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The Na/K ATPase pump, a signaling switch between life and death Biln, Perveen Kaur 2015

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The Na/K ATPase pump, a signaling switch between life and death by  Perveen Kaur Biln  BSc. Kinesiology, Simon Fraser University, 2001 MSc. Molecular Biology and Biochemistry, Simon Fraser University, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Cell and Developmental Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2015  © Perveen Kaur Biln, 2015   ii Abstract  This thesis is the first in-depth investigation into the roles of Na/K ATPase in assembly and maintenance of septate junctions (SJ) and tricellular junctions (TCJ). Together, these domains block the flow of fluids or pathogens across an epithelia. Loss of either domain (SJ or TCJ) leads to a loss of the permeability barriers. These domains contain large protein complexes that include both the alpha and beta subunits of Na/K ATPase of which the alpha subunit has the potential for scaffolding protein complexes and cell signaling. In cultured fibroblasts the alpha subunit binds and inhibits Src kinase and downstream signaling pathways, but the role in developing epithelia is unknown. Likewise how the Na/K ATPase interacts with other junctional components to establish, maintain and regulate SJs and TCJs is unknown.  We examined the consequences of loss of Na/K ATPase in the simple epithelium of the Drosophila wing imaginal disc to understand the role of the pump in mediating cell signaling and scaffolding of SJ and TCJ components. Using RNAi-mediated knockdown of either Na/K ATPase subunit, we observed an increase in activation of the tyrosine kinases, Src and Abl, leading to JNK-mediated apoptosis and apoptosis-induced proliferation. The role of Na/K ATPase in regulating Src activation is conserved between vertebrates and invertebrates and functions to regulate epithelia in vivo. An additional finding was that RNAi knockdown of Na/K ATPase in the imaginal wing disc led to changes in SJ and TCJ proteins that were distinct from knockdown of another core protein, NeurexinIV. In particular the Na/K ATPase led to a specific increase of the TCJ proteins Discs-large and Gliotactin at the TCJ. Overexpression of Gliotactin leads to JNK-mediated apoptosis and cell spreading and these were suppressed by loss of Na/K ATPase but enhanced by loss of NrxIV. This suggests Na/K ATPase has a unique role in both regulation and maintenance of the TCJ. Overall our data support a role for Na/K ATPase both as   iii a scaffolding protein that organizes a subset of SJ and TCJ proteins and as a signaling component in epithelial cell survival.    iv Preface Chapter 2: “Loss of the Na/K ATPase triggers apoptosis induced proliferation via JNK signaling”  Work in this chapter has produced a manuscript in preparation as: Biln, PK., Brown, K., and Auld, VA. Loss of the Na/K ATPase triggers apoptosis induced proliferation via JNK signaling.   For this publication I contributed to all aspects of the manuscript including study design, data collection, analysis and interpretation. Kristen Brown preformed the biochemical analysis and contributed to data collection and interpretation. Vanessa Auld contributed study conception and design, data interpretation, writing, and editing of the manuscript. Chapter 3: “Differential interactions between the core SJ domain proteins, NrxIV and Na/K ATPase, and the tricellular junction”  Work in this chapter has produced a manuscript in preparation as: Biln, PK., Brown, K., and Auld, VA. Differential interactions between the core SJ domain proteins, NrxIV and Na/K ATPase, and the tricellular junction.    For this publication I contributed to study design, data collection, analysis and interpretation. Kristen Brown contributed to the data collection and interpretation of the biochemical analysis. Vanessa Auld contributed to study conception and design, data interpretation and editing of this chapter.   v Table of Contents  Abstract.......................................................................................................................................... ii	  Preface........................................................................................................................................... iv	  Table of Contents ...........................................................................................................................v	  List of Tables ................................................................................................................................ ix	  List of Figures.................................................................................................................................x	  List of Abbreviations .................................................................................................................. xii	  Acknowledgments ...................................................................................................................... xiv	  Dedication .....................................................................................................................................xv	  Chapter 1: Introduction ................................................................................................................1	  1.1	   Structure and Function of Vertebrate Tight Junctions....................................................... 1	  1.2	   Structure and Function of Septate Junctions...................................................................... 5	  1.2.1	   Septate Junctions are Involved in Multiple Cellular Processes ................................ 12	  1.3	   Septate Junction Proteins: Core and Associated.............................................................. 14	  1.3.1	   Fasciclin3 (Fas3)....................................................................................................... 14	  1.3.2	   Disc Large (Dlg) ....................................................................................................... 15	  1.3.3	   Gliotactin (Gli).......................................................................................................... 17	  1.3.4	   Macroglobulin Complement Protein (Mcr) .............................................................. 19	  1.3.5	   Neurexin IV (NrxIV) and Coracle (Cora) in SJs ...................................................... 20	  1.3.5.1	   Coracle (Cora).................................................................................................... 21	  1.3.5.2	   Neurexin IV (NrxIV) in SJ ................................................................................ 22	  1.4	   The Na/K ATPase, an Ion Pump and Core SJ protein..................................................... 24	    vi 1.4.1	   Comparison of Vertebrate and Drosophila Na/K ATPase........................................ 25	  1.4.2	   Na/K ATPase as a Cell Adhesion Protein that Links to the Cytoskeleton ............... 28	  1.4.3	   Na/K ATPase as a Signaling Protein ........................................................................ 30	  1.4.4	   Src Kinase ................................................................................................................. 33	  1.4.5	   Src Kinase and Na/K ATPase in Drosophila............................................................ 37	  1.5	   Hypotheses....................................................................................................................... 37	  1.5.1	   Na/K ATPase as a Signaling Complex ..................................................................... 40	  1.5.2	   Na/K ATPase Associates with Dlg and Gli at the TCJ Subdomain ......................... 40	  Chapter 2: Loss of the Na/K ATPase Triggers Apoptosis and Apoptosis-induced Proliferation via JNK signaling ..................................................................................................41	  2.1	   Synopsis ........................................................................................................................... 41	  2.2	   Introduction...................................................................................................................... 42	  2.3	   Results.............................................................................................................................. 45	  2.3.1	   The Na/K ATPase is Important for Maintenance of SJs in the Imaginal Wing ....... 45	  2.3.2	   Loss of Na/K ATPase Leads to Cell Death and Delamination................................. 50	  2.3.3	   Increased Cell Proliferation with Loss of ATPα is Associated with an Increase in pSrc .................................................................................................................................. 53	  2.3.4	   Loss of ATPα Triggers JNK-Mediated Apoptosis................................................... 60	  2.4	   Discussion........................................................................................................................ 62	  2.4.1	   Na/K ATPase is Required for the Maintenance of SJs............................................. 62	  2.4.2	   Na/K ATPase is a Negative Regulator of Src........................................................... 64	  2.5	   Materials and Methods..................................................................................................... 67	  2.5.1	   Flystocks ................................................................................................................... 67	    vii 2.5.2	   Biochemistry............................................................................................................. 67	  2.5.3	   Immunolabeling ........................................................................................................ 68	  2.5.4	   Imaging ..................................................................................................................... 68	  2.5.5	   Statistical Analysis.................................................................................................... 69	  2.6	   Supplementary Data for Chapter 2 .................................................................................. 70	  Chapter 3: Differential Interactions Between the Core SJ domain Proteins, NrxIV and Na/K ATPase, and the Tricellular Junction..............................................................................73	  3.1	   Synopsis ........................................................................................................................... 73	  3.2	   Introduction...................................................................................................................... 74	  3.3	   Results.............................................................................................................................. 77	  3.3.1	   Na/K ATPase and NrxIV are Required for SJ Maintenance in the Imaginal Wing Disc .................................................................................................................................. 77	  3.3.2	   Na/K ATPase has a Unique Relationship With the TCJ Protein Gliotactin............. 85	  3.3.3	   Na/K ATPase Suppresses Migration of Apoptotic Cells in a Gli Overexpression Model .................................................................................................................................. 91	  3.4	   Discussion........................................................................................................................ 96	  3.4.1	   Na/K ATPase and NrxIV are Required for Correct Localization of Bicellular SJ Components. ......................................................................................................................... 97	  3.4.2	   Na/K ATPase Interacts with the TCJ proteins Gli and Dlg at the TCJ .................... 99	  3.4.3	   Na/K ATPase Regulates the Overexpression and Spread of Gli ............................ 101	  3.5	   Materials and Methods................................................................................................... 102	  3.5.1	   Flystocks ................................................................................................................. 102	  3.5.2	   Biochemistry........................................................................................................... 103	    viii 3.5.3	   Immunolabeling ...................................................................................................... 103	  3.5.4	   Imaging ................................................................................................................... 104	  3.5.5	   Statistical Analysis.................................................................................................. 104	  3.6	   Supplementary Data for Chapter 3 ................................................................................ 105	  Chapter 4: Conclusions and Discussion...................................................................................109	  4.1	   The Na/K ATPase and the Bicellular SJ........................................................................ 110	  4.1.1	   Na/K ATPase and NrxIV form Different Protein Complexes in the SJ ................. 111	  4.1.2	   Na/K ATPase and Dlg - Potential Cytoskeletal Interactions.................................. 114	  4.1.3	   Subdomain Interactions with ATPα and Nrv2 isoforms ........................................ 116	  4.2	   The Na/K ATPase as a Signaling Center....................................................................... 119	  4.3	   The Na/K ATPase and the Tricellular Junction Protein Gliotactin ............................... 123	  4.3.1	   Na/K ATPase and Gli at the TCJ............................................................................ 124	  4.3.2	   Na/K ATPase Can rescue Ectopic Gli in the Bicellular SJ..................................... 125	  4.4	   Conclusion ..................................................................................................................... 127	  Bibliography ...............................................................................................................................129	     ix List of Tables  Table 3.1: Transheterozygotes of GliDV3 with NrxIV4304 or ATPαDTSR3 display normal wing discs...................................................................................................................................................... 91	     x List of Figures  Figure 1.1: Structure and function of SJs versus TJs...................................................................... 6	  Figure 1.2: Morphology of the tricellular junction ......................................................................... 9	  Figure 1.3: Molecular organization of septate junctions .............................................................. 11	  Figure 1.4: Schematic representation of Na/K ATPase and its regulatory domains. ................... 26	  Figure 1.5: Na/K ATPase as a cell adhesion molecule................................................................. 29	  Figure 1.6: Schematic representation of the Na/K ATPase and its potential signaling partners. . 32	  Figure 1.7: Gal4/UAS expression system in the columnar epithelia. ........................................... 39	  Figure 2.1: ATPα is required for maintenance of SJs of the imaginal wing disc......................... 48	  Figure 2.2: RNAi knock down of Nrv2 phenocopies loss of ATPα and is required for maintenance of the imaginal wing disc SJs. ................................................................................. 50	  Figure 2.3: Loss of Na/K ATPase leads to cell death and compensatory proliferation................ 53	  Figure 2.4: Interactions of pSrc with the ATPα subunit .............................................................. 57	  Figure 2.5: Proximity ligation assay demonstrates a subset of ATPα and pSrc42A associate in vivo................................................................................................................................................ 59	  Figure 2.6: Low resolution (20X) images of 3rd instar wing discs ............................................... 61	  Figure 2.7: Three independent RNAi lines mediate the knockdown of ATPα ............................ 70	  Figure 2.8: Alignments of the second and third cytosolic domains of ATPα from a range of species........................................................................................................................................... 71	  Figure 2.9: Distribution of Src proteins and effect of ATPα on EGFR and FAK activation....... 72	    xi Figure 3.1: Expression of ATPα-RNAi or NrxIV-RNAi leads to mislocalization or loss of SJ proteins.......................................................................................................................................... 81	  Figure 3.2: RNAi knockdown of ATPα has differential effects on Dlg compared to RNAi knockdown of NrxIV. ................................................................................................................... 83	  Figure 3.3: RNAi knockdown of ATPα has differential effects on Gli compared to RNAi knockdown of NrxIV knockdown. ............................................................................................... 88	  Figure 3.4: RNAi knockdown of Gli increases the immunofluorescence of the SJ proteins ATPα, Nrv2 and NrxIV............................................................................................................................ 90	  Figure 3.5: Loss of function alleles of ATPα or nrv2 suppress the GliWT phenotype, while NrxIV loss of function enhance ............................................................................................................... 94	  Figure 3.6: Loss of function of Na/K ATPase reduces cell death in wing imaginal discs expressing ectopic Gli................................................................................................................... 95	  Figure 3.7: RNAi knockdown of ATPα has differential effects on Fas3 compared to RNAi knockdown of NrxIV knockdown. ............................................................................................. 105	  Figure 3.8: RNAi knockdown of Nrv2 phenocopies RNAi knockdown of ATPα. ................... 108	    xii List of Abbreviations α Subunit - ATPα AIP – Apoptosis Induced Proliferation AJ – Adherens Junction ap – apterous Abl – Abelson Kinase BSK - Basket Cas3 – Caspase3 CD – Cytosolic Domain Chk – Csk homologous Kinase CNS – Central Nervous System Cont – Contactin Cora – Coracle CTS – Cardiotonic Steroid Csk – C-terminal Src Kinase DAPI - 4',6-diamidino-2-phenylindole dCsk – Drosophila C-terminal Src Kinase Dlg - Discs Large dsRNA – double-stranded Ribonucleic Acid Ecad – E-cadherin EGFR – Epidermal Growth Factor Receptor ELM – Eukaryotic Linear Motifs FAK – Focal Adhesion Kinase FERM – Four.1, Ezrin, Radixin, Moesin FRAP – Fluorescence Recovery After Photobleaching GEF – Guanine nucleotide Exchange Factor GFP – Green Fluorescent Protein Gli – Gliotactin Grb2 - Growth Factor Receptor-bound Protein 2 GUK – Guanylate Kinase HRP – Horse Radish Peroxidase SJ – Septate Junction TJ – Tight Junction JAK/STAT - Janus Kinase/Signal Transducers and Activators of Transcription  JAM – Junctional Adhesion Molecule JNK – Jun kinase Lac – Lachesin MAGUK – Membrane Associated Guanylate Kinase MAPK - Mitogen-Activated Protein Kinase; MARVEL – MAL and Related proteins for Vesicle trafficking and membrane Linking Mcr – Macroglobulin complement-related Mega – Megatrachea MEK - MAPK–ERK Activating Kinase; MMP1 - Matrix MetalloProteinases Nrg – Neuroglian NrxIV – Neurexin IV Nrv2 – Nervana2 PDZ – PSD95, Dlg, ZO1 pHis3 – phospho-Histone3 PI3K - Phosphoinositide 3-kinase PKC – Protein Kinase 3 protein 2  PLA – Proximity Ligation Assay PLC - Phospholipase C PNS – Peripheral Nervous System PSF – Point Spread Function pSJ – pleated Septate Junction RNAi - Ribonucleic Acid interference   xiii ROS - Reactive Oxygen Species   SABD – Spectrin / Actin Binding Domain Scrib – Scribble SFK – Src Family Kinases Shc – Src homology and collagen protein SH2 – Src Homology domain 2 SH3 – Src Homology domain 3 Sinu- Sinuous SOS - Son of Sevenless Src - Proto-oncogene tyrosine-protein kinase  sSJ – smooth Septate Junction SsK – SnakeSkin  TAMP – TJ-associated Marvel domain Protein TCJ - Tricellular Junction TCP – Tricellular Plug  UAS – Upstream Activating Sequence UD – Unique Domain Vari – Varicose WT – WildType YFP- Yellow Fluorescent Protein ZO – Zonula Occludins           xiv Acknowledgments  I would like to thank my research supervisor Dr. Vanessa Auld for her guidance, mentorship and support in both science and life. I have grown as a person and as a scientist during my time in the Auld Lab. I would also like to thank my supervisory committee, Dr. Ninan Abraham, Dr. Calvin Roskelly, and Dr. Edwin Moore for the time and attention they devoted to my thesis and my growth as a scientist. I appreciate the encouragement to think about my data in different ways and their high scientific standards. A special thank you to Dr. Mary Gilbert for her council, support and encouragement in all aspects of life.   Research is not a solitary endeavour and although it is my name on this thesis, I had many people in the lab support me. This work could not be completed without them. Thank you to all Auld Lab Members, past and present for helping with flipping my flies, adding an antibody in a pinch, putting a vial in the cold room or entertaining my kids so I could get one last dissection done. I learned so much from our conversations about science, family and our lives.  Lastly and most importantly I would like to thank my husband Nick and my children, Bishan and Pritam for their love, support and encouragement.    xv Dedication         To my Nanaji, Pritam Singh Sekhon, your grand daughter is finally a Doctor. To my children, Bishan and Pritam, know that you can always follow your dreams    1 Chapter 1: Introduction  Permeability barriers that form between cells are essential to prevent fluid flow and/or pathogen invasion across tissues such as the intestine, brain, and other organs. These barriers are created by the adhesion of proteins that span the membrane and adhere to proteins or structures on neighbouring cells to create tight, impermeable junctions. Permeability barriers are conserved throughout all animals, they are created by tight junctions (TJs) in vertebrates and septate junctions (SJs) in insects. TJs and SJs have similar physiological roles as well as common components. Both types of junctions are composed of two distinct domains, the bicellular junction formed between two neighbouring cells and the tricellular junction (TCJ) created at the convergence of three cells. The TCJ blocks the flow of fluids and pathogens through the gap created by the meeting of three cells. Thus the TCJ is a continuous with the bicellular SJs. How bicellular SJs and TCJs are created and maintained to form intact SJs are not well understood. Therefore it is the overall goal of my research to investigate how bicellular SJ and TCJ proteins interact and to determine the mechanisms that underlie barrier formation during development. 1.1 Structure and Function of Vertebrate Tight Junctions  Vertebrate TJs form the adhesive contacts between cells and are critical to the formation and maintenance of epithelial and endothelial permeability barriers (Niessen, 2007; Shin et al., 2006). They were first identified in electron micrographs as adjacent cell membrane regions in extremely close proximity, called “kissing points,” and were later shown to consist of a continuous and branching network of protein strands that encircled the most apical part of the lateral membrane of cells like a belt (Figure 1.1). This architecture is critical to the three main functions of TJs: 1) a selectively permeable barrier with selectivity dependent on its molecular   2 composition 2) a molecular fence to restrict the mixing of apical and basolateral membrane components, 3) a signaling and transportation hub that both spatially confines signaling and polarity molecules and acts as a docking site for secretory vesicles. The known molecular components of TJs are similarly subdivided into categories: structural proteins and peripheral membrane proteins. Structural proteins are necessary for the initiation, formation and maintenance of TJs and the intercellular seal. Peripheral membrane proteins target all integral membrane proteins to the correct sub-compartment of the membrane, or bind to the cytoskeleton to form the fence or initiate and regulate signaling events (Miyoshi and Takai, 2008; Niessen, 2007; Shin et al., 2006).  There are three mains classes of transmembrane proteins in TJs: junctional adhesion molecules (JAMs), TJ-associated MARVEL domain proteins (TAMPs), and claudins. Claudins form the primary structure of TJs, while JAM and TAMP proteins regulate the assembly and organization of claudins into TJs. Claudin proteins bind to partners on adjacent cells to form a barrier across the intercellular space. Members of these three classes of proteins have been shown to interact with a variety of PDZ (PSD90/Dlg/ZO1) domain-containing peripheral membrane proteins, including the zonula occludins (ZO1, ZO2, ZO3) and the Crumbs polarity complex (Niessen, 2007; Sawada et al., 2003; Shin et al., 2006). These PDZ-containing proteins contribute to the formation of TJ strands, demarcate the apico- and baso-lateral membranes and link TJs to the cytoskeleton. JAMs, TAMPs, and claudins either have Drosophila homologues or have shared structural and functional properties with SJ proteins. Drosophila has three claudin family members and numerous SJ proteins with large extracellular Ig domains that could be considered functional homologues of JAMs. These molecular and functional similarities indicate   3 that studies of Drosophila SJ structure and function will have applicability to understanding of TJs as well.  The JAMs are a group of proteins important in regulating both the assembly and the permeability of TJs (Garrido-Urbani et al., 2014; Traweger, 2014). The JAM family of proteins can be separated into classical and non-classical subgroups. Classical JAMs have a short cytoplasmic tail and a type II PDZ motif. Non-classical JAMs have a longer cytoplasmic tail with a type I PDZ motif. These PDZ motifs are important in facilitating interactions with scaffolding proteins that can then modulate signaling molecules (Severson et al., 2009). For example JAM-A complexes with the two PDZ domain-containing molecules Afadin and guanine nucleotide exchange factor (GEF) 2, to mediate signaling events (Severson et al., 2009).  All claudin family members are single pass transmembrane proteins with large extracellular Ig domains that can form homophilic and heterophilic interactions in both cis and trans configurations. This dimerization assists JAMs in connecting different molecules together (Bazzoni et al., 2000; Traweger, 2014). JAMs are the first to localize to sites of cell-cell adhesion and may recruit TJ components, although they cannot induce formation of TJs when transfected into non-TJ containing cells. Support for this hypothesis is demonstrated by the ability of JAM A/B/C to directly interact with the cell polarity protein Par3 (Ebnet et al., 2001). Although there are no Drosophila JAM homologues, SJ protein complexes contain cell adhesion proteins with large extracellular Ig domains including Fasciclin3, Lachesin, Neuroglian, and Contactin (Dubreuil et al., 1996; Faivre-Sarrailh, 2004; Llimargas, 2004; Snow et al., 1989). All of these proteins are important in the assembly of SJs and are functionally similar to JAMs.  The second class of TJ-associated proteins, the TAMP family, has three members: the first identified TJ protein occludin (also called MarvelD1), MarvelD3 as well as the tricellular   4 junction protein Tricellulin (also called MarvelD2) (Furuse et al., 1993; Ikenouchi et al., 2005; Van Itallie and Anderson, 2014). TAMPs are characterized by the M-shaped topology formed by their four transmembrane regions and cytoplasmic N- and C- termini. Further, TAMPS contain the tetraspanin MARVEL (MAL and Related proteins for VEsicle trafficking and membrane Link) domain (Sánchez-Pulido et al., 2002). This domain appears to be important for establishing lipids rafts within the plasma membrane as well as having roles in trafficking. (Magal et al., 2009; Sánchez-Pulido et al., 2002; Yaffe et al., 2012). Interestingly, TAMP protein topology is shared with the claudin group of proteins and TAMPs interact with claudins to form TJs. However, unlike claudins, expression of any of the TAMPs in TJ free cells does not induce formation of TJs, though their loss does affect TJ function (Traweger, 2014). Drosophila has five MARVEL domain-containing proteins: two conceptual genes that have not been studied, one that has been studied in the context of innate immune response, one that functions in presynaptic vesicle docking and release (Flybase (St Pierre et al., 2014)), and one which functions in myoblast fusion (Estrada et al., 2007). Although none of the Drosophila MARVEL-containing proteins have been examined for a potential role in SJ formation and function, one or more of them may be a TAMP homolog required for SJ assembly and function.  The core structure of TJs is formed by the claudin family of proteins (Furuse and Tsukita, 2006). Although structurally similar to TAMPs, transfection of claudins into TJ-free cells induces de novo synthesis of TJ strands (Furuse et al., 1998). Like TAMPs, claudins have four transmembrane domains, two extracellular loops and intracellular N- and C – termini. The extracellular loops allow claudins to directly interact with TJ proteins on the opposing cell membrane. To date over 25 claudins have been identified with the greatest sequence diversity occurring within the C-terminus. In most claudins, the last three amino acids of the C-terminus   5 comprise a PDZ motif, which suggests the tissue specific functions of claudins arise from intracellular protein associations (Izumi and Furuse, 2014). Most claudins directly associate with ZO1, a multi-domain scaffolding protein, while others are also able to associate with ZO2 and ZO3 (Furuse and Tsukita, 2006; Günzel and Fromm, 2012; Traweger, 2014). Similar to vertebrate homologues, the Drosophila claudins Sinuous, Megatrachea and Kune Kune are necessary for correct organization of SJs. Loss of these proteins results in mislocalization of SJ proteins, incorrect assembly of SJ strands and a loss of the barrier function (Behr et al., 2003; Nelson et al., 2010; Wu et al., 2004). The shared structural and functional properties between vertebrate TJs and Drosophila SJs enable the use of Drosophila as a model organism to study cell junctions (Figure 1.1). The simpler genetics of Drosophila facilitates the analysis of the molecular mechanisms that underlie junction assembly and the consequences that occur when junction assembly is disrupted. 1.2 Structure and Function of Septate Junctions  Although SJs and TJs are functionally equivalent structures and share a number of components, they differ both in their morphology and their location within the cell. TJs are located above the AJs and are located where the cell membranes of two apposing cells appear to fuse, the kissing points of two cells. Freeze fracture electron microscopy images show TJs are a network of overlapping intramembranous strands that eliminate the paracellular space (Furuse and Tsukita, 2006). SJs are also in the apical domain of epithelial cells, but are located basal to the AJs. Additionally, electron microscopy has shown SJs strands have a more ordered appearance than TJs. In Freeze fracture images, SJs exhibit a 15 nm intracellular gap flanked by electron dense material at the plasma membranes that is crossed by a series of septae, or strands   6 of intramembranous material, giving SJs a ladder-like appearance (Figure 1.1, Figure 1.2) (Banerjee et al., 2006; Baumgartner et al., 1996; Fristrom, 1982; Tepass et al., 2001). These septa are present in the apical 1/3 to 2/3 of the lateral membrane and are typically organized into clusters. These multiple clusters of septae create an impermeable barrier (Tepass et al., 2001).   Figure 1.1: Structure and function of SJs versus TJs A) Permeability barriers form a tight seal between adjacent epithelial cells preventing fluid flow and/ or pathogen (yellow ovals) invasion. (B) Comparison of the morphology and cellular localization of the permeability barriers formed by invertebrate SJs and vertebrate TJs.   7  SJs have two distinct roles: 1) they function as barriers to the mixing of apical and basolateral membrane proteins throughout the plasma membrane, called the fence function, and 2) they seal neighbouring cells together so that water-soluble molecules and pathogens cannot leak between the cells, called the barrier function (Banerjee et al., 2011; Nelson and Beitel, 2009; Tepass et al., 2001). Beyond being a barrier, SJs, like TJs, are multifunctional platforms present in a variety of cells types and tissues, from imaginal discs and tracheal tubes to the peripheral and central nervous systems. In all these cell types, the core SJs proteins are present to form the occluding barrier but SJs also have cell specific roles. In the trachea, SJs control tube size and secretion of the matrix modifying proteins (Laprise et al., 2010; Paul et al., 2003; Wu et al., 2004); in glial cells they ensheath neurons and are involved in the establishment of the blood-nerve barrier (Auld et al., 1995); in imaginal wing discs they are important regulators of cell division (Charish, 2011); in imaginal eye discs they are required for ommatidial integrity and blood-eye barrier (Banerjee et al., 2008). The variety of functions requires a complex architecture of proteins to be involved in the regulation of  these tissue specific functions, as well as trafficking and modification of SJ proteins. How TJs and SJs are organized and formed, how TJ and SJ proteins are modified and trafficked is poorly understood.   Two types of SJs occur in Drosophila, smooth (sSJs) and pleated (pSJs), based on their morphology. Both pSJs and sSJs contain septae, which are morphologically distinct. pSJs are found in all ectodermaly derived epithelia and surround cells like threads on a screw with septae bridging the intermembrane space. sSJs are limited to the midgut and form parallel bands around the cell with septae fused together into ridges between cells (Yanagihashi et al., 2012). To date, no molecular similarity has been found between the two types of SJs suggesting they are fundamentally different structures (Banerjee et al., 2006). Up until recently, no known proteins   8 localized to sSJs. Using antibodies against silkworm sSJ membrane fraction, a protein complex made up of two proteins, SnakeSkin (Ssk) and Mesh, has been identified that localizes specifically to sSJs, and is required for development (Izumi et al., 2012; Yanagihashi et al., 2012). This project will focus on pSJs, the only SJ found in the imaginal disc. Additionally an increasing number of components and vertebrate homologs have been identified for pSJs rather than sSJs as no vertebrate homologs to Ssk and Mesh have been identified. Pleated septate junctions will be henceforward be referred to as SJs in this thesis.   SJs are electron dense, ladder-like structures with septae spanning the intercellular gap between two cell membranes. The septa have a spacing of 15-22 nm that connect adjacent cells and separate apical and basal intercellular spaces (Baumgartner et al., 1996; Fristrom, 1982; Noirot-Timothée et al., 1978; Tepass et al., 2001). The strands run circumferentially at the intersection of two cells, then turn abruptly basal at the intersection of three cells. These two subdomains of SJs are termed bicellular SJ and tri-cellular junctions (TCJ) respectively in this thesis (Figure 1.2). At TCJs, a stack of closely spaced diaphragms replace the septae to seal the resulting channel between the three cells. Of the numerous identified SJs components, only one protein, Gli, has been identified that specifically localizes to the TCJ.      9  Figure 1.2: Morphology of the tricellular junction A) TEM of the TCJ in Drosophila. Stage 17 embryos, pSJ are located in the apical 1/3 to 2/3 of the lateral membrane. Septa are present as clusters (brackets) with single or pairs of septa (arrows) sprinkled between them  B) At TCJ, septa are present at all bicellular junctions (arrowheads). Note septa join two adjacent cells until they reach a third cell. Scale Bar: 50 nm. (A-B: (Schulte et al., 2003) ©1979 Rockefeller University Press. Journal of Cell Biology. 161:991–1000. doi:10.1083/jcb.200303192. C) SJ strands (blue) surround epithelia (orange) like a belt connecting two cells together. SJ turn sharply basal at the intersection of three cells at the tri-cellular junction (TCJ). At the TCJ, a series of diaphragms (red) “plug” the channel. Reprinted with permission from (Browne, 2009).  Although SJs have been well characterized in terms of ultrastructure, the list of nearly 50 protein components remains incomplete. Genetic analysis has revealed some important components, including: varicose (vari), sinuous (sinu), megatrachea (mega), scribble (scrib), discs-large (dlg), Gliotactin (Gli), Lachesin (Lac), Contactin (Cont), Neurexin IV (NrxIV), coracle (cora), Neuroglian (Nrg), and both subunits of the Na/K ATPase (α subunit: ATPα (Atpα); (β subunit: nervana (nrv2)) (Faivre-Sarrailh, 2004; Genova and Fehon, 2003; Lamb et al., 1998; Llimargas, 2004; Moyer and Jacobs, 2008; Paul et al., 2003; Schulte et al., 2003; Woods and Bryant, 1991). These proteins can be sub-divided into either core components or SJ-associated proteins based on the phenotype observed in null mutant animals. The proteins NrxIV, C  10 Nrg, Cor, Cont, Vari, Nrv2 and ATPα are considered to be core SJ components. These core components form an interdependent complex as removal of any one of these proteins results in a reduction or absence of the others from the SJ (Figure 1.3)(Bachmann et al., 2008; Faivre-Sarrailh, 2004; Genova et al., 2000; Moyer and Jacobs, 2008; Woods and Bryant, 1991). Null mutations of core components result in a loss of both the barrier function and a loss of septae. The lack of proper SJ formation in null mutation results in embryos with numerous developmental defects including abnormal dorsal closure and head involution, overgrown trachea that do not inflate, and necrotic salivary glands (Baumgartner et al., 1996; Fehon et al., 1994; Genova and Fehon, 2003; Lamb et al., 1998; Paul et al., 2003). Numerous epidermal defects such as reduced number of denticles and thinner cuticles that are delaminated (Lamb et al., 1998). The other category, SJ-associated proteins, includes: Dlg, Scrib, Fas3, Gli, sinu, and mega. Null mutations of SJ-associated proteins result in a loss of barrier function only that is due to malformed septae. Importantly the septae are still present, but permeable. Both groups of proteins are embryonic lethal due to the compromised permeability barrier function, but mutants in associated components die at later stages of embryogenesis than core components (Tepass et al., 2001). These mutants also display abnormalities in epidermal organization and development including cuticles with holes, cell-cell contacts that either have gaps or complete separation as well as a reduction in peristaltic movements (Auld et al., 1995; Bilder and Perrimon, 2000; Schulte et al., 2003).      11  Figure 1.3: Molecular organization of septate junction proteins SJs are composed of a series of complexes between SJ core proteins, SJ-associated proteins and the actin cytoskeleton. The core SJ proteins are NrxIV, Cora, Nrg, Mcr, Vari, and Na/K ATPase subunits Nrv2 and ATPα. Nrv2, Nrg, NrxIV. Some of these proteins contain large extracellular domains that span the interstitial space to form either heterophilic or homophilic associations with binding partners on the opposing membrane. The large SJ core protein complex is likely composed of smaller sub-complexes. The subunits of the Na/K ATPase pump, Nrv2 and ATPα, are always found together. NrxIV and Cora are always associated. Vari and Cora are cytoplasmic proteins that directly bind to the cytoplasmic tail of NrxIV. NrxIV forms a complex that contains Nrv2 and ATPα but how these proteins associate is not known. NrxIV is likely to bind to Nrg on an opposing membrane. None of the SJ proteins comprising the core complex have been demonstrated to interact with the cytoskeleton. Vari or Dlg could provide a link to the cytoskeleton as MAGUK proteins with a HOOK domain. SJ-associated proteins, Fas3, Sinu, Mega, and Kune are required for SJ assembly and maturation but are not required formation of the intracellular septae. How these complexes interact with each other and the cytoskeleton remains to be discovered.    12 1.2.1 Septate Junctions are Involved in Multiple Cellular Processes  Similar to vertebrate TJs, SJs have roles in multiple cellular processes. In addition to the barrier function, SJs are required for controlling proper tracheal tube length during development, independent of their role in barrier formation (Behr et al., 2003; Nelson et al., 2010; Wu and Beitel, 2004; Wu et al., 2007). The mechanism through which this regulation occurs has yet to be determined. Additionally, SJ proteins including NrxIV, Cora, Nrv2, are required for adhesion of cardiac cells and cardiac performance (Yi et al., 2008). The Drosophila heart is a simple tube formed by a single layer of contractile cardiomyocytes and pericardial cells that align along each side of the heart wall. Neither cardiomyocytes or pericardial cell have SJs between them, though they do have spot AJs (Lehmacher et al., 2012). These SJ proteins are functioning independent of their canonical barrier function and are necessary for adhesion. This is strong evidence that the SJ proteins form tissue-specific, non-SJ complexes with distinct functions (Yi et al., 2008). Another example of SJ proteins participating in adhesive functions independent of the presence of SJs is the interaction between Nrx IV and a GPI-anchored Ig protein Wrapper in the CNS (Stork et al., 2009; Wheeler et al., 2009). A common feature of nervous systems is the separation of axons through ensheathment by glial cells, a process that requires an intricate series of glial-neuron interactions. Neuronal-expressed NrxIV acts as a heterophilic adhesion molecule by binding to glial expressed Wrapper to stabilize and attract glial processes (Stork et al., 2009; Wheeler et al., 2009). This interaction is independent of SJs as no other SJ proteins are recruited and requires NrxIV in a unique protein complex that includes Drosophila E-cadherin and Canoe, the Drosophila orthologue of AF-6 (Slovakova and Carmena, 2011). How these tissue-specific, non-SJ complexes are formed, localized or function is not clear. One possible avenue is through tissue specific isoforms of these proteins that form different protein complexes. An example of   13 this is found with the vertebrate Na/K ATPase, which has multiple α and β isoforms that assemble in a tissue-specific manner, enabling the pump to take on different functions (Lingrel, 2010). For example in cardiac cells, α1 is evenly distributed throughout the plasma membrane, while α2 is located close to the sarcoplasmic reticulum and the Na+/Ca2+ exchanger. This localization reflects α2’s ability to modulate contractility by altering Ca2+ flux through regulation of Na+ concentration which in turn modulates Na+/Ca2+ exchanger rates (James et al., 1999). Therefore SJ proteins can play multiple roles in different tissues. It is still unknown whether SJ proteins play multiple roles within the SJ itself. In this thesis, we will be asking if ATPα and Nrv2 have different roles in the wing imaginal disc in addition to the formation of septae and the physical permeability barrier.   A potential mechanism for controlling and regulating the various cellular roles of SJs is through the establishment of subcomplexes within the SJs. Recent work has raised the possibility of subcomplexes within the SJ domain. For instance co-immunoprecipitation experiments demonstrated that the β subunit of the Na/K ATPase, Nrv2, was able to pull down Cora and NrxIV but not Nrg suggesting that additional unidentified proteins may be part of this complex (Genova and Fehon, 2003). One of these may be Macroglobulin Complement-related protein (Mcr), a recently identified, novel SJ core protein that has a unique relationship with Nrg. Mutations in Nrg altered the distribution of Mcr from both the apical and SJ domains to primarily the apical domain. Mutations in Mcr led to a similar mislocalization of Nrg to the apical domain. In contrast, Cora is basolaterally mislocalized in either Mcr or Nrg mutants (Batz et al., 2014; Hall et al., 2014). Taken together these results support the hypothesis that additional proteins and/or complexes are involved in SJ organization and that distinct protein subcomplexes may exist within the SJ domain.   14  1.3 Septate Junction Proteins: Core and Associated  As outlined above, SJ proteins can be separated into two categories: core SJ proteins and SJ-associated proteins. In addition to Na/K ATPase, which will be discussed in section 1.4, this thesis will focus on a subset of these proteins, NrxIV, Mcr, Cora, Fas3, Dlg and Gli. We have chosen to focus on these proteins for a number of reasons. Firstly, during development, NrxIV and Cora are the first components recruited to the SJ domain and are essential to the genesis of SJs and for correct targeting of the other core components (Baumgartner et al., 1996; Fehon et al., 1994; Genova and Fehon, 2003). Mcr is known to form a distinct protein subcomplex (Batz et al., 2014; Hall et al., 2014). Fas3, Dlg and Gli are SJ-associated proteins required for the correct formation of the SJ barrier, and Dlg functionally associates with both Gli and Fas3. Additionally Dlg and Gli are part of the TCJ complex and co-regulate epithelial integrity (Padash-Barmchi et al., 2013; Schulte et al., 2003; Schulte et al., 2006). 1.3.1 Fasciclin3 (Fas3)  Fas3 is a glycoprotein belonging to the Ig super family (Snow et al., 1989). It is an integral membrane protein consisting of a short intracellular region, a single transmembrane domain and a large extracellular domain with three Ig folds (Castonguay et al., 1995; Snow et al., 1989). Fas3 was the first Drosophila molecule shown to mediate homophilic cell adhesion when transfected into cultured Drosophila S2 cells (Snow et al., 1989).  Fas3 has two distinct expression patterns within the ventral epidermis that occur at different embryonic development stages (Patel et al., 1987). Fas3 is transiently expressed on a subset of neuroepithelial cells in a segmentally repeated pattern (Patel et al., 1987). This   15 restricted expression, combined with its adhesion properties suggested that Fas3 may have a role as an axon recognition molecule (Patel et al., 1987; Snow et al., 1989). Through the formation of homophilic associations between motor neuron growth cones and muscles Fas3 is able to mediate synaptic target recognition (Chiba et al., 1995; Kose et al., 1997; Rose and Chiba, 1999). In a later stage of development, Fas3 is again expressed segmentally in the ectoderm, suggesting it may have a role in tissue morphogenesis (Patel et al., 1987). Evidence supporting this hypothesis is now beginning to emerge with the demonstration that Fas3 is required for correct hindgut curvature (Wells et al., 2013). Interestingly, the requirement of Fas3 for hindgut curvature occurs prior to the genesis of SJ strands, highlighting the multiplicity of functions played by individual SJ proteins. Whether Fas3 has different roles in the SJs and whether it associates into distinct protein subcomplexes has not been examined. We will be asking if Fas3 responds differently to the loss of Na/K ATPase versus the loss of NrxIV.  1.3.2  Disc Large (Dlg)  Dlg is a founding member of the membrane-associated guanylate kinase (MAGUK) superfamily of proteins. The modular organization of MAGUK proteins enables them to function as molecular hubs that localize proteins to specific subcellular domains and link signal transduction to the cytoskeleton. MAGUK proteins have a basic structure of one or more PDZ domains, a SH3 domain, and a non-catalytic GUK domain (Anderson, 1996; de Mendoza et al., 2010; Woods and Bryant, 1991). Some MAGUK proteins have additional protein interacting domains such as a HOOK domain, which is able to bind to the cytoskeleton. (de Mendoza et al., 2010; Hough et al., 1997; Oliva et al., 2011). PDZ domains are 80-100 amino acid long protein-binding elements that play a key role in localizing transmembrane and signaling proteins to   16 specific cellular subdomains (González-Mariscal et al., 2000). The SH3 domain has been less characterized than the PDZ domain, but it appears to be an important modulator of the GUK domain through intramolecular interactions (McGee and Bredt, 1999; Newman and Prehoda, 2009).The inactive-GUK domain has evolved into a protein-protein interaction domain, and in Drosophila, Dlg directly interacts with the protein GUK-holder via its GUK domain (Mathew et al., 2002). These multiple-protein interacting domains make MAGUK proteins a versatile platform for assembly of protein complexes.    The SJs require two known MAGUK proteins, Dlg and Varicose (Vari) for correct barrier formation (Bachmann et al., 2008; Moyer and Jacobs, 2008; Woods and Bryant, 1991; Wu et al., 2007). In addition to the core MAGUK structure, both Dlg and Vari have HOOK domains, which provide potential links between SJs and the cytoskeleton. The PDZ domain of Vari binds to the cytoplasmic domain of NrxIV, though if Vari also interacts with the cytoskeleton is unknown (Bachmann et al., 2008; Wu et al., 2007). Whether Dlg directly binds to any of the core components has not been tested but Dlg is in a complex with the TCJ protein Gli (Padash-Barmchi et al., 2013; Schulte et al., 2006).  Whether Dlg is affected when SJ protein mutations are present could be dependent on which tissue is being examined. For instance loss of ATPα from the trachea lead to a mislocalization of Dlg, however the effect of NrxIV mutants on Dlg in the trachea is unknown. Conversely loss of NrxIV or Cora in the salivary gland or the wing imaginal disc lead to a loss of Dlg but the effect of the Na/K ATPase mutants was not addressed.  (Genova and Fehon, 2003; Hijazi et al., 2011; Nelson et al., 2010; Paul et al., 2003; Ward et al., 1998) One of the questions this thesis will examine is whether the core SJ proteins NrxIV and Na/K ATPase interact with Dlg differently. We are focusing on Dlg for two reasons; its   17 demonstrated interaction with Gli at the TCJ and to address which core complex member holds Dlg at the SJ.  This thesis will be the first direct comparison of loss of NrxIV and the Na/K ATPase on Dlg within the same tissue, specifically the columnar epithelia of the wing imaginal disc.  1.3.3 Gliotactin (Gli)  SJs are commonly described as ladder-like structures that seal the intracellular space between two cells. However, in epithelial sheets composed of mostly hexagonal cells, septae formed between two cells and at the meeting of three cells, the TCJ, cannot form a barrier. To seal the intracellular space at the TCJ, a unique molecular structure is formed by the bicellular SJ strands of all three cells turning 90° to run basally along the lateral membrane and are anchored by a series of occluding diaphragms or tricellular plugs (TCP) (Fristrom, 1982; Lane and Swales, 1982; Schulte et al., 2003). How this unique organization of the TCJ, with TCPs anchoring SJ strands, occurs and how this specialized domain within SJs is connected to the bicellular SJ is unknown (Ikenouchi et al., 2005; Schulte et al., 2003). Understanding how TCJs and bicellular SJs interact with each other will provide insights into understanding how other potential subdomains within SJs interact with each other.    In Drosophila, Gli is the only protein shown to localize specifically to the TCJ in mature epithelial. During development Gli localizes to the ectoderm shortly after NrxIV localization and like NrxIV, is evenly distributed in early embryogenesis. However, during SJs genesis NrxIV is redistributed to the apical half of the lateral membrane, while Gli becomes restricted specifically to the TCJ. This unique localization of Gli suggests it has a role in SJs that is different from other SJ proteins. At the bicellular junctions of Gli null embryos, septae are correctly formed but they   18 do not form a uniformly spaced series, instead they are present as single or pairs of septae, resulting in an immature morphology and a loss of barrier function (Auld et al., 1995; Schulte et al., 2003). At the TCJ, the tricellular plugs are thought to anchor the septae at the bicellular junctions immediately bordering it (Fristrom, 1982; Lane and Swales, 1982). In Gli-/- embryos these plugs are absent leaving enlarged gaps at the TCJ, and the bicellular SJ closest to the TCJ are missing septae (Schulte et al., 2003). This demonstrates that Gli is required for maturation of both the TCJs and the bicellular SJs and suggests Gli plays a critical role in connecting the TCJ and bicellular SJ proteins complexes.   The molecular structure of Gli contains several domains that would enable it to form a bridge between the TCP and bicellular SJs (Auld et al., 1995; Schulte et al., 2006). Gli is a transmembrane cholinesterase-like protein with a large extracellular domain and a smaller intracellular tail (Auld et al., 1995). The extracellular domain contains a highly conserved non-enzymatic serine esterase-like domain that is able to form functional oligomers and could potentially bind to the TCP (Gilbert et al., 2001; Schulte et al., 2003; Schulte et al., 2006). The intracellular region contains a PDZ binding motif that could recognize the PDZ domain of a TCJ specific- or SJ-protein. At the TCJ, Gli is part of a larger complex with Dlg (Padash-Barmchi et al., 2013; Schulte et al., 2006). In addition, the PDZ motif, the intracellular domain of Gli has two conserved phospho-tyrosine sites (Auld et al., 1995; Schulte et al., 2006). These phospho-tyrosine sites are critical to ensure that Gli is restricted to the TCJ (Padash-Barmchi et al., 2010). Restriction of Gli protein to the TCJ is critical to epithelial cell survival. Over-expression of Gli results in Gli spreading around the entire cell membrane away from the TCJ and leads to ectopic folds, delamination, and apoptosis. Gliotactin levels are controlled by endocytosis and phosphorylation of the two conserved tyrosines Y760 and Y799. Blocking endocytosis or   19 blocking phosphorylation with Phe substitutions, Y760F and Y799F (GliFF), prevents Gli endocytosis and greatly enhances the deleterious effects seen with ectopic Gli (Padash-Barmchi et al., 2010). These results suggest that phosphorylation of Gli targets it for degradation as a mechanism to control protein levels and cell survival. The kinase responsible for Gli phosphorylation is unknown, although previous bioinformatics approaches have suggested Src as a candidate kinase and Src kinase is able to phosphorylate Gli in vitro (Padash-Barmchi et al., 2010).   In addition to being the only SJ protein that is localized specifically to the TCJ, Gli is associated with Dlg in a complex at the TCJ (Schulte et al., 2003; Schulte et al., 2006). Interestingly, a direct association between Gli and Dlg has not been demonstrated despite genetic analysis demonstrating they are co-regulated to maintain epithelial integrity (Padash-Barmchi et al., 2013). These data suggests that these proteins are part of a larger complex including unidentified components. Which components of the bicellular SJ interact with Gli at the TCJ to form a complete SJ are unknown. Gli does not associate with NrxIV nor does it associate with coracle (Schulte et al., 2006). However the potential interaction between Na/K ATPase and Gli to link the bicellular SJ to the TCJ has not been tested. In this thesis we test if Na/K ATPase is the key link between the bicellular SJs and the TCJ. 1.3.4 Macroglobulin Complement Protein (Mcr)   Mcr is the most recently identified member of the SJ family of proteins (Batz et al., 2014; Hall et al., 2014). Mcr is one of five members of the conserved thioester protein family (TEP) in Drosophila and includes many vertebrate complement factors important in innate immunity (Batz et al., 2014; Nonaka, 2004). All five Drosophila TEPs, including Mcr are expressed as   20 secreted proteins in hemocytes and are involved in preventing pathogen invasion (Bou Aoun et al., 2011; Lagueux et al., 2000; Stroschein-Stevenson et al., 2006). Mcr is unique among the five TEPs as it has a mutated thioester motif and an additional predicted transmembrane domain (Batz et al., 2014). Null mutations of Mcr results in a failure of the permeability barrier, embryonic lethality and a complete absence of septa within epithelial tissues demonstrating that Mcr is required for SJs organization and paracellular barrier function, placing it with the core SJ proteins (Batz et al., 2014; Hall et al., 2014).   Mcr is unique among SJ proteins in that it has two distinct apical localizations (Hall et al., 2014). In addition to its localization at the SJ domain, Mcr is also present in the apical plasma membrane above the AJs where it colocalizes with the apical marker uninflatable (Hall et al., 2014; Zhang and Ward, 2009). This suggests it may have significant non-junctional roles, although this remains to be tested. As discussed above, loss of Nrg has a different effect on Mcr compared to a loss of NrxIV. This is the first piece of evidence suggesting that protein subcomplexes that have distinct interactions may be present within SJs. Whether loss of Na/K ATPase also leads to a similar redistribution of Mcr has not been tested. In this thesis, we will be asking if loss of NrxIV or loss Na/K ATPase have similar effects on Mcr.  1.3.5 Neurexin IV (NrxIV) and Coracle (Cora) in SJs  NrxIV and Cora were the first components identified that participate in the formation of SJs and are the first proteins to localize to the SJ domain in developing embryos. Genetic and biological data suggest these proteins are tightly interdependent. Null mutations of these proteins phenocopy each other, they are interdependent for correct localization to the SJ domain and binding assays have demonstrated the cytoplasmic tail of NrxIV is sufficient and necessary for   21 binding of Cora (Baumgartner et al., 1996; Genova and Fehon, 2003; Paul et al., 2003; Ward et al., 1998). We have chosen to focus on NrxIV / Cora in this thesis because of the absolute requirement of NrxIV and Cora for SJs, along with the demonstration that Na/K ATPase participates in a complex with these two proteins but does not require Cora to correctly target to the SJ domain (Genova and Fehon, 2003).    1.3.5.1 Coracle (Cora)  Cora was the first core SJ to be identified and belongs to the Protein Band 4.1 superfamily (Fehon et al., 1994; Ward et al., 1998). This family includes a diverse group of proteins which link transmembrane proteins to the cytoskeleton as well as being involved in numerous cell functions from membrane stabilization to tumor suppression and signal transduction (Sun et al., 2002). Two conserved functional domains; one in the N-terminus and one in the C-terminus characterize membership in this superfamily. The N-terminal domain is the conserved FERM binding domain (Four.1, Ezrin, Radixin, Moesin) that binds to the cytoplasmic tail of a variety of transmembrane proteins. The C-terminal domain is important for interactions with the cytoskeleton and in many cases is a spectrin/actin (SABD) binding domain (Lamb et al., 1998; Sun and Salvaterra, 1995b; Ward et al., 2001).   Cora is one of many Drosophila homologues for the mammalian erythrocyte protein band 4.1 and shares 60% amino acid identity in the N-terminus FERM domain. It is through the FERM domain that Cora interacts with NrxIV. This domain is absolutely necessary for correct localization of both NrxIV and coracle to the SJ domain (Fehon et al., 1994; Ward et al., 1998). Ultrastructural and functional analyses of Cora null mutants have demonstrated defects in various developmental processes including embryonic dorsal closure, tracheal inflation and cell proliferation. Loss of Cora in epithelial cells results in a loss of septa and permeability barrier   22 function (Lamb et al., 1998; Ward et al., 1998). The expression of Coras’s FERM domain by itself can rescue mutant animals past embryogenesis. However, these animals did not survive past the larval stage suggesting one or more domains in addition to the FERM is necessary for complete rescue (Ward et al., 2001). Interestingly, the C-terminus of Cora, which has 30% homology with vertebrate Protein 4.1, does not have a consensus SABD domain suggesting that in Drosophila, Cora may not link NrxIV to the cytoskeleton but to other proteins that either scaffold the NrxIV/ Cora complex or restrict activation of another protein in the complex.   1.3.5.2 Neurexin IV (NrxIV) in SJ  The Neurexin family of proteins were originally identified as a synapse specific receptor for the black widow spider venom and has been subdivided based on their domain structure (Baumgartner et al., 1996; Bellen et al., 1998). All neurexin genes encode proteins with large extracellular regions that contain EGF and Laminin G repeats for forming protein-protein interactions. Two neurexin proteins, Drosophila NrxIV and the vertebrate Casp/Paranodin are subdivided into their own group because their extracellular domains contain a discoidin domain instead of a Laminin G repeat (Bellen et al., 1998). Drosophila NrxIV is a type I transmembrane protein. Its large extracellular region contains a signal peptide, a discoidin domain, a neurexin motif (laminin G domain- epidermal growth factor (EGF) domain-laminin G domain) repeat, the transmembrane domain and a short cytoplasmic tail, which has a conserved band 4.1 binding domain and a PDZ binding motif (Bachmann et al., 2008; Baumgartner et al., 1996; Bellen et al., 1998). The band 4.1 binding domain anchors NrxIV to Cora, while the PDZ motif binds to the MAGUK protein Vari (Bachmann et al., 2008). NrxIV and Cora are absolutely required for correct localization of each other. However while NrxIV is able to recruit Vari to the SJ domain (Baumgartner et al., 1996; Bellen et al., 1998), Vari is not sufficient for NrxIV recruitment   23 (Bachmann et al., 2008). This further supports the founding role of NrxIV and Cora in SJ genesis.    NrxIV is localized to all cells types that contain SJs, including ectodermally derived epithelial cells such as (but not limited to) the tracheal system, salivary glands and imaginal discs. In addition, NrxIV is found in glia and neurons in the peripheral and central nervous systems (PNS, CNS). Functional analysis of NrxIV null animals revealed a requirement of NrxIV for correct axonal insulation, blood-nerve barrier formation, defects in dorsal closure, and loss of septae of SJs (Baumgartner et al., 1996). Interestingly, NrxIV expression is detected immediately prior to the morphological appearance of SJs and it is required for correct localization of core SJ proteins and some SJ-associated proteins (Genova and Fehon, 2003; Laprise et al., 2009; Ward et al., 1998; Wu et al., 2007). This suggests NrxIV has a primary role in the establishment of these SJs. Although NrxIV and Cora are the first to localize along the lateral membrane and form a protein complex, how this complex functions as a single unit to recruit and establish SJs is not well understood. Unlike its vertebrate homologs, Cora does not have an actin binding domain to link the SJ complex to the cytoskeleton nor have Cora or NrxIV been demonstrated to complex with SJ-associated proteins such as Fas3 or Dlg.  Overall the ability to separate SJ proteins into core and SJ-associated proteins suggest that SJs are composed of a series of protein complexes or subcomplexes that form distinct interactions. These subcomplex interactions would be important in regulating the various cellular functions of SJs. Of the known SJ components, Na/K ATPase is the only component composed of two individually regulated subunits that have known cell adhesion properties, interactions with scaffolding proteins and interactions with the cytoskeleton (Vagin et al., 2012). Thus Na/K ATPase is a protein that could link two different protein complexes through interactions with its   24 alpha and beta subunits. The focus of this thesis is on how the Na/K ATPase may mediate SJ proteins interactions.   1.4 The Na/K ATPase, an Ion Pump and Core SJ protein   The core SJ protein that is the focus of this thesis is the Na/K ATPase, which has both ion transport and scaffolding functions. While much is known about the ion transport function of the Na/K ATPase, much less is known about how the Na/K ATPase functions as a scaffolding protein and as a signaling protein. The aim of this thesis was to address these two latter functions and their role in SJ formation.   The Na/K ATPase is a well-characterized ion transporter that hydrolyzes ATP to transport Na+ and K+ to establish and maintain their gradients across the cell membrane in both vertebrates and invertebrates. These ion gradients are important in maintaining a resting membrane potential, in driving solutes such as vitamins and amino acids into the cell and in driving co-transporters and exchangers (Jorgensen et al., 2003; Lingrel, 2010). In addition to its role in homeostasis, the Na/K ATPase has been identified as both an adhesion and signal modulating protein. Na/K ATPase is a core SJ protein involved in the formation of the septae as well as an important regulator of tracheal tube size (Genova and Fehon, 2003; Paul et al., 2003). Although its role in tube size regulation and septate junction formation has been established, its role in the maintenance of SJs and its role in SJ functions beyond tracheal tubulogenesis is largely unknown. The function of Na/K ATPase at the SJs in established columnar epithelial has not been examined. The focus of my thesis was to examine the role of Na/K ATPase in the imaginal wing disc, a simple tissue comprised of two epithelial sheets, a squamous and columnar epithelia (Weaver and Krasnow, 2008).    25  1.4.1 Comparison of Vertebrate and Drosophila Na/K ATPase   The minimal functional unit of Na/K ATPase is composed of two subunits, the large α subunit (~1000 amino acids, 110 kDa) that forms the pore and the smaller 370 amino acid β subunit (Figure 1.4) (Kaplan, 2002). A third subunit, which is important in modulating Na/K ATPase activity but is not required for channel formation or function, is the FXYD group of proteins (Geering, 2005; Geering, 2006). These small proteins bind to the alpha subunit and alter its affinity for the cations Na and K, thus changing the efficacy of ion transport and the intracellular concentrations of Na and K (Geering, 2006). As Na/K ATPase transport is dependent on the intracellular concentrations of Na, FXYD proteins alter the kinetics of Na/K ATPase transport via the alteration of the binding affinity for these cations. FXYD proteins are seven structurally similar proteins expressed in a cell and tissue-specific manner and are only found only in vertebrates (Geering, 2006).   The α subunit is composed of 10 transmembrane helices connected with five extracellular loops and 3 intracellular loops. It has 6 cytoplasmic domains that contain the ATP binding domain, the phosphorylation domains, the cation binding site as well as the Src, ankryin, and acetylated tubulin binding sites (Figure 1.4) (Nelson and Veshnock, 1987; Tian et al., 2006). Vertebrates have four different isoforms of the α subunit, each encoded by a different gene, with specific tissue distributions. α1 is present in all tissues including epithelia cells, α2 is seen in muscle cells, the brain, lung and adipocytes; α3 in neurons, ovaries, white blood cells and the developing rat and human heart, while α4 is exclusively in the sperm (Kaplan, 2002; Lingrel and Kuntzweiler, 1994). Despite tissue specific distributions, there are only minor sequence   26 differences between the four isoforms, suggesting that their dimerization with specific β subunits confers tissue specific functions. The β subunit ectodomain interacts most strongly with the α subunit at the extracellular loop connecting transmembrane domains 7/8, though they appear to interact in other regions as well (Jorgensen et al., 2003; Kaplan, 2002).  Figure 1.4: Schematic representation of Na/K ATPase and its regulatory domains. In Drosophila, the Na/K ATPase is made up of two subunits the ATPα or α subunit and Nrv or β subunit. The main core is formed by the α subunit, which has conserved binding sites for Src on the second and third cytosolic domains (blue bars), Ankryin (purple bar), ouabain (orange) and acetylated tubulin (red).  The β subunit is a small type II transmembrane protein with a molecular weight of 55 kDa that is required for a functional pump, even though the catalytic activity of the enzyme is in the α subunit. It is obligatory for normal pump maturation, trafficking and stabilization (Lingrel and Kuntzweiler, 1994). The β subunit has a single transmembrane region with the majority of   27 the protein existing in the extracellular space, ~300 amino acids form the ectodomain and ~30 amino acids the intracellular domain. Vertebrates have 3 different β isoforms (β1, β2 and β3) that can dimerize with any of the α subunits. Similar to α1, β1 is ubiquitously expressed while β2 appears to be mainly neuronal and β3 is unique to Xenopus oocytes. All three isoforms have disulfide bridges and consensus N-glycosylation sites in their extracellular regions. It has been suggested that the difference in the number of N-glycosylation sites may regulate the localization of the β subunit and consequently Na/K ATPase (Kaplan, 2002; Vagin et al., 2007).   Both subunits of Na/K ATPase have a high level of homology throughout the animal kingdom, with most species having multiple copies of each (Sun et al., 1998). In Drosophila melanogaster, there are two α subunits encoded by two different genes, ATPα and JYα. ATPα is ubiquitously expressed, sharing a high degree of homology with the vertebrate α1. JYα is 60% identical to the mouse α4 and, similar to vertebrates, its expression is restricted to sperm and is necessary for sperm motility (Masly et al., 2006). The β subunit is encoded by three genes each of which can be alternatively spliced: Nervana1 (Nrv1) Nervana 2 (Nrv2) and Nervana3 (Nrv3). All three proteins share a similar structure, but differ in their amino acid sequence, in their cellular distribution and in their function in SJs. Nrv2 is expressed in all ectodermally derived epithelial tissue, is the only Nrv protein specifically localized to the SJ domain and is required for both SJ and tube-size functionality in the trachea (Genova and Fehon, 2003; Paul et al., 2003). Nrv1 is expressed in most of the same tissues as Nrv2, with the exception of the hindgut and is excluded from the SJ domain even when overexpressed (Paul et al., 2007). In contrast to Nrv2, Nrv1 is also expressed in endodermally derived tissue and localizes to the basolateral domain (below the septate junctions). Nrv3 is expressed in the nervous system and is excluded from epithelial tissues (Paul et al., 2003; Paul et al., 2007).    28 1.4.2 Na/K ATPase as a Cell Adhesion Protein that Links to the Cytoskeleton  In addition to its role as a transporter, Na/K ATPase has been shown to function as a scaffolding protein and a ligand receptor participating in different signaling cascades (Arce et al., 2008; Haas et al., 2002; Tian et al., 2006; Xie and Cai, 2003). Interestingly, Na/K ATPase has been shown to play a critical role in junction formation in both invertebrates and vertebrates (Genova and Fehon, 2003; Madan et al., 2007; Paul et al., 2003; Violette et al., 2006). In vertebrate epithelial cells, the Na/K ATPase is localized along the lateral membrane where it colocalizes with both AJ and TJ proteins and inhibition of Na/K ATPase leads to alterations in TJ permeability and localization of TJs proteins (Contreras et al., 1999; Rajasekaran et al., 2006; Rajasekaran et al., 2003; Rajasekaran et al., 2001a) (Figure 1.5). The ability of Na/K ATPase to act as a cell adhesion molecule is facilitated by its multi-subunit composition. The beta subunit is capable of forming homophilic cell-cell associations as a heterodimer with the Na/K ATPase alpha subunit (Lemas et al., 1994; Lingrel and Kuntzweiler, 1994) and numerous studies have confirmed that the beta subunit acts as an adhesion molecule. Transfection of the canine β1 increases cell aggregation (Shoshani et al., 2005) and expression of β1 induces TJ formation in MDCK cells (Rajasekaran et al., 2001a). The beta subunit acts as a cell adhesion protein through the large extracellular domain (Lemas et al., 1994; Lingrel and Kuntzweiler, 1994) and requires N-glycosylation for junction integrity and stability (Vagin et al., 2009). The alpha subunit has a role in cell adhesion as well, specifically TJ formation. Inhibition of Na/K ATPase with toxic levels of ouabain (> 200 nM) leads to a loss of stress fibers, discontinuous staining of the scaffolding protein ZO1 and occludin, and increased barrier permeability (Rajasekaran and Rajasekaran, 2003; Rajasekaran et al., 2006; Violette et al., 2006). In contrast when cells are treated with physiological levels of ouabain (10-100 nM) such that Na/K ATPase transport   29 activity is unaffected, TJ seals are tightened and TJ proteins are recruited via Src and ERK 1/2 pathways (Larre et al., 2010). These data suggest that vertebrate Na/K ATPase complexes with ZO1 to recruit and bind other TJs components. The potential interaction of ZO1 and Na/K ATPase is of particular interest to us. ZO1 is a member of the membrane-associated guanylate kinase family (MAGUK), membership that is shared with the SJ-associated protein Dlg. ZO1 is required for the clustering of TJ claudins and provides a direct link between TJs and the actin cytoskeleton (Niessen, 2007). As Dlg is localized to the SJ, this suggests that it may associate with the Na/K ATPase and provide a direct link between SJ proteins and the actin cytoskeleton.    Figure 1.5: Na/K ATPase as a cell adhesion molecule. In vertebrates Na/K ATPase localizes to the TJs and the basolateral membrane where it generates ion gradients through active transport of Na and K. At the TJ, Na/K ATPase does not transport ions rather it is acting as cell adhesion molecule. The beta subunit mediates homophilic binding at these cell-cell junctions. TJ integrity is dependent on beta subunit dimerization, which requires N-glycosylation. Reprinted with permission from (Vagin et al., 2012).    30  Na/K ATPase is a part of the core complex of SJ proteins required for the formation of septae. We propose that Na/K ATPase may be the linker or organizing centre that mediates cell-cell adhesion and links to the underlying cytoskeleton. The alpha subunit has been shown to act as a scaffolding protein as it can bind to ankryin-fodrin and acetylated tubulin to link junction components to the cytoskeleton (Jordan et al., 1995; Zampar et al., 2009; Zhang et al., 1998). Ankryin is a universal adaptor protein that mediates linkage of membrane proteins to the cytoskeleton and ATPα has two conserved ankryin binding sequences in its cytoplasmic loops (Jordan et al., 1995; Paul et al., 2007; Zhang et al., 1998). Mammalian ATPα binds acetylated tubulin and microtubules through its fifth cytoplasmic domain, an interaction that inhibits pump enzymatic activity (Casale et al., 2005; Zampar et al., 2009). Together, these associations make Na/K ATPase a favorable candidate to link the SJ to the cytoskeleton.  Whether Drosophila Na/K ATPase is capable of linking SJs to the cytoskeleton is unknown. In this thesis we will be examining how Na/K ATPase interacts with SJ components and asking if it could be a scaffold on which SJ are built.  1.4.3 Na/K ATPase as a Signaling Protein  Beyond a role in ion transport and cell adhesion, Na/K ATPase also functions as a signaling protein. The cardiotonic steroids (CTS) or ouabain-binding site on the α subunit of Na/K ATPase is well conserved throughout evolution. Binding of CTS cause an increase in contractility and vasoconstriction of the heart and vasculature as well as up-regulation of certain genes (Lingrel, 2010). It has been established that these effects are actually due to activation and amplification of Src, a non-receptor tyrosine kinase, via Na/K ATPase. Studies using cell lines or myocyte cultures have demonstrated that a population of non-transporting Na/K ATPase is   31 present in caveolae and is associated with Src (Liang et al., 2007; Liu et al., 2003). In vitro binding assays have demonstrated that Srcs’ SH2 and kinase domains bind to the second and third cytosolic domains (CD2, CD3) of the alpha subunit, respectively maintaining Src in an inactive state (Lai et al., 2013; Tian et al., 2006). Upon binding of ouabain or another CTS, a conformational change occurs in this complex, which results in release of the Src kinase domain to increase Src kinase activity and translocation to downstream effectors including caveolin1, phosphoinositide3 kinase (PI3K) and epidermal growth factor (EGFR) (Figure 1.6). The association of Src with its effectors (i.e. EGFR) results in tyrosine phosphorylation of the effector, triggering a cascade of events that end with increased cell growth and proliferation (Li and Xie, 2009). In a similar vein, loss of the Na/K ATPase pump through RNAi knockdown leads to a global increase in Src activity (Liang et al., 2006).     32  Figure 1.6: Schematic representation of the Na/K ATPase and its potential signaling partners.  In vertebrates, a non-ion transporting subpopulation of Na/K ATPase is in complex with Src that is localized within caveolae, along with other signaling molecules. Binding of CTS such ouabain leads to a conformational change in Na/K ATPase that results in the release of activated Src. The released Src is than able to target multiple signal pathways. These molecules which are potential targets of Src include EGFR, epithelial growth factor receptor; PKC, protein kinase C; PI3K, phosphoinositide 3’ kinase; Grb2, growth factor receptor-bound protein 2; Sos, son of sevenless; PLC, phospholipase C; MAPK, mitogen-activated protein kinase; MEK, MAPK–ERK activating kinase; ROS, reactive oxygen species; Shc, src homology. Reprinted with permission from (Xie and Cai, 2003).  However, the in vivo role of the Na/K ATPase-Src complex has not been determined in any animal nor has the conservation of this signaling complex across different species been   33 tested. Understanding the in vivo function of a potential Na/K ATPase-Src complex is important as controlling phosphorylation through the actions of the Na/K ATPase has a range of potential impacts during epithelial development. Signaling through a Na/K ATPase signaling complex may control cascades for cell growth and proliferation or cell survival. Conversely signaling through a Na/K ATPase - Src complex may regulate the function of different SJ components which then regulate the location and protein levels of both core and associated SJ proteins. We asked if the Na/K ATPase / Src complex is present in Drosophila and what are the cellular consequences of Src activation via loss of Na/K ATPase.  1.4.4 Src Kinase  A key component of the Na/K ATPase signaling complex from cultured mammalian cells is the non-receptor tyrosine kinase Src. Src is important in a number of pathways including cell proliferation, cell motility, and cell adhesion (Bradshaw, 2010; Ingley, 2008; Roskoski, 2004). In vertebrates, the Src family of kinases (SFK) consists of 8 proteins expressed in all cell types, highlighting their importance in growth and development (Ingley, 2008). SFK are characterized by their conserved domain structure, which consists of a N-terminus localization motif or SH4 domain, a Unique domain (UD), a SH2 and SH3 domains and the catalytic SH1 domain (Songyang and Cantley, 1995; Tatosyan and Mizenina, 2000). As Src is a cytoplasmic protein, the N-terminal SH4 localization domain is important in targeting SFK to specific cellular compartments (Ingley, 2008; Songyang and Cantley, 1995). The UD links the SH2 and SH3 domains and was originally thought to be a spacer because of the absence of sequence similarity between SFK family members. However, the UD is well conserved within specific SFK members, and contains phosphorylation sites and lipid binding domains (Amata et al., 2014). For   34 example the UD of the SFK member, Lck is conserved between organisms and phosphorylation of Ser59 in this domain regulates the specificity of Lck’s SH2 domain. Thus the UD provides another level of controlling SFK localization and activity. Of all the SFK domains, the SH2 and SH3 domains have been extensively studied and both are important in SFK function. Together, these domains constrain the enzyme through intramolecular interactions, bind to adaptor proteins that contain SH2 and / SH3 motifs, and increase the activation of Src through displacement of their intramolecular binding (Ingley, 2008; Roskoski, 2004). Src is activated by phosphorylation of Tyr416 in the kinase domain and this domain is responsible for tyrosine kinase activation. Activation of Src can be modulated by a wide variety of pathways including receptor protein-tyrosine kinases such as EGFR, integrin receptors, and steroid hormone receptors such as the Na/K ATPase (Roskoski, 2004).   The participation of SFK’s in almost every cellular pathway requires precise control of Src activation levels which is made more challenging because Src activation is not simply on or off, but a step-wise activation. Src is kept fully inactive by binding of the SH2 domain to the phosphorylated regulatory tyrosine (Y527) on the C-terminal tail, binding of the SH3 domain to the kinase domain, and dephosphorylation of (Y416). Src can be partially activated by disrupting the SH2/SH3 binding or become fully activated by phosphorylation of Y416, even if Y527 is phosphorylated (Ingley, 2008; Roskoski, 2004). An additional level of complexity is that phosphorylation of Y416 requires trans-autophosphorylation, which occurs by “unlocking” of the inactivated kinase. SFKs are activated or unlocked by two mechanisms: displacement of the SH2/SH3 binding domain with another protein or phosphatase-mediated dephosphorylation of Y527 (Brown and Cooper, 1996). The requirement for trans-autophosphorylation after unlocking   35 of the kinase demonstrates SFKs are activated in clusters and that adaptor molecules such as Na/K ATPase have important regulatory roles.   Under normal physiological circumstances, Src is held in a quiet ground state and once activated either degraded or the regulatory c-terminal Y527 is phosphorylated to inactivate Src. C-terminal Kinase (CSK) and Csk homologue kinase (CHK) are the primary kinases responsible for inhibiting SFK activation by both phosphorylating Y527 and by binding to the Src SH2 domain, forming a stable inactive complex (Chong et al., 2005; Ia et al., 2010). Regulation of Src by Na/K ATPase involves a similar non-catalytic inhibitory mechanism. The alpha subunit of Na/K ATPase binds to Src in two different locations. One interaction occurs between the SH2 domain of Src and the second cytoplasmic domain (CD2) of the alpha subunit and the other occurs between the Src kinase domain and the third cytoplasmic domain (CD3) of the alpha subunit (Li et al., 2009; Tian et al., 2006).  The SFK is also an important component of signal pathways in Drosophila, but unlike the vertebrate system, the SFK is smaller and genetically simpler. Drosophila has two Src genes, Src64B and Src42A with critical roles in Drosophila growth and development (Ma et al., 2013a; O'Reilly et al., 2006; Shindo et al., 2008; Takahashi et al., 2005). Src42A and Src64B have been found to be co-expressed and functionally redundant in some tissues but also may have independent functions in others. For instance Src42A and Src64B are redundant in regulation of dorsal closure during embryogenesis (Takahashi et al., 2005). On the other hand, Src42A is preferentially activated during tracheal morphogenesis where it down regulates the adherens junction proteins E-cadherin and Armadillo to regulate cell adhesion and turnover (Shindo et al., 2008). Of note, the role of Src42A in tracheal development is thought to be independent of the SJ proteins that control tracheal elongation (Förster and Luschnig, 2012; Nelson et al., 2012).   36 Src64B is required for formation and growth of the ring canals during oogenesis, where it is regulated by Drosophila C-terminal Src Kinase (dCsk) (O'Reilly et al., 2006; Thomas, 2004). In the imaginal wing disc, Src42A has been implicated in control of cell invasion and cell death through activation of Drosophila Abelson Kinase, downstream of dCsk. Whether Src64B participates in this signal cascade is unknown and has not been tested (Singh et al., 2010; Vidal et al., 2006).   Drosophila Src kinase has a broad spectrum of roles in cell proliferation, apoptosis, and adhesion, dependent upon the cell type and its cellular environment (Cooper et al., 1996; Csiszar et al., 2010; Shindo et al., 2008; Vidal et al., 2007). Like vertebrates, dCsk is the primary negative regulator of Src and loss of dCsk function leads to overgrowth of multiple tissues (Read et al., 2004; Stewart et al., 2003). Paradoxically, Src over expression results in apoptosis and loss of developing tissues phenotypes, suggesting the cellular environment is important or that dCsk is not the only negative regulator (Pedraza et al., 2004; Takahashi et al., 1996). The evidence suggests that these scenarios are not mutually exclusive. Unlike vertebrate Csk/Chk which are highly specific for SFK Y527 (Chong et al., 2005; Ia et al., 2010), dCsk regulates kinases other than Src, including STAT (Stewart et al., 2003) and lats (Read et al., 2004), such that a broad reduction ends in overall cell proliferation. However, when dCsk was reduced in discrete patches (somatic null clones), cells with a reduced dCsk activity were removed from the epithelia and replaced via apoptosis and compensatory proliferation (Singh et al., 2010; Vidal et al., 2006; Vidal et al., 2007). In these cells Abl is activated by the increased Src activity that resulted from a reduction in dCsk (Singh et al., 2010). Once activated Abl creates a positive feedback loop by simultaneously re-activating Src while triggering Rac to control cell proliferation, invasion, and JNK to trigger apoptosis (Singh et al., 2010). Although dCsk is the primary negative regulator of   37 Src, studies on Drosophila oocytes suggests other modulators may be present. Src64 activity is required for proper ring canal growth, but dCsk is dispensable (O'Reilly et al., 2006). This suggests that other adaptor molecules might be present to control Src activity. One such adapter could be the Na/K ATPase binding to SH2/SH3 domain of Src to regulate its activity. In this thesis, we looked at the ability of Na/K ATPase to modulate Src activity in vivo and whether it is a negative regulator of Src.  1.4.5 Src Kinase and Na/K ATPase in Drosophila  The role of the different Src proteins and their distribution within the junctional domains of the wing imaginal disc is unknown. It is not known whether Src42A or Src64B is part of a protein complex with Na/K ATPase at the SJ domain nor are the consequences to Src signaling when Na/K ATPase is lost from the SJ domain. The focus of this thesis is to determine whether Na/K ATPase and Src are in a complex at the SJs domain. We will be assaying for changes in Src signaling when this potential Na/K ATPase/Src complex is disrupted by Na/K ATPase knockdown from the SJ domain.   1.5 Hypotheses  The following two hypotheses will be tested in this thesis: Hypothesis 1: I hypothesize that the Na/K ATPase organizes a signaling complex within the SJ domain and is able to activate Src and other downstream targets. Hypothesis 2:  I hypothesize that the Na/K ATPase has distinct interactions and functions from NrxIV within the SJ (bicellular and tricellular).    38 To test these hypotheses, I used the imaginal wing disc of Drosophila melanogaster as a model tissue. The imaginal wing disc is a simple epithelial tissue consisting of only two cell types; the tall and narrow, columnar cells and the flat and wide, squamous peripodial cells (Figure 1.7). Additionally during the third instar larval stage, these cells are undifferentiated and have no maternal RNA contributions. I focused on the columnar epithelial cells, as the SJs in these cells are large, well defined, easily identified structures and the cells have clear apical – basal polarity. In Drosophila, the GAL4/UAS system enables the selective expression of any cloned gene in a cell and tissue specific manner (Figure 1.7). In this way UAS-RNAi lines for a wide range of SJ and SJ-associated proteins can be crossed with different GAL4 lines to specifically knock down expression in the imaginal wing disc (Figure 1.7B). The apterous-Gal4 driver was used to drive expression in the dorsal portion of the imaginal wing disc. Thus the ventral portion served as an internal control for assaying and quantifying the effectiveness of the RNAi and cellular phenotypes.    39  Figure 1.7: Gal4/UAS expression system in the columnar epithelia. A) The wing imaginal disc is composed of a single layer of tall narrow columnar epithelial lying beneath a layer of wide flat squamous peripodial cells. B) The Gal4/UAS expression system utilizes a yeast transcriptional activator Gal4 inserted into the fly genome under the control of a specific promoter (such as apterous). When Gal4 binds to the target Upstream Activator Sequence (UAS), transcription of the downstream sequence is activated as outlined for the expression of the target gene GFP. This system enables the use of different promoters upstream of Gal4 to drive expression of the target gene in a tissue specific manner (Brand and Perrimon, 1993; Phelps and Brand, 1998). Conversely, a protein can be knocked down by using the UAS to drive the expression of a hairpin RNA (dsRNAs) (Brumby et al., 2011). BAXApGal4ApGal4/+ApGal4/UASGFP+/UASGFPUASGFP  40 1.5.1 Na/K ATPase as a Signaling Complex In Chapter 2, I examined the role of Na/K ATPase as a scaffold for a signaling complex. Vertebrate cell culture studies have shown Na/K ATPase forms a signaling complex with Src and regulates Src activity and affect a range of downstream signaling pathways (Liu and Xie, 2010). The degree of conservation, the biological significance and whether this signaling occurs in vivo, has not been determined. I examined whether Na/K ATPase can act as a signal organizer within the wing imaginal disc of Drosophila melanogaster. Specifically, I tested whether the Na/K ATPase and Src existed in a complex at the SJ domain, if this complex was able to initiate signal cascades via activation of downstream targets, and its effects on cell survival pathways.  1.5.2 Na/K ATPase Associates with Dlg and Gli at the TCJ Subdomain In Chapter 3, I examined if subdomains of the SJ have differential functions and specifically if Na/K ATPase has differential roles in the bicellular SJ and the TCJ compared to NrxIV. I used a combination of RNAi and genetic interactions assays to examine the differential roles of the Na/K ATPase compared to NrxIV with respect to the SJ-associated proteins Dlg and Fas3 and the TCJ protein Gli. My data suggests Na/K ATPase is unique from NrxIV and Na/K ATPase has specific interactions with the TCJ.       41 Chapter 2: Loss of the Na/K ATPase Triggers Apoptosis and Apoptosis-induced Proliferation via JNK signaling 2.1 Synopsis Permeability barriers are critical in blocking the flow of fluids or pathogens from crossing between epithelial cell boundaries. Permeability barrier function is fulfilled by tight junctions (TJ) in vertebrates and septate junctions (SJ) in invertebrates. Although many junctional components have been identified, how these essential junctions are assembled and maintained is not clearly understood. The Na/K ATPase pump is a critical component of both SJs and TJs and consists of highly conserved alpha and beta subunits. In vertebrates, inhibition of the Na/K ATPase ion transport activity or removal of either subunit compromises permeability barrier function and disrupts TJs. Within the SJ of invertebrates, the function of the Na/K ATPase is independent of its role as an ion transporter and provides the opportunity to study transport-independent functions of the Na/K ATPase. Within these junctional complexes, the alpha subunit of the pump has the potential for both scaffolding functions and cell signaling. Studies in cultured fibroblasts have determined the alpha subunit is capable of binding and inhibiting Src kinase. Binding of cardiotonic steroids such as ouabain, triggers the release of the kinase domain, activation of Src and activation of downstream signaling pathways. However the role of the Na/K ATPase/Src complex has not been investigated in polarized epithelia with established permeability barriers or in vivo to determine the cellular consequences of this complex in a living animal. The simplified epithelium of the Drosophila imaginal wing disc allows for a systematic analysis of the Na/K ATPase in scaffolding or organizing other SJ components and its potential as a regulator of Src signaling. Using RNAi-mediated knockdown of either Na/K ATPase   42 subunit, we observed disruption of the SJ components NrxIV and Dlg. We observed a concomitant increase in activated Src and Abl, ultimately leading to JNK-mediated apoptosis and apoptosis-induced proliferation. This study demonstrates an in vivo function of Na/K ATPase-mediated Src regulation of cell survival and proliferation.  2.2  Introduction  The Na/K ATPase is a well-characterized ion transporter composed of two subunits, the large alpha (α) subunit (~1000 amino acids, 110 kDa) that forms the pore and the smaller 370 amino acid beta (β) subunit (Kaplan, 2002). In addition to its role in the maintenance of ion gradients, the Na/K ATPase has been well established as an important hormone receptor for cardiotonic steroids and is capable of modulating physiological and pathophysiological processes including salt homeostasis, blood volume, blood pressure, and cell-cell communication (Cereijido et al., 2012; Lichtstein, 2014; Manunta et al., 2009). Extensive studies in vertebrate cell culture have demonstrated that the modulation of these signaling pathways occurs through an association between Src kinase and Na/K ATPase, and is independent of its ability to transport ions across the membrane (Lingrel, 2010). Given its critical role in ion balance, studying the transport-independent signaling functions of the Na/K ATPase in vivo has been challenging due to the difficulty in separating the signaling and ion-transporting functions.   The septate junctions (SJ) of the Drosophila imaginal wing disc are an ideal model tissue in which to study the non-transporting functions of Na/K ATPase. The wing is not essential for viability and is a simple epithelium with a well-characterized developmental program. Crucially, the two functions of Na/K ATPase can be separated thus permitting study of its role as a signal transducer. In this simple epithelium, the Na/K ATPase is specifically localized to the septate   43 junctions (SJ) where no ion exchange is observed. Furthermore, a mutation in the pore-forming α subunit of Na/K ATPase that abolishes ion exchange is able to form normal SJs (Genova and Fehon, 2003; Paul et al., 2003; Paul et al., 2007). Therefore, we can use this tissue as a model to study ion transport-independent functions of the Na/K ATPase.  Drosophila SJs are permeability barrier junctions functionally analogous to vertebrate tight junctions (TJ). In addition, both TJs and SJs have roles in cell adhesion, trafficking, and cell-cell signaling (Banerjee et al., 2006; Nelson and Beitel, 2009; Tepass et al., 2001). The Na/K ATPase associates with other SJ proteins, Neurexin-IV (NrxIV), Neuroglian (Nrg), Coracle (Cora) and Macroglobulin complement-related protein (Mcr) and form the essential, or core, structural complex (Batz et al., 2014; Genova and Fehon, 2003; Hall et al., 2014; Paul et al., 2003). Loss of any of these core proteins results in the loss of permeability barrier function and viability (Genova and Fehon, 2003; Paul et al., 2003).   The core complex of SJs contain numerous protein interacting motifs which are predicted to mediate the assembly of the core SJ complex, to anchor SJs to the cytoskeleton and to link to signaling mechanisms (Banerjee et al., 2006; Tepass et al., 2001). For instance, both subunits of Na/K ATPase contain binding domains for the cytoskeleton proteins ankryin and spectrin (Nelson and Veshnock, 1987; Vagin et al., 2007; Vagin et al., 2009). In addition to scaffolding domains, the ATPα subunit can bind and regulate the activity of Src kinase (Li et al., 2009; Tian et al., 2006). Vertebrate Na/K ATPase interacts directly with Src through two binding motifs, the second cytosolic domain of the α1 subunit binds the Src SH2 domain, and the third cytosolic domain binds and inhibits the Src kinase domain. Binding of ouabain to Na/K-ATPase disrupts this interaction, resulting in Src activation (Li et al., 2009; Tian et al., 2006). Src kinase is a well-established signaling molecule with key roles in development, proliferation and morphogenesis   44 (Roskoski, 2004; Shindo et al., 2008; Tateno et al., 2000; Vidal et al., 2007). Therefore the ability of Na/K ATPase to regulate Src activity allows Na/K ATPase and the SJs to control Src activation levels and cellular function; however the consequences of disrupting this regulatory interaction have not been tested within a developing epithelium or in vivo.   The genetic complexity of vertebrate Na/K ATPase subunits makes study of the in vivo roles of the Src-Na/K ATPase complex difficult. Vertebrate Na/K ATPase consists of three subunits: α, β, and a γ (FXYD family member). Each subunit is encoded by multiple genes, many of which can compensate for each other in null mutations: four genes for the α subunit, three for the β and seven FXYD, providing extensive tissue- and function-specific diversity (Geering, 2006; Lingrel and Kuntzweiler, 1994). Drosophila Na/K ATPase is simpler, consisting of only α and β subunits and lacking the FXYD family member. Furthermore, each subunit is encoded by fewer genes: two α subunits genes ATPα and JYα, and three β subunits Nervana (Nrv) 1, 2 (2.1, 2.2) and 3 (DeTomaso et al., 1993; Lebovitz et al., 1989; Masly et al., 2006; Sun and Salvaterra, 1995b; Sun et al., 1998). The ATPα protein is expressed in all tissue types, sharing a high degree of homology with vertebrate α1. In the imaginal wing disc, ATPα is localized specifically to the SJ domain. JYα expression is restricted to sperm and is necessary for sperm motility (Masly et al., 2006; Sun et al., 1998). Three genes, each of which can be alternatively spliced, encode the β subunit. Nrv1 and Nrv2 are expressed in ectodermally derived epithelial tissue, but only Nrv2 is localized to the SJ and is required for SJ and tube-size functionality (Genova and Fehon, 2003; Paul et al., 2003). Nrv3 is exclusively expressed in the nervous system (Paul et al., 2003; Paul et al., 2007). Thus in imaginal wings discs, Na/K ATPase is specifically localized to the SJs and consists only of ATPα and Nrv2 subunits.    45  In the present study we investigated the function of the Na/K ATPase at the SJs using the imaginal wing disc as an in vivo model to determine whether it is able to function as a signal-transducing module. RNAi mediated knock down of either the α or Nrv-2 subunits in the imaginal wing disc demonstrated that Na/K ATPase is required for the formation and maintenance of SJs. Significantly, we found that RNAi-mediated degradation of either subunit led to activation of Src/Abl signaling and culminated in JNK directed apoptosis and apoptosis-induced proliferation. These data demonstrate that the SJs are critical in regulating cell proliferation and apoptosis likely through the restriction of Src activation by Na/K ATPase. 2.3 Results 2.3.1 The Na/K ATPase is Important for Maintenance of SJs in the Imaginal Wing  Whether the Na/K ATPase is important for the maintenance of SJs and whether it has other functional roles in the Drosophila wing epithelia has been difficult to study as somatic null clones are cell lethal and eliminated from the epithelia (Genova and Fehon, 2003). We used RNAi to temporally and spatially degrade the mRNA of each subunit to study the role of Na/K ATPase in the SJs of the wing imaginal disc. Specifically, we used the Gal4/UAS system (Brand and Perrimon, 1993) and apterous-Gal4 to ectopically express RNAi specific to either ATPα οr Nrv2 in the dorsal compartment of the imaginal wing disc (hereafter referred to as the apterous (ap) compartment). To ensure observed phenotypes were due to loss of either subunit and not due to degradation of off-target mRNA, three different UAS-ATPα RNAi and two UAS-Nrv2 RNAi constructs were tested. Each RNAi targeted a unique region of the ATPα or the Nrv2 mRNA. The Drosophila nervana2 gene has two splice isoforms (Nrv2.1, Nrv2.2) that are   46 functionally indistinguishable (Paul et al., 2003; Sun and Salvaterra, 1995a; Sun and Salvaterra, 1995b). As both isoforms localize to the SJ domains and are targeted by the RNAi expression constructs, the two proteins will be collectively referred to as Nrv2. We confirmed the efficiency of each RNAi by expressing them in flies with ATPα or Nrv2 endogenously tagged with GFP (ATPα::GFP and Nrv2::GFP). All wing discs ectopically expressing the RNAi transgenes effectively removed the GFP-tagged ATPα and Nrv2 from the SJ domain (Figure 2.1, Figure 2.2, Figure 2.7A, B). As expected from prior analysis of ATPα mutants (Paul et al., 2003), loss of ATPα led to the complete absence of Nrv2::GFP from the SJ domain (Figure 2.1B). Instead Nrv2::GFP accumulated intracellularly. Both subunits must form a functional complex prior to transport to the membrane (Geering et al., 1996; Rodriguez-Boulan et al., 2004), therefore the accumulation of Nrv2::GFP is likely in the ER due to the lack of ATPα with which to heterodimerize. RNAi knock down of ATPα led to a reduction in intensity and a basolateral spread of both the core SJ protein NrxIV and the SJ-associated protein Dlg, when compared to the wild type side of the wing disc (Figure 2.1C). RNAi knock down of Nrv2 phenocopied these results; both immunolabeled NrxIV and Dlg decreased and spread basolaterally in the apterous compartment of the disc, but not in the wild type compartment (Figure 2.2B). Reduction of Nrv2 resulted in a loss of ATPα from the membrane, as expected due to the requirement of Nrv2 for correct transport of the Na/K ATPase (Figure 2.2C) (Geering et al., 1996). Loss of either subunit did not affect formation or placement of the adherens junctions (AJ) as there was no change in the distribution of the AJ markers, E-cadherin and phosphorylated Src (pSrc) (Figure 2.7C, Figure 2.2B). Overall loss of either subunit of the Na/K ATPase led to a mislocalization of both core and associated SJ proteins. These results demonstrate that the Na/K ATPase is required for the maintenance of SJs in the wing imaginal disc. Furthermore, this confirms the validity of our   47 RNAi-mediated knock down model system for studying the relationship between Na/K ATPase and other SJ components and regulatory proteins.    48  Figure 2.1: ATPα  is required for maintenance of SJs of the imaginal wing disc. En face projections and side projections of 3rd instar imaginal wing discs with apterous-Gal4 driving ATPα-RNAi. The dashed line indicates the apterous compartment border. Arrowhead indicates the SJ domain in side projections. (A) A wild type control disc with ATPα::GFP (A’, green) immunolabeled for pSrc64B to mark the AJs (A’’, red) and Dlg (A’’’, blue). ATPα and Dlg localized to the SJ domain, which are basal to the AJs.  (B) Expression of ATPα-RNAi (GD12330) with Nrv2::GFP (B’, green) led to a loss of immunolabeled ATPα (B’’, red). Nrv2GFP is produced but cannot reach the membrane and accumulates in the cytosol. DAPI (B’’’, blue) labeled nuclei.  (C) Expression of ATPα-RNAi (KK100619) with UAS-mCD8::GFP to distinguish the apterous compartment (C’’’, blue). RNAi knock down of ATPα caused a decrease and basal spread of immunolabeled Dlg (C’, green) and NrxIV (C’’, red) at the SJ domain.  Scale bars 15um.    49  Merge Nrv2::GFP Dlg DAPINrv2::GFP NrxIV Dlg-------------- -------------- -------------- -------------- MergeMerge Nrv2::GFP ATPA pSrc42A-------------- -------------- -------------- Nrv2RNAiJFNrv2RNAiGDWildtypeA A’ A’’ A’’’B B’ B’’ B’’’C C’ C’’ C’’’  50  Figure 2.2: RNAi knock down of Nrv2 phenocopies loss of ATPα  and is required for maintenance of the imaginal wing disc SJs.  En face projections and side projections of 3rd instar imaginal wing discs from apterous-Gal4 driving Nrv2-RNAi. The dashed line indicates the apterous compartment border. Arrowheads indicate the SJ domain. (A) Control disc with Nrv2::GFP (A’, green) immunolabeled with Dlg (A’’, red) and DAPI (A’’’, blue) to mark the nuclei. Nrv2::GFP is localized to the SJ domain with Dlg.  (B) Expression of Nrv2-RNAi (JF03081) with Nrv2::GFP (B’, green) and immunolabeled for ATPα (B’’, red). AJs are marked with pSrc42A (B’’’, blue). RNAi knock down of Nrv2 led to a concomitant loss of immunolabeled ATPα from the SJ domain. The localization and distribution of pSrc42A did not change, indicating the AJs were not affected.  (C) Expression of Nrv2-RNAi (GD2660) with Nrv2::GFP (C’, green) and immunolabeled for NrxIV (C’’, red) and Dlg (C’’’, blue). Expression of Nrv2-RNAi caused a decrease and a basal spread of the immunolabeling for Dlg and NrxIV at the SJ domain. Scale bars 15um. 2.3.2 Loss of Na/K ATPase Leads to Cell Death and Delamination  The apterous-Gal4 driven expression of ATPα-RNAi or Nrv2-RNAi is lethal. This was not unexpected as apterous-Gal4 is expressed in a range of tissues including neurons and the Na/K ATPase is a critical component of most cells. Within the wing imaginal disc, we observed pyknotic nuclei of delaminated cells in the basal region of columnar epithelia expressing either ATPα-RNAi or Nrv2-RNAi. We analyzed the ATPα-RNAi expressing discs for the presence of activated Caspase3 (Cas3), an indicator of apoptosis (Song, 1997). Clusters of Cas3 positive cells were observed in the basal side of the apterous compartments and corresponded to delaminated cells with pyknotic nuclei (Figure 2.3A, B). Cells expressing ATPα-RNAi were also positive for matrix metalloprotease1 (MMP1) (Figure 2.3A, B). Activation of MMP1 occurs via the Jun kinase (JNK) pathway, and increased MMP1 activity is associated with increased cell mobility (McCawley and Matrisian, 2000; Page-McCaw et al., 2007). We also observed some Cas3 positive cells with more intense MMP1 immunolabeling scattered within the apterous   51 compartment (Figure 2.3A, B arrowheads). Our data suggest that knock down of ATPα and Nrv2 leads to apoptosis likely mediated by the JNK pathway.    Despite the extensive cell death and delamination in response to loss of ATPα or Nrv2 in the apterous compartment, ATPα-RNAi wing discs were the same size as wildtype discs (Figure 2.3A, Figure 2.6A). Previous studies in Drosophila have demonstrated the ability of imaginal disc epithelial cells to recover from injury or induced apoptosis through increased cell proliferation and mitosis (Milán et al., 1997). We assayed cell proliferation rates to see if they differed between the apterous and wild type compartments of the wing disc pouch. The frequency of cells positive for a mitotic marker, phospho-histone3 (pHis3), was significantly greater (37.33 vs. 23.82, p<0.0007) in ATPα-RNAi expressing region than the wild type (Tapia et al., 2005)(Figure 2.3C and Figure 2.3D). As epithelial cells in the apterous compartment had increased rates of cell proliferation compared to the control compartment, this suggests that loss of ATPα and Nrv2 leads to cell death and compensatory proliferation.    52    53 Figure 2.3: Loss of Na/K ATPase leads to cell death and compensatory proliferation. apterous-Gal4 was used to drive expression of ATPα−RNAi (GD12330)(ATPα-RNAiGD) (A-B) Wing disc with ATPα::GFP and ATPα−RNAiGD immunolabeled with activated Caspase3 (Cas3) (green, A’, B’), MMP1 indicating extracellular matrix degradation (A’’, B’’, red) and ATP::GFP (blue, A’’’) and DAPI (blue, B’). (A) 20x magnification image. (B) High resolution (60x) image of a single z-slice 10µm below the SJ domain. Arrows indicate colocalization of Cas3 with pyknotic nuclei confirming apoptosis. Arrowheads indicated cells positive for both MMP1 and Cas3. Scale bar: 15 µm.  (C) Wing disc with apterous-Gal4 driving ATPα−RNAiGD and UAS-mCD8::GFP to indicate the apterous compartment (green) immunolabeled with phosphoHistone3 (pHis3) (red) to mark cells entering mitosis and DAPI (blue) to mark nuclei. The circle indicates the wing pouch where the pHis3 positive cells were counted in the apterous and wildtype compartment to compare proliferation.  (D) Statistical analysis of cell proliferation in the apterous and control compartments of ATPα-RNAiGD. Significantly more mitotic cells (p=0.0007, n=11 discs) were present in the apterous compartment. Error bars represent SEM. 2.3.3 Increased Cell Proliferation with Loss of ATPα  is Associated with an Increase in pSrc    The increase in cell proliferation in response to loss of ATPα raised the question of whether Na/K ATPase is able to modulate cell proliferation. In vitro studies of vertebrate cells have shown that loss of Na/K ATPase (Tian et al., 2009) or partial inhibition of Na/K ATPase ion transport function with ouabain triggers cell proliferation (Aydemir-Koksoy et al., 2001; Haas et al., 2000; Kometiani et al., 2000). Mechanistically, in vitro knock down of the α1 subunit or inhibition of the ion transporting function leads to increased Src activation (pSrc) and triggers downstream growth pathways including Ras/Raf/Erk1/2 cascades through EGFR and PKC (Liang et al., 2006; Lingrel, 2010; Liu and Xie, 2010). However, the activation of the Src signaling pathway has not been demonstrated to occur in vivo or within polarized epithelia, (Lingrel, 2010). The Drosophila wing disc epithelial model is well suited to test for the developmental requirement of this pathway.    54  The vertebrate Na/K-ATPase interacts directly with Src through two intracellular domains. The second cytosolic domain binds the Src SH2 domain and the third cytosolic domain binds and inhibits the Src kinase domain. A specific 20-amino acid peptide (NaKtide) from the third domain was identified that targets and disrupts the formation of the Na/K-ATPase/Src receptor complex (Li et al., 2009). Both intracellular domains and the NaKtide peptide sequence are highly conserved in the Drosophila ATPα protein (Figure 2.8). Prior to testing for the presence of an Src-Na/K ATPase signalling pathway, we wanted to test the presence of Src within the SJ domain and determine whether it was capable of complexing with the Na/K ATPase. Drosophila has two Src homologs, Src42A and Src64B and both are present in the wing imaginal disc. Src42A is mainly cytosolic while Src64B is both cytosolic and localized in puncta within the membrane (Figure 2.9A-B). In the wing imaginal disc, phosphorylated Src42A (pSrc42A) is localized to the AJs (Singh et al., 2010). We used antibodies specific to the phospho-tyrosine residues of activated Src (Y400Src42A, Y434Src64B) (O'Reilly et al., 2006; Shindo et al., 2008) and confirmed the presence of pSrc42A and determined that pSrc64B was also present (Figure 2.1A, Figure 2.2B). Both forms of activated Src were primarily localized at the AJs. Using biochemical approaches, we were unable to immunoprecipitate either Src42A or Src64B with ATPα in a complex, while Nrv2 and ATPα were readily co-immunoprecipitated (Figure 2.4D, Figure 2.9E). This result may reflect that the Src-Na/K ATPase interaction is either at very low levels or is of a transient nature. Src42A and Src64B have been shown to be functionally redundant (Takahashi et al., 2005; Tateno et al., 2000) and did not exhibit any differences in association or localization with ATPα, therefore we focused our subsequent analysis on pSrc42A.    55  The inability to detect Na/K ATPase in a stable complex with Src suggests the relationship between these proteins is either indirect or highly transient. If this true, loss of ATPα or Nrv2 should still result in a change in either the distribution and/or immunolabeling intensity of pSrc. We examined changes in both the distribution and immunolabeling intensity of pSrc in response to ATPα−RNAi. Reduction of ATPα did not alter the pattern of pSrc42A distribution at AJs (Figure 2.2B, Figure 2.4A). We next quantified pSrc42A for differences in the signal intensity between matched regions in the wildtype and apterous compartments (see Material and Methods). When the mean gray value intensity of pSrc42A was expressed as ratio of the intensity in the apterous compartment / WT compartments, the ratio was significantly greater than 1 using either RNAi construct (1.37x, p=0.0082 or 1.22x, p=0.0190) (Figure 2.4E). This demonstrates an increase in pSrc and these results support our hypothesis that the stoichiometry of Src-Na/K ATPase association may not be detectable due to sensitivity limitations inherent to immunoprecipitation methodology or the interaction may be transitory. Therefore, we used proximity ligation assay (PLA), a more sensitive assay to test for an association between pSrc42A and ATPα (Weibrecht et al., 2010). This assay generates a fluorescent product only when two proteins are less than 40nm apart. We observed PLA signal between pSrc42A and ATPα at the apical domain in the wild type compartment that was absent in cells expressing ATPα -RNAi in the apterous compartment (Figure 2.5C, D). A PLA signal that decreases concomitantly with a reduction in ATPα was observed within the AJ and SJs domains, which is strongly supportive of a protein-protein interaction between pSrc42A and ATPα. In vertebrate studies, the Src - Na/K ATPase complex was localized to the caveolae microdomain (Liu et al., 2003) and not at TJs. Our PLA results suggest that similarly, a subpopulation of pSrc42A is associating with ATPα within a discrete region of the cell. PLA between ATPα and Nrv2   56 showed punctate PLA signal around the cell membrane at the depth of the SJ domain, which was reduced in the ATPα-RNAi background (Figure 2.5A, B). This punctate pattern of PLA between proteins known to associate in junctional complexes has been also been observed with AJs components (Figueiredo et al., 2012; Van Itallie et al., 2014). Taken together, these results suggest that in Drosophila epithelia a subpopulation of the Na/K ATPase is less than 40 nm from Src Kinase and may be functioning as a signaling complex.       57  Figure 2.4: Interactions of pSrc with the ATPα  subunit  (A-C) Single z-slice en face view and side projections of 3rd instar wing disc with apterous driving ATPα-RNAiGD with ATPα::GFP. Dotted lines indicate the apterous boundary, yellow boxes indicate the area used to quantify the mean gray value of the WT and apterous compartments (E). Scale bars: 15 µm (A, B) Knock down of ATPα (A’, B’ green) led to an increase in pSrc42A (B’’, C’’, red).  (C) Knock down of ATPα (C’’ green) led to an increase in pAbl (C’’, red), a downstream target of Src.  (D) Immunoprecipitations (IP) from embryo extracts of control (w1118) or ATPα::GFP using GFP-Trap magnetic beads. Western blots of lysates or IPs were probed with antibodies to ATPα, Nrv2, pSrc42A (Src), and GFP. Nrv2 was immunoprecipitated in a complex with ATPα::GFP, but pSrc42A was not detected.  w1118ATPA::GFPw1118ATPA::GFPLysate IPATPASrcNrv2GFPMerge ATPA::GFP pSrc----------------------Merge ATPA::GFP pSrc----------------------Merge ATPA::GFP pAbl0.00.51.01.52.02.53.0GDATPA KKATPA Ratio of ap / WT p = 0.0082 p = 0.01900.00.51.01.52.02.53.0ATPAGDRatio of ap / WTp = 0.0103D EA A’ A’’C C’ C’’B’B B’’pSrc42A pAbl  58 (E) The fluorescence intensity of pSrc42A and pAbl in the apterous compartment was quantified and expressed as a ratio of the wildtype (WT) compartment. The ratio of apterous: WT was significantly greater than 1 for pSrc42A (ATPα-RNAi GD: n=21 discs; KK: n=17 discs) and pAbl (n=10 discs). Error bars represent SEM.   One hypothesis arising from studies in mammalian tissue culture, is that activated epidermal growth factor receptor (pEGFR) and phosphorylated focal adhesion kinase (pFAK) levels increase as a consequence of the release of activated Src from Na/K ATPase (Haas et al., 2002; Liang et al., 2006). We tested this hypothesis in our in vivo Drosophila wing epithelia system but were unable to detect any changes in activated pEGFR or pFAK with loss of ATPα (Figure 2.9C). In Drosophila, Src family kinases have dual roles; increased Src activation triggers both cell death and cell proliferation through independent pathways (Pedraza et al., 2004) (Vidal et al., 2007). Drosophila Abelson (dAbl), a non-receptor tyrosine kinase, is activated downstream of Src. Src dependent activation of dAbl generates cell proliferation, apoptosis and invasion through activation of Rac, a Rho family GTPase (Singh et al., 2010). Rac activation triggers two independent pathways initiated respectively by ERK and JNK to produce cell proliferation and apoptosis (Singh et al., 2010). The increased cell proliferation observed with apoptosis in ATPα-RNAi wing discs suggested increased pSrc could be acting upstream of dAbl. If true, we would expect phosphorylated dAbl levels to increase as well in response to loss of ATPα. We observed an increase in phosphorylated dAbl (pAbl) in response to ATPα knock down (Figure 2.4E). When we analyzed differences in the mean gray value between matched regions, we observed a significant increase (1.38x, p=0.0135) (Figure 2.4E). Further, we observed that the loss of ATPα led to an increase in activated JNK (pJNK) (Figure 2.6E). These data confirm that Na/K ATPase   59 can associate with Src in Drosophila and the Na/K ATPase may act as a signal transducer to activate downstream signaling cascades through Abl.    Figure 2.5: Proximity ligation assay demonstrates a subset of ATPα  and pSrc42A associate in vivo. apterous-Gal4 was used to drive expression of ATPα−RNAiGD in 3rd instar discs endogenously tagged with Nrv2::GFP. The white dotted lines mark the WT / apterous border. Scale bars are 15 µm.  (A) Enface maximum intensity projections of the SJ region. The PLA signal (red) is detected in the WT compartment when using antibodies to ATPα and GFP. The SJ domain is marked by Nrv2::GFP (green). The PLA signal was reduced or absent from the apterous side. (B) Side projections show the PLA occurs at the level of the SJ domain labelled with Nrv2::GFP (arrowhead).   (C) Enface maximum intensity projections of the SJ domain. The PLA signal (red) is detected in the WT compartment when using antibodies to GFP and pSrc (red). The SJ domain is marked by Merge Nrv2::GFP--------------PLAPLAMerge Nrv2::GFP-----------------------PLAPLAATPA-RNAiGDATPA-RNAiGDA A’ A’’B’ B’’BC’ C’’CD’ D’’D  60 Nrv2::GFP (green). In the apterous compartment, Nrv2::GFP accumulated in the cytosol and the PLA signal in the SJ domain was lost.  2.3.4 Loss of ATPα  Triggers JNK-Mediated Apoptosis  The activation of the pAbl-RAC-JNK pathway suggested the cells in the ATPα RNAi expressing compartment were proliferating in response to death and delamination, a process known as compensatory proliferation (Pérez-Garijo et al., 2009; Ryoo et al., 2004). This hypothesis was tested by blocking apoptotic cell death by expressing baculovirus p35 protein, an inhibitor of Cas3 activation (Hay et al., 1994), alone or with either ATPα or Nrv2-RNAi. Blocking cell death led to overgrowth of the apterous compartment of the imaginal wing disc when p35 was expressed with ATPα or Nrv2-RNAi (Figure 2.6C) but not when p35 was expressed alone (Figure 2.6B). The observed hyperplasic overgrowth results from the persistence of  “undead” apoptotic cells kept alive by p35 expression that continue to secrete mitogens mediated by JNK signaling (Mollereau et al., 2012; Pérez-Garijo et al., 2009; Ryoo et al., 2004). We expressed a dominant negative form of JNK (BskDN) in the RNAi background to confirm that both apoptosis and the resulting compensatory proliferation were mediated by JNK. Expression of BskDN with ATPα-RNAi, blocked both the apoptosis and the overproliferation (Figure 2.6G). The discs were no longer Cas3 positive, did not exhibit pyknotic nuclei in the basal compartments (data not shown) and did not have the excessive number of folds or outgrowth observed in the discs expressing either ATPα-RNAi or Nrv2-RNAi with UAS-p35 (Figure 2.6B, C). These results show that loss of Na/K ATPase pump leads to JNK-mediated apoptosis and compensatory proliferation.    The ability of pSrc to activate the JNK signaling pathway has been well established (Ma et al., 2013b; Singh et al., 2010; Tateno et al., 2000; Vidal et al., 2006) and thus it is likely that   61 activation of Src is upstream of JNK. Alternatively the involvement of JNK in triggering apoptosis and its requirement in compensatory proliferation raises the possibility that the increases in Src and Abl activation may be a consequence of JNK activation within apoptotic cells and not due to loss of Na/K ATPase. We tested whether the activation of Abl is upstream of JNK by analyzing pAbl immunolabeling in wing discs expressing BskDN in conjunction with ATPα-RNAi. The mean gray value of immunolabeled pAbl increased (1.2x) in the apterous compartment (Figure 2.6H) even when BskDN was co-expressed with ATPα-RNAi. Our results suggest that Abl may be the trigger that links loss of the Na/K ATPase to the induction of JNK mediated apoptosis.  Figure 2.6: Low resolution (20X) images of 3rd instar wing discs  (A) A control wing disc with Dlg::GFP showing the normal size and fold of an imaginal wing. Controlap>p35ap>ATPARNAi ap>ATPARNAi ,BskDN p35p35p35Dlg::GFPATPA::GFPap>Nrv2-RNAi, p35ap>ATPARNAi, p35ap>BskDNATPA::GFPpJNKA B C DE F G HBskDN;ATPAKK0.00.51.01.52.02.53.0Ratio of ap / WTpAbl  62 (B) apterous-Gal4 driving the expression of the baculovirus p35 protein (ap>p35) to block apoptosis, immunolabeled for p35. Expression of p35 by itself does not affect disc size, shape or number of folds.  (C,D) apterous-Gal4 driving the expression of p35 and ATPα−RNAiKK (C) or driving the expression of p35 and Nrv2-RNAiGD (D) both immunolabeled for p35. Expression of the baculovirus p35 with RNAi to either subunit of the Na/K ATPase led to increased proliferation and an increase in the number of folds, overtaking the ventral compartment.  (E) apterous-Gal4 driving the expression of ATPα-RNAiGD immunolabeled with phosphorylated JNK (pJNK). Loss of ATPα led to increase in activated pJNK labeling in the dorsal compartment.  (F) apterous-Gal4 driving the expression of a dominant negative JNK (BasketDN: BskDN). Expression of BskDN alone had no effect on the wing disc. (G) apterous-Gal4 driving the expression of ATPα-RNAi and BskDN. BskDN suppressed the AIP observed with co-expression of p35 demonstrating that apoptosis is not occurring.  (H) The fluorescence intensity of pAbl was quantified from discs expressing BskDN and ATPα-RNAiKK. The mean gray value was quantified in the apterous and wild type compartments and expressed as a ratio with the WT compartment (n=5 discs). Error bars are SEM.  2.4  Discussion  The Na/K ATPase, studied for more than 20 years as an important regulator of homeostasis, is required for the formation of both invertebrate SJs and vertebrate TJs (Madan et al., 2007; Paul et al., 2003; Paul et al., 2007; Rajasekaran et al., 2006; Violette et al., 2006). Cells junctions are important sites of cell signaling, yet to date there are few studies to specifically link SJs to signal transduction. This study demonstrates that the core SJ component, Na/K ATPase, is necessary for the stability of the SJ within the wing imaginal disc and is capable of signal transduction in addition to its structural role in vivo. Na/K ATPase is a part of a signaling cascade that includes Src and Abl to activate JNK pathway and modulate apoptosis and cell proliferation in the Drosophila wing imaginal disc.  2.4.1 Na/K ATPase is Required for the Maintenance of SJs  SJs are formed through highly organized protein-protein interactions to form tight impenetrable barriers between cells in epithelial sheets. Genetic null mutant analysis and somatic   63 mosaic studies have identified core components of SJs and suggested their interdependence for correct localization. However, analysis of the role of individual core components is made more difficult by the elimination of somatic null clones of SJ gene mutants from the wing epithelium (Genova and Fehon, 2003). In vitro studies have suggested that α and β subunits of Na/K ATPase must be correctly paired prior to exit from the ER and for retention of the protein at the membrane (Brand and Perrimon, 1993; Lingrel and Kuntzweiler, 1994; Yoshimura et al., 2008). We have confirmed that ATPα and Nrv2 absolutely require each other to correctly localize to the SJ domain in Drosophila epithelia. In addition to demonstrating the co-dependence of the ATPα and Nrv2, RNAi-mediated knock down also confirmed the requirement of Na/K ATPase for correct localization of the core SJ protein NrxIV (Genova and Fehon, 2003; Paul et al., 2003). However, unlike what has been observed in somatic clones of ATPα mutants, we observed a disruption in the distribution of Dlg, which is normally distributed in somatic clones but spread basolaterally in our RNAi experiments. The difference in Dlg localization in somatic clones vs. RNAi suggested that part of the SJ domain was intact in these mutant backgrounds. A possible reason for this difference could be the small size of the somatic clones compared to the large compartments affected by RNAi mediated knockdown. As somatic clones are surrounded by wild type tissue, the SJ components in neighboring wild type cells may be able to form trans-interactions with SJ components in the mutant clone and maintain the presence of some SJ components. A possibility is Nrv2, a known homophilic adhesion protein with the potential to organize the SJ complex through its extracellular domain and linkages to the cytoskeleton intracellularly (Paul et al., 2007; Rajasekaran et al., 2001b; Shoshani et al., 2005; Sun and Salvaterra, 1995a).    64  The displacement of Dlg in the Na/K ATPase RNAi-mediated knock down raises the intriguing possibility that Na/K ATPase forms a link between the core SJ proteins and SJ-associated proteins. Loss of vertebrate Na/K ATPase leads to a loss of stress fibers and discontinuous staining of the TJ proteins ZO1 and Occludin (Madan et al., 2007; Rajasekaran et al., 2006; Violette et al., 2006). The removal of TJs proteins in response to loss of Na/K ATPase is associated with alterations in the phosphorylation status of TJ proteins via activation of Src and Erk1/2 (Contreras et al., 1999; Giannatselis et al., 2011; Larre et al., 2010; Rajasekaran et al., 2006; Rincon-Heredia et al., 2013). Phosphorylation of Occludin at specific residues prevents its interaction with ZO1 and destabilizes its localization to the TJ (Elias et al., 2008). ZO1, a membrane-associated guanylate kinase (MAGUK) family member, functions as a scaffolding and an adaptor protein. Through its PDZ and SH3 domains, ZO1 binds to the actin cytoskeleton and TJ proteins to mediate TJ dynamics (González-Mariscal et al., 2000; Shen, 2012). Likewise Drosophila Dlg, a MAGUK protein, has a similar structure and role to ZO1 in SJs. RNAi mediated knock down of Na/K ATPase from SJs led to a loss of Dlg and concomitant increases in Src activation. These data suggests that Na/K ATPase may complex with Dlg to maintain it and other SJs components at the SJ domain via regulation of their phosphorylation status. This presents a model similar to the Na/K ATPase maintenance of TJs proteins in mammalian tissues (Giannatselis et al., 2011; Violette et al., 2006).  2.4.2 Na/K ATPase is a Negative Regulator of Src  One clue that Na/K ATPase has multiple functions in epithelial tissues, in addition to the well-characterized ion transport function, comes from studies of tracheal development in Drosophila embryos. The Na/K ATPase is necessary for maintaining proper embryonic tracheal   65 tube size independent of the formation of SJs (Genova and Fehon, 2003; Paul et al., 2003; Paul et al., 2007). Further, the Na/K ATPase co-operates with Src42A in late embryogenesis to determine correct tracheal tube lengths, raising the possibility that the restriction of tube length by the Na/K ATPase is through its regulation of Src42A activation (Nelson et al., 2012). Vertebrate cell culture and ex-vivo studies demonstrated a clear role for Na/K ATPase in maintaining junction integrity and as a modulator of cell growth via its association with pSrc. Whether this complex signals cell proliferation or cell growth inhibition appears to be cell type specific and dependent on both the concentration of Na/K ATPase and the activated second messengers (Tian et al., 2009). Correct regulation of Src activation levels is required for normal tissue growth and development as misregulation of Src activation leads to deleterious effects such as cancer (Pedraza et al., 2004; Read et al., 2004; Tateno et al., 2000; Vidal et al., 2007). The cellular outcomes associated with Src activation, cell proliferation and apoptosis, are dependent upon the level of Src signaling, which is controlled by the balance between activation of positive and negative regulators (Roskoski, 2004; Vidal et al., 2007). A biologically relevant function for this mechanism has been difficult to verify in vivo in vertebrates due to the large number of the genes encoding the Na/K ATPase subunits. In contrast, the Drosophila genome encodes only one somatic cell Na/K ATPase alpha subunit, three beta subunits, and two Src genes. A previously identified negative regulator of Src is Drosophila C-terminal Src kinase (dCsk) (Chong et al., 2005; O'Reilly et al., 2006; Pedraza et al., 2004; Read et al., 2004). dCsk regulates Src through phosphorylation of a conserved C-terminal tyrosine residue and loss of this residue or the C-terminal domain removes Csk regulation of Src. Though Csk is thought to be the main regulator, Src can also be regulated by ligands binding to its SH3-SH2 domains (Bjorge et al., 2000; Gonfloni et al., 2000). Our data builds on prior work and suggests that Drosophila   66 Na/K ATPase regulates Src in vivo. In vitro studies identified a motif in the third cytosolic domain (NaKtide) that binds and maintains Src in an inactive state (Tian et al., 2006). The close proximity (less than 40 nm) of pSrc with a subpopulation of ATPα and the increase in pSrc with loss of the Na/K ATPase suggests this complex is present in Drosophila. The cellular consequences of loss of Na/K ATPase results in JNK mediated apoptosis and our data strongly suggest that Src42A functions with pAbl to trigger JNK. The link between the pSrc-dAbl loop and activation of MAP kinases (ERK and JNK) in the imaginal disc is well established (Singh et al., 2010). Loss of the Na/K ATPase alone leads to JNK mediated apoptosis and compensatory proliferation, but interestingly does not lead to increased migration or invasion of the dying cells into the non-apterous compartment of the wing disc. This is different from the phenotypes observed with loss of Csk, where there was substantive over proliferation, apoptosis and cell migration (Pedraza et al., 2004; Read et al., 2004; Vidal et al., 2006). This may be because dCsk is also known to regulate other kinases such as the lats/warts family of Ser/Thr kinases (Stewart et al., 2003) and the Jak/Stat pathway (Read et al., 2004). We propose that the Na/K ATPase is another regulator of the Src-pAbl pathway and loss of the ion transporter leads to the activation of the Src/Abl signaling cascade to trigger JNK-mediated apoptosis.   In summary our data has confirmed the importance of Na/K ATPase as a core component of the SJs. We have demonstrated an association between Na/K ATPase with pSrc in vivo and identified a potential mechanism for regulating SJ dynamics. Further we have demonstrated a physiological role for the Src-Na/K ATPase complex in Drosophila to control early development through negative regulation of pSrc.     67 2.5 Materials and Methods 2.5.1 Flystocks  The following fly lines were obtained from Vienna Drosophila Resource Center (VDRC): UAS-ATPα−RNAi stocks: v12330, v100619; UAS-Nrv2-RNAi stock: v2660; UAS-NrxIV RNAi stocks: v8353, v108128 (Brumby et al., 2011; Grzeschik et al., 2010). The following fly lines: UAS-Nrv2-RNAi (JF03081), Dlg::GFP (CC01936), ATPα::GFP (G00109), Nrv2.2::GFP (ZCL1649), apterous-GAL4, UAS-p35 were from the Bloomington stock centre. UAS-BskDN (Weber et al., 2000) was from the DRGC stock centre.   2.5.2 Biochemistry  Membrane preparations were carried out essentially as described (Schulte et al., 2006). Immunoprecipitations were carried out using GFP-Trap coupled to magnetic particles (Chromotek). Embryos were collected from: w1118 or ATPα::GFP fly lines (150 µg) and homogenized in 200-250 µl lysis buffer (10 mM Tris-HCl, 0.5 mM EDTA, 0.5 mM EGTA, 150 mM NaCl, 0.5% NP-40, 1x phoSTOP complete (Roche). Immunoprecipitations were carried according to the manufacturer’s protocol and then subjected to western blot analysis using the following primary antibodies: mouse anti-α5 (5ug/ml), mouse anti-Nrv (5F7)(1ug/ml), rabbit anti-pSrc42A (1/5000) (Abcam), rabbit anti-Src64B (1/500) (O'Reilly et al., 2006). Secondary antibodies used included HRP-conjugated goat anti-mouse (1:10,000) and HRP-conjugated goat anti-rabbit (1:5000) (Jackson Immunoresearch).   68 2.5.3 Immunolabeling  Imaginal discs were dissected from third instar wandering larvae into ice-cold PBS before being fixed in warm 4% Paraformaldhyde (PFA). Fixed discs were washed three times in PBST (PBS plus 0.1% Triton-X100) and blocked either for 1 hour at room temperature (RT) or overnight at 4oC with 2% normal goat serum. Discs were immunolabeled with primary antibodies for 2 hours at RT or overnight at 4oC degrees. Discs were rinsed three times, incubated with DAPI (company, 1/1000) before being mounted onto a slide with Vectashield (Invitrogen) and visualized.  Antibodies used were: Cell signaling (Massachusetts) - rabbit anti-pSrc (1/300), rabbit anti-pJNK (1/300), rabbit anti-cleaved Caspase3 (1/300); Abcam (Cambridge, UK): rabbit anti-pAbl (1/300), rabbit anti-phosphoHistone3 (1/600); Novus Biologicals (Oakville, Ca) - rabbit anti-p35 (1/1000); rabbit anti-NrxIV (1/300) (Baumgartner et al., 1996), rabbit anti-pSrc64B (1/100) (O'Reilly et al., 2006). The following monoclonal antibodies were obtained from Developmental Hybridoma Bank: anti-α5 (1/20), anti-Cora (C615-16 and C566.9)(1/600 each), anti-Dlg (4F7)(1/200), anti-Fas3 (1/100), anti-MMP1 (1/200). The following secondary antibodies were used: AlexaFluor secondary antibodies (Alexa488, 568, 647)(1/300)(Invitrogen).   Proximity ligation assay (PLA) was carried according to the manufacturer’s specifications (Sigma). Prior to PLA, wing imaginal discs were isolated, fixed in 4% PFA and incubated with primary antibodies as described above.   2.5.4 Imaging  Imaginal discs were imaged using a DeltaVision Spectris (Applied Precision, GE) with a 60x NA1.4 oil immersion lens and 200 nm Z steps. Image stacks were deconvolved by a   69 maximum likelihood algorithm using a PSF measured from a 200 nm bead conjugated to Alexa488 (Invitrogen) via SoftWorx 2.0 Software. SoftWorx was used to create side projections. ImageJ (Schneider et al., 2012) was used for image analysis and processing before being exported as tiff images for compilation in Adobe Photoshop / Illustrator CS3. Low magnification images were collected on a Zeiss Axioskop with a 20x NA 0.50 air lens using Northern Eclipse Software 8.0. 2.5.5 Statistical Analysis Cell proliferation: Maximum intensity projections of the SJ region were used with ImageJ cell counter to count the number of phosphohistone3 positive cells in the dorsal and ventral compartments of the wing pouch. Immunolabeling intensity: Using ImageJ, two equal ROI boxes were drawn around the signal on the apterous and wildtype compartment. Mean gray value was calculated on side projections and expressed as ratio of the RNAi/Wildtype. All Data was exported to Prism 6.0f for statistical analysis using a Students T-test.   70 2.6 Supplementary Data for Chapter 2  Figure 2.7: Three independent RNAi lines mediate the knockdown of ATPα  (A, B, C) En face single z-slice view and side projections of apterous-Gal4 driving three different ATPα-RNAi lines in imaginal wing discs with ATPα::GFP. Side projections of each are shown (A’, B’, C’). (A) VDRC line: v12330 (GD), (B) VDRC line: v100619 (KK), (C) TRiP line 28073 (Trp). Arrows indicate ATPα::GFP remaining at the SJ in ap>ATPα-RNAiTrp and this weaker line was not used in the analysis.  (D) Side projections of apterous-Gal4 driving ATPα-RNAiGD imaginal wing disc immunolabeled with ATPα (D’, green) and Ecad to mark the AJs (D’’, red).  ap>ATP_RNAiGD ap>ATP_RNAiKK ap>ATP_RNAiTrpATP_::GFP ATP_::GFP ATP_::GFPMerge ATP Ecad----------------------------A B CA’ B’ C’D D’ D’’  71  A) Second cytosolic domain Hs  YQEAKSSKIMESFKNMVPQQALVIRNGEKMSINAEEVVVGDLVEVKGGDRI 199 Am  YQESKSSKIMESFKNMVPQIAIVIREGEKLTLKAEELVLGDVVEVKFGDRI 211 Tc  YQESKSSKIMESFKNMVPQFATVIREGEKLTLRAEDLVLGDVVEVKFGDRI 241 Lp  YQEAKSSKIMDSFKNMVPQYAVVVRSGEKLNVRAEELVVGDMVEVKFGDRI 204 Dm  YQESKSSKIMESFKNMVPQFATVIREGEKLTLRAEDLVLGDVVEVKFGDRI 217     YQEAKSSKIMESFKNMVPQ A VIR GEKM I AEEVVVGDLVEVK GDRI  Hs  VEGTARGIVVYTGDRTVMGRIATLASGLEGGQTPIAAEIEHFIHIITGVA 299 Am  VEGTAKGVVICCGDQTVMGRIAGLASGLDTGETPIAKEIHHFIHLITGVA 311 Tc  VEGTAKGVVISCGDNTVMGRIAGLASGLDTGETPIAKEIHHFIHLITGVA 341 Lp  VEGTCVGLVVKTGDKTVMGRIANLASGLEVGETPIAKEIAHFIHLITGVA 304 Dm  VEGTAKGVVISCGDHTVMGRIAGLASGLDTGETPIAKEIHHFIHLITGVA 317     VEGT  GIVV  GD TVMGRIA LASGLE G TPIAKEI HFIHLITGVA  B) Third cytosolic domain NaKtide               SATWLALSRIAGLCNRAVFQ  Hs     ADTTENQSGVSFDKTSATWLALSRIAGLCNRAVFQANQENLPILKRA 446 Am     ADTTEDQSGLQYDRTSPGFKALAKIATLCNRAEFKAGQEDKPILKRE 458 Tc     ADTTEDQSGVQYDRTSPGFKALSRIATLCNRAEFKGGQNDVPILKRE 488 Lp     ADTSEDQSNATYNKDTADWIALSRIAMLCNRAEFKAGQDNVPVLKKE 451 Dm     ADTTEDQSGVQYDRTSPGFKALSRIATLCNRAEFKGGQDGVPILKKE 464        ADTTENQS   FD  S  W ALSRIA LCNRA F A QE  PILKR Figure 2.8: Alignments of the second and third cytosolic domains of ATPα  from a range of species  Comparison of the amino acid sequence of (A) the second cytoplasmic domain and (B) third cytoplasmic domain of the Na/K ATPase α subunit sequence between a range of invertebrate α subunits: Ap: Apis mellifera (honeybee), Tc: Tribolium casteneum (beetle), Lp: Loligo pealei (squid), Dm: Drosophila melanogaster (fruit fly) and the human α1a (Hs). Conserved amino acids are aligned below. Alignment through Clustal omega. Identical amino acids are bold. A420 and A425, required for Src interaction with the α subunit, are indicated in red. Subunits mutant for the conserved Ala residues (A420P, A425P) are incapable of binding and regulating Src (Lai et al., 2013). The identified NaKtide peptide that binds Src and prevent the formation of Src-Na/K ATPase complex (Li et al., 2009) is also shown.     72  Figure 2.9: Distribution of Src proteins and effect of ATPα  on EGFR and FAK activation (A) En face single z-slice view of a 3rd instar imaginal discs with Src64B endogenously tagged with YFP (Src64B::YFP) (A’, green) and immunolabeled with Dlg to show the membrane (A’’, red). Src64B appears in puncta within the AJ and SJ domains.   (B) En face single z-slice view of a 3rd instar imaginal discs with ATPα::GFP (A’, green) and immunolabeled with Src42A (A’’, red). Src42A immunolabeling appears mainly cytosolic.  (C,D) Side projections of apterous-Gal4 driving ATPα-RNAiGD with ATPα::GFP (C) or Scrib::GFP (D) to mark the SJ domain with immunolabeling for activated EGFR (C, red) or phosphorylated FAK (D, red).  (E) Immunoprecipitations (IP) from embryo extracts of control (w1118), ATPα::GFP or Nrv2::GFP using GFP-Trap magnetic beads. Western blots from lysates or IPs were probed with antibodies to the Src64B C-terminus (Src64BCT). Src64B was detected in the lysates but undetectable in IP complexes with ATPα or Nrv2.  Src64B::YFPSrc64::YFP DlgMergeATPA::GFPATPA::GFP Src42Aw1118ATPA::GFPNrv2::GFPw1118ATPA::GFPNrv2::GFPLysateSrc64BCTIPMerge ATPA::GFP pEGFRap>ATPA-RNAiGD--------------Merge Scrib::GFP pFAKDA A’ A’’ E’’B B’ B’’C C’ C’’250150100755037  73 Chapter 3: Differential Interactions Between the Core SJ domain Proteins, NrxIV and Na/K ATPase, and the Tricellular Junction 3.1 Synopsis  Permeability barriers form between cells and are essential to prevent both fluid flow and pathogen invasion across tissues including the epidermis, intestine, and brain. These barriers are called tight junctions (TJ) in vertebrates and septate junctions (SJ) in invertebrates. Spanning the membranes of two adjacent cells are protein strands that adhere to one another to create tight, impermeable junctions. Na/K ATPase is part of the core complex of proteins along with Coracle, Neuroglian, Neurexin IV (NrxIV), and Macroglobulin complement-related that are required for the formation of SJs. At the sites where the SJs of three or more cells meet, SJ components overlap with proteins that constitute the Tricellular Junctions (TCJs) of which the only known component is Gliotactin (Gli). However, the mechanism by which Na/K ATPase interacts with other barrier components to establish, maintain and regulate these junctions is unknown. We have examined the consequences of loss of Na/K ATPase in the context of several barrier components and developed a model by which Na/K ATPase may be contributing to SJ function. We show that RNAi knockdown of Na/K ATPase in the imaginal wing disc of Drosophila leads to increased immunolabeling of Gli and Dlg and an expansion of their basolateral domain at the TCJ, a relationship unique to the Na+/K+ ATPase. Loss of the canonical core component NrxIV results in a basolateral spread and reduction of Gli immunolabeling. These differential effects also extend to interactions with ectopic Gli. The deleterious consequences triggered by spread of Gli away from the TCJ were suppressed by loss of Na/K ATPase and enhanced by loss of NrxIV.   74 These data suggests that Na/K ATPase has a unique role in both the regulation and maintenance of the TCJ.  3.2 Introduction  Invertebrate SJs are classically defined as structures important in both the establishment and maintenance of the paracellular barrier and apical-basal polarity. In addition, these junctions are also recognized as multi-functional platforms required for a variety of cell functions including cell adhesion, cell proliferation, and tubulogenesis (reviewed in (Izumi and Furuse, 2014; Tepass et al., 2001)). SJs are present in all ectodermally derived epithelia as well as the central and peripheral nervous system. In each of these cell types the SJs are organized and formed through a sophisticated complex of macromolecular interactions to regulate their tissue specific functions, trafficking and modification of the SJ proteins. However, the basic mechanisms of how SJs are organized and formed, and how SJ proteins are modified and trafficked, have not been described.    Over 20 different genes have been identified to be important in structure and /or function of SJs, yet little is known about how these proteins interact with each other. SJ proteins can be grouped into core SJ proteins, which are required for both formation of the septae and correct organization, and SJ-associated proteins, which are required for correct organization of SJs (Tepass et al., 2001). The core SJ proteins are Neurexin-IV (NrxIV), Coracle (Cora), Neuroglian (Nrg), Varicose, the α and β subunits of Na/K ATPase (ATPα and Nrv2), Macroglobulin complement-related protein (Mcr) and Contactin (Cont) (Bachmann et al., 2008; Batz et al., 2014; Baumgartner et al., 1996; Faivre-Sarrailh, 2004; Genova and Fehon, 2003; Hall et al., 2014; Moyer and Jacobs, 2008; Ward et al., 1998). The SJ-associated group of proteins include:   75 the claudins Megatrachea (Mega), Sinuous (Sinu), and Kune Kune (Kune); Ly6 proteins Crooked (Crok), Crimpled (Crimp), and Coiled (Cold), Boudin (Bou); and the basolateral polarity complex members Scribble (Scrib), Discs Large (Dlg), and Lethal giant larvae (Lgl) (Behr et al., 2003; Bilder et al., 2002; Hijazi et al., 2009; Nelson et al., 2010; Nilton et al., 2010; Tepass et al., 2001; Wu et al., 2004).  Genetic, biochemical and FRAP analyses have demonstrated that the core transmembrane proteins, NrxIV, Nrg, ATPα and Nrv2, form an interdependent complex with a very slow turnover of components (Genova and Fehon, 2003; Oshima and Fehon, 2011). Genetic analysis has suggested these proteins are mutually dependent on each other for correct targeting to the SJ domain and for formation of SJs. However, while the Na/K ATPase interacts in a complex with NrxIV and Cora, it does not require Cora for localization nor does it associate directly with Nrg (Genova and Fehon, 2003; Paul et al., 2003). Conversely, NrxIV is considered a canonical SJ structural protein. It is the first known protein to accumulate at the SJ domain and NrxIV is required for correct trafficking of SJ components and interacts in a complex with Cora, Nrg, and Na/K ATPase (Baumgartner et al., 1996; Genova and Fehon, 2003; Paul et al., 2003; Tepass and Hartenstein, 1994). Recent studies demonstrated that loss of individual core proteins had distinct effects further supporting the possibility of subdomains within the bicellular SJ architecture (Hall et al., 2014; Nelson et al., 2010). Null mutations in Mcr led to apical relocalization of Nrg and removal from the SJ domain (Batz et al., 2014; Hall et al., 2014). In contrast, Cora is basolaterally mislocalized in either Mcr or Nrg mutants (Hall et al., 2014; Nelson et al., 2010; Wu et al., 2007). Similarly, mutations in each of the Drosophila SJ-associated claudin family members have distinct effects on the levels and localization of Cora (Wu et al., 2004; Wu et al., 2007). Whether subdomains exist within the bicellular junction remains to be confirmed, but SJs   76 do contain a unique subdomain at the contact site of three or more epithelial cells called the tricellular junction (TCJ).   The TCJ is formed by SJ strands that turn 90o at the corner of the cell and then run basally, parallel to the lateral membrane creating a small channel between the cells. This channel is plugged by a series of lens-shaped diaphragms called tricellular plugs (Fristrom, 1982; Noirot-Timothée et al., 1978). Despite ultrastructural identification of the TCJ over 50 years ago, much of its molecular composition remains unknown. Only one Drosophila protein, Gliotactin (Gli), has been identified specifically at the TCJ. Mutations in Gli lead to mislocalization of SJ proteins, a failure of septae to compact, loss of the permeability barrier, and embryonic lethality (Schulte et al., 2003). The TCJ is a unique subdomain of the SJ as it is made by the convergence of three SJs (Fristrom, 1982) and contains the SJ-associated protein Dlg along with Gliotactin (Schulte et al., 2003; Schulte et al., 2006). While Gliotactin is not required for the formation of the core complex, it is required for proper maturation and assembly of proteins into SJs through unidentified protein associations (Schulte et al., 2003). How the TCJ interacts with the bicellular junctions and the core SJ proteins is unknown. While Gli does not directly bind with either the structural SJ proteins, NrxIV, Cor, or Nrg, the interaction with the Na/K ATPase has not been investigated.    Overall the composition and differential function of the SJ subdomain(s) and how SJ proteins interact with the TCJ is not well studied. Biochemical and genetic analyses suggest that Na/K ATPase and NrxIV may have distinct functions in SJ assembly and this may extend to the interaction with the TCJ. However, the roles played by the Na/K ATPase or NrxIV in recruitment of TCJ proteins such as Gliotactin and the SJ-associated protein Dlg are unknown.      77  In this study, we have begun to determine if there are differences in how the Na/K ATPase and NrxIV interact with the other components of the bicellular SJ and the tricellular junction proteins Gli, and the MAGUK protein Dlg. Using a combination of RNAi and genetic interactions, we demonstrated that the loss of Na/K ATPase has distinct effects on Gli and Dlg specifically at the TCJ. Both Gli and Dlg levels increased at the TCJ when Na/K ATPase was reduced. Conversely, loss of NrxIV led to a uniform reduction and mislocalization of Dlg from the SJ domain and reduction of Gli from the TCJ. We found that loss of ATPα versus NrxIV also had differential effects when Gliotactin was overexpressed and spread within the bicellular SJ domain. Overall all our results point to a function for the Na/K ATPase with the TCJ complex distinct from NrxIV, and suggest that Na/K ATPase has unique roles in organizing SJ-associated proteins into the SJ domain and regulating the endocytosis of Gli.  3.3 Results 3.3.1 Na/K ATPase and NrxIV are Required for SJ Maintenance in the Imaginal Wing Disc   NrxIV and Na/K ATPase are part of the core complex proteins required for the establishment of SJs, but as Na/K ATPase has the potential to mediate signal transduction cascades, we investigated if these two proteins had differential associations and roles within the SJ domain. The requirement of NrxIV and Na/K ATPase for the formation of SJs and the interdependence of the core complex limited the use of null mutants. Null mutants do not survive past embryogenesis and somatic clones of null alleles are cell lethal and removed from epithelium (Baumgartner et al., 1996; Genova and Fehon, 2003; Lamb et al., 1998; Paul et al., 2003). To overcome these issues, we used the Gal4/UAS system (Brand and Perrimon, 1993)   78 together with RNAi transgenes to knock down NrxIV or Na/K ATPase in the columnar epithelia of the imaginal wing disc. Specifically, we used apterous-GAL4 (ap-Gal4) to drive expression of RNAi in the dorsal half of 3rd instar wing imaginal discs to knock down either NrxIV or each subunit of the Na/K ATPase pump (ATPα or Nrv2) individually. In these experiments the unmodified ventral portion of the wing disc served as an internal control. In Drosophila, the beta subunit nervana2 has two isoforms both of which localize to the SJ domains in the wing disc. The two proteins (Nrv2.1, Nrv2.2) have not been shown to have any functional differences to date and will be referred collectively as Nrv2 (Paul et al., 2003; Paul et al., 2007; Sun and Salvaterra, 1995a; Sun and Salvaterra, 1995b). For all three proteins (NrxIV, ATPα and Nrv2), two different UAS-RNAi constructs targeting distinct regions of each mRNA were used to ensure observed phenotypes were due to specific knockdown of the target mRNA.   Initial experiments were done to test the hypothesis that the RNAi-mediated knockdown phenotypes would be the same as those observed in somatic null clones and mutant embryonic epithelium. RNAi knockdown of NrxIV or ATPα effectively removed each protein and its respective binding partner (Cora, Nrv2) from the SJ domain (Figure 3.1A, G, I). In addition to depletion of its binding partner, Cora, RNAi knockdown of NrxIV leads to a complete loss of the other core SJ proteins, ATPα and Nrv2, from the SJ domain and the plasma membrane. These results confirm the requirement of NrxIV for the recruitment and localization of other core components and validate use of the RNAi system (Figure 3.1F, G). Loss of ATPα leads to a loss of the apical enrichment of SJ proteins creating a basolateral spread of the core SJs proteins, NrxIV and Cora, and an overall reduction of the immunofluorescence signal (Figure 3.1C, D). RNAi knockdown of Nrv2 phenocopied ATPα − RNAi and led to a loss of both NrxIV and Cora from the SJ domain, along with a basolateral spread (data not shown). Mcr is a newly identified   79 SJ protein required for junction formation and epithelial barrier function (Batz et al., 2014; Hall et al., 2014). When we examined Mcr localization in either NrxIV-RNAi or ATPα-RNAi expressing wing discs, Mcr immunofluorescence was no longer apically enriched as previously observed (Batz et al., 2014; Hall et al., 2014) (Figure 3.1E, I). These data demonstrate that NrxIV is required for correct localization of the core SJ proteins to the SJ domain and for assembly of SJs. In contrast, loss of Na/K ATPase leads to a basolateral expansion of the SJ domain suggesting that the Na/K ATPase is dispensable for localization of the SJ complex to the membrane but is required for their correct assembly within the SJ domain. The data supports the suggestion (Hall et al., 2014) that each individual core SJ protein is not strictly interdependent on the other, as originally thought, but that SJ sub-complexes exist. Further, the data demonstrates a high degree of interdependence among sub-complex members but less dependence between sub-complexes.  Core SJ proteins were differentially affected when NrxIV was knocked down compared to Na/K ATPase. These differential effects on the core components led us to question whether Na/K ATPase and NrxIV would have different effects on SJ-associated proteins. We looked at the effect of loss of either NrxIV or ATPα on the SJ-associated proteins Dlg and Fas3. NrxIV RNAi knockdown led to a uniform reduction of immunolabeled Fas3, but Fas3 was still observed in the SJ domain (Figure 3.1H, Figure 3.7). This phenocopies the previous observations with cora loss of function somatic clones (Batz et al., 2014; Baumgartner et al., 1996; Genova and Fehon, 2003; Hall et al., 2014). Fas3 is a transmembrane protein with homophilic adhesion properties but is not critical to the formation of SJ domains as null alleles are viable (Han et al., 2000; Snow et al., 1989). In contrast to NrxIV-RNAi, ATPα RNAi knockdown led to a complete loss of Fas3 from the SJ domain (Figure 3.1B, Figure 3.7). Localization of Fas3 to the SJ domain   80 is dependent on Dlg (Woods et al., 1996), therefore the loss of Fas3 in response to ATPα-RNAi raised the possibility that Na/K ATPase may interact with Dlg and Fas3. We compared the effects of RNAi knockdown of NrxIV or ATPα on Dlg to determine whether loss of Na/K ATPase has a differential relationship with Dlg. Wing epithelia expressing NrxIV-RNAi had a reduction in immunolabeled Dlg within the SJ domain (Figure 3.2B). Examination of surface plots of the SJ region indicated that Dlg staining is uniformly reduced in voxel intensity at the SJ domain, with a basolateral spread of Dlg (Figure 3.2D). RNAi mediated knock down of ATPα had a differential effect on Dlg. Intriguingly, Dlg was lost or reduced from the bicellular SJ but was retained at the tricellular junction at the corner of the cells (Figure 3.2A). With ATPα-RNAi expression, Dlg spread basolaterally with an increase in voxel intensity that was greatest at the tricellular corners (Figure 3.2C). We measured the mean gray value of immunolabeled Dlg at the SJ region in WT and ATPα-RNAi compartments (Figure 3.2E) and expressed it as ratio of apterous:WT. The ratio of Dlg intensity between the apterous and WT compartments did not significantly differ from 1, despite a reduction in the immunolabeling of Dlg at the bicellular junction. Since the overall mean gray value of Dlg at the SJ domain did not change even though Dlg signal was lost from the bicellular junction, these results suggest that Dlg is increasing at the TCJ and not simply retained there. This novel finding of Dlg recruitment to the TCJ in response to loss of Na/K ATPase (compared to the loss of NrxIV) suggests that the Na/K ATPase has a unique function or set of interactions in the TCJ subdomain.   81  Figure 3.1: Expression of ATPα-RNAi or NrxIV-RNAi leads to mislocalization or loss of SJ proteins.  Side projections of the SJ region from 3rd instar imaginal wing discs with apterous-GAL4 driving the expression of ATPα-RNAi (GD12330: A,B; KK100619: C-E) and NrxIV-RNAi (GD8353: F-H; KK108128: I). Scale bar 15um. Merge---------- Nrv2::GFP---------- ATPAMerge--------- ATPA::GFP--------- FasIIIMerge--------- mCD8::GFP NrxIV---------Merge--------- mCD8::GFP Cora---------Merge--------- mCD8::GFP--------- McrATPAKKATPAGD--------- Nrv2::GFP--------- NrxIVMergeMerge------------------Nrv2::GFP FasIIIMerge Mcr Cora------------------Merge ATPA Ecad------------------NrxIVKKNrxIVGDA’’A A’B B’ B’’C C’ C’’D D’ D’’E E’ E’’F F’ F’’G G’ G’’H H’ H’’I I’ I’’  82 (A-B) RNAi knockdown of ATPα removed both ATPα  (A’’, red) immunolabeling and Nrv2::GFP (A’, green) from the SJ domain. ATPα-RNAi removed ATPα endogenously tagged with GFP (ATPα::GFP) (B’) and Fas3 (B’’, red) immunolabeling from the SJ domain.  (C-E) Expression of ATPα-RNAi with UAS-mCD8::GFP (C’,D’,E’, green). Knockdown of ATPα led to a reduction and basal lateral spread of NrxIV (C’, red), Cora (D’, red) and the loss of Mcr (E’ red).  (F) RNAi knockdown of NrxIV removed ATPα (F’’, green) immunolabeling but did not alter the localization or intensity of E-cadherin immunolabeling (F’, red).  (G,H) RNAi knockdown of NrxIV in a Nrv2::GFP (G’, green) wing disc immunolabeled with NrxIV (G’’, red) or Fas3 (H’’, red). NrxIV-RNAi expression removed Nrv2::GFP (G’,H’, green), immunolabeling for NrxIV (G’’, red) and Fas3 from the SJ domain (H’’, red).  (I) RNAi knockdown of NrxIV led to loss of Mcr (I’, green) and Cora (I’, red) immunolabeling.    83  Figure 3.2: RNAi knockdown of ATPα  has differential effects on Dlg compared to RNAi knockdown of NrxIV. (A-B) Maximum intensity projections and side projections of the SJ region of 3rd instar imaginal wing discs using apterous-GAL4 to drive the expression of ATPα-RNAi (A) or NrxIV RNAi (B). Boxes indicate the areas used for surfaces plots in C and D. Scale bar 15um. Merge NrxIV Dlg--------------NrxIVGDMerge ATPA::GFP Dlg--------------ATPAGDDlg DlgPixel Intensityz Depthx axis positionATPAGDPixel Intensityz Depthx axis positionNrxIVGD ATPA  ATPA 0.00.51.01.52.0 Ratio of ap/WT DlgKKGDA A’ A’’DCB B’ B’’E  84 (A) Expression of ATPα-RNAi in a ATPα::GFP (A’, green) wing disc and immunolabeled with Dlg (A’’, red). Dlg was reduced and spread basolaterally in the bicellular junction, but increased at the TCJ (arrowhead).  (B) RNAi knockdown of NrxIV (B’, green) leads to a reduction and basolateral spread of Dlg immunolabeling (B’’, red) without increased signal intensity (arrow).  (C,D) Surface plots of the pixels intensity plotted as a function of the z-depth and x-axis position for Dlg in an ATPα-RNAi (C) or NrxIV-RNAi (D) wing disc. Arrows indicate the apterous boundary between the RNAi and WT compartments.  (C) With ATPα-RNAi, Dlg signal intensity remained the same or higher with a periodicity similar to the TCJ. (D) With NrxIV-RNAi, Dlg signal intensity was reduced and spread along the z-axis.  (E) Statistical analysis of the Dlg changes with two different ATPα-RNAi lines (GD and KK). The mean gray value of Dlg immunolabeling from equivalent areas of the RNAi and WT compartments expressed as ratio of apterous/WT. A students t-test was used and the ratio did not significantly differ from 1 indicating there is no significant difference in Dlg immunofluorescence between the WT and RNAi compartments (GD, mean= 1.098, n=12 discs; KK, mean=0.9873, n=9 discs)(means + SEM).    85  3.3.2 Na/K ATPase has a Unique Relationship With the TCJ Protein Gliotactin  The differential results of loss of ATPα versus NrxIV on Dlg at the TCJ led us to test if these results extended to the TCJ protein Gliotactin (Gli). Gli is in a protein complex with Dlg and is recruited to the TCJ by Dlg (Padash-Barmchi et al., 2013; Schulte et al., 2003; Schulte et al., 2006). NrxIV-RNAi resulted in a basolateral spread of the immunolabeled Gli similar to the other SJ-associated proteins, Dlg and Fas3 (Figure 3.3B). Surface plots of the SJ region show a decrease in voxel intensity of Gli immunolabeling along the basolateral axis (Figure 3.3D), suggesting the same amount of protein spread to occupy a larger area. Surprisingly, ATPα-RNAi elicited an increase of immunolabeled Gli at the TCJ along with basal spreading (Figure 3.3A). Surface plots of the SJ regions showed an increase in voxel intensity in Gli along the basolateral axis of the TCJ (Figure 3.3C) suggesting more Gli was present. We measured the mean gray value of Gli in both compartments of the wing discs and compared the intensity levels between the apterous and WT compartment. The ratio of Gli mean gray value in apterous / WT was significantly greater than 1, demonstrating a significant 2-fold increase in the level of Gli (p=0.0006 GD-RNAi, and p=0.0015 KK-RNAi) (Figure 3.3E). The recruitment of Dlg to the TCJ and increase in Gli voxel intensity was also seen with Nrv2-RNAi knockdown (Figure 3.8). These data suggests that the Na/K ATPase may be part of the mechanism regulating the level of Gli at the TCJ.   The increase in Dlg and Gli at the TCJ in response to RNAi knockdown of either subunit of Na/K ATPase, suggests the Na/K ATPase may be a critical component of the regulatory mechanism that controls Gliotactin and Dlg levels at the TCJ. To see if this interaction was reciprocal we next tested if there were differential effects of loss of Gli on NrxIV versus the   86 Na/K ATPase. We utilized a Gli-RNAi transgene to knock down Gli in wing discs where either Nrv2 or ATPα was endogenously tagged with GFP (ATPα::GFP, Nrv2::GFP) and were immunolabeled for NrxIV (Figure 3.4) . RNAi mediated knockdown of Gli was highly effective (Figure 3.4A”) and led to increased levels of ATPα::GFP, Nrv2::GFP and NrxIV (Figure 3.4A, B) along with the basolateral spread of all three proteins. Surface plots of the SJ region showed an increase in voxel intensity of all three SJ proteins along the basolateral axis (Figure 3.4C-E).  Loss of Gli in embryonic epithelia led to the basolateral spread of SJ proteins and septa (Schulte et al., 2003) but did not affect the stability and turnover of the core SJ complex (Oshima and Fehon, 2011), suggesting that Gli is necessary to consolidate the SJ strands in the apical domain but not the formation of the core complex. The similar effect of Gli knockdown on all three SJ proteins supports this hypothesis. The increase in the levels of the SJ proteins was unexpected and may indicate that Gli has a role in the trafficking or endocytosis of SJ proteins during the maturation and maintenance of SJs (Oshima and Fehon, 2011; Padash-Barmchi et al., 2010).    Na/K ATPase and Gli are important in the formation, maturation and maintenance of SJs as null mutants of ATPα, nrv2 or Gli lead to a loss of the permeability barrier due to either absent or malformed SJs (Genova and Fehon, 2003; Schulte et al., 2003). To test for a genetic interaction between Na/K ATPase and Gliotactin, we examined trans-heterozygotes of GliDV3 with ATPαDTS2R3 or NrxIV4304 loss of function alleles and found no defects in the imaginal wing morphology or SJ protein localization (Table 3.1). This suggests that there is tolerance for reduction of both gene products and that their genetic relationship is neither epistatic nor at the point of convergence of two genetic pathways. An alternative possibility is that both Gli and ATPα act at the same level of a genetic pathway in a cooperative balance.     87  We have previously reported that Dlg is the only known SJ protein shown to associate with Gli in a complex, but Gli does not associate with NrxIV, Cora or Nrg at the TCJ (Schulte et al., 2006). The changes observed in Gli intensity levels in response to RNAi knockdown of either Na/K ATPase subunit, suggested that the Na/K ATPase might be capable of associating with Gli. We tested the hypothesis that Gli is in a complex with either ATPα or Nrv2 subunits by co-immunoprecipitation analysis. Anti-GFP conjugated magnetic beads were used to immunoprecipitate proteins from late stage embryonic extracts of either ATPα::GFP or Gli::YFP, and isolated complexes were assayed for the presence of either Nrv2 or Gli. We were unable to detect an association between Gli and the ATPα subunit of the Na/K ATPase (Figure 3.4F), while we were able to successfully detect the association between Nrv2 and ATPα (Figure 3.4F). These data suggest that Gli may not be part of a complex with the Na/K ATPase or that any association is below the level of detection.    88  Figure 3.3: RNAi knockdown of ATPα  has differential effects on Gli compared to RNAi knockdown of NrxIV knockdown.  (A-B) Maximum intensity projections and z-projections of the SJ region of 3rd instar imaginal wing discs with apterous-GAL4 driving ATPα-RNAiGD (A) NrxIV RNAiGD (B). Boxes indicate the area used for surface plots in C and D. Scale bar 15µm. Merge ATPA::GFP Gli---------------------Merge Nrv2::GFP Gli-------Pixel Intensityz Depthx axis positionATPAGDGlix axis positionz DepthPixel IntensityGliA A’ A’’DCB B’ B’’ENrxIVGDATPAGDNrxIVGD012345 ATPA  ATPA KKGDRatio of ap/WTGlip=0.0006 p=0.0015   89 (A) Wing disc from an ATPα::GFP (green) wing disc immunolabeled with Gli (A”, red). Expression of ATPα-RNAiGD led to a basolateral spread and increase in Gli immunolabeling at the TCJ (arrowhead).  (B) Nrv2 endogenously tagged with GFP (Nrv2::GFP)(B’, green) and immunolabeled for Gli (B’’, red). RNAi knockdown of NrxIVGD led to a reduction and basolateral spread of Gli immunolabeling (arrowhead).  (C,D) Surface plots of the pixels intensity plotted as a function of z –axis and the x-axis position for Gli in an ATPα-RNAiGD (C) or NrxIV-RNAiGD (D) wing disc. Arrows indicate the apterous boundary between the RNAi and WT compartments.  (C) Within the ATPα-RNAi compartment, Gli signal intensity remained the same or increased as it spreads along the basolateral axis of the TCJ.  (D) Within the NrxIV-RNAi compartment, Gli signal intensity was reduced and spread along the z-axis.  (E) Statistical analysis of Gli immunofluorescence in ATPα-RNAi wing discs. The mean gray value from equivalent areas of the RNAi and WT compartments was measured, and the RNAi compartment was normalized to the WT. There was a significant difference (students t-test) between the RNAi vs. WT compartments in both ATPα-RNAi lines tested (GD, mean= 1.749, n=14 discs; KK, mean=2.596, n=9 discs)(means + SEM).    90  Figure 3.4: RNAi knockdown of Gli increases the immunofluorescence of the SJ proteins ATPα , Nrv2 and NrxIV Side projections of the SJ region from 3rd instar imaginal wing discs with apterous-GAL4 driving the expression of Gli-RNAi. (A) Wing disc with endogenously tagged ATPα::GFP (A’, green) immunolabeled with Gli (A”, red). Gli-RNAi knocked down Gli immunolabeling and led to a basolateral spread plus increased fluorescence of ATPα::GFP (arrow). (B) Wing disc with endogenously tagged Nrv2::GFP (A’, green) immunolabeled with NrxIV (A”, red). Expression of Gli-RNAi led to a basolateral spread and increased signal intensity of Nrv2::GFP and NrxIV immunolabeling (arrow). (C-E) Surface plots of the pixels intensity plotted as a function of z –axis and the x-axis position for ATPα::GFP (C), Nrv2::GFP (D) or NrxIV (E) in a Gli-RNAi wing disc. Arrows indicate the apterous boundary between the RNAi and WT compartments. Within the Gli-RNAi Merge Gli---------------- ATPA::GFPMerge--------- Nrv2::GFP NrxIV---------Pixel Intensityz Depthx axis positionPixel Intensityz Depthx axis positionPixel Intensityz Depthx axis positionATPA::GFP Nrv2::GFP NrxIVGliRNAiGDA A’ A’’B B’ B’’CFD EGli::YFPATPA::GFPGliYFPATPA::GFPGliYFPATPA::GFPGlianti-Glianti-Nrv2Nrv2Lysate IP IP250150100755037  91 compartment, ATPα-GFP(C), Nrv2::GFP (D) and NrxIV (E) signal intensity remained the same or increased and spread along the basolateral axis. (F) Immunoprecipitations (IP) from embryo extracts of Gli endogenously tagged with YFP (Gli::YFP) or ATPα endogenously tagged with GFP (ATPα::GFP) using GFP-Trap magnetic beads. Western blots from lysates or IPs were probed with antibodies to Nrv2 and Gli. Nrv2 was immunoprecipitated in a complex with ATPα::GFP, but Gli was not detected. Table 3.1: Transheterozygotes of GliDV3 with NrxIV4304 or ATPαDTSR3 display normal wing discs  Genotype Number of discs Wing defects Gli[dv3]/+; NrxIV[4304]/+ 8 no Gli[dv3]/+; ATPα[DTSR3]/+ 9 no Wing imaginal discs from transheterozygotes of GliDV3 with NrxIV4304 or ATPαDTSR3 were dissected and examined for alterations in the localization of SJ proteins. SJ protein localization and wing disc morphology of transheterozygotes were indistinguishable from wildtype.  3.3.3 Na/K ATPase Suppresses Migration of Apoptotic Cells in a Gli Overexpression Model  The unique changes to Gli levels with loss of Na/K ATPase subunits compared to NrxIV, suggested Na/K ATPase is capable of modulating Gli levels. As the Gli-RNAi knockdown experiments failed to show differential effects on NrxIV versus Na/K ATPase, we chose to address this question using a Gli overexpression assay. Overexpression of Gli using apterous-GAL4 in the columnar epithelial of the wing imaginal disc leads to the spread of Gli into the bicellular SJ domain. This triggers tyrosine phosphorylation and ubiquitin mediated endocytosis of Gli with subsequent targeting for lysosomal degradation (Padash-Barmchi et al., 2010). The cellular consequences of Gli overexpression include over-proliferation, apoptosis and delamination, and leads to the migration of Gli overexpressing cells into the non-apterous ventral side of the wing disc. The degree of cell migration into the ventral half of the disc can be   92 quantified and has been utilized to identify genes that modulate Gli levels including the JNK pathway and Dlg (Padash-Barmchi et al., 2010; Padash-Barmchi et al., 2013)(Figure 3.5B). To test the effects that loss of SJ proteins have on Gli overexpression, we over-expressed a full-length Gli transgene (GliWT) in heterozygous mutants where ATPα, Nrv2, or NrxIV were reduced by 50% using loss of function alleles (ATPαDTSR3, nrv2nwu5 or NrxIV4304). We utilized a temperature sensitive GAL80 to temporally control Gli expression and overcome embryonic lethality by shifting to 29oC to permit GAL4 expression starting in the first larval instar. After 48 hr at 29oC, the spread of Gli overexpressing cells into ventral portion of the wing disc averaged 25% of the overall ventral compartment length (Figure 3.5B, F). Both ATPαDTSR3/+ or nrv2nwu5/+ heterozygotes fully suppressed the ectopic Gli phenotypes. We observed no Gli expressing cells beyond the apterous boundary (0% migration) (Figure 3.5D-F). Conversely, NrxIV4304/+ heterozygotes showed enhancement of the GliWT phenotype with Gli overexpressing cells migrating significantly farther distances into the ventral compartment, 40.14% of the ventral compartment (Figure 3.5C, F). These data suggest that Na/K ATPase has differential interactions with Gli within the bicellular septate junction compared to NrxIV.   Ectopic GliWT expression in an otherwise wildtype genetic background leads to increased cell proliferation, and consequently to ectopic folds, in addition to the apoptosis and migration of cells into the wildtype compartment. Therefore, we also examined imaginal wing discs of GliWT within an nrv2 or ATPα heterozygous mutant for phenotypic changes and compared them to control discs with GliWT overexpression in a wildtype background. E-cadherin immunolabeling was unchanged on both dorsal and ventral sides of all the wing discs (Figure 3.6C, D), indicating that AJs are unaffected, regardless of the presence or absence of loss of function alleles. We observed no ectopic folds and decreased numbers of pyknotic nuclei,   93 suggesting a reduction of cell proliferation and apoptosis, respectively, when GliWT is overexpressed in either ATPα or nrv2 heterozygotes (Figure 3.6C’’’, D’’’). We also observed an increase in the number and size of Gli-containing vesicles compared to GliWT alone (Figure 3.6E’’, F’’). This data suggests the reduction of Na/K ATPase by 50% rescues the Gli-overexpression mediated proliferation, apoptosis and cell migration by enhancing the mechanism by which Gli is phosphorylated and endocytosis.     94 Figure 3.5: Loss of function alleles of ATPα  or nrv2 suppress the GliWT phenotype, while NrxIV loss of function enhance Ectopic expression of GliWT transgene with apterous-GAL4 (ap>GliWT) in SJ mutant backgrounds. (A) WT, (B) ap>GliWT, (C-E) ap>GliWT in trans with either (C) NrxIV4304, (D) nrv2nwu5 or (E) ATPαDTS1R3 loss of function alleles.   (B) Ectopic expression of Gli led to ectopic folds, cell death and migration of the Gli expressing cells beyond the apterous border. The yellow double arrow line indicates the distance migrated from the D-V boundary. The white line indicates the distance from D-V boundary to the ventral edge of the disc.  (C) GliWT driven in trans with NrxIV4304 loss of function alleles enhanced the migration phenotype.  (D, E) Ectopic expression of GliWT in trans with loss function alleles for (D) nrv2 or (E) ATPα  completely suppressed the migration of Gli expressing cells.  (F) Statistical analysis (pairwise comparison) of the distance travelled beyond the D-V boundary by Gli expressing cells. Expression of Gli in trans with NrxIV4304 almost doubled the distance migrated from an average of 25% of the ventral compartment to 40%, a significant increase (n=10 discs). There was no migration in either the nrv2nwu5 (n=6 discs) or ATPαDTS1R3 (n=4 discs), a significant different compared to GliWT alone. Bars represent means + SEM.    95  Figure 3.6: Loss of function of Na/K ATPase reduces cell death in wing imaginal discs expressing ectopic Gli.  Imaginal wing discs with apterous-GAL4 driving GliWT (ap>GliWT) alone (A, C) and in a nrv2nwu5 heterozygote (B, D). wg-lacZ (blue, A,C) marks the non-mutant discs. (A,B) 20x images of wing disc immunolabeled for Ecad (green) and Gli (red). (A) GliWT alone triggers migration of cells away from the D-V boundary (double arrow). (B) Removal of 50% of nrv2 suppresses the migration.  (C,D) Single slice of the SJ domain (C,D) or 10 um basal to the SJ domain (C’’’,D’’’) in a wing imaginal disc immunolabeled for Ecad (green), Gli (red) and either wg-lacZ (blue, C) or DAPI (blue, D).  (C) ap>GliWT. Ectopic Gli spread away from the TCJ and into the bicellular junction. Gli expressing cells migrated into the ventral compartment (arrowhead). (C’’’) DAPI labeling showed extensive pyknotic nuclei.    96 (D) ap>GliWT, nrv2nwu5. nrv2nwu5 suppressed the migration of Gli expressing the cells (n=5 discs) but did not prevent Gli from spreading into the bicellular junction. (D’’’) Single slice 10 um below the SJ domain indicated less pyknotic nuclei were present in a suppression of cell death.  (E) 350x digital magnification of the boxed regions in C’’. Gli positive vesicles are less frequent and smaller.  (F) 350x digital magnification of the boxed region in D’’. Endocytic vesicles are larger and more frequent, making them more readily identifiable. 3.4 Discussion  SJs are present in all ectodermally derived epithelia including the central and peripheral nervous systems where they play both occluding and non-occluding roles. Despite the importance of SJs in normal growth and development, and the identification of many of the components, little is known about how SJ proteins interact with each other to assemble and maintain the tight impermeable seal while undergoing morphogenesis. In addition to structural roles in the formation of the SJs strands, some SJ proteins have identified roles independent of barrier formation. NrxIV is important in trafficking of components to the SJ domain, and Na/K ATPase is required for tracheal tubulogenesis (Jaspers et al., 2012; Nakajima et al., 2014; Nilton et al., 2010; Paul et al., 2003; Paul et al., 2007). Similarly, how the components of the SJ converge and coordinate with the TCJ subdomain is not known. Here we provide evidence that the core SJ proteins Na/K ATPase and NrxIV have different interactions with SJ and TCJ components. Loss of NrxIV leads to loss of the entire SJ complex and the loss of the TCJ components Gli and Dlg from the TJC. Loss of the Na/K ATPase, however, leads to a spread and reduction in SJ proteins along with an increase in Gli and Dlg at the TCJs. Finally, Na/K ATPase mutants are able to suppress the migration, apoptosis and overgrowth characteristic of the Gli overexpression phenotype while NrxIV mutants enhanced the phenotype. These results provide   97 new insight into how components of the SJs associate and coordinate with one another within distinct subdomains of SJs. 3.4.1 Na/K ATPase and NrxIV are Required for Correct Localization of Bicellular SJ Components.   NrxIV and the Na/K ATPase have been shown to be required for both SJ formation and maintenance. Furthermore, they have been shown to be a highly stable complex interdependent for localization to the SJ domain during establishment of the SJs (Genova and Fehon, 2003; Oshima and Fehon, 2011). Somatic clone analysis and studies of mutant animals have clearly demonstrated an interdependence of the core proteins for SJ formation that is not the case for SJ-associated proteins (Genova and Fehon, 2003; Ward et al., 1998). Targeted knockdown of NrxIV, ATPα or Nrv2 in the imaginal wing disc confirmed the absolute necessity of NrxIV for correct targeting of core SJ proteins and supports the importance of NrxIV in trafficking SJ proteins to the SJ domain (Jaspers et al., 2012; Nilton et al., 2010). In comparison, our results found that the loss of ATPα or Nrv2 altered the assembly of core proteins into SJ strands at the SJ domain. The correct targeting but basolateral spread of the NrxIV/Cora complex at the SJ domain demonstrates the requirement of Na/K ATPase to correctly integrate protein complexes into the SJ. Further, the loss of Fas3 demonstrates a novel association between a SJ-protein and a SJ-associated protein. Fas3 localization is altered in nrv2 and ATPα mutants and nrv2, ATPα and Fas3 mutants have similar tracheal defects supporting their interaction as a protein complex (Hemphälä et al., 2003; Paul et al., 2003). This was intriguing for two reasons: 1) Fas3 is a homophilic adhesion molecule that regulates tissue curvature through the formation of cell-cell contacts during localized JAK/STAT signalling (Wells et al., 2013); and 2) correct localization of Fas3 to the SJ domain is dependent on Dlg (Woods et al., 1996). It is not unusual for proteins   98 involved in signalling pathways to be localized within microdomains. We propose that the loss of Fas3 seen with Na/K ATPase RNAi is likely due to Fas3 being a potential target of a signal cascade. Loss of Na/K ATPase leads to in an increase in pSrc activation, which in turn may trigger an increase in intracellular signal activation through JAK/STAT or another signalling pathway leading to a decrease in Fas3 either at the protein or mRNA level. This might explain why the loss of NrxIV had no effect on Fas3 localization. Interestingly, mutants in varicose, another MAGUK protein involved in SJ formation and organization, have displaced Fas3 (Moyer and Jacobs, 2008). Furthermore, Varicose binds to NrxIV in vivo (Bachmann et al., 2008). Whether Varicose, Dlg, and Fas3 complex together is unknown, but it could be a possible model for how one subdomain of SJ components communicates with another.   As a coordinator for the integration of proteins into SJs, Na/K ATPase would be capable of directing proteins into different sub-complexes. Currently, Na/K ATPase is present in 3 distinct complexes at the SJ: 1) with NrxIV and Cora to establish SJs (Genova and Fehon, 2003; Laprise et al., 2009), 2) with Yurt, Cora and NrxIV to regulate epithelial polarity and 3) as we have demonstrated here, with Dlg and Gli to modulate the TCJ. How does Na/K ATPase assemble these different complexes? A possible mechanism through which Na/K ATPase is able to direct the formation of sub-complexes is with the SJ specific β subunit, Nrv2. Nrv2 has two isoforms, Nrv2.1 and Nrv 2.2, with identical extracellular and transmembrane domains, and both are required for SJ formation and tube size control (Paul et al., 2003; Paul et al., 2007). While junction formation depends on the identical extracellular domains of Nrv2.1 and Nrv2.2, these two proteins differ dramatically in their intracellular regions and potentially could form distinct intracellular connections. Paul et al (2007) made chimeras of Nrv2.1 and Nrv3 and demonstrated that the Nrv2.1 intracellular region had the ability to both localize to the SJ domain and to   99 associate with SJs (Paul et al., 2007). Nrv2.1 contains a SH2 binding motif and potential phosphorylation sites, while Nrv2.2 only has phosphorylation regulatory regions (Dinkel et al., 2013). Additionally, ATPα has multiple isoforms (Long A, B, C and Short A, B, C, D) and expression of any one of the long isoforms in null animals fully rescues SJ defects. As all combinations of ATPα and Nrv2 isoforms form a functional Na/K ATPase, forming protein-protein associations with specific isoforms of either subunit could create sub-complexes with various proteins combinations. Whether Na/K ATPase is in a complex with NrxIV, Nrg and Cora to form SJs, or with Yurt, NrxIV, and Cora to regulate epithelial polarity, or is associated with Dlg and Gli to regulate the TCJ, would depend on the specific ATPα/Nrv2 combination. How Na/K ATPase directs integration of different components into SJs and whether Nrv2.1 and Nrv2.2 have different intracellular proteins associations requires additional studies.  3.4.2 Na/K ATPase Interacts with the TCJ proteins Gli and Dlg at the TCJ  RNAi knockdown of the core proteins NrxIV and Na/K ATPase revealed that the loss of these proteins had differential effects on the TCJ proteins Gli and Dlg at the TCJ. The TCJ is important in maintaining epithelial integrity, the permeability barrier and the maturation of SJs, yet little is known about its molecular structure and how this domain links to the bicellular SJ. At the TCJ, Gli is recruited by and forms a complex with Dlg but in vitro binding assays have demonstrated that this interaction is not direct, suggesting the presence of additional protein(s) involved in the TCJ complex formation and Gli stabilization (Padash-Barmchi et al., 2010; Schulte et al., 2006). Neither Na/K ATPase nor NrxIV associate with Gli, though they may interact with unidentified TCJ proteins. We have previously shown that when the degree of Dlg knockdown is titrated, Dlg is lost from the bicellular junction and accumulates along with Gli at   100 the TCJ without a loss of global polarity (Padash-Barmchi et al., 2013). When Dlg was lost from the TCJ, Gliotactin was also lost, suggesting Dlg stabilizes Gli at the TCJ (Padash-Barmchi et al., 2013). The recruitment of Gli and Dlg to the TCJ in response to ATPα-RNAi knockdown suggests the Na/K ATPase is part of a signaling microdomain that participates in the regulation of endocytosis at the TCJ. Recycling of SJ components are important in the maturation and maintenance of SJs (Hildebrandt et al., 2015; Jaspers et al., 2012; Tiklová et al., 2010). The endosomal markers Rab5, Rab11 and Clathrin heavy chain (Chc) localize to the SJ domain and colocalizes with Bark Beetle (bark), a newly identified SJ-associated protein that functions in SJ maturation with the endocytic machinery. In Chc mutants, NrxIV is mislocalized from the SJ but the stability of the complex is maintained, leading to a model where the subcellular localization of the SJ core complex depends on endocytosis (Oshima and Fehon, 2011). Colocalization of Bark with Rab5, Rab11, and Chc appears to occur near or at the TCJ (Figure 6 in (Hildebrandt et al., 2015)), raising the possibility that endocytosis of SJ proteins occur at the TCJ. The increase in Gli and Dlg when Na/K ATPase was knocked down suggests that the Na/K ATPase may play a role at the TCJ in controlling or regulating endocytosis possibly through interactions with Bark. Loss of the Na/K ATPase could disrupt the integration of the endocytic machinery at the TCJ, leading to the retention and recruitment of Gli and Dlg paired with the spread of the other SJ components within the membrane.  In contrast, loss of NrxIV leads to a block in the assembly of the entire core complex at the TCJ, including Gli and Dlg, leading to a reduction and spread of the TCJ components.    101 3.4.3 Na/K ATPase Regulates the Overexpression and Spread of Gli  Gliotactin localizes specifically to the TCJ and its restriction to the TCJ is critical for survival of polarized epithelium in the imaginal wing disc (Padash-Barmchi et al., 2010). The spread of Gli beyond the TCJ into the bicellular junction leads to phosphorylation and endocytosis of ectopic Gli. Too much ectopic Gli triggers a JNK mediated signaling cascade that ends in cell delamination, migration and apoptosis (Lingrel, 2010; Padash-Barmchi et al., 2010). Normally, when Gli extends beyond the TCJ and into the bicellular junction, it is phosphorylated on two conserved intracellular tyrosine residues by an unidentified kinase and is rapidly endocytosed. Phosphorylation of Gli mediates the formation of a Gli-Dlg complex within the bicellular SJ domain leading to overproliferation and apoptosis (Padash-Barmchi et al., 2013). The ability of the ATPα and Nrv2 loss of function transgenes to suppress the ectopic Gli phenotypes suggests that a 50% loss of ATPα or Nrv2 may lead to a reduction in the ability of Gli and Dlg to interact due to less Dlg at the bicellular junction (Padash-Barmchi et al., 2013). Alternatively, the reduction of either subunit could result in an increase in Gli phosphorylation, possibly through increased Src activation as activated Src kinase can phosphorylate Gli in vitro (Padash-Barmchi et al., 2010). The loss of the Na/K ATPase leads to increased Src and Abl activation in Drosophila (Chapter 2). The observation of larger and more frequent Gliotactin vesicles combined with less cell death when Gli is overexpressed in ATPα or nrv2 heterozygotes suggest a greater degree of Gli phosphorylation and endocytosis.  On the other hand, the increased cell death observed when Gli was expressed in NrxIV4304 heterozygotes suggests that when Gli spreads into the bicellular junction, NrxIV may sequester Gli to prevent its interaction with Dlg. Although NrxIV does not normally interact with Gli, it does bind ectopic Gli (Schulte et al., 2006). A 50% genetic reduction in NrxIV combined with   102 the overexpression of Gli would reduce the availability of NrxIV to bind and sequester Gli in the bicellular junction. Overall, these data demonstrate that Na/K ATPase has a distinct role from NrxIV as a regulator of TCJ integrity and also may participate in the regulation of Gli endocytosis to suppress the ectopic Gliotactin phenotypes.  In conclusion we have demonstrated that the Na/K ATPase has distinct interactions with both the SJ and TCJ components, Fas3, Dlg and Gli. Na/K ATPase, Fas3 and Dlg have demonstrated roles in developmental signaling pathways, raising the intriguing possibility that these proteins may associate in a signaling subdomain of the bicellular portion of SJs. Na/K ATPase modulation of Gli and Dlg at the TCJ further supports a differential role of the Na/K ATPase in a subdomain within SJs. The presence of protein subdomains within the SJ region has been hypothesized by multiple groups (Hall et al., 2014; Nelson et al., 2010). The means by which this is reconciled with the demonstrated high stability of the SJ components (Oshima and Fehon, 2011) remains to be discovered. If these highly stable complexes were able to form distinct subdomains it would provide a mechanism for the SJs to adapt to the cellular shape changes known to affect SJs that occur during dynamic events such as morphogenesis (Bahri et al., 2010; Fristrom, 1982). Signal-directed interactions of subdomains between the bicellular and tricellular junction components would maintain a tight, impenetrable seal between cells of the epithelial sheet during these dynamic events. 3.5 Materials and Methods 3.5.1 Flystocks  The following fly strains were used: UAS-GliWT (Padash-Barmchi et al., 2010), nrv2nwu5 (Paul et al., 2003), ATPαDTSR2 (Palladino et al., 2003), NrxIV4304 (Baumgartner et al., 1996). The   103 following fly lines were obtained from: Vienna Drosophila Resource Center (VDRC) - UAS-ATPα−RNAi (v12330, v100619), UAS-Nrv2-RNAi (v2660), UAS-NrxIV-RNAi (v8353, v108128); Bloomington stock centre - UAS-Nrv2-RNAi (JF03081), UAS-mCD8::GFP, ATPα::GFP (G00109) (Morin et al., 2001), Nrv2.2::GFP (ZCL1649) (Morin et al., 2001), apterous-GAL4, UAS-p35; Kyoto DGRC stock centre - Gli::YFP (Lowe et al., 2014), UAS-BskDN (Weber et al., 2000).  3.5.2 Biochemistry  Membrane preparations were carried out essentially as described (Schulte et al., 2006). Immunoprecipitations were carried out using GFP-Trap coupled to magnetic particles (Chromotek). Embryos were collected from: w1118, ATPα::GFP, or Gli::YFP (150 µg) and homogenized in 200-250 µl lysis buffer (10 mM Tris-HCl, 0.5 mM EDTA, 0.5 mM EGTA, 150 mM NaCl, 0.5% NP-40, 1x phoSTOP complete (Roche). Immunoprecipitations were carried according to the manufacturer’s protocol and then subjected to Western blot analysis using the following primary antibodies: mouse anti-α5 (5ug/ml), mouse anti-Nrv (5F7)(1ug/ml), rabbit anti-Gli (Venema et al., 2004). Secondary antibodies used included HRP-conjugated goat anti-mouse (1:10,000) and HRP-conjugated goat anti-rabbit (1:5000) (Jackson Immunoresearch). 3.5.3 Immunolabeling  Imaginal discs were dissected from third instar wandering larvae into ice-cold PBS before being fixed in 4% paraformaldehyde (PFA). Fixed discs were washed three times in PBS-T (PBS plus 0.1% Triton-X100) and blocked either for 1 hour at room temperature (RT) or overnight at 4oC with 2% normal goat serum. Discs were immunolabeled with primary antibodies for 2 hours   104 at RT or overnight at 4oC. Discs were rinsed 3 times, incubated with DAPI (Thermo Scientific, 1/1000) before being mounted onto a slide with Vectashield (Invitrogen).  Antibodies used were: rabbit anti-cleaved Caspase3 (1/300)(Cell Signaling); rabbit anti-Mcr (1/600)(Hall et al., 2014), rabbit anti-NrxIV (1/300)(Baumgartner et al., 1996), rabbit anti-Gliotactin at 1:300 (Venema et al., 2004). The following monoclonal antibodies were obtained from Developmental Hybridoma Bank: anti-α5 (1/20), anti-Cora (C615-16 and C566.9)(1/600), anti-Dlg (4F7)(1/200), anti-Fas3 (1/100), anti-MMP1 (1/200), anti-Gliotactin 1F6.3 (1:100) (Auld et al., 1995), anti-DE-cadherin (1:50). The following secondary antibodies were used: AlexaFluor secondary antibodies Alexa488, 568, 647(1/300)(Invitrogen).  3.5.4 Imaging  Imaginal discs were imaged using a DeltaVision Spectris (Applied Precision, GE) with a 60x NA1.4 oil immersion lens and 200 nm Z steps. Image stacks were deconvolved by a maximum likelihood algorithm using a PSF measured from a 200 nm bead conjugated to Alexa488 (Invitrogen) and Softworx 2.0 Software. SoftWorx was used to create side projections. ImageJ (Schneider et al., 2012) was used for image analysis and processing before being exported as tiff images for compilation in Adobe Photoshop / Illustrator CS3. Low magnification images were collected on a Zeiss Axioskop with a 20x NA 0.50 air lens using Northern Eclipse Software 8.0. 3.5.5 Statistical Analysis  Immunolabeling intensity: Using ImageJ, two equal ROI boxes were drawn around the signal on the apterous and wildtype compartment. Mean gray value was calculated on side   105 projections and expressed as ratio of the RNAi/wildtype. Data was exported to Prism 6.0f and a Students T-test was used to compare ratios across genotypes.  Percentage Migration: The distance travelled by migrating cells from the D–V boundary was measured using the linear tool in ImageJ. The distance travelled was expressed as percentage of the distance from the D-V boundary to the ventral edge of the wing disc.   All data was exported to Prism 6.0f and one-way ANOVA with a pairwise comparison was used to compare migration distance between genotypes.  3.6 Supplementary Data for Chapter 3  Figure 3.7: RNAi knockdown of ATPα  has differential effects on Fas3 compared to RNAi knockdown of NrxIV knockdown. (A-B) Maximum intensity projections of the SJ region of 3rd instar imaginal wing discs with apterous-GAL4 driving ATPα-RNAiGD (A) NrxIV RNAiGD. Scale bar 15um. ATPAGDNrxIVGDFas3Nrv2::GFPMergeMerge ATPA::GFP Fas3A A’ A’’B B’ B’’  106 (A) Wing disc from an ATPα::GFP (green) wing disc immunolabeled with Fas3 (A”, red). Expression of ATPα-RNAiGD led to a loss of Fas3 immunolabeling from the SJ domain.  (B) Nrv2 endogenously tagged with GFP (Nrv2::GFP)(B’, green) and immunolabeled for Fas3 (B’’, red). RNAi knockdown of NrxIVGD led to a reduction of Fas3 immunolabeling at the SJ domain.    107  x axis positionz DepthPixel IntensityPixel Intensityz Depthx axis positionNrv2GDGli DlgMerge Nrv2::GFP Gli--------------01234Nrv2 Nrv2Ratio of ap/WTGlip=0.0086 p=0.0096 GD JFMerge Nrv2::GFP Dlg--------------0.00.51.01.52.0Ratio of ap/WTDlg Nrv2 Nrv2GD JFp=0.0074 A A’ A’’DCFB B’ B’’ENrv2GDNrv2GD  108 Figure 3.8: RNAi knockdown of Nrv2 phenocopies RNAi knockdown of ATPα .  (A-B) Maximum intensity projections and z-projections of the SJ region of 3rd instar imaginal wing discs with apterous-GAL4 driving Nrv2-RNAiGD. Boxes indicate the area used for surface plots in C and D. Scale bar 15um. (A) Wing disc from an Nrv2::GFP (green) wing disc immunolabeled with Gli (A”, red). Expression of Nrv2-RNAiGD led to a basolateral spread and increase in Gli immunolabeling at the TCJ (arrowhead).  (B) Nrv2 endogenously tagged with GFP (Nrv2::GFP)(B’, green) and immunolabeled for Dlg (B’’, red). RNAi knockdown of Nrv2GD led to a reduction and basolateral spread of Dlg at the bicellular junction, but increased at the TCJ (arrowhead).  (C,D) Surface plots of the pixels intensity plotted as a function of z –axis and the x-axis position for Gli (C) or Dlg (D) in an Nrv2-RNAiGD wing disc. Arrows indicate the apterous boundary between the RNAi and WT compartments.  (C) Within the Nrv2-RNAi compartment, Gli signal intensity remained the same or increased as it spreads along the basolateral axis of the TCJ.  (D) Within Nrv2-RNAi, Dlg signal intensity remained the same or higher with a periodicity similar to the TCJ. (E) Statistical analysis of Gli immunofluorescence with two different Nrv2-RNAi lines (GD and JF). The mean gray value from equivalent areas of the RNAi and WT compartments was measured, and the RNAi compartment was normalized to the WT. There was a significant difference (students t-test) between the RNAi vs. WT compartments in both Nrv2-RNAi lines tested (GD, mean= 2.203, n=9 discs; JF, mean=1.798, n=9 discs)(means + SEM) (F) Statistical analysis of the Dlg changes in Nrv2-RNAi lines (GD and KK). The mean gray value of Dlg immunolabeling from equivalent areas of the RNAi and WT compartments was measured and normalized to WT. A students t-test was used and a significant decrease in Dlg immunofluorescence between the WT and RNAi compartments was observed in one line (GD, mean= 0.6643, n=6 discs, p=0.0074 ; JF, mean=1.165, n=5 discs, non-significant)(means + SEM).    109 Chapter 4: Conclusions and Discussion  Cell junctions including septate junctions play critical roles not only in mediating cell-cell adhesion, but also in creating apical basal polarity, permeability barriers and signaling platforms. Much of the knowledge about Drosophila septate junctions (SJs) has been gained from studies of epithelial sheets undergoing morphogenesis, whether in the embryo during dorsal closure or in the trachea during tubulogenesis. Thus it is difficult to distinguish the molecular interactions involved in SJ maintenance from morphogenesis. The wing imaginal disc from the 3rd instar larvae is an ideal model tissue to study how SJs are formed and maintained as well as the non-occluding roles of SJs. The wing disc is created by a pair of epithelial sheets with an established apical – basal polarity that facilitates easy analysis of changes to SJ structure, composition and function.   The Na/K ATPase pump, studied for more than 20 years as an important regulator of homeostasis, is a critical structural component of SJs. The Na/K ATPase is a core SJ protein required for establishment of SJs and regulation of tracheal tube size (Genova and Fehon, 2003; Paul et al., 2003; Paul et al., 2007). Somatic null clone analysis demonstrated that Na/K ATPase-negative cells were eliminated from the epithelium (Genova and Fehon, 2003). This suggested the Na/K ATPase is required for maintenance of SJs in addition to their establishment. In addition to its role in establishing an occluding barrier, vertebrate cell culture studies have demonstrated that the Na/K ATPase regulates numerous signaling pathways including promotion of cell growth, apoptosis and tumor invasion (Laprise et al., 2009; Liu et al., 2007; Rajasekaran et al., 2001a; Rincon-Heredia et al., 2013; Sun et al., 2013; Tian et al., 2006). Whether the Drosophila Na/K ATPase has a similar multiplicity of roles had not been determined prior to this thesis. We utilized a combination of RNAi and genetic techniques to study the role(s) of the   110 Na/K ATPase in the columnar epithelia of the wing imaginal disc. The imaginal wing disc paired with SJ protein -RNAi transgenes expressed with the Gal4/UAS system provides a powerful tool to further dissect how Na/K ATPase interacts with septate and tricellular junction components.  In Chapter Two we identified a novel link to apoptotic pathways through which the Na/K ATPase may mediate cell death in the imaginal wing disc. Although the Na/K ATPase has been identified as member of the SJ core complex, it was thought to function only as a structural protein. Here we demonstrated the ability of the Na/K ATPase to activate Src and JNK mediated apoptosis. Although the Na/K ATPase has been identified as a signal transducer in mammalian cell culture studies, this thesis work is the first to establish Na/K ATPase as an activator of the Src/JNK signaling pathway in vivo.   In Chapter Three, we identified the Na/K ATPase as a regulator of the tricellular junction (TCJ) protein complex. RNAi knockdown and genetic interactions demonstrate the Na/K ATPase, along with the MAGUK protein Dlg, is important in the restriction and stabilization of Gli at the TCJ. The regulation of TCJs by Na/K ATPase likely requires additional molecular components and provides new insights into how the components of the SJ domain interact and communicate.   4.1 The Na/K ATPase and the Bicellular SJ  This thesis investigates the role of the Na/K ATPase in the SJ and TCJ with the aim to further our understanding of the ability of the Na/K ATPase to act as an adhesion protein or a scaffold, critical for the maintenance and function of the SJs. In Chapter 3, we demonstrated the Na/K ATPase is critical for correct assembly of SJ components into SJ strands. Further, we demonstrated the Na/K ATPase has a different relationship with the TCJs proteins Gli and Dlg   111 than NrxIV. Loss of Na/K ATPase through RNAi knockdown of either subunit caused the incorrect localization of the core SJ proteins NrxIV and Cora and a basolateral spread of SJ-associated proteins. In contrast, loss of NrxIV, which is involved in targeting components to the SJ domain, completely eliminated the immunofluorescence signal for Cora, Nrv2 and ATPα. Similarly, a comparison of the effects of Na/K ATPase versus NrxIV knockdown on SJ-associated proteins Gli, Dlg and Fas3 supports a differential role for Na/K ATPase. Loss of NrxIV leads to a loss of SJ integrity due to loss of the core components and an expansion of the domain occupied by Gli, Fas3 and Dlg. In contrast, loss of Na/K ATPase removed Fas3 immunolocalization, depleted Dlg from the bicellular junction, and recruited additional Gli and Dlg immunofluorescence to the TCJ. This modulation and redistribution of these SJ-associated proteins, in contrast to their simple spreading resulting from loss of proper associations with core SJ components, is consistent with a unique role for Na/K ATPase in assembly of SJs and TCJs. 4.1.1 Na/K ATPase and NrxIV form Different Protein Complexes in the SJ   We demonstrated a clear difference between NrxIV and Na/K ATPase in their relationship with SJ-associated proteins and the TCJ. The question then becomes what is the basis for this difference?    A formal possibility is that the RNAi approaches utilized were the source of the observed difference. This thesis utilized the UAS-GAL4 tissue-specific expression of RNAi constructs to reduce production of specific gene products. This was done to bypass the embryonic lethality of mutants in SJ genes proteins and the cell lethality observed with somatic clones in the wing imaginal disc. RNAi mediated knockdown is not necessarily the equivalent of a null mutation. The use of genetic nulls ensures that neither transcript nor protein is present in the   112 cells/tissue/animals being studied. In contrast, during RNAi knockdown, mRNA is still produced and is then subsequently targeted for degradation. Therefore in the RNAi protocol, the lack of a detectable protein is not equivalent to the lack of mRNA and transcriptional regulation could occur. Genova and Fehon (2003) demonstrated that loss of individual SJs proteins does not affect the protein stability of other SJ components, suggesting SJ proteins do not transcriptionally regulate each other and that compensatory upregulation of other SJ components does not occur. The demonstration in Chapter Two that RNAi knockdown of ATPα in the imaginal wing disc leads to the intracellular retention of Nrv2 but not its removal, supports the hypothesis that transcriptional regulation between SJs proteins does not occur. Further, the retention of Nrv2 in the cytosol affirms that protein-protein interactions are required for correct localization and function of SJ proteins. It is unlikely that the differences we observed are due to differences in the efficiency of RNAi mediated knockdown of the Na/K ATPase and NrxIV RNAi transcripts. We observed these differences using a total of six different RNAi transgenes and observed similar effects with RNAi mediated knockdown of both Nrv2 and ATPα. In addition, the effects of RNAi knockdown of NrxIV we observed in the wing imaginal disc mirrored those seen in somatic clones of NrxIV null alleles (Genova and Fehon, 2003).    Overall our data points to a distinct role for Na/K ATPase compared to NrxIV in SJ and TCJ formation. Prior work has pointed to the presence of subdomains within the SJ domain and it is possible that NrxIV and Na/K ATPase form different protein complexes within the SJ. NrxIV is in a protein complex with both Cora and Vari and is the first SJ protein to be localized to the SJ domain. NrxIV/Cora complex has demonstrated roles in the correct targeting of SJ components to the domain (Baumgartner et al., 1996; Hildebrandt et al., 2015; Jaspers et al., 2012). In NrxIV mutants SJ components cannot reach the SJ domain to be assembled leading to   113 the loss of most SJ components. In contrast, a Na/K ATPase protein complex has been less well established and our results suggest that this complex has unique interactions compared to NrxIV.   The presence of distinct protein complexes within the SJ domain has been previously noted (Hall et al., 2014). It is likely that the Na/K ATPase is forming unique complexes with other adhesion molecules within the SJ and Fas3 is a strong candidate as complex partner. Fas3, a SJ-associated protein component with known adhesion properties (Snow et al., 1989; Wells et al., 2013), was lost from the SJ domain in response to RNAi knockdown of Na/K ATPase but not with NrxIV RNAi. Fas3 could be participating in a complex with Na/K ATPase and forming homophilic associations with neighboring cells. Fas3 contains extracellular Ig repeats that are important for cell – cell adhesion. Similar to Nrv2, Fas3 is alternatively spliced to produce proteins of different lengths, which differ in their cytoplasmic domains (St Pierre et al., 2014) flybase). Elm sequence analysis (Dinkel et al., 2013) of cytoplasmic regions from both the short and long Fas3 isoforms revealed both isoforms contain numerous features including multiple phosphorylation sites. Additionally the long Fas3 isoform contains a class I PDZ domain and SH3 and SH2 binding motifs. These domains have the potential to help create unique signaling domains within the SJ in conjunction with the Na/K ATPase. Whether Fas3 is interacting with Na/K ATPase directly or indirectly through binding with another scaffolding protein such as Dlg has not been determined. A proximity ligation assay between Na/K ATPase and Fas3 would be a simple test to determine if these two proteins are in a complex together. An alternative model is that the loss of Fas3 observed in ATPα-RNAi wing discs is a downstream effect of Na/K ATPase/Src signaling as Fas3 mRNA levels are regulated by localized JAK/STAT signaling (Wells et al., 2013). This later model would also explain why the loss of NrxIV did not lead to Fas3 disruption while the loss of Na/K ATPase did. To test this possibility we could block Src   114 activity by ectopically expressing Drosophila C-terminal Src kinase to inhibit Src using (UAS-dCsk) or examine Src mutants in wing discs expressing ATPα-RNAi. If loss of Fas3 is due to cross-talk between pSrc and the Jak/STAT pathway, blocking Src activation should prevent the loss of Fas3.  4.1.2 Na/K ATPase and Dlg - Potential Cytoskeletal Interactions   The redistribution of Dlg in the Na/K ATPase RNAi-mediated knock down raises the intriguing possibility that Na/K ATPase may form a link between the core SJ proteins and SJ-associated proteins to the cytoskeleton within the cell. Loss of vertebrate Na/K ATPase leads to a loss of stress fibers and discontinuous staining of the TJ proteins ZO1 and occludin (Madan et al., 2007; Rajasekaran et al., 2006; Violette et al., 2006). This data suggests the vertebrate Na/K ATPase may complex with ZO1 to recruit other TJs components. However, it is unknown if the alpha or beta subunit directly bind ZO1. ZO1, like Dlg, functions as a scaffolding protein through its PDZ and SH3 domains and is a member of the membrane-associated guanylate kinase family (MAGUK). Drosophila Dlg has a similar structure and plays a similar role to ZO1 in SJs. Additionally, Dlg is required for barrier formation and is a member of the polarity complex along with Scrib and Lgl (Tepass et al., 2001). Of the two Na/K ATPase subunits, it is more likely that Dlg could bind Nrv2. Dlg has a SH3 domain that could potentially bind the SH3 motif (KPVPMSP) on the cytoplasmic tail of the beta subunit of Na/K ATPase, Nrv2 (ELM sequence analysis (Dinkel et al., 2013)). If Dlg and Nrv2 are associating, this is one possible connection to the cytoskeleton via the Dlg HOOK domain, which is known to bind actin (Hough et al., 1997). Another means by which Na/K ATPase could interact with the cytoskeleton is through the acetylated tubulin and ankyrin-binding domains of the alpha subunit  (Jordan et al.,   115 1995; Zampar et al., 2009; Zhang et al., 1998). These domains are conserved between vertebrates and Drosophila (Paul et al., 2007). Ankryin is an universal adaptor protein that mediates linkage of membrane proteins to the cytoskeleton and ATPα has two conserved ankryin binding sequences in its cytoplasmic loops (Jordan et al., 1995; Paul et al., 2007; Zhang et al., 1998). In vitro, ATPα complexes with microtubules through its fifth cytoplasmic domain that binds to acetylated tubulin (Zampar et al., 2009). Interestingly binding of ATPα to acetylated tubulin inhibits enzymatic activity of Na/K ATPase (Casale et al., 2005; Zampar et al., 2009). Thus the association of ATPα with acetylated tubulin could serve two roles, to keep the transporter inactive and to provide an anchor site for microtubules with SJs (Zampar et al., 2009). Together, these associations make Na/K ATPase a favorable candidate to link the SJ to the cytoskeleton.   To define the relationship between ATPα in cytoskeletal stability and potential cell shape changes, we could carry out a functional analysis of the ankryin and acetylated tubulin domains in vivo. To determine if these domains effect Na/K ATPase localization or cell shape, we can create FLAG epitope-tagged ATPα derivatives that are deleted for these domains. These proteins would be expressed in the imaginal wing disc by placing them under the control of the Gal4/UAS system. Initial analysis would be in wild type tissues to examine the effect of these mutations on Na/K ATPase localization, SJ protein complex formation and SJ organization. Next we would evaluate the ability of the ATPα deletion mutants (ATPα - Δ) to rescue ATPα-RNAi imaginal discs. We would use the wild type ATPα tagged with GFP paired to GFP-RNAi to knockdown endogenous ATPα and co-express the ATPα deletion mutants. If ATPα is anchoring SJs through its cytoskeleton binding domains, we would expect the SJs of imaginal wings expressing ATPα − Δ to have lose their apical localization and exhibit an expanded domain.    116 4.1.3 Subdomain Interactions with ATPα  and Nrv2 isoforms  We and others have raised the potential for distinct subdomains within the SJ domain, the composition of which may be dependent on the different isoforms of ATPα and Nrv2 (Hall et al., 2014). Vertebrates possess multiple genes encoding the α subunit allowing great flexibility in spatial and temporal control of expression levels. In Drosophila, only one gene encodes the ubiquitously expressed α subunit, ATPα that is alternatively spliced to generate structurally diverse ATPα proteins. These proteins can be broadly split based on size, into long, medium and short isoforms that are the result of different translation initiation sites (Palladino et al., 2003; St Pierre et al., 2014). Of the three length categories, only the long isoform, which contain an additional 39aa in the N-terminus, is capable of fully rescuing all tracheal defects of the ATPα null mutant while the short isoforms are capable of partial rescue. This leads to the possibility that different isoforms are partnering within different SJ complexes. In vertebrates, two isoforms of the alpha subunit are expressed in cardiac cells that clearly have different functional roles in vivo (He et al., 2001; James et al., 1999) and localize to distinct domains of the plasma membrane (Liang et al., 2007). The RNAi lines used in this thesis targeted all potential ATPα isoforms, while the GFP trap lines we used tagged only the long ATPα isoform (G0010). Through the use of a GFP specific-RNAi, we can target the long isoform of ATPα for degradation and use the ATPα antibody to immunolabel the remaining short isoforms to examine the effect of loss of a single isoforms on SJ assembly, pSrc activation and recruitment of the Gli/Dlg complex to the TCJ.   The differential localization of ATPα isoforms might reflect the need to diversify the control of signaling pathways within the SJ domain. For instance, the additional 39aa in the long isoforms contain an N-terminal SH2 motif that would be available for binding to SH2 domain   117 proteins, suggesting the ATPα might be important for regulating signal transduction. This is distinct form the internal Src binding site found in all isoforms. Additional ATPα variation occurs at exon 6, which encodes the part of the sixth transmembrane segment and the entire cytoplasmic loop between transmembrane segments six and seven. The existence of multiple, alternatively spliced versions of exon 6 in the ATPα gene suggests that the sequence differences encoded by these alternative exons could have profound functional consequences on pump kinetics, ion selectivity, or regulatory properties. These differences could provide a means to have a non-transporting isoform localized within the SJ domain. Assaying the ability of expression of transgenes that include these exon 6 splice isoforms to rescue imaginal disc where the long isoform was knocked down using GFP-specific RNAi could test the implications of these different alpha isoforms.  Nrv2 also has two splice variants, adding another degree of flexibility for directing protein-protein interactions. The Nrv2 locus has alternative splicing in the 5’ region, giving rise to two transcripts, Nrv2.1 and Nrv2.2, that encode proteins which differ only in their N-terminus cytoplasmic domains (Genova and Fehon, 2003; Paul et al., 2003; St Pierre et al., 2014; Sun and Salvaterra, 1995b). In Drosophila, Nrv2 specifically is required for barrier formation as neither expression of Nrv1 nor the neuronal Nrv3 are able to rescue SJ defects in the trachea, while expression of cDNA for Nrv2.1 or Nrv2.2 was able to fully rescue the tracheal and / or barrier function phenotypes in embryos (Genova and Fehon, 2003; Paul et al., 2007). This leads to the question of whether the two Nrv2 isoforms are functionally redundant. The available GFP lines for Nrv2 tagged either Nrv2.1 (ZCL1649) or both Nrv2.2 and Nrv2.1 (ZCL2903) while the RNAi lines targeted both isoforms (Morin et al., 2001; St Pierre et al., 2014). The inability to detect localization differences between the two Nrv2::GFP lines does not preclude them from   118 having distinct roles in non-occluding function of SJs. For example, in the developing eye, Nrv2::GFP was detected in the developing eye only in line ZCL2903, suggesting Nrv2.2 is the dominant isoform in this tissue (Baumann and Salvaterra, 2010).  It is likely that in terms of barrier function and SJ complex assembly, the proteins are redundant as it is the extracellular region of Nrv2 that is necessary for junctional activity, but the differences in their cytoplasmic region may be important in regulation of the interaction of Na/K ATPase with SJ subdomains. ELM analysis (Dinkel et al., 2013) of the cytoplasmic domain of Nrv2.1 revealed a SH3 motif that could potentially interact with the Dlg SH3 binding domain or another SH3 containing protein. As the alpha subunit has no likely Dlg binding sites (neither SH3 domains nor PDZ binding motifs), this suggests that it is the Nrv2 subunit isoforms that may interact with Dlg. As well the alternative splicing of Nrv2 could lead to the creation of unique protein complexes with Dlg.   These unique Na/K ATPase complexes allow for different interactions between the bicellular and tricellular junctions. Loss of the Na/K ATPase leads to an increase in both Dlg and Gli at the TCJ. To determine if the increase in Gli observed with RNAi knockdown is due specifically to loss of one or both Nrv2 isoforms, we can rescue Nrv2 null clones by driving expression of cDNA for either Nrv2.1 or Nrv2.2. Further, the ability to differentiate between the two Nrv2 isoforms using the GFP tag in ZCL1649 would allow us to look at how Dlg associates with each these two proteins using a proximity ligation assay (PLA). If Nrv2.1 were associated with Dlg, we would expect to see a positive PLA only between Nrv2.1 and Dlg. The use of a GFP specific –RNAi to knockdown ZCL1649 would confirm that Nrv2.1 is mediating sub-complex formation. The alternative splicing that leads to the nrv2.1 and nrv2.2 transcripts, would allow us to create an RNAi to be directed against either nrv2.1 or nrv2.2 specific exons as a   119 secondary test. Analysis of the cellular consequences of RNAi knockdown will allow us to distinguish if there are differential effects on Gli and Dlg at the TCJ. These experiments will clarify how Na/K ATPase proteins complexes are formed within SJs  We have found loss of the Na/K ATPase affects SJ complexes in a manner distinct from loss of NrxIV. Since Na/K ATPase is composed of two subunits, the alpha and beta, each subunit can associate with a different protein complexes and mediate different SJ or TCJ interactions.  The Na/K ATPase provides potential mechanism through which the SJs link to the cytoskeleton and how the bicellular junction links to the TCJ.  4.2 The Na/K ATPase as a Signaling Center  In this thesis we have demonstrated Na/K ATPase is important for negatively regulating growth and survival in the imaginal wing disc epithelium. Loss of either the ATPα or Nrv2 subunits via RNAi knock down resulted in apoptosis, delamination and increased compensatory proliferation. The observed cellular consequences required the JNK signaling pathway and mimics results observed with the loss of C-terminal Src kinase (dCsk), the only previously identified Src negative regulator in Drosophila (Read et al., 2004). Further, recent reports have shown Src activates JNK through Abl to control tumorigenesis in the imaginal disc (Enomoto and Igaki, 2012; Rudrapatna et al., 2013; Singh et al., 2010). In this thesis we have shown that loss of Na/K ATPase resulted in significant increases in the levels of pSrc and pAbl, accompanied by apoptosis and compensatory proliferation. Further, PLA, demonstrates Na/K ATPase / pSrc exist in proximity to each other (40nm). Together these results demonstrate the ability of Na/K ATPase to regulate a pSrc-signaling pathway in vivo.   120  The ability of Na/K ATPase to bind and regulate Src has been well established in vertebrate cell culture studies (Lingrel and Kuntzweiler, 1994; Xie, 2003). In vertebrates, the Na/K-ATPase isoform that binds Src is localized to caveolae through two caveolin binding motifs (Liang et al., 2006; Liu and Shapiro, 2007; Wang et al., 2004). Caveolin proteins interact with a variety of signaling molecules including Src family kinases, ion channels, and mitogen-activated kinases (MAPKs) (Fridolfsson et al., 2014; Shvets et al., 2014). Vertebrate Na/K ATPase along with its signaling partners must be localized within the caveolar microdomain for Src-dependent signal transduction to occur and to keep the ion-transporter function inactive (Liang et al., 2006; Wang et al., 2004). In addition, the caveolae are also sites of organization of the signaling molecules Src would target upon release from Na/K ATPase (Fridolfsson et al., 2014; Shvets et al., 2014).  Drosophila does not have caveolin homologs in its genome, nor does it have a functional equivalent of the caveolae structure in its plasma membranes (Gupta et al., 2009). We hypothesize that SJs are the sites in Drosophila in which Na/K ATPase-mediated Src signaling occurs. In Drosophila, the localization of the Na/K ATPase at the SJs would also facilitate association with phosphorylation targets. Potential SJ and TCJ targets include SJ proteins and SJ-associated proteins such as Dlg and Gli, which are important regulators of epithelial integrity (Padash-Barmchi et al., 2010; Padash-Barmchi et al., 2013). In addition to SJ proteins and SJ-associated proteins, Src has been demonstrated to be upstream of signaling molecules in a variety of signal pathways including the Rho family of GTPase to regulate actin dynamics and cell adhesion, as a regulator of the interactions of the apical determinant Par3/Bazooka complex, in growth and development via the Hippo pathway (Brumby et al., 2011; Kwon et al., 2015; Langton et al., 2009; Sotillos et al., 2013). These are just few examples of the large and varied   121 number of potential Src targets. Whether components of these pathways are located within SJs or if they interact with SJs proteins remains to be determined. Rho1 is of particular interest as it has been shown to accumulate in the SJ region and potentially binds to Mega, a SJ-associated protein involved in trafficking of SJ components with NrxIV (Fox, 2005; Jaspers et al., 2012). Rho1 is also upstream of two JNKKKs, Tak1 and Slipper and membrane localization of Rho1 triggers JNK signaling in the imaginal disc (Neisch et al., 2010). How SJ or SJ components regulate signal pathways has not been examined. The demonstration of a Na/K ATPase / Src complex is the first evidence of a SJ component mediating signal transduction. As discussed below, our model would enable us to easily and quickly test for components in a Na/K ATPase/Src signal pathway. We can use our model in a genetic modifier screen to test known targets of Src such the Rho family of GTPases, or other JNK pathway components and to also identify novel components that enhance or suppress the ATPα-RNAi phenotype.  Whether pSrc is physically bound to the ATPα subunit in Drosophila was not clearly established in this thesis, as we were unable to isolate Src in a complex with ATPα. PLA demonstrated that Na/K ATPase and pSrc42A are localized within 40nm of each other. This combined with the increased levels of pSrc and a downstream target, pAbl, with loss of Na/K ATPase demonstrates a functional interaction between the two proteins and strongly suggests they are forming a complex. A clear future avenue of research would be to use genetics to confirm the involvement of these two proteins in a signaling pathway. We have obtained loss of function mutants for the Src42A and Src64B genes (Strong and Thomas, 2011; Tateno et al., 2000). We would expect that expression of ATPα-RNAi heterozygous with any one of these mutations should result in imaginal wings discs showing a decrease in JNK mediated apoptosis. A reduction in the JNK pathway could be measured by analysis of phosphorylated JNK (pJNK)   122 or MMP1 immunofluorescence and a reduction in the presence Cas3 positive cells. The removal of a single copy of either Src42A or Src64B may only result in a partial loss of the signal transduction pathway. Expression of ATPα-RNAi in an Src42A; Src64 double mutant should demonstrate a strong reduction in JNK activation, MMP1 or Cas3 immunofluorescence. As a secondary confirmation, Src activation can be blocked by expressing Drosophila C-terminal Src kinase (dCsk (Read et al., 2004)) in the ATPα-RNAi background. Discs expressing both dCsk and ATPα-RNAi should phenocopy Src loss of function with ATPα-RNAi wing discs. These experiments would demonstrate Src is epistatic to Na/K ATPase and the Na/K ATPase –Src complex is an important regulator of cell survival.  In vertebrate cell culture, a ouabain sensitive isoform of the α1 subunit complexes with Src in response to ouabain, in a dose dependent manner (Haas et al., 2002; Tian et al., 2006) but only within caveolae  (Liu et al., 2003; Wang et al., 2004). This suggests that other proteins are involved in recruiting the Src-Na/K ATPase complex, which is consistent with Src having multiple adaptor proteins and regulatory sites (Ingley, 2008). The ability to immunoprecipitate the pSrc-Na/K ATPase complex requires activation of Na/K ATPase signaling pathway via administration of cardiotonic steroids (Haas et al., 2000; Tian et al., 2006). Ouabain-dependent Src complexing with Na/K ATPase has not been demonstrated in Drosophila as it is difficult to administer sufficient ouabain in vivo to trigger cell responses due to the efficiency of the malpighian tubules to rapidly clear ouabain (Torrie et al., 2004). The simplest approach to establish a direct association between pSrc and Na/K ATPase in Drosophila would be to culture isolated imaginal wing discs in ouabain-containing media prior to completing a PLA between pSrc and ATPα. We would expect an increase in the PLA signal after treatment with ouabain.    123  A diverse range of cellular pathways coalesce at JNK, as such control of JNK activation is a multilevel process (Ríos-Barrera and Riesgo-Escovar, 2012). In Drosophila, only one JNKK has been identified but at least six JNKKK are known which may be activated by the Na/K ATPase/Src complex (Neisch et al., 2010; Ríos-Barrera and Riesgo-Escovar, 2012). Of these, Tak1 is the most likely target, as we know it is present in the SJ domain and functionally interacts with Gli (Gayathri Samarasekera, personal communication). The elimination of Cas3 and MMP1 expression when a dominant negative JNK, Basket (Bsk), was co-expressed with ATPα-RNAi. provides a simple readout for assaying JNK activation. To determine if the Na/K ATPase-Src complex is signaling directly to the JNK signal transduction cascade or through alternate components, we can express ATPα-RNAi with dominant-negative or null alleles of a variety of JNKKKs including Slipper, Tak1 and Mekk1 and determine the degree of Cas3 and MMP1 immunofluorescence observed. These experiments would further clarify how Na/K ATPase fits within established signaling pathways.   Our studies on the activation of pSrc in response to RNAi knockdown of either subunit of the Na/K ATPase, ATPα or Nrv2, has established the presence of a Na/K ATPase-Src signaling pathway in Drosophila. Our proposed experiments build upon these results and will clarify the contributions of the Na/K ATPase to Src activation as well as a potential regulator of JNK mediated apoptosis.  4.3 The Na/K ATPase and the Tricellular Junction Protein Gliotactin  In this thesis we have observed two distinct and novel functions of the Na/K ATPase. In chapter 2, we described how the Na/K ATPase could be a regulator of activated pSrc levels, and in chapter 3 we described how the Na/K ATPase restricts the interaction of Dlg and Gli to the   124 TCJ.  The TCJ forms at the convergence of three epithelial cells and is critical for maintaining the permeability barrier. To date only one protein, Gli, has been identified to localize specifically to the TCJ and together with Dlg, it is regulated to maintain epithelial integrity (Padash-Barmchi et al., 2010; Padash-Barmchi et al., 2013; Schulte et al., 2003). We observed two different interactions between Gli and Na/K ATPase at the TCJ and the bicellular SJ domain respectively.  At the TCJ Gli immunofluorescence increased in response to Na/K ATPase RNAi knock down. When Gli spread into the bicellular SJ in an overexpression model, a 50% genetic reduction in Na/K ATPase suppressed the Gli overexpression phenotype. This suggests that the Na/K ATPase may regulate Gli both at the TCJ and when Gli spreads into the bicellular SJ domain.   4.3.1 Na/K ATPase and Gli at the TCJ   We have demonstrated that RNAi knockdown of Na/K ATPase resulted in the recruitment of Gli and Dlg to the TCJ. This recruitment was not due to a general loss of SJ integrity as no recruitment occurred with knockdown of NrxIV. This suggests the Na/K ATPase is part of a signaling subdomain that is involved in the regulation of endocytosis at the TCJ. Maturation and maintenance of SJs relies on endocytosis and recycling of SJ components (Hildebrandt et al., 2015; Jaspers et al., 2012; Tiklová et al., 2010). Analysis of mutants in the endosomal pathway component Clathrin heavy chain (Chc) demonstrated that NrxIV is mislocalized from the SJ but the stability of the core complex was maintained. This leads to a model where the subcellular localization of the SJ core complex depends on endocytosis (Oshima and Fehon, 2011). Bark Beetle (bark), has been recently identified as a SJ-associated protein that functions with the endocytic machinery in SJ maturation (Hildebrandt et al., 2015). Bark colocalizes with Chc and the endosomal markers Rab5, Rab11 near or at the TCJ (Figure 6   125 (Hildebrandt et al., 2015)) and suggest that endocytosis of SJ proteins occur at the TCJ. Na/K ATPase may play a role at the TCJ in controlling or regulating endocytosis possibly through interactions with Bark. Loss of the Na/K ATPase could prevent the proper assembly of the endocytic apparatus at the TCJ preventing endocytosis of SJ components. This would result in the observed retention and recruitment of Gli and Dlg along with the spread of the other SJ components. In contrast, loss of NrxIV leads to a collapse of the entire core complex at the TCJ, including Gli and Dlg, leading to a reduction and mislocalization of the TCJ components. So while the Na/K ATPase does not directly associate with Gli, it may interact with unidentified TCJ proteins such as Bark to regulate endocytosis of Gli and Dlg, as well as other SJ components.   4.3.2 Na/K ATPase Can rescue Ectopic Gli in the Bicellular SJ   Spreading of Gli into the bicellular junction leads to phosphorylation and endocytosis of Gli with Dlg, but the components involved in this pathway are unknown. In analyzing the relationship between Gli and the Na/K ATPase we used a model of ectopic Gli expression and demonstrated that the Na/K ATPase genetically interacts with the TCJ complex proteins Gli, possibly functioning with Dlg to regulate and maintain epithelial integrity. Suppression of the ectopic GliWT phenotype by nrv2 and ATPα indicates that the Na/K ATPase functions in a pathway to regulate Gli levels and localization of Gli to the TCJ. In contrast, the enhancement by NrxIV loss of function alleles demonstrates that NrxIV functions in a parallel pathway.   The ability of nrv2 or ATPα loss of function alleles to suppress the Gli phenotype leads to two possible mechanisms.    126 (1) Dlg is key to the ectopic phenotypes generated when Gliotactin is overexpressed (Padash-Barmchi et al., 2013). Indeed dlg heterozygous mutants in trans to GliWT can also rescue the Gli phenotypes (Gayathri Samarasekera, personal communication). Therefore a decrease in the available Dlg at the bicellular domain resulting from a 50% loss of Na/K ATPase could explain the rescue of the ectopic Gliotactin phenotypes. If the loss of Na/K ATPase is decreasing the interaction between Dlg and Gli at the bicellular junction, then either subunit may be directly associated with Dlg. We can test for Na/K ATPase – Dlg interactions through immunoprecipitation assays. We can also use a proximity ligation assay between Dlg and Na/K ATPase to assess the localization of any Dlg – Na/K ATPase complexes. We can test for the strength of the PLA interaction within the context of reduced Na/K ATPase in the ATPαDTSr3 or nrv2nwu5 heterozygotes.  (2) Alternatively the observed increase in Gliotactin vesicles with a 50% reduction in the Na/K ATPase suggests an increase in Gliotactin endocytosis perhaps due to increased phosphorylation.  A mutant with both intracellular tyrosines of Gliotactin mutated to Asp to mimic phosphorylation (GliDD) also had increased levels of endocytosis (Padash-Barmchi et al., 2010). As Na/K ATPase can control Src levels, the suppression of GliWT by loss of Na/K ATPase may be due to an increase in pSrc levels. Gli can be phosphorylated by Src in vitro (Padash-Barmchi et al., 2010), suggesting that the increase in Src activation due to loss of the Na/K ATPase may trigger Gli downregulation through increased phosphorylation. We can test the role of Src in Gli phosphorylation by expressing GliWT in trans with loss of function alleles to nrv2 in concert with Src42A or Src42B mutants. If Src is involved, a 50% reduction in either Src should remove the suppression observed with nrv2 / ATPα in trans with GliWT. Alternatively, because the Src42A and Src64B may be redundant, overexpression of Src C-terminal kinase (dCsk), a   127 negative regulator of Src, in trans with GliWT and nrv2nwu5 would also remove the suppression of the GliWT phenotypes.  The TCJ forms at the convergence of three epithelial cells and is critical for maintaining the permeability barrier. To date only one protein, Gli, has been identified to localize specifically to the TCJ and together with Dlg, it is regulated to maintain epithelial integrity (Padash-Barmchi et al., 2010; Padash-Barmchi et al., 2013; Schulte et al., 2003). The components involved in this regulation and the mechanism through which the bicellular components interact with the TCJ remains uncovered. We have demonstrated Na/K ATPase is a novel component of the pathway regulating the TCJ with Gli and have provided a potential mechanism through which this regulation may occur. 4.4 Conclusion  The aim of this thesis was to examine how the Na/K ATPase, an ion transport molecule, functions as both a scaffolding and signal transduction molecule at the SJ of imaginal wing discs. We have demonstrated a clear role for Na/K ATPase in the correct assembly of SJs. We have demonstrated the relationship between Na/K ATPase and select SJ-associated proteins is different from the relationship of these proteins with NrxIV. These data provides strong evidence for the existence of microdomains within the SJs and suggests a model for how Na/K ATPase is regulating the establishment of microdomains within the SJs. In addition, this thesis work has provided in vivo evidence of the presence of Na/K ATPase/Src signaling complex and points to the pathway through which loss of the Na/K ATPase leads to JNK-mediated apoptosis. Src kinases are important regulators of many cellular processes including cell adhesion, and deregulation of its activation level has dramatic cellular consequences. Despite its crucial role in   128 development, little is known about how activated Src levels are maintained in Drosophila. In this thesis we have identified and described a mechanism by which the Na/K ATPase functions as a negative regulator of Src activation in the imaginal wing disc to regulate cell growth and development. While many questions remain unanswered, we have provided a foundation from which we can investigate this mechanism further.   129 Bibliography   Amata, I., Maffei, M. and Pons, M. (2014). Phosphorylation of unique domains of Src family kinases. Front Genet 5, 181. Anderson, J. M. (1996). 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