven distribution of sialic acid in the plasma membrane of Nup153 KD cells. This will result in viral agglomerations where the sialic acids were concentrated and explain the reduction in IAV uptake in Nup153 KD cells (Figures 3-4A and 3-4B). Additional reduction of IAV cellular uptake in Nup153 KD cells could be attributed to the presence of plasma membrane blebs and defects in F-actin organization in these cells. The latter predicts defects in clathrin-mediated endocytosis and macropinocytosis, both of which are used by IAV to enter non-polarized cells such as HeLa cells (reviewed by Sun & Whittaker, 2013).  In addition to reduction in viral uptake, the few virions that enter the cells encounter defects in microtubule organization. Thus, virion-containing endosomes do not move towards the nucleus of Nup153 KD cells and when acidification of late endosomes occurs, the vRNPs are released away from the nuclear periphery (Figure 6-1). The combined defects on cellular uptake and trafficking of virion-containing endosome could be enough to explain reduction in the production of infectious particles in the Nup153 KD cells compare to control cells. However, Nup153 depletion results in additional defects in late steps of the IAV infective cycle (Figure 6-2). First, defects in microtubule organization might affect vRNP cytoplasmic transport in Nup153 KD cells, 219  which might result in defects in incorporation of vRNPs into progeny virions. Our evidence for defects at this step is the cytoplasmic accumulation of progeny vRNPs observed in Nup153 KD cells but not in control cells at 8 and 12 hours after infection (Figure 3-8). Additionally, transport of HA towards the plasma membrane (and probably neuraminidase, which also follows the secretory pathway) is affected (Figure 6-2). Our detection of HA by immunofluorescence microscopy in Nup153 KD cells after transfection with a HA plasmid or infection with IAV (Figures 4-14 and 5-9) confirm defects in the targeting of HA to the plasma membrane.  Furthermore, the shifts in the localization of EGFR, TfR, and HA subsequent to Nup153 depletion led us to consider the possibility that the targeting of the other two IAV transmembrane proteins (NA and M2) to the plasma membrane might also be defective. In addition, our preliminary results obtained in the study of autophagy flux in Nup153 KD cells (discussed in Chapter 4), suggest an alteration of autophagy pathways in these cells. In this context, virion stability might be compromised in IAV infected Nup153 KD cells since IAV subverts autophagy through interaction of M2 with LC3B (Beale et al., 2014). Altogether, we speculate that it is possible that the transport of vRNPs to the plasma membrane along microtubules and defects in the composition of viral membrane proteins resulted in defective viral particles and/or reduced infective particles (summarized in Figure 6-2). This explains the observed reduction of viral titer in Nup153 KD cells (Figure 3-1C). 220   221  Figure 6-1. Proposed model of alterations in the internalization and endosomal trafficking of IAV in Nup153 KD cells. A) Representation of early infection steps in Control siRNA treated cells. First the virus attaches to the sialic acid moieties in the plasma membrane, and then enters the cell by endocytosis. The virus in the early endosomes and then late endosomes traffics towards the cell periphery. Endosomal acidification occurs in late endosomes resulting in release of vRNPs. B) Representation of early infection steps in Nup153 siRNA treated cells. First the virus attaches to the clustered sialic acid moieties in the plasma membrane, and then, a reduced number of viruses enter the cell by endocytosis due to defects in F-actin organization and plasma membrane blebbing. The IAV is found in early endosomes and then late endosomes, both dispersed throughout the cell due to defects in microtubule organization. Endosomal acidification occurs in late endosomes resulting in release of vRNPs away from the nuclear periphery. 222    223  Figure 6-2. Proposed model of alterations in late steps of IAV infection in Nup153 KD cells. A) Representation of late infection steps in Control siRNA treated cells. The vRNPs are exported out of the nucleus and accumulate at the MTOC where they are transported to the plasma membrane along microtubules by “piggybacking” in Rab11 positive recycling endosomes. At the plasma membrane the vRNPs are incorporated into the progeny virion, which is mediated by packaging signals present in the vRNP. The plasma membrane area where budding occurs contains viral proteins HA, NA, and M1. After M2 mediates the scission of the budding virus and NA prevents aggregation of the virus at the surface, new viral particles are release. B) Representation of late infection steps in Nup153 siRNA treated cells. The (?) character denotes unknown defective steps that might be present in Nup153 KD cells. Due to defects in Nup153 depleted cells, post-translational modifications of viral proteins might be affected. The vRNP is then exported out of the nucleus, but might not accumulate at the MTOC and “piggybacking” in Rab11 positive recycling along microtubules, because Nup153 KD induces defects in microtubule organization. This transport alteration might result in defects in incorporation of vRNPs into the progeny virion. Major changes observed in the distribution of HA in Nup153 depleted cells are indicated in the diagram, these include: HA in large filled or empty vesicles, at the nuclear membrane periphery, and accumulated in “patches” at the plasma membrane. We propose that since NA also follows the secretory pathway, the distribution of NA might also be affected. Altogether, the membrane composition of the new virion might be altered and the new viral particles might be defective   224  6.2. Biology of the IAV: lessons learned in the absence of Nup153 Our study following IAV infection in Nup153 KD cells has provided important lessons regarding the cellular requirements of IAV. The entry of IAV into non-polarized cells occurred despite changes in the distribution of F-actin and changes to the plasma membrane such as blebbing that otherwise affected the uptake of extracellular ligands dependent on clathrin-mediated endocytosis and macropinocytosis. Furthermore, changes to microtubule organization in Nup153 KD cells did not completely hinder vRNP traffic towards the nucleus and the depletion of Nup153 reduced NLS-dependent nuclear import, but did not obstruct vRNP translocation through the NPC. This suggests the participation of compensatory mechanisms and other Nups. Unexpectedly, our findings suggest that a lack of Nup153 affects the IAV infective cycle during or after vRNP assembly. It is possible that Nup153 facilitates post-translational modifications to viral proteins or vRNAs inside the nucleus, or that the defects observed in microtubule organization − and possibly the secretory pathway − act in concert to reduce the IAV titer by 4-fold in Nup153 KD cells.  Nup153 depletion has been associated with a reduction of viral infection not only for IAV (Benitez et al., 2015; König et al., 2010; Watanabe et al., 2014; York et al., 2014), but also for HIV-1 (Brass et al., 2008; Koh et al., 2013; König et al., 2008; Matreyek & Engelman, 2011). In this case, however, Nup153 interacts directly with the HIV-1 viral capsid protein, which forms the capsid core that houses the viral genome (reviewed by Campbell & Hope, 2015). In addition, it has been shown that Nup153 is important for the nuclear import of the PIC and is involved in guiding the PIC to areas of integration in the chromosome (Koh et al., 2013). Moreover, HIV-1 infection affects the NPC composition by 225  displacing Nup62 and reducing the abundance of other Nups such as Nup153 (Monette et al., 2011). Interestingly, HIV-1 virion assembly, like IAV virion assembly, takes place at the cell’s plasma membrane, where the Gag protein mediates essential steps in viral assembly and budding (reviewed by Sundquist & Krӓusslich, 2012). Additionally, the HIV-1 viral membrane proteins Env and Vpu are targeted to the plasma membrane via the secretory pathway (reviewed by Sundquist & Krӓusslich, 2012). It would be interesting to determine whether the defects observed in HA targeting to the plasma membrane in the absence of Nup153 would also affect Env and Vpu transport to the plasma membrane and consequently, affect HIV-1 virus production due to defects in virion assembly or budding.  Other Nups have also been considered important for viral infectivity during IAV infection. Genome‐wide siRNA screens identified Nup214, Nup98, and Nup205 as host cell proteins required for IAV replication and infectivity (Karlas et al., 2010; König et al., 2010). Additionally, Nup358 (Figure A-1) and Nup62 (Morita et al., 2013) depletion also affect IAV viral titer. It is possible that the localization of the FG nucleoporins Nup214 and Nup358 at the cytoplasmic filaments of the NPC enable these nucleoporins to aid in the nuclear export of vRNPs due to their interaction with CRM1 (reviewed by Hutten & Kehlenbach, 2007). Moreover, Nup62 is displaced in IAV infected cells and is required during viral mRNA and vRNA export, which is not the case for Nup214 or Nup98 (Morita et al., 2013). However, other findings showed that Nup98 interacts with IAV NS2/NEP through its FG motif and that its overexpression ultimately results in decreased in virus propagation (Chen et al., 2010). In accordance, the expression of Nup98 is downregulated during IAV infection (Satterly et al., 2007). It is therefore possible that IAV alters the NPC in strategic ways that have not yet been described yet and merit further investigation. New advances in IAV treatments could 226  include targeting the nuclear transport of IAV components, which is a very important and well-conserved process. 6.3. Nup153 and the intracellular domino effect  Based on our discoveries of a wide variety of cellular changes upon Nup153 depletion including intracellular organization of organelles, alterations in the cytoskeleton, and changes in nuclear morphology and deformations of the nuclear envelope, we propose that depletion of Nup153 by RNAi triggers a domino effect illustrated in figure 6-3. In this model Nup153 is required to maintain nuclear architecture and that its absence results in deformation in the nucleoskeleton, which in turn causes alterations of the cytoskeleton and unleashes defects in intracellular communication.  The domino effect in Nup153 KD cells starts with the redistribution of the LINC complex and alterations in the ONM such as nuclear herniation, which originate changes in the nuclear morphology. As a consequence, deformation in nuclear shape impacts the organization of the cytoskeleton through the connections between the nucleoskeleton and the cytoskeleton. Then, cytoskeletal rearrangements create tensions that modify the cell morphology and gives rise to plasma membrane blebbing. A modified cytoskeleton also results in the spatial redistribution of endosomal markers, which together with plasma membrane blebbing, negatively impacts the uptake of cellular cargo. In addition, the changes in HA localization and the presence of unidentified vesicles suggest defects in protein targeting and vesicle formation in the secretory pathway.  227  As a basis for our domino effect model, previous research has shown that Nup153 is necessary for NPC assembly during interphase through interactions with the INM (Vollmer et al., 2015) and that Nup153 depletion results in alterations to the organization of the nuclear lamina (lamin A/C) and Sun1 (Zhou & Panté, 2010). These changes are not surprising since Nup153 binds to lamin A/C and Sun1 through both its C-terminus and N-terminus (reviewed by Duheron & Fahrenkrog, 2004; Li & Noegel, 2015).  The ONM and the INM curve at the NPC, hence, defects in anchoring the NPCs to the nuclear lamina could generate deformities in the nuclear envelope and nuclear morphology. Accordingly, our findings showed deformed nuclei in Nup153 depleted cells after 72 h of RNAi treatment. Mackay et al. (2009) likewise found abnormal nuclear morphology in Nup153 depleted cells. In contrast, Duheron et al. (2014) found drastic changes to nuclear morphology and chromatin organization when Nup153 was overexpressed. On the whole, it appears that even modest variations in Nup153 expression in different cell types would result in altered nuclear morphology. Nucleoskeleton components such as lamin A/C and the LINC complex proteins nesprin-2 giant and nesprin-3 have been shown to play roles in anchoring the actin cap fibers to the nucleus (Chambliss et al., 2013). Furthermore, through the regulation of the expression of Nesprin-3 and its connection with actomyosin, the cell nucleus drives lamellipodia-independent migration in a 3D environment (Petrie et al., 2014). Interestingly, Nup153 depleted cells exhibited impaired cell migration and polarization (Zhou & Panté, 2010). It is therefore possible to speculate that Nup153 becomes a functional member of the nucleoskeleton, through its interactions with the INM, lamin A/C, and Sun1 and therefore its 228  absence affects the organization of the cytoskeleton (Figure 6-3). As a consequence of a modified cytoskeleton, plasma membrane dynamics are altered, as well as the spatial distribution of organelles dependent on the cytoskeleton for intracellular traffic (Figure 6-3). Although we have emphasized that the cellular defects observed in Chapters 4 and 5 could be due to consequences of the cell’s altered nuclear and cellular morphology, it is also possible that Nup153 depletion results in altered gene expression and therefore disrupts cellular homeostasis. There is an established relationship between nuclear architecture and gene expression. An increasing amount of evidence demonstrates that Nup153 expression affects not only nuclear architecture, but also chromatin organization (Duheron et al., 2014), which is important for gene expression. For example, in Drosophila, the KD of Nup153 alters the expression of over 5,000 genes and Nup153 binds to active chromosomal regions (Vaquerizas et al., 2010). More recently, Nup153 was found bound to genes in order to repress expression and prevent stem cell differentiation (Jacinto et al., 2015). These findings showed that Nup153 localization at the nuclear basket – where it can directly or indirectly interact with chromatin – could be required to control gene expression.     229   230  Figure 6-3. The domino effect: summary model of the major cellular defects observed in Nup153 KD cells. Representation of major intracellular alterations in cells treated with Nup153 siRNA after 72 h as found in Chapters 4 and 5. The domino effect model proposes nuclear membrane alterations/defects give results in deformed nucleus. Changes in the nuclear morphology impact the cytoskeleton and leads to rearrangements of vimentin, microtubules and actin. This changes lead to plasma membrane blebbing, changes in the spatial distribution of early endosomes, late endosomes and lysosomes. Additionally, HA distribution suggests defects in the secretory pathway in Nup153KD cells. Lastly, autophagosome-like vesicles could be indicative of alterations in the autophagy flux in Nup153 KD cells. Altogether, the changes in the nuclear morphology give rise to cytoskeletal changes that in turn negatively impact intracellular traffic. ONM: outer nuclear membrane. INM: inner nuclear membrane. HA: hemagglutinin. EEA1: early endosome antigen 1. CI-M6PR: cation independent mannose-6-phosphate receptor.     231  6.4. Future directions  Recent findings showing that Nup153 regulates stem cell pluripotency (Jacinto et al., 2015) highlight the need to study Nup153 from a wider perspective. Ideally, the creation of a Nup153 deficient mouse model would allow us to study Nup153 requirements by using different tissues to build a better picture of the role of this nucleoporin in the cell. However, it is likely that the deletion of such an important protein would be lethal. In the absence of such animal model (to the best of our knowledge) a genome-wide associated study in a human population identified a polymorphism near the NUP153 gene (Datta et al., 2012). The polymorphism in NUP153 was associated to the nuclear-cytoplasmic traffic of biliverdin reductase, a key enzyme involved in the bilirubin metabolism (Datta et al., 2012).  A matter that is yet to be explored is the precise role of Nup153 during IAV infection. In pursue of this goal, several approaches would be necessary. First, it would be of interest to study post-translational modifications of IAV proteins that could be defective in Nup153 KD cells. Second, we propose to determine if the formation of new virions is compromised in Nup153 KD cells; for this, we would use embedding-sectioning EM to examine Nup153 KD cells infected with IAV at different stages of the infective cycle. Third, to address viral infectivity, we suggest to perform a comparative analysis of the glycosylation profile of HA and NA from infected cells. Through such experiment, we would determine whether alterations in the glycosylation of HA and/or NA contribute to reduction of IAV infectivity. Lastly, it would be interesting to determine the distribution of the autophagy marker LC3B in Nup153 KD cells infected with IAV; this information would allow us to predict whether the formation of new virions is stable. 232  Although our results show that Nup153 is of importance during IAV, it is imperative to emphasize that further experiments in physiologically relevant systems are necessary to further validate the role of this protein during IAV infection. The use of systems such as primary human airways cultures and air-liquid cultures to study the role of Nup153 and other nucleoporins during IAV infection, would allow the scientific community to establish findings that could be of clinical relevance.  A major contribution from this work focuses on using IAV as a molecular tool to reveal the cellular role of Nup153. By following IAV infection in Nup153 KD cells we documented the effects that Nup153 depletion have in intracellular organization and traffic. To continue this line of research, it would be of interest to determine if the cargo size is relevant when studying cellular entry by macropinocytosis or receptor-mediated endocytosis in a Nup153 KD cells. Additionally, further research is required to determine effects of Nup153 depletion not only in the endocytic pathway but also in the exocytic pathway; for example, by studying the release of exosomes from Nup153 KD cells.  Finally, it would be interesting to further discern the impact of Nup153’s location in the relationship between the LINC complex protein nesprin and the various cytoskeletal elements. The answers to these questions and others expressed throughout this thesis would further increase the understanding of the non-canonical roles of nucleoporins and expand their implications for the understanding of human diseases.  6.5. Concluding remarks  233  In conclusion, infection of Nup153 depleted mammalian cells with IAV led us to find the role of this nucleoporin during IAV infection. Based on our results we propose that Nup153 depletion negatively affects IAV viral titers through a combination of defects in viral entry, vRNP intracellular traffic, and virus assembly, all of which are consequences of alterations in the cytoskeleton and the secretory pathway in Nup153 KD cells. The observed changes in the distribution of HA during infection and transfection in Nup153 depleted cells suggest defects in the secretory pathway that were never associated with nucleoporins depletion before. Interestingly, these findings reveal the possibility of the defective targeting of viral envelope proteins that follow the secretory pathway during other viral infections. 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Depletion of Nup358 and Nup214 in HeLa result in a decrease of IAV viral titer. HeLa cells treated with Nup358 siRNA (A) or Nup214 siRNA (B), control siRNA, transfection reagent or untreated were infected with IAV for 24 h at 37°C. Supernatants were then collected and subjected to plaque assay. PFU= plaque forming unit. Mean values are shown (bars) and error bars represent the S.E.M. Plaque assays shows significant (ANOVA, ***p>0.05) difference of the viral titer between Nup153 siRNA and the rest of the conditions. Results shown are representative of the plaque assay, which was repeated 3 times independently. 253     Figure A-2. Subcellular localization of NP cannot be reliably quantified in NP-transfected Nup153 KD cells. Quantification of nuclear and cytoplasmic fluorescence 254  intensity from Figure 3-3 where HeLa cells were treated with either Control siRNA or Nup153 siRNA for 72 h were transfected for 24 h with NP construct 48 h after beginning of siRNA treatment. Leptomycin B was added 8 h post NP transfection to a final concentration of 11 nM. Each graph (A, B, or C) represents the quantification of independent experiments (3 totals). Mean values are shown (bars) and error bars represent the S.E.M. Statistical significance was assayed through ANOVA and Tukey's Multiple Comparison Test (ANOVA, ***p>0.05).     255  Appendix B – Supplementary results and figures from Chapter 4 B.1. Quantification of changes in the distribution of EEA1, CI-M6PR, and LAMP-1 in Nup153 KD cells  To quantify the changes observed in the distribution of the cellular markers EEA1, CI-M6PR, and LAMP1 in Nup153 depleted cells (results presented in Figures 4-7A, 4-9A, and 4-9E), we used an statistical approach of point distribution within the confines of a compartment of any size and shape to analyze the endosomal marker data (Andrey et al. 2010). After image acquisition through confocal microscopy, each cell was delineated, to analyze the spatial distribution of the EEA1 immunostaining, the images were reduced to binary (presence or absence) of pixels, establishing then the first noise reduction criteria. For each cell, three distance functions were computed F, G, and H. The F-function represents the CDF of the distance between EEA1 dots and the surrounding space not containing other EEA1 dots (Figure B-1B), more specifically, the distance between a typical position within the studied compartment and its closest point in the pattern of EEA1 signal. The G-function represents the CDF of the distance between EEA1 dots and the closer nearby EEA1 dots, its nearest neighbor (Figure B-1C). The H-function represents the CDF of the distance between a typical EEA1 dots and any other EEA1 dot (Figure B-1D) within the cell. In each of these spatial distribution functions, the resultant cumulative distribution from the spatial analysis of EEA1 signal from each cells (blue curve) were compared with a computer generated randomly distributed point pattern (same number of points as observed EEA1 points) (red curve) in what it is called a completely random binomial point process (CRBPP). In the CRBPP, points are distributed in an independent manner of each other and 256  evenly/non-clustered. The distance values found using the CRBPP curve with the one obtained from the EEA1 signal, were scored using p-values involving Monte-Carlo simulations (Andrey et al., 2010). Those p-values are referred as spatial distribution index (SDI), in these indexes a value closer to 0 refers to a clustered pattern while an evenly space signal pattern produce values closer to 1. As observed in Figure B-1B, both EEA1 (blue) curves in the control siRNA and Nup153 siRNA treated cells deviate from the CRBPP curves and its 95% confidence interval (green curves). However, in the curves in the G-function (Figure B-1C) and H-function (Figure B-1D) we observe clearly that the curve representing EEA1 (blue) appears closer to the CRBPP curve in Nup153 depleted cells than in control siRNA treated cells. This means that the EEA1 signal in the Nup153 depleted cell (pointed by an arrow in Figure B-1A) follows a spatial distribution pattern more closely related to a random distribution pattern, instead of the aggregated one observed in the examined control siRNA treated cell (pointed by an arrow in Figure B-1A). In addition to obtaining the SDI values for each function after studying the EEA1 pattern distribution in individual cells, we also evaluated the physical distance reached when the cumulative frequency of EEA1 signal reached 50% in each cell. We evaluated those values from F, G, and H function; the results show that in Nup153 depleted cells, the EEA1 signal was significantly further from its nearest neighbor (G function). The equivalent analysis from the other distance function (F and H) showed not significant differences (graphs not shown). Thus, confirming that EEA1 spatial distribution is more spread-out in HeLa Nup153 depleted cell than in control cells.  257  Further analysis led us to create a frequency histogram with the F-function–related SDIs for each experimental condition. The SDI values obtained if the spatial distribution was random would be equally distributed between 0 and 1 throughout the histogram (same % of cells for each value). However, that is not the case when we examined the F-function related histogram (Figure B-1F) displaying the SDI values for control and Nup153 depleted cells. In these, there is a strong accumulation towards 1. In contrast, when we examined the G-function related frequency histogram (Figure B-1G), Nup153 depleted cells appeared to have more SDI values closer to the middle of the distribution. When both conditions were examined using a Chi-square test for trend performed in the frequency distribution histogram, we found that there is a significant difference in the distribution of SDI values between Nup153 depleted cells and control cells. The observed difference in the G-function means that EEA1 positive endosomes nearest-neighbor are distributed more randomly in Nup153 depleted cells than in control cells.  Lastly, the same analysis was repeated with the H-function SDI values (Figure B-1H); where they were arranged in a frequency histogram and differences in distribution were evaluated using a Chi-square test for trend. The results in Figure B-1H also show a difference in the distribution of SDI values between the population of control and Nup153 depleted cells. In control cells, the SDI values are accumulated at 0 while dispersed between 0 and 1 in Nup153 depleted cells. This indicates that in control cells, compared to a random distribution, the EEA1 signal has a regularly space pattern and it is not random, while the distribution observed in Nup153 depleted cells suggests that in these cells EEA1 signal can be unevenly distributed and overall more randomly distributed.  258    259    Figure B-1. Quantification of changes observed in the distribution of the cellular markers EEA1 presented in Figure 4-7A. A) This is the same figure shown in Figure 4-7A and shows confocal images of HeLa cells transfected with control siRNA or Nup153 siRNA for 72 h and immunolabelled with antibodies against EEA1 (green) and Nup153 (white). Cells were also labelled with Dapi (blue) to observe the nucleus and fluorescently-labelled phalloidin to visualize F-actin (not shown); the latter was used to delineate cell boundaries. Scale bar, 10 μm. B-D) Statistical analysis of spatial distribution of EEA1 from the cells indicated by arrows in (A). Shown are the results for the distance F-function (B), G-function (C) and H-function (D) from the distribution of EEA1 (blue) compared with a randomly distribution (red) of the same number of EEA1 dots within the cell boundaries with a 95% confidence interval (green). The F-function represents the cumulative distribution function (CDF) of the distance between EEA1 dots and the surrounding space not containing other EEA1 dots. The G-function represents the CDF of the distance between EEA1 dots and the closer nearby EEA1 dots. The H-function represents the CDF of the distance between a 260  typical EEA1 dots and any other EEA1 dot. The red lines in (B) and (C) represent the measured distance of 50% of the cells in the CDF. E) Mean distance between the EEA1 dots and its nearest neighbor, which was found to be significantly difference (Student’s t-test, **P<0.05) during the analysis of the G-function (C). Error bars in (E) represent S.E.M. Equivalent analysis done for F-function and H-function showed not significant difference when analyzed as in (E). F-H) Frequency distribution histograms (in % of cells) of the spatial distribution index (SDI) for the F-function (F), G-function (G), and H-function (H). SDI is a predictor of clustering (closer to 1) or repulsion (closer to 0). An n >20 cells from each condition (Control or Nup153 siRNA) were analyzed for each function. The frequency distribution of SDI values in the histograms (F-H) between both experimental conditions (Control and Nup153 siRNA) were analyzed using Chi-square test for trend with a p-value < 0.001. (F) and (H) distribution between Control and Nup153 siRNA SDI values were significantly difference.   Similar quantification of the distribution of the endosome marker CI-M6PR showed that the CI-M6PR immunostaining in control cells was punctate and located in an area closer to the nucleus (Figure B-2A). Because of its non-uniform distribution tendencies, we performed the spatial distribution analysis previously used to evaluate the distribution pattern of EEA1 (Figure B-1). All three functions (F, G, and H functions) were obtained and analyzed as performed previously with EEA1. Shown in Figure B-2B is an example graph obtained as a result of the CDF of CI-M6PR signal (blue) in control and Nup153 depleted cells as well as a CRBPP red curve (random signal distribution) and its 95% interval (green curves). Although the CI-M6PR signal appeared very different, no major differences were observed between the F-function graphs in control and Nup153 depleted cells. Likewise for the G-function (Figure B-2C) and H-function (Figure B-2D) no major differences were detected. However, when studying the distance when the cumulative frequency of the CI-261  M6PR signal reached 50% in each cell (Figure B-2E), the results showed that the CI-M6PR signal (presumably vesicles) were located closer to its nearest neighbor in Nup153 depleted cells. These results are consistent with the images show in the enlargement panel in Figure B-2A, were M6PR clusters are seen in Nup153 depleted cells (second and third row). To describe the SDIs in the whole population of examined cells, each function (F, G and H) SDIs are shown as frequency histogram. The results for the F-function (Figure B-2F) show an accumulation of SDI values towards 1 while the G-function show a distribution of values towards extremes (0 and 1). The statistical analysis used for the frequency histogram to evaluate the differences in distribution was a Chi-square test for trend, and resulted in no differences between control and Nup153 siRNA treated cells. However, the distribution of the SDI values from the H-function (Figure B-2H) was found closer to 0 in control siRNA treated cells while a large percentage of Nup153 depleted cells have an SDI value, which means each dot is closer to any other CI-M6PR dot (not only to its closest neighbor).    262  263   Figure B-2. Quantification of changes observed in the distribution of the cellular markers CI-M6PR presented in Figure 4-9A. A) This is the same figure shown in Figure 4-9A and shows confocal images of HeLa cells transfected with control siRNA or Nup153 siRNA for 72 h and immunolabelled with antibodies against CI-M6PR (green) and Nup153 (white). Cells were also labelled with Dapi (blue) to observe the nucleus and fluorescently-labelled phalloidin to visualize F-actin (not shown), which was used to delineate the cell periphery. Right panels in (A) are enlargements of boxed areas in merge panels; it compares the distribution pattern observed in Nup153 depleted cells with control siRNA treated cells. Scale bar, 10 μm, 5 μm (enlargement). B-D) Statistical analysis of spatial distribution of CI-M6PR from the cells indicated by arrows in (A). Shown are the results for the distance F-function (B), G-function (C) and H-function (D) from the distribution of CI-M6PR (blue) compared with a randomly distribution (red) of the same number of CI-M6PR dots within the cell boundary with a 95% confidence interval (green). The F-function represents the cumulative distribution function (CDF) of the distance between CI-M6PR signal and the 264  surrounding space not containing other CI-M6PR signal. The G-function represents the CDF of the distance between CI-M6PR signal and the closer nearby CI-M6PR signal. The H-function represents the CDF of the distance between a typical CI-M6PR signal and any other CI-M6PR signal. The red lines in (B) and (C) represent the measured distance of 50% of the cells in the CDF. (E) Mean distance between the CI-M6PR signal and its nearest neighbor, which was found to be significantly difference (Student’s t-test * p<0.05) during the analysis of the G-function (C). Error bars in (E) represent S.E.M. Equivalent analysis done for F-function and H-function showed not significant difference when analyzed as in (E). F-H) Frequency distribution histograms (in % of cells) of the spatial distribution index (SDI) for the F-function (F), G-function (G), and H-function (H). SDI is a predictor of clustering (closer to 1) or repulsion (closer to 0). An n >20 cells from each condition (Control or Nup153 siRNA) were analyzed for each function. The frequency distribution of SDI values in the histograms (F-H) between both experimental conditions (Control and Nup153 siRNA) were analyzed using Chi-square test for trend with a p-value < 0.001. (H) Distribution between Control and Nup153 siRNA SDI values was significantly difference.   Once again, we made use of the spatial distribution analysis (previously used for EEA1 and CI-M6PR) to determine whether there were major differences in LAMP-1 distribution between both experimental conditions. All three functions (F, G and H functions) were obtained and analyzed as performed previously; however this time, only the F-function (Figure B-3B) and the H-function (Figure B-3C) are shown. The G-function results were omitted as they showed not significantly different in both experimental conditions and the SDI values were almost all entirely 1, which means that the LAMP-1 fluorescence signal was clustered in both conditions equally when compared to a random CRBPP distribution. In Figure B-3B the F-function graph (obtained from analyzing the selected cell, arrow) show very similar LAMP-1 signal (blue) in control and Nup153 depleted cells (arrow, 265  Figure B-3A) as well as a CRBPP red curve (random signal distribution) and its 95% interval (green curves). The curves originated from the CDF product of the H-function show that in Nup153 depleted cell the results is mostly similar to the distribution of CRBPP, which means it has a rather random distribution. The distance of the cumulative distribution of LAMP-1 when signal reached 50% of dots did not show any difference in any of the three studied parameters (F, G, or H functions; not shown). Finally, the SDI frequency distribution histogram of F-function shows a tendency towards the extremes, 60% of the cells in Nup153 depleted cells showed SDI values of 0 (Figure B-3D). A large population with SDI values of 0 results in more equidistantly spaced out signal than expected by the binomial distribution model in the Nup153 depleted cells, which is contrary to what it is observed in the control treated cells.  Lastly, the frequency histogram of SDIs obtained through the H-function (Figure B-3E) is also distributed throughout all the values for Nup153 KD cells and shows a pattern more likely observed when there is no signal clustered or when there is random distribution of the feature at the population level. The significance of these differences was determined using a Chi-square for trend test.  266     267  Figure B-3. Quantification of changes observed in the distribution of the late endocytic marker/lysosome LAMP-1 presented in Figure 4-9E. A) This is the same figure shown in Figure 4-9E and shows confocal images of HeLa cells transfected with control siRNA or Nup153 siRNA for 72 h and immunolabelled with antibodies against CI-M6PR (green) and Nup153 (white). Cells were also labelled with Dapi (blue) to observe the nucleus. Right panels in (A) are enlargements of boxed areas in merge panels; through the enlargement panels we compare the LAMP-1 distribution pattern observed in Nup153 depleted cells with control siRNA treated cells. Scale bar, 10 μm. B-D) Statistical analysis of spatial distribution of LAMP-1 from the cells indicated by arrows in (A). Shown are the results for the distance F-function (B), H-function (C) from the distribution of LAMP-1 (blue) compared with a randomly distribution (red) of the same number of LAMP-1 dots within the cell boundary with a 95% confidence interval (green). The F-function represents the cumulative distribution function (CDF) of the distance between LAMP-1 dots and the surrounding space not containing other LAMP-1 dots. The H-function represents the CDF of the distance between a typical LAMP-1 dots and any other LAMP-1 dots signal. D-E) Frequency distribution histograms (in % of cells) of the spatial distribution index (SDI) for the F-function (D), and H-function (E). SDI is a predictor of clustering (closer to 1) or repulsion (closer to 0). An n >20 cells from each condition (Control or Nup153 siRNA) were analyzed for each function. The frequency distribution of SDI values in the histograms (D-E) between both experimental conditions (Control and Nup153 siRNA) was analyzed using Chi-square test for trend with a p-value <0.001 (D) and p-value < 0.05 (E). (D) and (E) distribution between Control and Nup153 siRNA SDI values was significantly difference.     268  Appendix C – Supplementary results and figures from Chapter 5   Figure C-1. Phosphorylation or accumulation of myosin light chain might be related to changes in the actin cytoskeleton observed in Nup153 depleted cells. HeLa cells were transfected with control siRNA or Nup153 siRNA for 72 h and prepared for immunofluorescence microscopy. Immunofluorescence of cells labelled with antibodies against phospho-myosin light chain 2 (Ser18/Thr19; P-MLC2) (green), rhodamine-phalloidin to visualize F-actin (red), Nup153 antibody (white), and Dapi (blue) to observe the nucleus. Control siRNA and Nup153 siRNA cells were treated with the ROCK inhibitor Y-27632 (10 μM) for 1 h. Cells were prepared for immunofluorescence and re-examined for presence of blebs and actin stress fibers visualized through labelling of F-actin. Arrows in (left) point to condensed filamentous F-actin overlapping with PMLC2 fluorescence signal. Scale bar, 10 μm.   

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