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Molecular mechanisms underlying the crosstalk between autophagy and apoptosis Hou, Ying Chen 2009

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Molecular mechanisms underlying the crosstalk between autophagy and apoptosis  by  Ying-Chen Hou B.Sc. (Honours), Simon Fraser University, 2003  A THESIS SUBMITED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies (Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August, 2009 © Ying-Chen Hou, 2009  ABSTRACT Macroautophagy (hereafter referred to as autophagy) is an evolutionary conserved mechanism for the degradation of long-lived proteins and organelles inside lysosomes. Autophagy functions as an adaptive survival response to starvation and other cellular stresses, but recent studies also demonstrated a role for autophagy in cell death. Functional studies indicate that a complex relationship exists between autophagy and apoptosis, the most common form of cell death, but the regulatory mechanisms underlying their interactions are largely unknown. To advance our understanding of the links between these two processes, I conducted a RNAi screen of Drosophila melanogaster cell death-related genes for their requirement in the regulation of starvation-induced autophagy. I discovered that six cell death genes, Dcp-1, hid, Bruce, Buffy, debcl, and p53 as well as Ras/Raf/MAPK signaling pathway components function in autophagy regulation in Drosophila l(2)mbn cells. I used Drosophila genetics to investigate a role for effector caspase Dcp-1 and inhibitor of apoptosis protein Bruce during autophagy in vivo. During Drosophila oogenesis, I found that autophagy is induced at two nutrient status checkpoints, the germarium and mid-oogenesis. Degenerating mid-stage egg chambers in DmAtg1 and DmAtg7 mutants exhibited reduced DNA fragmentation, suggesting autophagy may contribute to cell death during oogenesis. At the two nutrient status checkpoints in the developing ovary, Dcp-1 and Bruce function to regulate both autophagy and starvation-induced cell death, and epistasis analysis showed that Dcp-1 is downstream of Bruce, indicating that Bruce may negatively regulate Dcp-1 activity. In addition, I found that the catalytic activity of  ii  Dcp-1 is essential for autophagy regulation, suggesting that Dcp-1 dependent proteolysis may serve as a regulatory mechanism by which Dcp-1 mediates a cellular switch between autophagy and apoptosis. To identify potential substrates of Dcp-1, I employed an immuno-precipitation and mass spectrometry assay, the results of which can be used to generate a working model for how Dcp-1 controls autophagy. In summary, I developed an efficient screening method that resulted in the identification of several cell death-related regulators of autophagy. Further genetic and biochemical analyses of the effector caspase Dcp-1 in autophagy regulation provided new insights into the relationships between autophagy and apoptosis.  iii  TABLE OF CONTENTS ABSTRACT........................................................................................................................ ii TABLE OF CONTENTS................................................................................................... iv LIST OF TABLES............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii ACKNOWLEDGEMENTS............................................................................................. viii DEDICATION................................................................................................................... xi CO-AUTHORSHIP STATEMENT.................................................................................. xii Chapter 1 Introduction..................................................................................................... 1 1.1 The autophagy process.............................................................................................. 1 1.2 The molecular machinery of autophagy ................................................................... 2 1.3 Roles of autophagy in Drosophila melanogaster development................................. 4 1.4 Role of autophagy in cancer ..................................................................................... 5 1.5 Apoptosis .................................................................................................................. 6 1.6 Caspase activation and substrate recognition ........................................................... 9 1.7 The Drosophila melanogaster ovary model system and starvation-induced cell death.............................................................................................................................. 11 1.8 Relationship between autophagy and apoptosis ..................................................... 12 1.9 Objectives and hypotheses...................................................................................... 14 1.9.1 Rationale .......................................................................................................... 14 1.9.2 Hypotheses and specific aims .......................................................................... 15 1.10 References............................................................................................................. 27 Chapter 2 Effector caspase Dcp-1 and IAP protein Bruce regulate starvationinduced autophagy during Drosophila oogenesis ......................................................... 43 2.1 Introduction............................................................................................................. 43 2.2 Materials and methods ............................................................................................ 46 2.2.1 Cell culture conditions ..................................................................................... 46 2.2.2 dsRNA synthesis.............................................................................................. 47 2.2.3 RNA interference (RNAi)................................................................................ 47 2.2.4 Flow cytometry based LysoTracker Green (LTG) assay................................. 48 2.2.5 GFP-LC3 detection .......................................................................................... 48 2.2.6 Statistical analysis............................................................................................ 49 2.2.7 Generation of transgenic flies .......................................................................... 49 2.2.8 Fly strains......................................................................................................... 50 2.2.9 Generation of Atg 1 germline clones (GLCs).................................................. 50 2.2.10 LysoTracker Red staining .............................................................................. 50 2.2.11 TUNEL assay................................................................................................. 51 2.2.12 Quantitative RT-PCR..................................................................................... 52 2.3 Results..................................................................................................................... 53 2.3.1 RNAi screening assay identifies known positive and negative regulators of starvation-induced autophagy in Drosophila l(2)mbn cells...................................... 53 2.3.2 Identification of cell death-related genes that regulate starvation-induced autophagy in l(2)mbn cells........................................................................................ 56 2.3.3 Autophagy occurs in response to nutrient deprivation in germaria and midstage egg chambers in the Drosophila melanogaster ovary. .................................... 58 iv  2.3.4 Dcp-1 and Bruce regulate autophagy in germaria and degenerating mid-stage egg chambers. ........................................................................................................... 59 2.3.5 Dcp-1 and Bruce mutants have altered TUNEL staining in germaria and degenerating mid-stage egg chambers. ..................................................................... 61 2.3.6 Autophagy contributes to cell death in nutrient-deprived ovaries. .................. 62 2.4 Discussion ................................................................................................................... 63 2.5 References................................................................................................................... 91 Chapter 3 The effector caspase Dcp-1 catalytically regulates starvation-induced autophagy......................................................................................................................... 98 3.1 Introduction............................................................................................................. 98 3.2 Materials and methods .......................................................................................... 101 3.2.1 Cell culture and transfection .......................................................................... 101 3.2.2 Immunofluorescence...................................................................................... 102 3.2.3 LysoTracker Red (LTR) and DAPI staining.................................................. 103 3.2.4 Immunoprecipitation (IP) and MS/MS analysis ............................................ 103 3.2.5 Western Blot .................................................................................................. 105 3.2.6 Computational analyses of protein sequences ............................................... 105 3.3 Results................................................................................................................... 106 3.3.1 Effector caspase Dcp-1 genetically interacts with IAP protein Bruce and functions downstream of Bruce .............................................................................. 106 3.3.2 IAP protein Bruce delays starvation induced autophagic responses in the larval fat body and midgut ................................................................................................ 107 3.3.3 The effector caspase Dcp-1 accelerates the starvation-induced autophagic response but is not required for autophagy in the larval fat body........................... 108 3.3.4 Starvation has a rapid effect on activation of Dcp-1 but not drICE............... 109 3.3.5 The catalytic activity of Dcp-1 but not drICE is required for the induction of starvation-induced autophagy ................................................................................. 110 3.3.6 Potential regulators or substrates of Dcp-1 in cells undergoing autophagy... 111 3.4 Discussion ............................................................................................................. 112 3.5 References............................................................................................................. 134 Chapter 4 Conclusions and future research............................................................... 140 4.1 Overall summary and significance of the thesis research..................................... 140 4.2 Current limitations and summary of future research directions............................ 150 4.3 Potential applications of the research findings ..................................................... 151 4.4 References............................................................................................................. 159 Appendices..................................................................................................................... 166 Appendix A: abbreviations list ................................................................................... 166  v  LIST OF TABLES Table 2.1 Primer sequences for the preparation of dsRNAs............................................. 85 Table 2.2 Comparison of essential autophagy genes in the Drosophila larval fat body and l(2)mbn cells.............................................................................................................. 86 Table 2.3 Quantification of autophagy in region 2 germaria............................................ 87 Table 2.4 Quantification of autophagy in stage 8 degenerating egg chambers ................ 88 Table 2.5 Quantification of cell death in region 2 germaria ............................................. 89 Table 2.6 Quantification of cell death in stage 8 degenerating egg chambers ................. 90 Table 3.1 Potential Dcp-1 substrates .............................................................................. 133  vi  LIST OF FIGURES Figure 1.1 Model of the autophagy process...................................................................... 17 Figure 1.2 Molecular machinery of autophagy................................................................. 19 Figure 1.3 Core apoptosis signaling pathways in nematodes, fruitflies and mammals. ... 21 Figure 1.4 Conserved features of effector caspases in Drosophila melanogaster and mechanism of caspase 7 activation ........................................................................... 23 Figure 1.5 Drosophila melanogaster ovariole structure ................................................... 25 Figure 2.1 Quantification of starvation induced autophagy in Drosophila l(2)mbn cells. 68 Figure 2.2 Identification of known cell death related genes in autophagy regulation in l(2)mbn cells using RNAi. ........................................................................................ 70 Figure 2.3 Validation using 2nd set of dsRNAs................................................................. 73 Figure 2.4 Nutrient deprivation induces autophagy at region 2 within the germarium and in dying mid-stage egg chambers. ............................................................................ 75 Figure 2.5 The effector caspase Dcp-1 is not only required for nutrient starvation induced autophagy but also is sufficient for the induction of autophagy during Drosophila oogenesis................................................................................................................... 77 Figure 2.6 Bruce suppresses autophagy at region 2 within germarium and in dying stage 8 egg chambers. ........................................................................................................... 79 Figure 2.7 Dcp-1 is required for nutrient starvation induced germarium cell death and IAP protein Bruce inhibits germarium and mid-oogenesis cell death. ..................... 81 Figure 2.8 Lack of Atg7 or Atg1 function reduces DNA fragmentation during midoogenesis cell death. ................................................................................................. 83 Figure 3.1 Ovarian atrophy phenotype resulting from Bruce mutations is rescued by Dcp1............................................................................................................................... 119 Figure 3.2 Bruce postpones starvation-induced autophagy in the larval fat body and midgut. .................................................................................................................... 121 Figure 3.3 Expression of Dcp-1 accelerates autophagy in response to nutrient withdrawal but is not essential for autophagy in the fat body and midgut ................................ 124 Figure 3.4 Time course analyses of Dcp-1 and drICE cleavage at the inter-domain site during nutrient deprivation. .................................................................................... 126 Figure 3.5 The catalytic activity of Dcp-1 but not drICE is required for starvation-induced autophagy................................................................................................................ 128 Figure 3.6 Putative model of Dcp-1 mediated autophagy .............................................. 130 Figure 3.7 Bruce expression increase in fat body in response to nutrient withdrawl…..131 Figure 4.1 A hypothetical pathway for the regulation of sensitivity thresholds leading to autophagy or apoptosis. .......................................................................................... 153 Figure 4.2 The effector caspase Dcp-1 is sufficient for the induction of autophagy during Drosophila oogenesis.............................................................................................. 155 Figure 4.3 Possible relationships between autophagy and DNA degradation in Drosophila oogenesis................................................................................................................. 157  vii  ACKNOWLEDGEMENTS I would like to thank the following people who supported me during my graduate studies.  First, I would like to thank my supervisor, Dr. Sharon Gorski, whose constant support, and advice were indispensable in accomplishing the work presented here.  She has  challenged my knowledge and critical thinking and gave me such a wonderful opportunity to be independent. When I joined her lab, her encouragement motivated me to stay in research and become a scientist. I will remind myself of her encouragement when I face difficulties in the future.  My very special thanks to my committee member, Dr. Marco Marra, who has always challenged me to be better, and whose advice helped me to develop my scientific career. He also introduced me to espresso which has become my morning ritual and addiction.  I would also like to thank my committee member, Dr. Isabella Tai for her valuable advice and guidance throughout my studies. I will never forget our chats. Thank you also to my committee member, Dr. Marianne Sadar, for her time and valuable advice with regards to my studies.  I would like to thank members of the Gorski and Marra labs at the Genome Sciences Center for making it such a friendly and stimulating environment. In particular, I would like to thank Dr. Suganthi Chittaranjan, Doug Freeman, Dr. Ian Bosdet, Melissa McConecky, James Wilton, Brian Kwok, Adrienne Hannigan, Amy Leung and Lindsay  viii  deVorkin for all their assistance during my research and for being wonderful people to work with. I want to thank Malachi Griffith for his constant support during my technical problems with computer software and computational analysis, and his advice and discussions on cloning. Thank you also to Trevor Pugh for the help with sequencing activities.  I would like to thank Dr. G. Morin and Dr. A. Moradian for their collaborations in the proteomic study, and Dr. G. Cheng and Carri-Lyn Mead for their suggestions and help. Thank you to A. Dorn for l(2)mbn cells, T.E. Rusten and H. Stenmark for the eGFPLC3 plasmid and the UASp-GFP-LC3; nanos-GAL4 Drosophila strain, P. Meier for plasmids and antibodies of Dcp-1 and drICE, J.E. Beliz’ario for CasPredictor software and T. Grigliatti for the p2ZOp2F plasmid. Thank you to K. McCall, B. Hay, T. Neufeld, and H. Steller for fly stocks. Thank you to A.H. Tien and C. Helgason for allowing me to use the flow cytometer and for training and technical support on the flow cytometer. I want to thank members of McCall’s lab including S González Barbosa, Mateos San Martin, E. Tanner, J. Chang, and B. Laundrie Stahl, for their collaborations on my first manuscript.  My special thank to Drs. Eric Baehrecke, Andreas Bergman, Bruce Hay, Thomas Neufeld, Pascal Meier, Kim McCall, and Denise Clark for their wonderful advice and suggestion on my project.  ix  Last but not least, I would like to thank my parents, Peter and Lisa, for their unconditional love and support which are essential in accomplishing this work and for their understanding during the busiest period of my life. Also I want to thank my parents for always encouraging me to pursue my PhD. I want to thank my sister, Lilith, who is my best friend and is always there for me when I am down.  x  DEDICATION  To my parents, Peter and Lisa, for their love, wisdom and inspiration. Dad, thank you so much for your support. Now it is your turn. I will support you to pursue your dream of getting a PhD degree.  xi  CO-AUTHORSHIP STATEMENT Hou Y-C. C, Chittaranjan S, González Barbosa SN, McCall K, Gorski S.M. Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila oogenesis. Journal of Cell Biology 2009; 182: 1127-39. I performed 90% of the experiments presented in this paper. Dr. S. Chittaranjan assisted in the construction of the Drosophila GFP-LC3 transfected l(2)mbn cell line. Analyses of ectopic expression of Dcp-1 during mid-oogenesis using the LysoTracker Red assay, and generation of Atg1 germline clones were conducted in collaboration with Dr. S. González Barbosa and Dr. K. McCall. I prepared the manuscript in collaboration with Dr. S. Gorski.  Hou Y-C. C, Moradian A, Morin GB, Gorski SM. 2009. The effector caspase Dcp-1 catalytically regulates starvation-induced autophagy. In preparation. I performed 95% of the experiments presented in this paper. Protein interaction studies were conducted in collaboration with Dr. A. Moradian and Dr. G. Morin. I prepared the manuscript in collaboration with Dr. S. Gorski.  Hou Y-C. C, Hannigan A.M, Gorski S.M. An executioner caspase regulates autophagy. Autophagy 2009; 5: 530-33. A. Hannigan assisted in preparation of the last paragraph of the manuscript. I prepared the manuscript in collaboration with Dr. S. Gorski.  xii  Chapter 1 Introduction 1.1 The autophagy process Autophagy (self eating) is a process for the degradation of cytosolic components inside lysosomes.  Based on the mechanisms by which substrates are delivered to  lysosomes, three major forms of autophagy, macroautophagy, microautophagy and chaperone-mediated  autophagy  (CMA)  have  been  described.1  During  the  macroautophagy process, cytosolic components and intracellular organelles are first sequestered within double-membrane vesicles called autophagosomes.2, 3 The origin of the pre-autophagosomal structure, also known as the phagophore assembly site, has been a topic of debate; however, two models for its origin, termed the maturation or assembly models, have been proposed.4  Based on the maturation model, the origin of the  autophagosomal membrane is the endoplasmic reticulum (ER), whereas the assembly model suggests that the autophagosomal membrane assembles de novo from localized lipid synthesis.4 The recent identification of a novel double FYVE domain containing protein 1 (DFCP1) that co-localizes with ER membrane and autophagosomal proteins provides support for the maturation model.5 The autophagosome subsequently fuses with a lysosome to become an autolysosome where the cellular components are degraded (Figure 1.1). Microautophagy is involved in a direct uptake of inclusions (e.g., glycogen) and organelles (e.g., ribosomes and peroxisomes) at the lysosomes by invagination of the lysosomal membrane without the formation of intermediate transport vesicles.1 CMA involves the recognition and transport of protein substrates to the lysosome, and is responsible for degradation of approximately 30% of cytosolic proteins in response to  1  nutrient deprivation.1,  6  Substrate proteins of CMA contain a particular pentapeptide  motif (KFERQ) that are recognized by a cytosolic chaperone hsc70 and subsequently bind to a receptor, lysosome-associated membrane protein type 2a (lamp2a), at the lysosomal membrane which mediates lamp2a protein complex translocation into the lysosomes for degradation.1, 6 Macroautophagy is the most studied form of autophagy and hereafter is referred to as autophagy. Autophagy occurs at basal levels in normal conditions and is induced by various stimuli including starvation, hypoxia, intracellular stress, hormones and growth factor deprivation.7 The most fundamental role of autophagy is to provide an internal source of nutrients under starvation conditions.1 Nucleotides, amino acids, and free fatty acids are generated from the degradation of the sequestered materials and further recycled for the synthesis of macromolecules and ATP production.1 Basal autophagy also plays a key role in eliminating defective organelles (e.g. mitochondria)8 or aggregated proteins that may be resistant to the ubiquitin-proteosome degradation pathway.9-11 In addition, autophagy is involved in other processes including cellular differentiation, tissue remodeling, growth control, elimination of pathogens, and clearance of apoptotic cells.1215  1.2 The molecular machinery of autophagy The discovery and characterization of autophagy-related (Atg) genes in yeast, Saccharomyces cerevisiae, have contributed to the understanding of the molecular mechanisms involved in autophagy. There are 31 autophagy-related genes in yeast, and 18 Atg proteins are essential for autophagosome formation.3 The Atg genes encode proteins which are required for the induction of autophagy, autophagosome nucleation,  2  vesicle expansion and completion, and final retrieval of Atg proteins from mature autophagosomes.16 Most of the Atg genes are conserved in higher eukaryotes, including mammals. The molecular mechanisms of autophagy are illustrated in Figure 1.2. TOR kinase and class III phosphatidylinositol 3 kinase (PI3K) are two important regulators of autophagy.17, 18 TOR kinase is a sensor of amino acid deprivation, and is believed to be a “gatekeeper” for the induction of autophagy.17 Under nutrient-rich conditions, TOR kinase phosphorylates Atg13 (autophagy-related gene-13).19  The  hyperphosphorylated form of Atg13 has a low affinity for its interacting proteins Atg1 and Atg17.19 This results in a reduction in Atg1 kinase activity which ultimately results in inhibition of autophagy.19, 20 Conversely, during amino acid deprivation, TOR activity is inhibited, resulting in Atg 13 interaction with Atg1 and Atg17, which activates autophagy (Figure 1.2 A).19, 20 The initial step of vesicle nucleation is the activation of the class III PI3K, also known as Vps34, to generate phosphatidylinositol 3-phosphate (PI3P) in both yeast and mammals.18 In yeast, Vps34 activation depends on the formation of a multi-protein complex that includes Atg6, the myristoylated Vps15 kinase, and Atg14 (Figure 1.2 B).1 In mammals, activation of Vps34 is dependent on the formation of a multi-protein complex that consists of Atg6/Beclin-1, Ultraviolet irradiation resistance-associated tumour suppressor gene (UVRAG), Bif-1, Ambra1, and a myristylated serine kinase Vps15 (Figure 1.2 B).21-23 Two ubiquitin-like conjugation systems are involved in autophagosome membrane elongation and completion.  Atg12 is activated by an E1-like enzyme called Atg7,  followed by the transfer of Atg12 by Atg7 to an E2-like enzyme, Atg10.24-26 Later,  3  Atg12 is conjugated to Atg5 and forms a larger protein complex with Atg16 through oligomerization  of  Atg16  monomers.24-26  The  Atg5/Atg12/Atg16  complex  asymmetrically localizes to the outer side of the autophagosomal membrane throughout the elongation process and dissociates from the autophagosome upon completion (Figure 1.2 C).26 Atg8 is cleaved by Atg4 to produce the cytosolic form Atg8-glycine and is activated by Atg7. Another E2-like protein Atg3 is responsible for Atg8 conjugation.26 Finally Atg8 is lipidated with phosphatidylethanolamine (PE) and inserted in the autophagosome membrane.27 In contrast to the Atg5/Atg12/Atg16 system, Atg8 remains on the autophagosomal membrane after the completion of autophagosome formation.27 Microtubule associated protein 1 light chain 3B (MAP1LC3B or LC3) is the functional human homologue of Atg8.28 Based on the observations that LC3 displays different intracellular locations and an increased electrophoretic mobility on SDS-polyacrylamide gels during autophagic membrane recruitment, it has been employed as a marker for autophagosomes and autophagic activity.28, 29 A recycling pathway that is composed of Atg9 and Atg18 mediates the disassembly of Atg proteins from mature autophagosomes (Figure 1.2 D).30, 31 However, the retrieval mechanism which the Atg9-Atg18 complex mediates is still poorly understood.  1.3 Roles of autophagy in Drosophila melanogaster development Autophagy is observed in several Drosophila melanogaster larval tissues, including salivary glands, fat body and midgut, and genetic studies in flies provided evidence that autophagy plays a role in Drosophila melanogaster development. Mutations in some of the autophagy genes result in lethality at various stages of Drosophila melanogaster development.7 For example, null mutations in Atg1 exhibit a  4  highly penetrant lethal phenotype in the pupal stage, prior to eclosion.32 Lethality at the larval stage is observed in flies carrying mutations in Atg18 or Atg6 genes.32 However, flies carrying mutations in Atg7 or Atg8a develop to adulthood, despite an essential role for both genes in autophagy.32-34 Autophagy also plays an important role in adaptation to starvation in the larval fat body. The larval fat body is a nutrient storage organ that stores lipids and glycogen.35 Nutrients in the larval fat body are degraded by autophagy to support development of imaginal tissues which turn into adult fly tissues36 and to sustain larval survival for two or more weeks in the absence of amino acids.37 Atg1, Atg5, and Atg7 mutants were shown to die earlier than wild-type flies in response to starvation conditions.32,  33  Autophagy also promotes survival in response to a number of other  stresses, including oxidative stress, chill-induced coma and CO2 anesthesia.33 Further, autophagy appears to contribute to cell death in several tissues of Drosophila melanogaster. Deficiencies in Atg genes prevent complete destruction of larval salivary glands38 and suppress cell death of the aminoserosa which is eliminated during embryogenesis.39 In Atg7 mutants, the larval midgut shows reduced DNA fragmentation, an indicator of cell death, and its complete destruction is delayed for four hours during metamorphosis.33 Overexpression of Atg1 results in cell death in both the larval fat body and salivary glands.34,38 Together, these examples illustrate multiple roles for autophagy during Drosophila melanogaster development.  1.4 Role of autophagy in cancer Autophagy has been linked to various human diseases, including cancer. Autophagy functions as a survival mechanism when cells encounter nutrient deprivation. When cancer cells encounter hypoxia, metabolic stress, or nutrient starvation, autophagy may  5  aid in cell survival by providing nutrients and/or energy, and thus promote tumorigenesis.40 Autophagy has been shown to maintain tumour survival in response to metabolic stress in vitro and in hypoxic regions of tumours in vivo.41-44 Paradoxically, genetic studies show that autophagy can act as a tumour suppression mechanism. Beclin1 is the human orthologue of Atg6 in yeast and is involved in the early steps of autophagic vesicle formation.  Allelic loss of Beclin1 is observed in human breast,  ovarian and prostate cancers, and Beclin1 heterozygous mice have a high incidence of spontaneous tumours, indicating that beclin-1 is a haplo-insufficient tumour suppressor gene.45, 46  Autophagy related-4C (Atg4C) deficient mice are prone to tumour formation  following exposure to chemical carcinogens.47 Autophagy is known to help degradation of damaged organelles such as depolarized mitochondria.48 Defective mitochondria lead to accumulation of reactive oxygen species leading to genotoxic damage, which may explain the potential tumor-suppressive effects of autophagy.49 A recently described role of autophagy in maintaining genome stability could also explain the tumour suppression mechanism of autophagy.42 Alternatively autophagy may promote cell death as reported in some systems.38, 39, 50, 51  The survival and death promoting roles of autophagy in  tumour progression are still far from being understood. A better understanding of the roles of autophagy in both tumour survival and tumour suppression may ultimately provide novel approaches for cancer prevention and treatment strategies.  1.5 Apoptosis Programmed cell death maintains the homeostasis of metazoans by removing unwanted or damaged cells during development and adulthood.52,  53  Apoptosis, also  known as type I programmed cell death, has morphological characteristics of membrane  6  blebbing, chromatin condensation, cell shrinkage and DNA fragmentation.54,  55  Cells  undergoing apoptosis become fragmented and form apoptotic bodies which are engulfed by neighboring phagocytotic cells.56 Apoptosis is important for developmental processes, including  embryogenesis,  differentiation.57-59  nervous  system  development,  and  immune  system  In addition, apoptosis is involved in disease processes such as  autoimmune diseases, neurodegenerative disorders, immunologic deficiencies, and cancers.52 A family of proteases known as caspases is at the core of the apoptosis machinery.59,  60  Caspases are expressed ubiquitously and are synthesized as inactive  zymogens.60 In response to various death signals, caspases become activated, cleave multiple cellular substrates and lead to cell death.61, 62 Proteins which induce caspasedependent cell death and control levels of active caspases in cells are evolutionarily conserved (Figure 1.3).60, 63  In Caenorhabditis elegans, the sole caspase CED-3 and its  activator, the adaptor protein CED-4 are expressed ubiquitously.64 Activation of CED-3 is controlled by CED-4, but no inhibitors of activated CED-3 have been identified.64 Expression of the anti-apoptotic multi-domain Bcl-2 family protein, CED-9, prevents cell death by sequestering CED-4 at mitochondria and preventing CED-4 oligomerization, which is required for CED-3 activation.65 In dying cells, a small pro-apoptotic protein EGL-1 (a member of the BH3 domain only Bcl-2 family) binds to CED-9 and changes its conformation resulting in the release of CED-4 from the inhibitory CED-4-CED-9 complex.65 In Drosophila melanogaster, molecular mechanisms governing apoptosis have been studied extensively. There are seven caspases, and three of them, Dronc, Strica, and  7  Dredd, are initiator caspases, which are molecularly characterized by the presence of a long N-terminal prodomain.66-70 The remaining four caspases, Dcp-1, drICE, Decay and Daydream contain a short prodomain suggesting that they function as effector caspases.7174  Cell death is triggered by activation of the apical caspase Dronc (encoded by Nc) and  is mediated by the adaptor protein Dark, the fly homologue of CED-4 in worms and Apaf-1 in mammals.75, 76 Expression of the anti-apoptotic protein DIAP1 (encoded by th) suppresses Dronc activity77 and also downstream effector caspases (Dcp-1 and drICE).78, 79  The pro-apoptotic proteins, Reaper (Rpr), Grim, Hid, and Sickle, disrupt the  interaction between DIAP1 and caspases.80 Further, Drosophila melanogaster has two Bcl-2 family proteins, encoded by debcl and buffy, and these proteins have pro- and antiapoptotic activity respectively, suggesting that they might be the upstream regulators of caspase activation, but direct evidence for this interaction is still lacking.81-85  In  addition, several upstream signaling pathways have been shown to regulate the cell death machinery. For example, the EGF receptor/Ras pathway specifically inhibits Hid activity through MAPK-mediated phosphorylation.86 DNA damage up-regulates expression of Drosophila melanogaster p53 which then binds to the cis-regulatory region of rpr and induces apoptosis.87 In mammals, activation of caspase 9, the mammalian homolog of Dronc in Drosophila melanogaster, is regulated by Bcl-2 family proteins and dependent on Apaf1.88,  89  Cell death stimuli activate BH3-only family members which facilitate pro-  apoptotic Bcl-2 family member BAX and BAK-dependent release of pro-apoptotic mitochondria proteins, including cytochrome c and Smac (also known as DIABLO).88 Cytochrome c binds to and activates Apaf-1 in the cytoplasm which results in a  8  conformational change in Apaf-1. Apaf-1 further binds to ATP/dATP and forms the apoptosome which mediates the activation of caspase-9 and triggers a cascade of caspase activation.90-92 The tetrapeptide, Ala-Val-Pro-Ile, at the N terminus of mature Smac is an IAP-binding motif which binds to XIAP and relieves XIAP-mediated inhibition of caspase 9.93-95  The tetrapeptide is conserved between flies and humans, since the  Drosophila melanogaster RHG proteins, Reaper, Hid, Grim and Sickle, all contain a conserved IAP-binding tetrapeptide motif.60 Defective apoptosis is a hallmark of most, if not all, types of cancers and activation of apoptosis is one of the current strategies to eliminate cancer cells.96 The tumour suppressive role of apoptosis is well established, and pathways and genes associated with apoptosis have been studied extensively. Overexpression of the apoptosis inhibitors (e.g. Bcl-2 or IAP family members) or deletion of apoptosis effectors (e.g. caspases) suppresses apoptosis and allows tumour cells to survive the stresses of oncogene activation, uncontrolled proliferation and chemotherapy.97,  98  Antagonists of  Bcl-2 and IAP proteins have either entered the clinic or are under clinical trails as therapeutics to restore the apoptotic pathway of resistant tumours.99, 100 The finding that the IAP binding motif of Smac acts as a potent mediator of caspase activation makes this tetrapeptide a promising candidate for a therapeutic agent and efforts have been put into the development of small molecule Smac mimetics for cancer treatment.100  1.6 Caspase activation and substrate recognition Caspases are the executioners of the apoptotic response. Caspases are a family of cysteine proteases that cleave after an aspartate residue in their substrates.101 Each caspase monomer contains an N-terminal pro-domain followed by a 20kDa (p20) and a  9  10kDa (p10) subunit. Caspases exist constitutively as homodimers. Caspases can be divided into two classes: the initiator caspases have extended N terminal pro-domains which contain caspase recruitment domains (CARD) or death effector domains (DED) to interact with other molecules, while the effector caspases have a pro-domain that is relatively short.60 All caspases are synthesized as inactive zymogens and their catalytic activities are increased by several orders of magnitude after inter-domain cleavages.60, 102 In Drosophila melanogaster, Dcp-1 was shown to undergo inter-domain cleavage at Asp33 and Asp215, and the proteolytic cleavage of drICE occurs at Asp28 and Asp230 during Rpr-mediated apoptosis (Figure 1.4 A).103 The backbone configuration of all caspases is highly conserved and their catalytic groove is composed of four surface loops (L1-L4) as revealed by crystal structures of several active caspases (for review see Shiozaki 2004).102 Analyses of the human caspase-7 zymogen (Figure 1.4 B) and active caspase-7 (Figure 1.4 C) helped to reveal the mechanisms of caspase activation and substrate binding.104 The L1 and L4 loops constitute two parallel sides of the substrate binding groove and L3 serves as the base. The inter-domain cleavage allows the L2 loop and the L2’ loop of the neighboring caspase monomer to switch to an open conformation and expose the catalytic residue cysteine.104 A similar conformation change was also observed in Drosophila melanogaster effector caspase drICE.80 Caspases recognize at least four amino acids, P4-P3-P2-P1, in their substrates and cleave after the C-terminal residue (P1) which is usually an Asp residue. However, the initiator caspase Dronc has an equal specificity for either an Asp or Glu residue in P1.105 The most frequent residues at the P4-P1 position are Asp, Glu, Val, and Asp (DEVD), the canonical caspase cleavage motif, as determined with in vitro positional scanning peptide libraries.  However, there  10  are significant differences between the actual cellular caspase substrate cleavage sites and the in vitro substrate specificity profiles;106, 107 thus, the prediction of a caspase substrate based on caspase cleavage motifs in their amino acid sequences needs to be carefully validated.  1.7 The Drosophila melanogaster ovary model system and starvationinduced cell death Cell death in the Drosophila melanogaster ovary has been studied for more than a century.108 The appearance of vacuoles in the dying cells109 and the induction of cell death in nurse and follicle cells in response to nutritional changes at distinct stages during oogenesis make the ovary a potential model system to study the relationship between autophagy and cell death.110 Each adult Drosophila melanogaster ovary contains 15 to 20 ovarioles (for review see Spradling 1993)108. Each ovariole contains a series of developing egg chambers that consist of 16 germline cells (15 nurse cells and 1 oocyte) surrounded by a layer of somatic follicle cells (Figure 1.5). Germline stem cells are located at the anterior end of each ovariole in a specialized region called the germarium. The germline stem cells develop into cystoblasts which undergo four rounds of mitosis to form 16-cell cysts.  These cysts then undergo incomplete cytokinesis and remain  connected with each other through ring canals.  Somatic follicle cells surround the  germline cysts when they migrate into region 2b (Figure 1.5). Each germline cyst then becomes a complete egg chamber, moves out of the germarium, and progresses through 14-defined developmental stages. During pre-vitellogenic stages, the relative sizes of the oocyte and nurse cells remain similar. At stage 8, the oocyte increases its volume and undergoes vitellogenesis, a process of yolk protein synthesis and uptake.  Toward the 11  late stage of oogenesis, the nurse cells support the development of the oocyte by transferring to it their cytoplasmic contents via the ring canals.  The nurse cells  eventually undergo cell death and dying cells are engulfed by the surrounding follicle cells during late oogenesis.108, 109 When animals encounter nutrient deprivation, chemical insults or hormonal changes, egg chambers undergo cell death at two earlier stages, germarium and mid-oogenesis.110-112  Thus, cell death at these two stages has been  proposed to act as checkpoints, where the nutrient and/or environmental status of egg chambers are monitored prior to investing energy into egg production.110, 111, 113 The core apoptotic activators of Drosophila melanogaster, rpr, hid, grim and sickle, are not required for cell death in mid-oogenesis, and cell death genes such as debcl, ark and p53 also are not required for mid-oogenesis cell death.114 However, one essential player in mid-oogenesis cell death is the effector caspase Dcp-1.115 Thus, mid-oogenesis cell death has been described as “non-canonical”111, and the genes required for cell death at this stage are still largely unknown.  1.8 Relationship between autophagy and apoptosis A complex inter-connection exists between components of apoptosis and autophagy. Two apoptotic inducers, sphingolipid and ceramide, have been shown to activate autophagy in mammalian cells.116,  117  Components of the apoptotic pathway,  including TRAIL, FADD, Bad and DAPK, were shown to be positive regulators of autophagy.118-122  In contrast, the class I PI3K/Akt/Tor signaling pathway, an anti-  apoptotic pathway, acts to suppress autophagy (for reviews see Levine 2005 and Lum 2005).16,  41  Anti-apoptotic proteins, Bcl-2 and Bcl-XL, were shown to suppress  autophagy through a direct interaction with Beclin-1 which contains a BH3 domain.123, 124  12  Pro-apoptotic BH3-only proteins such as BNIP3, Bad and Puma, as well as pharmacological BH3 mimetics, also function to induce autophagy through the competitive disruption of the interaction between Beclin1 and Bcl-2 or Bcl- XL.124-127 An Atg5 cleavage product, which is generated by calpain cleavage, moves to mitochondria and associates with Bcl-xL leading to caspase activation.128 In addition, autophagy was shown to contribute to cell death during developmental programs in Drosophila melanogaster as discussed previously, and in apoptosis-deficient mammalian cells in response to cytotoxic stress.50,  51  Mouse embryonic fibroblasts (MEFs) from double-  knockout Bax-/- Bak-/- mice are resistant to various apoptosis inducers, and when Bax-/Bak-/- MEFs are treated with DNA damaging agents such as etoposide (a topoisomerase2 inhibitor), autophagy is induced in Bax-/- Bak-/- MEFs followed by non-apoptotic cell death.50 Knockdown of Atg5 or Atg6/Beclin1 by RNAi reduces the etoposide-induced cell death of Bax-/- Bak-/- MEFs.50 Inhibition of caspase 8 by treatment with the pancaspase inhibitor z-VAD or RNAi triggers autophagy and cell death concurrently in L929 mouse fibrosarcoma cells.51 z-VAD-induced cell death is rescued by knockdown of key autophagy genes such as Atg6/Beclin1 or Atg7 by RNAi.51 Cell death induced by zVAD was shown to be associated with the accumulation of reactive oxygen species (ROS) that is caused by the autophagy mediated- depletion of the major enzymatic ROS scavenger, catalase, and RNAi of Atg7 or Atg8 blocks both ROS accumulation and cell death.129 Autophagy also plays a role in eliminating apoptotic corpses by providing an energy source (ATP) to facilitate the generation of engulfment signals, including lysophosphatidylcholine secretion (come-get-me signal) and phosphatidylserine exposure (eat-me signal) in a mouse embryoid body cavitation model15 and in a mouse  13  neuroepithelium model.130  These findings clearly show a complex inter-connection  between apoptosis and autophagy; however, the molecular mechanisms underlying the relationships between apoptosis and autophagy pathways are still poorly understood.  1.9 Objectives and hypotheses 1.9.1 Rationale Several apoptotic components have been shown to play a role in the regulation of autophagy.118-122 Autophagy was shown to contribute to cell death during development or in apoptosis-deficient mammalian cells in response to cytotoxic stress (for reviews see Baehrecke 2002, Levine 2005, and Thorburn 2008).16, 131, 132  Autophagy has also been  linked to cancer; however, the survival and death promoting roles of autophagy in tumour progression are still far from being resolved. A better understanding of the interplay between autophagy and apoptosis will provide insight into their relationship during both development and disease, and aid in the design of cancer treatments. Numerous studies have focused on characterizing the function of Atg genes in cell death, but a systematic approach to determine the involvement of cell death genes in autophagy has not been employed. To address this question, I used Drosophila melanogaster as a model system and performed an RNAi gene silencing screen on cell death genes. I chose Drosophila melanogaster as the model system on the basis of its less redundant but evolutionary conserved cell death pathway and the availability of numerous molecular/genetic tools. For example, during evolution, an expansion of caspase family members took place. There are 11 caspases that play a critical role in apoptosis in mammals, but only 4 caspases are required for apoptosis in Drosophila melanogaster.59 Carrying out the  14  RNAi screen in a less complex model organism may thus provide a higher probability of identifying evolutionary conserved genes/pathways that regulate autophagy, since there is a less chance of functional redundancy in any given tissue of cell line. Findings in Drosophila melanogaster will also benefit our understanding of the crosstalk between autophagy and apoptosis in mammals. 1.9.2 Hypotheses and specific aims Hypothesis 1: Given that some apoptotic genes were shown to play a role in autophagy regulation, I hypothesized that a systematic screening approach would identify additional cell death genes involved in autophagy regulation in Drosophila l(2)mbn cells Specific Aim 1: Design a systematic approach to identify cell death genes involved in the regulation of autophagy A systematic RNAi screen coupled with flow cytometry-based LysoTracker Green and cell-based GFP-LC3 assays was employed to investigate the involvement of cell death genes in starvation induced autophagy in Drosophila l(2)mbn cells, which are haemocytes derived from lethal (2) malignant blood neoplasm mutants. Hypotheses 2: Given that amino acid starvation was shown to trigger cell death in the Drosophila melanogaster ovary, I hypothesized that starvation would induce the autophagic response and autophagy related genes would contribute to cell death in the ovary. Genes that were required for regulation of autophagy in l(2)mbn cells were predicted to have a similar function in autophagy regulation during Drosophila melanogaster oogenesis.  15  Specific Aim 2: Determine the roles of Atg genes (Atg1 and Atg7) in starvationinduced cell death and the roles of Dcp-1 and Bruce in autophagy regulation during Drosophila melanogaster oogenesis. LysoTracker Red (LTR) and GFP-LC3 assays were employed to determine whether autophagy was induced in response to nutrient deprivation in the germaria and midoogenesis egg chambers in the ovary.  The TUNEL assay, an indication of DNA  fragmentation, was employed to assess the function of two autophagy related genes, Atg1 and Atg7, in germarium and mid-oogenesis cell death. The effects of Dcp-1 and Bruce mutations on autophagy and cell death were examined in germaria and mid-oogenesis egg chambers. Hypotheses 3: First, I hypothesized that Bruce and Dcp-1 interact genetically and that Bruce functions as a negative regulator of Dcp-1 activity. Second, I hypothesized that the catalytic activity of Dcp-1 plays a regulatory function in the autophagy process, and substrates of Dcp-1 have a functional significance in mediating autophagy. Specific Aim 3: Determine the genetic interaction between Dcp-1 and Bruce and characterize the molecular mechanisms of Dcp-1 in the autophagy process To examine the genetic relationship between Dcp-1 and Bruce, I created Dcp-1; Bruce double mutants. Analyses of double mutants during starvation-induced autophagy and cell death in the ovary were performed. I employed the catalytically inactive construct of Dcp-1 to determine whether Dcp-1 dependent proteolytic events were required for starvation-induced autophagy. To identify potential substrates of Dcp-1 in l(2)mbn cells undergoing starvation-induced autophagy, I employed an immunoprecipitation and mass spectrometry (IP-MS) strategy.  16  Figure 1.1 Model of the autophagy process. Cytoplasmic components and organelles are sequestered into a double-membrane structure called an autophagosome. The autophagosome fuses with a lysosome to create an autolysosome where the inner membrane and cellular contents are degraded by lysosomal enzymes. During autophagy, Atg8 (LC3 in mammals) is cleaved, lipidated and conjugated to autophagosomes. The membrane translocation event of Atg8 makes it a useful marker to monitor autophagy. [Figure adapted from Klionsky 2008].29  17  Figure 1.1  18  Figure 1.2 Molecular machinery of autophagy. The formation of an autophagosome can be divided into four distinct steps: induction, vesicle nucleation, vesicle elongation, and membrane retrieval. (A) The phosphorylation (P) state of Atg13 is controlled by Tor kinase. Inactivation of Tor results in the dephosphorylation of Atg13, and the subsequent formation of a protein complex that includes Atg1, Atg13 and Atg17 for the induction of autophagy. (B) The activation of class III phophatidylinositol 3 kinase (PI3K), also known as Vps34, generates phosphatidylinositol 3-phosphate (PI3P) and is important for the vesicle nucleation step. In yeast, Atg6, Atg14, and Vps15, are part of the class III PI3K complex. In mammals, the PI3K complex consists of Beclin1/Atg6, Vps15, UVRAG, Ambra1, and Bif-1. Bcl-2 and Bcl-XL interact constitutively with Beclin1, and dissociation of Beclin1 from its inhibitors Bcl-2 or Bcl-XL is required for autophagy induction. (C) Two ubiquitin-like conjugation systems, the Atg12 conjugation system (Atg12, Atg5, and Atg16) and the Atg8 lipidation system (Atg8, Atg3 and Atg7) are involved in vesicle expansion. (D) The Atg9-Atg18 dependent membrane retrieval complex is required for the disassembly of Atg protein complexes from mature autophagosomes. [Figure adapted from Maiuri 2007 and Melendez 2008].7, 131  19  Figure 1.2  20  Figure 1.3 Core apoptosis signaling pathways in nematodes, fruitflies and mammals. In all three species, the core apoptotic components are conserved. However, mammals and the fruitfly Drosophila melanogaster have both initiator (purple) and effector (orange) caspases, and there is only a single caspase CED-3 that carries out both functions (initiator and effector) in the nematode worm Caenorhabditis elegans. The activity of caspases is restrained by inhibitor of apoptosis proteins (IAP) (grey). IAP binding proteins (blue) remove the IAP-mediated negative regulation of caspases. The adaptor protein CED-4/Dark/Apaf-1 (green) promotes the activation of caspases. CED4/Dark/Apaf-1 activity is inhibited by anti-apoptotic Bcl-2 family members (light yellow). The activation of Dark might be regulated by the Drosophila melanogaster Bcl2 family members, Debcl and Buffy, but direct evidence for this interaction is still lacking. [Figure adapted from Hay 2004].67  21  Figure 1.3  22  Figure 1.4 Conserved features of effector caspases in Drosophila melanogaster and mechanism of caspase 7 activation (A) A schematic representation of Drosophila melanogaster Dcp-1 and drICE. The red ovals indicate the catalytic cysteine residue. The four surface loops (L1-L4) and the p20 and p10 subunits are indicated for each caspase. Arrows indicate the sites of interdomain cleavage that results in generation of the p20 and p10 subunits. Caspases exist as homo-dimers and their catalytic activity is increased after cleavage at the inter-domain sites that result in an allosteric conformational change of surface loop 2 (L2).  L2  contains the catalytic residue cysteine and L2’ stabilizes the catalytic site of the adjacent caspase monomer. The conformation of the L2’ loop is dramatically different between mammalian inhibitor (I) bound procaspase-7 zymogen (B) and active caspase-7 (C). The L2’ loop of the procaspase-7 zymogen is locked in a closed conformation by covalent linkage.  After inter-domain cleavage, the L2’ and L2 loops switch to their open  conformation allowing substrate binding. L1 and L4 constitute two parallel sides of the substrate binding groove and L3 serves as the base. [Figure 1.4 B and C from (Shiozaki and Shi, 2004)]102  23  Figure 1.4  24  Figure 1.5 Drosophila melanogaster ovariole structure A schematic representation of a Drosophila melanogaster ovariole. Each ovariole is composed of the germarium in the anterior, followed by a row of progressively developing stages (2-14) of egg chambers. A cystoblast (cb) is derived from a germline stem cell (gsc) and undergoes four rounds of mitosis to form a 16-cell cyst (cc). While the germline cyst migrates from region 2a to 2b, it loses contact with the inner germarium sheath cells (isc) and is surrounded by a layer of follicle cells (fc) which are derived from the somatic stem cells (ssc). In region 3, a germline cyst surrounded by follicle cells moves out of the germarium and becomes an egg chamber. In each egg chamber, the most posterior germline cell becomes the oocyte (oo) and the remaining 15 cells are nurse cells (nc). 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Laundrie B, Peterson JS, Baum JS, Chang JC, Fileppo D, Thompson SR, McCall  K. Germline cell death is inhibited by P-element insertions disrupting the dcp-1/pita nested gene pair in Drosophila. Genetics 2003; 165:1881-8. 116.  Ghidoni R, Houri JJ, Giuliani A, Ogier-Denis E, Parolari E, Botti S, Bauvy C,  Codogno P. The metabolism of sphingo(glyco)lipids is correlated with the differentiation-dependent autophagic pathway in HT-29 cells. Eur J Biochem 1996; 237:454-9. 117.  Scarlatti F, Bauvy C, Ventruti A, Sala G, Cluzeaud F, Vandewalle A, Ghidoni R,  Codogno P. Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1. J Biol Chem 2004; 279:18384-91. 118.  Prins JB, Ledgerwood EC, Ameloot P, Vandenabeele P, Faraco PR, Bright NA,  O'Rahilly S, Bradley JR. Tumor necrosis factor-induced cytotoxicity is not related to rates of mitochondrial morphological abnormalities or autophagy-changes that can be mediated by TNFR-I or TNFR-II. 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Apoptosis 2008; 13:1-9.  42  Chapter 2 Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila oogenesis 2.1 Introduction Autophagy is an evolutionarily conserved mechanism for the degradation of longlived proteins and organelles.  During autophagy, cytoplasmic components are  sequestered into double membrane structures called autophagosomes which then fuse with lysosomes to form autolysosomes, where degradation occurs.1 Currently, there are 31 Atg (autophagy-related) genes in yeast, and 18 Atg proteins are essential for autophagosome formation.2 Most yeast Atg genes have orthologues in higher eukaryotes and encode proteins required for autophagy induction, autophagosome  nucleation,  expansion and completion, and final retrieval of Atg protein complexes from mature autophagosomes (for review see Levine and Yuan, 2005).3 Depending on the physiological and pathological conditions, autophagy has been shown to act as a pro-survival or pro-death mechanism in vertebrates.3, 4 In the case of growth factor withdrawal, starvation and neurodegeneration, autophagy has been shown  A version of this chapter has been published. Hou Y-C. C, Chittaranjan S, González Barbosa SN, McCall K, Gorski S.M. Effector caspase Dcp-1 and IAP protein Bruce regulate starvationinduced autophagy during Drosophila oogenesis. Journal of Cell Biology 2008; 182: 1127-39.  43  to function in cell survival.  5-7  In contrast, autophagy has been reported to act as a cell  death mechanism in derived cell lines where caspases or apoptotic regulators are impaired. 8, 9 The nature and perhaps level of the stress stimulus may also be important in determining whether autophagy promotes cell survival or cell death.10, 11 Overlaps between components in apoptosis and autophagic pathways have been described. Upstream signal transducers in apoptotic pathways, including TRAIL, TNF, FADD and DAPK, have been shown to play a role in autophagy regulation,12-15 and two apoptotic inducers, including sphingolipid and ceramide, can activate autophagy in mammalian cells.16,  17  In addition, two recent studies demonstrate physical and  functional interactions between components of apoptosis and autophagy. First, the antiapoptosis protein, Bcl-2, suppresses autophagy through a direct interaction with Beclin 1, a protein required for autophagy.18 Second, the autophagy related protein 5 (Atg5), which is cleaved by calpain, associates with Bcl-XL leading to cytochrome c release and caspase activation.19  Further examples and discussion of the connections between  apoptosis and autophagy can be found in several recent reviews on this topic.4, 20, 21 The current findings indicate that there is a complex relationship between apoptosis and autophagy, but the regulatory mechanisms underlying the cross-talk between the two processes are still largely unknown. Autophagy is observed in several Drosophila tissues during development, and thus Drosophila is useful as a model to study autophagy in the context of a living organism. Fourteen Drosophila annotated genes share significant sequence identity with the yeast Atg genes and, overall, 8 Drosophila Atg homologues have already been shown to be required for autophagy function.22, 23 In addition, recent studies demonstrated the  44  role of autophagy in Drosophila physiological cell death. Loss of Atg genes, including Atg1, Atg2, Atg3, Atg6, Atg7, Atg8, Atg12 and Atg18, inhibited proper degradation of salivary glands during development. Overexpression of Atg1 induced premature salivary gland cell death in a caspase-independent manner.23 In contrast, caspase activity was required for Atg1-mediated apoptotic death in the fat body.24 Mutation of Atg7 resulted in an inhibition of DNA fragmentation in the midgut but led to an increase of DNA fragmentation in the adult Drosophila brain.25 Together these results suggested that the mechanistic role of autophagy in cell death and the interrelations between autophagy and apoptosis may be tissue and/or context dependent. The adult Drosophila ovary contains 15 to 20 ovarioles comprised of developing egg chambers which consist of 16 germ line cells (15 nurse cells and 1 oocyte) surrounded by a layer of somatic follicle cells. The germline cells originate from stem cells which undergo mitosis to form 16-cell cysts in a specialized region called the germarium. In the late stage of oogenesis, the nurse cells support the development of the oocyte by transferring to it their cytoplasmic contents. After this “dumping” event, the nurse cells undergo cell death and their remnants are engulfed by the surrounding follicle cells.26, 27 In addition to this late stage developmental cell death, egg chambers can be induced to die at two earlier stages, during germarium formation (in region 2) and midoogenesis, by factors such as nutrient deprivation, chemical insults, and altered hormonal signaling.28-30 In some respects, cell death during Drosophila oogenesis is similar to the death of Drosophila larval salivary glands. Both nurse cells and salivary gland cells are large and polyploid, and the entire tissues undergo cell death simultaneously.28 Notably, morphological features of autophagy have been described during mid-oogenesis cell  45  death in a related species, Drosophila virilis,31 suggesting that the cell death process in ovaries and salivary glands share additional similarities. Previous studies have focused on characterizing the role of autophagy genes in cell death and determining the paradoxical functions of autophagy (pro-survival and prodeath) in various cell lines and organisms.  However, a systematic approach that  investigates the involvement of cell death genes in starvation-induced autophagy has not been conducted. Here we present RNAi analyses to determine whether known cell death related genes in Drosophila play a role in autophagy regulation in the l(2)mbn cell line. We chose the l(2)mbn cell line based on the following reasons. First, unlike other Drosophila cell lines (S2 and Kc), the origin tissue of the l(2)mbn cell line is known and was a sample of haemocytes from the Drosophila mutant lethal malignant blood neoplasm.32  Second, l(2)mbn cells showed lysosome bodies and autophagosome like  structures after addition of the steroid hormone 20-hydroxyecdysone.32 Finally our QRTPCR results showed the expression of core apoptotic and autophagic genes in l(2)mbn cells indicating both apoptosis and autophagy pathways are competent in the l(2)mbn cell line. We also utilize Drosophila genetics to investigate a role for the effector caspase Dcp-1 and the IAP family member Bruce in autophagy regulation in vivo during Drosophila oogenesis. Further, we examine the function of autophagy genes Atg7 and Atg1 in starvation-induced germline cell death in the Drosophila ovary.  2.2 Materials and methods 2.2.1 Cell culture conditions Drosophila l(2)mbn cells were maintained in Schneider’s Drosophila medium (Gibco-Invitrogen) supplemented with 10% fetal bovine serum (FBS) in 25-cm2  46  suspension flasks (Sarstedt) at 25°C.32 All the experiments were carried out 3 days after passage and the cells were discarded after 25 passages. 2.2.2 dsRNA synthesis Individual PCR products containing coding sequences for the transcripts to be targeted were generated by RT-PCR using the Superscript one-step RT-PCR kit with platinum taq (Invitrogen).  Each primer used in RT-PCR contained a 5' T7 RNA  polymerase binding site (TAATACGACTCACTATAGG) followed by sequences specific for the targeted genes (Table 2.1). For in vitro transcription reactions, 50µl of each RT-PCR product was ethanol-precipitated and resuspended in 8µl of nuclease free water and then used as a template. In vitro transcription reactions were carried out using T7 RiboMax Express RNAi systems (Promega) according to the manufacturer’s instructions. dsRNAs were ethanol precipitated and resuspended in 50µl of nuclease free water. A 5µl aliquot of 1/100 dilution was analysed by 1% agarose gel electrophoresis to determine the quality of dsRNA. The dsRNA was quantitated using the PicoGreen assay (Invitrogen) and adjusted to 200ng/µl with nuclease free water. 2.2.3 RNA interference (RNAi) 66μl of cells (1 x 106 cells/mL) in SFM922 medium were seeded into each well of a 96 well plate. 2μg of dsRNA (20μM) was added into each well.  After one hour  incubation at room temperature, the cells received Schneider’s medium supplemented with 10% FBS to achieve a final 200µl volume. Cells were incubated for 72 hours at 25°C.  Since RNAi of th triggered a massive amount of apoptosis at the standard  incubation time of 72 hours, we instead used a 24 hour incubation period. After 24 hours  47  of th-dsRNA treatment, there was already a significant number of apoptotic cells present indicating an efficient knock-down by RNA interference, but a sufficient number of healthy cells (more than 10,000) remained for LTG analysis. 2.2.4 Flow cytometry based LysoTracker Green (LTG) assay For drug treatments, 3-methyladenine (3MA; 10mM) or Bafilomycin A1 (Baf; 0.1μM) was added when nutrient full medium was replaced with 2mg/ml glucose in PBS. After 4 hours incubation at 25°C, cells were incubated for 20 minutes at room temperature with LysoTracker Green (LTG; 50nM) for quantification of autophagy levels and propidium iodide (PI; 2μg/mL) to eliminate dead cells. The cells were then analyzed using flow cytometry (FACSCalibur; Becton Dickinson). A minimum of 10,000 cells per sample were acquired for triplicate samples per experiment. LTG fluorescence levels of cells (excluding PI positive cells) were analyzed using Flowjo software. For RNAi experiments, the RNAi-treated cells after 72hrs incubation were transferred into a U bottom 96 well plate and centrifuged at 800 rpm for 5 minutes. Nutrient full medium was replaced with 2mg/mL glucose/PBS with dsRNA (20μM) for 4 hour starvation treatment and cells were labeled with LTG and PI for 20 minutes at room temperature, and analyzed as described above. 2.2.5 GFP-LC3 detection The p2ZOp2F-eGFP-LC3 plasmid was generated by restriction digestion of eGFP-LC3 from pUASP-eGFP-LC3  33  and cloning into the p2ZOp2F vector  34  . To  create a stable cell line, Drosophila l(2)mbn cells were transfected with p2ZOp2F-eGFPLC3 and selected for the presence of the construct using zeocin. The resulting p2ZOp2F-  48  eGFP-LC3 stable (GFP-LC3) l(2)mbn cells were maintained in Schneider’s Drosophila medium (Gibco-Invitrogen) supplemented with 5% fetal bovine serum (FBS) and zeocin (10μg/mL).  66μl of these GFP-LC3 l(2)mbn cells (2.5 x 106 cells/mL) in SFM922  medium were seeded into each well of an 8 well CC2 coated chamber slide (LabTek system). Cells were incubated with dsRNAs as described above, followed by 4 hour starvation treatment. Cells were fixed with 2% paraformaldehyde for 20 minutes and incubated with anti-GFP antibody (1:200; JL8; Clontech), followed by anti-mouse immunoglobulin Alexa 488 conjugates (Molecular Probes). Cells were mounted with SlowFade Gold antifade reagent with DAPI at room temperature (Invitrogen). Images were obtained using a 63x objective on a Zeiss Axioplan2 microscope and captured with a cooled mono 12 bit camera (Qimaging) and Northern Eclipse image analysis software (Empix Imaging Inc.)  Cells with more than three GFP-LC3 punctate dots were  considered as GFP-LC3 positive cells. A minimum of 200 cells per sample were counted manually for triplicate samples per experiment. 2.2.6 Statistical analysis Two tailed student’s t-test (equal variances) was used to compare mean levels. n=3. A value of P < 0.05 was considered statistically significant. 2.2.7 Generation of transgenic flies The UASp-full-length-Dcp-1 construct was generated by PCR amplification of the coding region from a Dcp-1 cDNA clone35 and subsequent cloning of the amplicon into the UASp vector.36 Transgenic flies were generated using standard procedures. To express full-length Dcp-1 in the germline, flies were crossed to NGT; nanosGAL4 flies 37  49  and resulting progeny were analyzed. To express truncated Dcp-1 and GFP-LC3 in the germline, yw; nanosGAL4 UASp-tDcp-1 were crossed to UASp-GFP-LC3; nanos GAL4 and resulting progeny were analyzed.38 2.2.8 Fly strains w1118 was used as the wild-type stock. Other fly stocks used were as follows: Dcp-1Prev1 and UASp-GFP-LC3; nanos-GAL4, Bruce  E81  , Bruce  E16  , Atg7d77, Atg7d14,  CG5335d30, and Atg1 Δ3D. 2.2.9 Generation of Atg 1 germline clones (GLCs) To generate germline clones, FRT2A was recombined onto the Atg1 chromosome.  Δ3D  Correct recombinants were confirmed by failure to complement  Df(3L)Bsc10 and Atg1  00305  . Germline clones were generated with the FLP/FRT/ovoD  technique as described 39. Larvae of the genotype HSflp; ovoD FRT2A/ Atg1 Δ3D FRT2A were heatshocked on day 4 and 5 for 1 hour at 37°C. 2.2.10 LysoTracker Red staining For nutrient deprivation experiments, flies were conditioned on yeast paste for 2 days and then placed in a dry vial with access to a 10% sucrose solution for 4-5 days 38. Ovaries were dissected in PBS and immediately transferred into PBS containing 0.8 μM LysoTracker Red (LTR) (Invitrogen) for 5 min at room temperature in the dark. The ovaries were then stained with 0.1mg/mL DAPI for 30 sec. The ovaries were washed three times with PBS, and mounted with SlowFade antifade reagent (Invitrogen) at room temperature. Images were obtained using a 20 or 40x objective on a Zeiss Axioplan2 microscope and captured with a cooled mono 12 bit camera (Qimaging) and Northern  50  Eclipse image analysis software (Empix Imaging Inc.). Egg chambers with more than ten LTR positive spots were considered as LTR positive.  Stage 8 degenerating egg  chambers in w1118 flies were scored by the presence of condensed nurse cell nuclei. The degenerating egg chambers in Dcp-1  Prev  files were characterized by a disappearance of  follicle cells and a persistence of nurse cell nuclei, as reported previously.40 For expression of Dcp-1 in ovaries, NGT/+; nanos-GAL4/UASp-fl-Dcp-1 flies were conditioned on wet yeast paste for 2-4 days and dissected in Ringers  41  . Ovaries  were incubated with 50µM Lysotracker Red DND-99 in PBS for three minutes, washed three times for five minutes each time in PBS, fixed ten minutes in 1:1 heptane:3.7% formaldehyde in Pipes buffer (0.1M Pipes, 2mM MgSO4, 1mM EGTA, pH 6.9), washed three times for five minutes each time in PBT (PBS +0.1% Triton-X), and mounted in anti-fade + 1.5µg/ml Hoechst 33258. Egg chambers were viewed at room temperature using an Olympus UPlanFl 20X, 0.50 objective on an Olympus BX50 confocal microscope.  Images were captured using Olympus Magnafire SP model#S99810  (Hoechst) and Olympus Fluoview (LTR) cameras. DABCO was used as the imaging medium. Egg chambers with more than ten LTR positive spots were considered as LTR positive. All figures were processed with Photoshop 7.0 (Adobe). Color was added on LysoTracker image using ImageJ. 2.2.11 TUNEL assay Ovaries were dissected in Schneider’s Drosophila medium (Gibco-Invitrogen) supplemented with 10% fetal bovine serum (FBS). The ovaries were fixed in PBS containing 4% paraformaldehyde. The ovaries were then washed two times (5 min each) in PBS, permeabilized with 0.2% triton X-100 for 5 minutes, followed by two washes in  51  PBS. TUNEL reaction was carried out with the DeadEnd fluorometric TUNEL system (Promega). The ovaries were then stained with 0.1mg/mL DAPI for 30 sec at room temperature, mounted in SlowFade antifade reagent (Invitrogen) and observed under a Zeiss Axioplan 2 microscope. Images were obtained using a 10, 20 or 40x objective on a Zeiss Axioplan2 microscope and captured with a cooled mono 12 bit camera (Qimaging) and Northern Eclipse image analysis software (Empix Imaging Inc.) 2.2.12 Quantitative RT-PCR Cells (approximately 2x105 cells in 600μL) were incubated with dsRNAs as described above at 25°C for 72 hours, followed by 4 hour starvation treatment. Cell cultures were transferred to RNAse free eppendorf tubes (Ambion) and cells were pelleted at 1000 rpm for 10 min. Cells were lysed in 1ml Trizol (Invitrogen) and total RNA was extracted according to manufacturer’s instructions. Isolated RNA was treated with RNAse free DNAse and 50ng of total RNA was used in a 15μL QRT-PCR reaction. QRT-PCR was performed using the one-step SYBR green RT-PCR Reagent Kit (Applied Biosystems) on an Applied Biosystems 7900 Sequence Detection System. Expression levels were calculated using the Comparative CT method with Drosophila rp49 as the reference. All samples were analyzed in triplicate. The knock-down efficiency was determined by comparing the fold change in expression between target dsRNA treated and untreated cells.  52  2.3 Results 2.3.1 RNAi screening assay identifies known positive and negative regulators of starvation-induced autophagy in Drosophila l(2)mbn cells. To quantify starvation induced autophagy we used a Drosophila tumorous larval hemocyte cell line, lethal (2) malignant blood neoplasm (l(2)mbn)32, and employed LysoTracker Green dye (LTG) shown previously to label lysosomes and autolysosomes in Drosophila.22,  42  Flow cytometry was employed to acquire LTG fluorescence of  individual cells. Under nutrient full medium conditions, we detected a basal level of LTG labeling in l(2)mbn cells (Figure 2.1A). When cells were transferred into amino acid deprived medium for 2 hours, we observed a detectable increase in LTG labeling. After 4 hours of amino acid deprivation, a further increase in the percentage of cells with high LTG fluorescence levels (LTGhigh population) was observed (Figure 2.1A).  To  confirm that autophagy is indeed upregulated under nutrient deprived conditions in l(2)mbn cells, we constructed a stable l(2)mbn cell line expressing mammalian microtubule associated protein 1 light chain 3 (LC3)/ATG8 fused to GFP protein, a widely used marker for autophagy.  During autophagy, LC3 conjugates to  phosphatidylethanolamine (PE) which then inserts into the autophagosomal membrane. Thus, localization of GFP-LC3 changes from a diffuse cytoplasmic pattern to a punctate autophagosomal membrane-bound pattern that can be monitored by microscopy.42, 43 As expected, the percentage of cells with more than three GFP-LC3 puncta (GFP-LC3 positive) was increased from 9% (n=216) in the nutrient-full condition to 32% (n=200) in the nutrient-starved condition for 2 hours (Figure 2.1B). Further, to confirm that LTG labeling correlates with autophagy levels in l(2)mbn cells, we  employed the  53  pharmacological autophagy inhibitors, 3-methyladenine (3MA) and Bafilomycin A1 (Baf). 3MA blocks autophagy by inhibiting PI3-kinase activity.44 Baf is a specific inhibitor of lysosomal proton pumps and prevents the fusion of autophagosomes with lysosomes.45 In l(2)mbn cells, both autophagy inhibitors significantly reduced LTG fluorescence levels following starvation treatment (Figure 2.1C and D). Consistently, addition of 3MA also decreased the numbers of GFP-LC3 positive cells following starvation treatment. As expected, the addition of Baf, which is known to increase the numbers of autophagosomes by preventing their lysosomal degradation, resulted in increased GFP-LC3 puncta in starved cells (Figure 2.1B). These results indicate that our flow cytometry based LTG assay is able to detect changes in the autophagy levels of l(2)mbn cells in response to starvation and autophagy-inhibiting drug treatments. To further validate our flow cytometry based LTG assay and determine its sensitivity, we used RNAi to specifically inhibit Drosophila autophagy genes. Currently, there are 14 Drosophila genes that share significant sequence identity with yeast Atg genes, and 7 Drosophila Atg homologues have already been shown to be essential for starvation-induced autophagy in the larval fat body.22  In our assay, RNAi of 11  Drosophila Atg homologues individually resulted in a statistically significant reduction in the LTGhigh population following starvation treatment (Figure 2.1E and Table 2.2). The effects on LTG staining by RNAi of Atg genes or 3-MA were modest in size (e.g. 3048% change relative to the Hs-RNAi negative control in Figure 2.1E), but reproducible and statistically significant. The limited magnitude of the detectable effects may be due, at least in part, to the nature of this dye as an acidophilic probe which detects autolysosomes but also background lysosomal staining. As with any RNAi-based screen,  54  incomplete target knockdown may also be a contributing factor. Comparison of our RNAi-mediated results with previous in vivo results from the Drosophila larval fat body (Table 2.2) indicates that l(2)mbn cells require the same autophagy genes as the fat body.22 In higher eukaryotes, starvation induced autophagy is suppressed by components of the insulin/class I phosphoinositide 3-kinase (PI3K) and TOR pathways (for reviews see Klionsky, 2007; Levine and Yuan, 2005; Maiuri et al., 2007).1,  3, 4  To determine  whether PI3K and TOR pathways are required for starvation-induced autophagy in l(2)mbn cells, we designed dsRNAs against several genes in these pathways. RNAi of Tor or RheB, negative regulators of autophagy, showed an increase in LTG  high  cells  compared to Hs-dsRNA (negative control) treated cells following starvation treatment (Figure 2.1F). In contrast, RNAi of Pten, Tsc1 Tsc2 and S6k, positive regulators of autophagy, showed a reduction in the LTG  high  population (Figure 2.1F). These results  indicate that components of TOR and PI3K pathway are essential to regulate starvationinduced autophagy in Drosophila l(2)mbn cells. These results also demonstrate that our primary screening method, a flow cytometry-based LTG assay, is capable of detecting alterations induced by RNAi-mediated knockdown of positive and negative regulators of autophagy. Thus, this method can be employed to identify novel components in starvation-induced autophagy. To ensure the changes in LTG fluorescence levels were due to alterations in autophagy, we used GFP-LC3 to track changes in autophagosome formation in cells. RNAi of Tor showed an increase in the numbers of GFP-LC3 positive cells following starvation treatment (Figure 2.2G).  In contrast, reduction of Pten  expression by RNAi resulted in a decrease in the number of the GFP-LC3 positive cells  55  (Figure 2.2G). Together these two methods allow us to monitor the dynamic steps, formation of autophagosomes (GFP-LC3) and autophagosome-lysosome fusion (LTG), during autophagy. 2.3.2 Identification of cell death-related genes that regulate starvation-induced autophagy in l(2)mbn cells. To better understand the relationship between autophagy and apoptosis, we investigated whether known cell death genes were required for starvation-induced autophagy in l(2)mbn cells. dsRNAs were designed against the Drosophila core cell death effectors, rpr,, hid, grim and skl, and autophagy was evaluated by the flow cytometry based LTG assay. Only dsRNA corresponding to hid, but not rpr, grim or skl showed an effect on autophagy by this assay. RNAi of hid decreased the percentage of LTGhigh cells following starvation treatment (Figure 2.2A). Previous studies showed that the Ras/Raf/MAPK pathway specifically inhibits the pro-apoptotic activity of hid.46 To determine whether the Ras/Raf/MAPK pathway also plays a regulatory role in autophagy in l(2)mbn cells, we designed dsRNAs to target these three components. RNAi of Ras, phl (also known as raf) or rl (also known as MAPK) all further enhanced the LTG fluorescence levels suggesting that, like in apoptosis, they have an inhibitory role in autophagy regulation (Figure 2.2B).  A second set of dsRNAs, non-overlapping with the  first set of dsRNAs, was designed to validate these new findings and consistent results were observed (Figure 2.3A). In addition, GFP-LC3 was employed to track changes in autophagosome formation in cells. RNAi of hid showed a decrease in the numbers of cells with GFP-LC3 puncta (Figure 2.2F and G), while RNAi of Ras, phl or rl all resulted  56  in a significant increase in the numbers of GFP-LC3 positive cells following starvation treatment (Figure 2.2G). All RHG family members, Rpr, Hid, Grim and Skl, bind to Drosophila Inhibitor of Apoptosis Protein-1 (DIAP1) and inhibit its anti-apoptotic activities.47 To test whether DIAP1 (encoded by th) is a putative downstream mediator of Hid-dependent autophagy in l(2)mbn cells, dsRNA was designed specifically to target th. We found that th-dsRNA treated cells showed no difference in LTG fluorescence levels compared to Hs-dsRNA (negative control) treated cells (Figure 2.2C). Interestingly, our data showed that reduced expression of Bruce, another IAP family member protein, further increased the LTG fluorescence levels following starvation treatment (Figure 2.2C) (confirmed using nonoverlapping dsRNAs; see Figure 2.3B). RNAi of Bruce expression also resulted in an increase in GFP-LC3 puncta following starvation treatment (Figure 2.2G). These results suggest that Bruce, instead of DIAP1, could be the downstream target of Hid during starvation induced autophagy in l(2)mbn cells. Next we investigated whether the transducers of apoptotic signals, Ark, Buffy, and debcl are required for starvation- induced autophagy. Reduced expression of Ark, the Drosophila homologue of mammalian Apaf-1, did not affect the LTG fluorescence levels (Figure 2.2D). RNAi of two Bcl-2 family members Buffy or debcl, resulted in a decrease in the percentage of LTGhigh cells following starvation treatment (Figure 2.2D). Consistently, reduction of Buffy and debcl expression by RNAi decreased the percentage of GFP-LC3 positive cells following starvation treatment (Figure 2F and G). Reduced expression of Ark, Buffy, and debcl was determined using quantitative RT-PCR (Figure 2.3C). In addition, we reduced expression of the tumor suppressor p53 by RNAi and  57  found that starvation-induced autophagy was inhibited (Figure 2.2D).  Results were  further confirmed using non-overlapping dsRNAs (Figure 2.3B). To investigate the requirement of caspases, the final effectors of apoptosis, in starvation-induced autophagy we designed gene specific dsRNAs corresponding to seven different Drosophila caspases. RNAi of just one caspase, Dcp-1 but not others resulted in a decrease in the percentage of LTGhigh cells following starvation treatment (Figure 2.2 E). A second dsRNA against Dcp-1, non-overlapping with the first dsRNA, yielded a similar result (Figure 2.3B). Reduction of Dcp-1 expression by RNAi was determined using quantitative RT-PCR (Figure 2.3C). Consistent with the LTG derived data, RNAimediated knock-down of Dcp-1 resulted in a decrease in GFP-LC3 positive cells following starvation treatment (Figure 2.2F and G). These results indicate that Dcp-1 functions as a positive regulator of autophagy in Drosophila l(2)mbn cells. 2.3.3 Autophagy occurs in response to nutrient deprivation in germaria and midstage egg chambers in the Drosophila melanogaster ovary. To further characterize the requirement of Dcp-1 and Bruce in autophagy regulation, we studied Drosophila melanogaster oogenesis in vivo. We used a transgenic fly line which expresses a GFP-LC3 fusion protein under the control of the UASp promoter.33 Co-expression of UASp-GFP-LC3 with the germline-specific nanos-GAL4 driver resulted in detectable GFP-LC3 expression in the germline (nurse cells and oocyte) cells but not in somatic (follicle) cells (Figure 2.4A).33, 36 When UASp-GFP-LC3; nanosGAL4 flies were subjected to nutrient deprivation, we observed numerous GFP-LC3 puncta in region 2 within the germarium (Figure 2.4B). In contrast, flies raised in the presence of yeast paste (well-fed) had a diffuse GFP-LC3 pattern (Figure 2.4B). In  58  addition, we found an increase in punctate Lysotracker Red (LTR) staining in germaria of nutrient deprived wild-type (w1118) flies compared to well-fed wild-type flies (Figure 2.4B and Table 2.3). We also observed numerous GFP-LC3 puncta in nutrient-deprived degenerating stage 8 chambers, but a diffuse GFP-LC3 pattern was detected in healthy egg chambers (Figure 2.4 C).  Similarly, degenerating stage 8 egg chambers had  numerous LTR positive dots in the nurse cells, while healthy egg chambers had a low level of LTR staining (Figure 2.4 C and Table 2.4).  In starved Atg7 mutants  (Atg7d77/Atg7d14), there was a significant decrease in punctate LTR staining in region 2 of the germarium and in stage 8 degenerating egg chambers compared to flies with the genotype CG5335d30/Atg7d14, employed previously as controls in Juhasz et al. 2007 (Figure 2.4D, and Table 2.3 and 2.4). These results indicated that nurse cells lacking the core autophagy regulator Atg7 failed to induce autophagy in response to nutrient deprivation.  Overall, our observations showed that nutrient deprivation induces  autophagy in region 2 germaria and in degenerating stage 8 egg chambers in Drosophila melanogaster. 2.3.4 Dcp-1 and Bruce regulate autophagy in germaria and degenerating mid-stage egg chambers. To determine whether Dcp-1 is required for autophagy in germaria and degenerating mid-stage egg chambers during oogenesis, we employed LTR staining in nutrient- deprived Dcp-1  Prev  mutant flies. We observed a decrease in punctate LTR  staining in region 2 of the germarium and in stage 8 degenerating egg chambers (Figure 2.5A and B, and Table 2.3 and 2.4) compared to nutrient-deprived wild-type flies. Consistent results were observed using GFP-LC3. Degenerating stage 8 egg chambers in  59  nutrient – deprived Dcp-1  Prev  mutants containing the GFP-LC3 transgene had a diffuse  GFP-LC3 pattern instead of punctate GFP-LC3 structures (Figure 2.5C). Together, these results indicate that nurse cells lacking Dcp-1 function are severely impaired in the ability to induce autophagy in response to starvation. To determine whether Dcp-1 was also sufficient to induce autophagy in vivo, we generated transgenic flies that express the full length Dcp-1 (fl-Dcp-1) under the control of the UASp promoter. In the presence of a nutrient rich food source, degenerating stage 8 egg chambers are observed only rarely in wild-type flies.30 However, under nutrient rich conditions, we observed an abundance of degenerating stage 8 egg chambers in nanos-GAL4/UASp-fl-Dcp-1 flies with increased levels of punctate LTR staining (Figure 2.5D and Table 2.4). Further, we expressed an activated form of Dcp-1 (missing the prodomain) and GFP-LC3 in the germline using the UASp/nanos-GAL4 system and observed numerous degenerating stage 8 egg chambers with GFP-LC3 puncta (Figure 2.5E), indicating that activity of effector caspase Dcp-1 is sufficient to induce autophagy during mid-oogenesis even under nutrient rich conditions. We identified the IAP protein Bruce as a negative regulator of autophagy in l(2)mbn cells.  We next asked whether Bruce is able to inhibit autophagy during  Drosophila oogenesis. We monitored the LTR staining in ovaries of BruceE81 flies, which have a deletion in the Baculoviral IAP Repeat (BIR) domain that binds to caspases.48  In the presence of a nutrient rich food source, we observed an increase in  punctate LTR staining in region 2 of the germarium in BruceE81 flies compared to controls (BruceE81/TM3) (Figure 2.6A and Table 2.3). Similarly, we observed numerous degenerating stage 8 egg chambers with increased levels of punctate LTR staining in  60  BruceE81 flies, resembling overexpression of Dcp-1 (Figure 2.6B and Table 2.4).  In  well-fed conditions, we observed no degenerating stage 8 egg chamber in control BruceE81//TM3 flies (n=187 ovarioles; Table 2.4). Punctate LTR staining was similarly observed in region 2 germaria (Table 2.3) and degenerating stage 8 egg chambers (data not shown) in well-fed Bruce  E16  flies which have a 10kb deletion in the 3’ end of the  Bruce gene sequence.49 Our results demonstrate that Bruce is normally required to inhibit autophagy under nutrient rich conditions. 2.3.5 Dcp-1 and Bruce mutants have altered TUNEL staining in germaria and degenerating mid-stage egg chambers. Our previous work showed that nutrient deprived Dcp-1 mutants (Dcp-1 Prev) have defects in mid-oogenesis germline cell death.40 To determine whether Dcp-1 is also required for germline cell death in region 2 within the germarium, we employed the TUNEL assay to detect levels of DNA fragmentation as an indication of cell death. We found that nutrient deprived Dcp-1 mutants had decreased levels of TUNEL positive cells in region 2 within the germarium compared to nutrient deprived wild-type flies (Figure 2.7A and Table 2.5), indicating that Dcp-1 is also required for germarium stage cell death. We also investigated the role of Bruce in cell death during oogenesis. In well-fed BruceE81 flies, we observed a degenerating ovary phenotype which has been shown previously in ovaries with partial loss of another IAP protein, DIAP1.50 This ovary phenotype may be a consequence of excess cell death. Consistent with this possibility, we observed an increased number of cells with TUNEL positive staining in region 2 within the germarium compared to controls (Figure 2.7A and Table 2.5). Similar results were  61  obtained with Bruce  E16  flies (Table 2.5). Numerous TUNEL positive dots were also  observed in degenerating stage 8 egg chambers of BruceE81 well-fed flies (Figure 2.7B). These findings demonstrate that Bruce acts as an inhibitor of cell death in germaria and mid-stage egg chambers. 2.3.6 Autophagy contributes to cell death in nutrient-deprived ovaries. To assess the role of autophagy that we observed during the germarium and midoogenesis stages, we employed the TUNEL assay to detect DNA fragmentation in two Atg gene mutants. Most Atg gene knockouts in Drosophila result in pupal or larval lethality, thus we first analyzed the fully viable Atg7 mutant flies.22, 25 We found that nutrient deprived Atg7 mutants had reduced levels of TUNEL positive cells in region 2 within the germarium compared to control flies (Table 2.5). Further, degenerating stage 8 egg chambers in starved Atg7 mutants showed low or no TUNEL positive staining compared to controls (Figure 2.8A and B and Table 2.6). However, nuclear DNA condensation was still observed in the degenerating stage 8 egg chambers of starved Atg7 mutants (Figure 2.8B). To further investigate the role of autophagy in starvation-induced germline cell death, we generated Atg1 germline clones (GLCs), since mutations in Atg1 result in lethality at the pupal stage of development.22, 24 Consistent with our Atg7 mutant observations, nutrient deprived Atg1 GLC ovaries had decreased levels of TUNEL staining in both germaria and degenerating stage 8 egg chambers, indicating a suppression of DNA fragmentation (Figure 2.8C and D and Table 2.5 and 2.6). Also consistent with Atg7, we observed nuclear DNA condensation in the Atg1 GLC degenerating stage 8 egg chambers (Figure 2.8D). Our results show that lack of autophagy results in a reduction of DNA fragmentation following starvation-induced cell  62  death in the germaria and mid-stage egg chambers, suggesting that autophagy contributes to the cell death process at these stages.  2.4 Discussion Key outstanding questions that need to be addressed are how autophagy and apoptosis pathways interact with each other, and whether common regulatory mechanisms exist between these two processes. I have shown here that six known cell death genes and the Ras/Raf/MAPK signaling pathway not only function in apoptosis but also act to regulate autophagy in Drosophila l(2)mbn cells.  I cannot rule out the  possibility that additional cell death genes that we screened may also function in autophagy but were not detected in our assay due to insufficient knockdown by RNAi, long half-life of the corresponding proteins, and/or functional redundancy. Consistent with our in vitro data, the involvement of Hid in autophagy regulation has been demonstrated in Drosophila. Overexpression of Hid induced autophagy in the fat body, larval epidermis, midgut, salivary gland, Malpighian tubules, and trachea epithelium.51 Further, expression of the constitutively active Ras form (Ras  V12  ), which  has been shown to inhibit Hid activity in apoptosis46, can also block Hid-induced autophagy.51 In Drosophila salivary glands, the Ras signaling pathway has also been shown to inhibit the autophagy process.52 Based on our loss-of-function findings and these previous gain-of-function studies, we speculate that the Ras/Raf/MAPK pathway acts upstream to inhibit Hid activity in autophagy. Poor nutrition has a dramatic effect on egg production in Drosophila. Flies fed on a protein-deprived diet showed an increase in cell death in germaria and midstage egg chambers.30 These two stages have been proposed to serve as nutrient status checkpoints  63  where defective egg chambers are removed prior to the investment of energy into them. The molecular mechanisms of germarium cell death are still largely unknown, and Daughterless, a helix-loop-helix transcription factor, was the only known regulator involved in cell death of germaria.53 Nurse cell death during mid-oogenesis is also different from most developmental cell death in other Drosophila tissues, since apoptotic regulators such as rpr, hid or grim are not required for cell death in these cells.54 However, the activity of caspases, particularly Dcp-1, was shown to be required for midoogenesis cell death.40, 55 My findings implicate several additional genes, Dcp-1, Bruce, Atg7, and Atg1, in nutrient deprivation induced cell death in the germarium, and also during mid-oogenesis. Other forms of cell death, such as autophagic cell death, have been proposed previously to be involved in the elimination of defective egg chambers during midoogenesis. Known signaling pathways including insulin and ecdysone pathways have been shown to be required not only for the survival of nurse cells in mid-oogenesis, but are known to also regulate the autophagy process, supporting the notion that autophagy plays a role in mid-oogenesis cell death.28,  30  Features of autophagy were observed  during Drosophila virilis mid-oogenesis cell death as shown by monodansylcadaverine staining and transmission electron microscopy.31 Our results using GFP-LC3 and LTG demonstrate that autophagy occurs in degenerating mid-stage egg chambers and also in germaria of nutrient deprived Drosophila melanogaster. We found that mutation of Atg7 results in a significant decrease of autophagy in dying mid-stage egg chambers and in germaria of starved flies, further supporting the presence of autophagy during these stages.  64  The role of autophagy in cell survival or cell death is still not well resolved and likely to be context dependent. Our results show that autophagy contributes to the cell death process in the ovary. Loss of Atg7 or Atg1 activity in both dying mid-stage egg chambers and germaria leads to decreased TUNEL staining, indicating a reduction in DNA fragmentation. Consistent results were observed previously in the larval midguts of Atg7 mutants, which also showed an inhibition of DNA fragmentation.25 Interestingly, lack of autophagy function does not appear to affect nuclear DNA condensation in nurse cells. Nurse cells in degenerating stage 8 egg chambers of starved Atg7 mutants or Atg1 GLCs appeared to still have condensed nuclei as shown by DAPI staining (Figure 2.8 B and D). Thus, based on Atg7 and Atg1 mutant analyses, autophagy contributes to DNA fragmentation but not all aspects of nurse cell death.  Future studies are required to  determine how autophagy is connected to known pathways leading to DNA fragmentation and chromatin condensation during cell death. The IAP family member Bruce was shown previously to repress cell death in the Drosophila eye.49  Bruce was also shown to protect against excessive nuclear  condensation and degeneration, perhaps by limiting excessive caspase activity, during sperm differentiation.48 Other IAP family members have been shown to bind caspases via a BIR domain and inhibit apoptosis.56 The presence of a BIR domain in Bruce suggests that it may also have caspase-binding activity. We found that lack of Bruce function resulted in an increase in both LTR and TUNEL staining in germaria and degenerating mid-stage egg chambers. Thus, the Bruce mutant degenerating phenotype in ovaries suggests that Bruce might function normally to restrain or limit caspase activity in this tissue. Since we found that Dcp-1 and Bruce are both required for the regulation of  65  autophagy and DNA fragmentation in germaria and dying mid-stage egg chambers, it is possible that Bruce acts to bind and degrade Dcp-1 in nurse cells under nutrient rich conditions. Future studies employing epistasis and protein interaction analyses will be required to test this prediction. We cannot rule out the possibility that other IAP proteins, such as DIAP1, and other caspases also play a role during these stages. However, at least in response to starvation signals, Bruce and Dcp-1 play a non-redundant dual role in the regulation of autophagy and cell death in the ovary. Numerous studies have linked caspase function to apoptosis, but recent findings indicate that caspases are also required for non-apoptotic processes including immunity and cell-fate determination.57,  58  We have shown here that Dcp-1 is also required for  starvation-induced autophagy. In the ovary, it appears that both apoptotic and autophagic events occur in the germaria and mid-stage egg chambers following nutrient deprivation. It is possible that Dcp-1 coordinates autophagy and apoptosis at these two nutrient status checkpoints to ensure elimination of defective egg chambers in the most efficient manner possible. Dcp-1 mutants exhibit intact nuclei in stage 8 defective egg chambers, indicating a block in both DNA fragmentation and nuclear condensation, and further supporting a dual regulatory role for Dcp-1 in mid-oogenesis cell death. Dcp-1 might function to induce autophagosome formation while coordinately acting upon alternate proteolytic targets to complete execution of apoptosis.  Future studies to elucidate  upstream regulators and downstream substrates of Dcp-1 in cells undergoing autophagy or apoptosis will help to establish the regulatory mechanisms governing the crosstalk between these two cellular processes. Given the multiple cellular effects associated with  66  autophagy, our results also have important therapeutic implications for the use of modulators of caspase or IAP activity in the treatment of cancer and other diseases.  67  Figure 2.1 Quantification of starvation induced autophagy in Drosophila l(2)mbn cells. A. Flow cytometry analysis of l(2)mbn cells starved for 2 hours (2hr S; blue) or 4 hours (4hr S; red) showed an increase in LysoTracker Green (LTG) fluorescence levels (x axis) compared to control cells in full nutrient medium (C; brown). The gate shown on the histogram represents the LTG high population. B. Representative images of GFP-LC3 puncta in fed control (C) l(2)mbn cells, 2hr starved (S) cells, and starved cells treated either with 3 methyladenine (3MA) or Bafilomycin A1 (Baf). Note the increased accumulation of GFPLC3 puncta in Baf treated cells. Scale bar, 10μm. C. Flow cytometry analysis of 4 hour starved cells were incubated with 3 methyladenine (4hr S+3MA; blue) or Bafilomycin A1 (4hr S+Baf; green). Both autophagy inhibitors reduced the LTG fluorescence levels compared to starved cells (4hr S; red) Control cells in nutrient full medium (C; brown) are represented by the brown line. D. Both autophagy inhibitors, 3MA and Baf, reduced the LTG high population significantly. (3MA; P=0.00001 and Baf; P=0.00006). E. RNAi of representative Atg genes decreased the LTR fluorescence levels compared to control. (Atg1; P=0.01, Atg5; P=0.01, Atg7; P=0.002, Atg8a; P=0.008, Atg8b; P=0.006 and Atg12; P=0.02) F. RNAi of all tested genes in the TOR/PI3K pathways had a statistically significant effect on LTG fluorescence levels. Known negative regulators of autophagy are shown with grey bars; positive regulators are shown with white bars. (Pten; P=0.007, Tsc1; P=0.027, Tsc2; P=0.025, RheB; P=0.005, Tor; P=0.016, and S6k; P=0.012). Results represent the mean value ± standard deviation (S.D.) from at least three independent experiments. dsRNA corresponding to a human gene (Hs) was employed as a negative control in (E) and (F).  68  Figure 2.1  69  Figure 2.2 Identification of known cell death related genes in autophagy regulation in l(2)mbn cells using RNAi. A. The percentage of LTGhigh cells was reduced by hid-RNAi (P=0.006) but not by rpr, grim and skl-RNAi. B. Knockdown of Ras, phl and rl expression by RNAi resulted in an increase in the percentage of LTGhigh cells.  (Ras; P= 0.003, phl; P=0.001, and rl;  P=0.028). C. th-RNAi treatment (24 hours) had no significant effect on LTG levels; in contrast, RNAi of Bruce resulted in an increase in LTG fluorescence levels (P=0.01). D. Reduction of debcl, Buffy or p53 expression by RNAi resulted in a decrease in LTG fluorescence levels.  (debcl; P=0.018, Buffy;  P=0.006, and p53;  P=0.004). E. RNAi of effector caspase Dcp-1 resulted in a significant decrease in the LTGhigh population (P =0.001). F. Representative images of GFP-LC3 puncta in cells treated with the indicated RNAi following 2 hours starvation treatment. Scale bar, 10μm. G. Quantification of cells with GFP-LC3 puncta following RNAi treatment. Cells with more than three GFP-LC3 punctate dots were considered GFP-LC3 positive cells.  Cells treated with the RNAi indicated here all showed a  significant difference (P < 0.05) in percentage of GFP-LC3 positive cells compared to the human (Hs) RNAi control. (Pten; P=0.006, Tor; P=0.034, Buffy; P=0.005, debcl; P=0.003, Bruce; P=0.003, Dcp-1; P=0.007, hid; P=0.002, Ras; P=0.006, and phl; P=0.050). Results represent the mean value ± S.D. from three independent experiments.  70  Figure 2.2  71  Figure 2.2 (continued)  72  Figure 2.3 Validation using 2nd set of dsRNAs A. Independent dsRNAs corresponding to Hid and Ras pathway components produced consistent results compared to those shown in Figure 2.2A and B. hid_2; P=0.005, Ras_2; P=0.007, phl_2; P=0.032, and rl_2; P=0.019. Results represent the mean value ± S.D. from three independent experiments. B. Independent dsRNAs, non-overlapping with the first set of dsRNAs, yielded reproducible results compared to those shown in Figure 2.2 C-E. dcp-1_2; P=0.024, debcl_2; P=0.0001, Buffy_2; P=0.030, p53_2; P=0.002, and Bruce_2; P=0.038. Asterix indicate P value <0.05 compared to Hs- RNAi control. Results represent the mean value ± S.D. from three independent experiments. C. mRNA levels of the representative genes were analyzed by QRT-PCR. RNAi knockdown ranged from 61% to 96%. The comparative CT method was used to calculate fold differences relative to non-starved cells. rp49 was employed as an endogenous control. S = Starvation  73  Figure 2.3  74  Figure 2.4 Nutrient deprivation induces autophagy at region 2 within the germarium and in dying mid-stage egg chambers. A. GFP-LC3 proteins were expressed in nurse cells (NC) but not in follicle cells (FC) by using the UASp/nanos Gal4 system. DAPI staining of nuclei is shown in blue. Scale bar, 20 μm. B. UASp-GFP-LC3; nanos GAL4 flies were conditioned on yeast paste and had a diffuse GFP-LC3 pattern. Numerous GFP-LC3 puncta (green) at region 2 within germarium were observed in nutrient deprived flies. Ovaries were stained with LysoTracker Red (LTR) in w1118 flies. Germarium of nutrient deprived w1118 flies had an increase in punctate LTR staining (red) compared to well-fed germarium. Scale bar, 20 μm. C. Degenerating stage 8 egg chambers (arrows) had numerous GFP-LC3 puncta (green) and an increase in LTR positive dots (red) compared to healthy egg chambers (arrowheads). DAPI (white) staining of nuclei is shown in the two panels on the right. Scale bar, 50 μm. D. Degenerating stage 8 egg chambers (arrows) of nutrient deprived Atg7 mutants (Atg7d77/Atg7d14 ) showed a dramatic decrease in LTR staining. Scale bar, 50 μm. DAPI staining of nuclei is shown in white. At least seven different animals from each strain were examined for each condition.  75  Figure 2.4  76  Figure 2.5 The effector caspase Dcp-1 is not only required for nutrient starvation induced autophagy but also is sufficient for the induction of autophagy during Drosophila oogenesis. A. Germaria of the nutrient deprived Dcp-1 Prev files showed a dramatic decrease in LTR staining compared to nutrient deprived wild type flies shown in Fig. 2.4 B. Scale bar, 20 μm. B. Degenerating stage 8 egg chambers (arrows) of nutrient deprived Dcp-1  Prev  files showed a dramatic decrease in LTR staining compared to nutrient deprived wild type flies shown in Fig. 2.4 C. Scale bar, 50 μm. C. Lack of Dcp-1 function (UASp-GFP-LC3 Dcp-1  Prev  /Dcp-1  Prev  ; nanos-  GAL4/+) resulted in uniform diffuse staining of GFP-LC3, rather than the punctate pattern observed in wild type degenerating stage 8 egg chambers shown in Fig 2.4 C. Scale bar, 50 μm. D. Dying egg chambers (arrows) of NGT/+; nanos-GAL4/UASp-fl-Dcp-1 flies that were conditioned on yeast paste showed a significant increase in punctate LTR staining (red) compared to healthy egg chambers (arrowheads). Scale bar, 50 μm. E. Expression of activated Dcp-1 (a truncated form) and GFP-LC3 in the germline  (UASp-GFP-LC3/+;  nanos-GAL4/nanos-GAL4  UASp-tDcp-1)  resulted in abundant degenerating stage 8 egg chambers (arrows) with numerous GFP-LC3 puncta (green). Scale bar, 50 μm. DAPI staining of nuclei is shown in white. At least seven different animals from each strain were examined for each condition.  77  Figure 2.5  78  Figure 2.6 Bruce suppresses autophagy at region 2 within germarium and in dying stage 8 egg chambers. A. The germarium in well-fed BruceE81 flies showed an increase in LTR staining (red) compared to wild type well-fed flies shown in Fig. 2.4 B (upper right). DAPI staining (white) of nuclei is shown on the right. Scale bar, 20 μm. B. In well-fed wild type flies, mid-oogenesis nurse cell death is a rare event. Lack of Bruce function resulted in an increase in dying stage 8 egg chambers (arrows) in ovaries under well fed condition and these degenerating stage 8 egg chambers had numerous LTR (red) punctate dots. DAPI staining (white) is shown on the right. Scale bar, 50 μm. At least seven different animals were examined.  79  Figure 2.6  80  Figure 2.7 Dcp-1 is required for nutrient starvation induced germarium cell death and IAP protein Bruce inhibits germarium and mid-oogenesis cell death. A. Ovaries were stained with TUNEL (green) to detect DNA fragmentation. Clusters of cysts with TUNEL staining were observed in region 2 in nutrient deprived w1118 files. In Dcp-1 Prev flies, fewer TUNEL positive cysts in region 2 were observed. Under well-fed condition, numerous TUNEL positive cysts were observed in BruceE81 flies. DAPI staining of nuclei is shown in white. Scale bar, 20 μm. B. Numerous degenerating stage 8 egg chambers (arrows) with TUNEL positive staining (green) were observed in well fed BruceE81 flies. DAPI staining of nuclei (white) is shown on the right. Scale bar, 50 μm. At least seven different animals from each strain were examined for each condition.  81  Figure 2.7  82  Figure 2.8 Lack of Atg7 or Atg1 function reduces DNA fragmentation during midoogenesis cell death. A. TUNEL positive staining was observed in dying stage 8 egg chambers (arrows) of starved control flies (CG5335d30/Atg7d14 ).  DAPI staining of  nuclei (white) is shown on the right. B. In nutrient deprived Atg7 mutants (Atg7d77/Atg7d14 ), degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation, detected by DAPI, was still observed. C. Dying stage 8 egg chambers from nutrient deprived control siblings (Atg1 Δ3D  /TM3) generated from the same cross in D had abundant TUNEL positive  staining. D. In nutrient deprived Atg1 GLCs, degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation (DAPI, right) in degenerating egg chambers appeared to occur as in the controls. Scale bar, 50μm. At least seven different animals from each strain were examined.  83  Figure 2.8  84  Table 2.1 Primer sequences for the preparation of dsRNAs  85  Table 2.2 Comparison of essential autophagy genes in the Drosophila larval fat body and l(2)mbn cells RNAi of 11 DmAtg genes showed a significant reduction in LTG high cells (P<0.05), indicating that these DmAtg genes are required for autophagy in l(2)mbn cells.  a  The fat body data was generated from a study by Scott et al.22  86  Table 2.3 Quantification of autophagy in region 2 germaria Nutritional Status and Autophagy at Region 2 within Germarium (n>7) LTR Genotype Nutritional Status Number positive w1118 w1118 Dcp-1 Prev Bruce E81 Bruce E81/TM3 Bruce E16 Bruce E16/TM3 Atg7d77/Atg7d14 CG5335d30/Atg7d14  Fed Nutrient deprivation Nutrient deprivation Fed Fed Fed Fed Nutrient deprivation Nutrient deprivation  8 25 17 67 18 35 13 14 37  30 36 53 116 55 63 71 65 68  % of Autophagy 27 69 32 58 33 56 18 22 54  Numbers in the fourth column refer to the numbers of individual germarium scored in at least seven different animals.  87  Table 2.4 Quantification of autophagy in stage 8 degenerating egg chambers Quantification of Autophagy in Stage 8 Degenerating Egg Chambers (n>7) LTR Genotype Nutritional Status Number positive w1118 Dcp-1 Prev Atg7d77/Atg7d14 CG5335d30/Atg7d14 nanos-GAL4/UASp-fl-Dcp-1 Bruce E81 Bruce E81/TM3  Nutrient deprivation Nutrient deprivation Nutrient deprivation Nutrient deprivation Fed Fed Fed  29 8 13 7 62 43 0  40 54 51 *14 74 52 **0  % of Autophagy 73 15 25 50 84 83 N/A  Number in column 4 refers to the number of individual individual degenerating stage 8 egg chambers scored in at least 7 different animals. * n=4 animals scored for this genotype; ** No degenerating stage 8 egg chambers detected  88  Table 2.5 Quantification of cell death in region 2 germaria Nutrional Status and Cell Death at Region 2 within Germarium (n>7) Genotype Nutritional Status TUNEL positive number 1118  w w1118 Dcp-1 Prev Bruce E81 Bruce E81/TM3 Bruce E16 Bruce E16/TM3 Atg7d77/Atg7d14 CG5335d30/Atg7d14 Atg1 GLC Atg1 Δ3D/TM3  Fed Nutrient deprivation Nutrient deprivation Fed Fed Fed Fed Nutrient deprivation Nutrient deprivation Nutrient deprivation Nutrient deprivation  9 34 22 26 12 20 14 21 95 22 53  50 51 69 70 69 54 80 77 202 77 64  % of TUNEL 18 67 32 37 17 37 18 27 47 29 83  Number in column 4 refers to the number of individual germarium scored in at least 7 different animals.  89  Table 2.6 Quantification of cell death in stage 8 degenerating egg chambers Quantification of Cell Death in Stage 8 Degenerating Egg Chambers (n>7) Genotype Nutritional Status TUNEL positive number d77  d14  Atg7 /Atg7 CG5335d30/Atg7d14 Atg1 GLC Atg1 Δ3D/TM3  Nutrient deprivation Nutrient deprivation Nutrient deprivation Nutrient deprivation  17 39 3 19  % of TUNEL  69 62 16 37  25 63 19 51  Number in column 4 refers to the number of individual degenerating stage 8 egg chambers scored in at least 7 different animals.  90  2.5 References 1.  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Atg7-dependent autophagy promotes  neuronal health, stress tolerance, and longevity but is dispensable for metamorphosis in Drosophila. Genes Dev 2007; 21:3061-6. 26.  Spradling AC. The Development of Drosophila melanogaster. Cold Spring  Harbor, NY: Cold Spring Harbor Laboratory Press, 1993. 27.  King RC. Ovarian Development in Drosophila melanogaster. New York:  Academic Press, 1970. 28.  McCall K. Eggs over easy: cell death in the Drosophila ovary. Dev Biol 2004;  274:3-14. 29.  Drummond-Barbosa D, Spradling AC. Alpha-endosulfine, a potential regulator of  insulin secretion, is required for adult tissue growth control in Drosophila. Dev Biol 2004; 266:310-21. 30.  Drummond-Barbosa D, Spradling AC. Stem cells and their progeny respond to  nutritional changes during Drosophila oogenesis. Dev Biol 2001; 231:265-78. 31.  Velentzas AD, Nezis IP, Stravopodis DJ, Papassideri IS, Margaritis LH.  Mechanisms of programmed cell death during oogenesis in Drosophila virilis. Cell Tissue Res 2007; 327:399-414. 32.  Ress C, Holtmann M, Maas U, Sofsky J, Dorn A. 20-Hydroxyecdysone-induced  differentiation and apoptosis in the Drosophila cell line, l(2)mbn. Tissue Cell 2000; 32:464-77. 33.  Rusten TE, Lindmo K, Juhasz G, Sass M, Seglen PO, Brech A, Stenmark H.  Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway. Dev Cell 2004; 7:179-92.  94  34.  Hegedus DD, Pfeifer TA, Hendry J, Theilmann DA, Grigliatti TA. A series of  broad host range shuttle vectors for constitutive and inducible expression of heterologous proteins in insect cell lines. Gene 1998; 207:241-9. 35.  Song Z, McCall K, Steller H. DCP-1, a Drosophila cell death protease essential  for development. Science 1997; 275:536-40. 36.  Rorth P. Gal4 in the Drosophila female germline. Mech Dev 1998; 78:113-8.  37.  Cox RT, Spradling AC. A Balbiani body and the fusome mediate mitochondrial  inheritance during Drosophila oogenesis. Development 2003; 130:1579-90. 38.  Peterson JS, Barkett M, McCall K. Stage-specific regulation of caspase activity in  drosophila oogenesis. Dev Biol 2003; 260:113-23. 39.  Chou TB, Perrimon N. The autosomal FLP-DFS technique for generating  germline mosaics in Drosophila melanogaster. Genetics 1996; 144:1673-9. 40.  Laundrie B, Peterson JS, Baum JS, Chang JC, Fileppo D, Thompson SR, McCall  K. Germline cell death is inhibited by P-element insertions disrupting the dcp-1/pita nested gene pair in Drosophila. Genetics 2003; 165:1881-8. 41.  Verheyen E, Cooley L. Looking at oogenesis. Methods Cell Biol 1994; 44:545-  61. 42.  Klionsky DJ, Cuervo AM, Seglen PO. Methods for monitoring autophagy from  yeast to human. Autophagy 2007; 3:181-206. 43.  Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K,  Tokuhisa T, Ohsumi Y, Yoshimori T. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol 2001; 152:657-68.  95  44.  Seglen PO, Gordon PB. 3-Methyladenine: specific inhibitor of  autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci U S A 1982; 79:1889-92. 45.  Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y.  Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct 1998; 23:33-42. 46.  Bergmann A, Agapite J, McCall K, Steller H. The Drosophila gene hid is a direct  molecular target of Ras-dependent survival signaling. Cell 1998; 95:331-41. 47.  Hay BA, Huh JR, Guo M. The genetics of cell death: approaches, insights and  opportunities in Drosophila. Nat Rev Genet 2004; 5:911-22. 48.  Arama E, Agapite J, Steller H. Caspase activity and a specific cytochrome C are  required for sperm differentiation in Drosophila. Dev Cell 2003; 4:687-97. 49.  Vernooy SY, Chow V, Su J, Verbrugghe K, Yang J, Cole S, Olson MR, Hay BA.  Drosophila Bruce can potently suppress Rpr- and Grim-dependent but not Hid-dependent cell death. Curr Biol 2002; 12:1164-8. 50.  Xu D, Li Y, Arcaro M, Lackey M, Bergmann A. The CARD-carrying caspase  Dronc is essential for most, but not all, developmental cell death in Drosophila. Development 2005; 132:2125-34. 51.  Juhasz G, Sass M. Hid can induce, but is not required for autophagy in polyploid  larval Drosophila tissues. Eur J Cell Biol 2005; 84:491-502. 52.  Berry DL, Baehrecke EH. Autophagy functions in programmed cell death.  Autophagy 2008; 4:359-60.  96  53.  Smith JE, 3rd, Cummings CA, Cronmiller C. Daughterless coordinates somatic  cell proliferation, differentiation and germline cyst survival during follicle formation in Drosophila. Development 2002; 129:3255-67. 54.  Peterson JS, Bass BP, Jue D, Rodriguez A, Abrams JM, McCall K. Noncanonical  cell death pathways act during Drosophila oogenesis. Genesis 2007; 45:396-404. 55.  Baum JS, Arama E, Steller H, McCall K. The Drosophila caspases Strica and  Dronc function redundantly in programmed cell death during oogenesis. Cell Death Differ 2007; 14:1508-17. 56.  Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis.  Nat Rev Mol Cell Biol 2004; 5:897-907. 57.  Kumar S. Migrate, differentiate, proliferate, or die: pleiotropic functions of an  apical "apoptotic caspase". Sci STKE 2004; 2004:pe49. 58.  Kuranaga E, Miura M. Nonapoptotic functions of caspases: caspases as regulatory  molecules for immunity and cell-fate determination. Trends Cell Biol 2007; 17:135-44.  97  Chapter 3 The effector caspase Dcp-1 catalytically regulates starvation-induced autophagy 3.1 Introduction Autophagy is a lysosomal-mediated bulk degradation process that functions to provide the building blocks for ATP production or protein synthesis for cell survival in response to starvation. In addition, autophagy functions to eliminate damaged organelles such as mitochondria1 or aggregated proteins.2-4 Autophagy-related genes (Atg genes) are involved in the molecular machinery of autophagy, including autophagy induction, nucleation, membrane expansion and membrane retrieval, and many of the yeast Atg genes have orthologues in mammals and other higher eukaryotes.5  Model organism  studies have provided evidence that autophagy is associated with several human diseases, including cancer, neurodegeneration, pathogenic infection and aging. Recent findings show that autophagy not only plays a fundamental role in cell survival in response to starvation but also has a role in cell death in response to various stimuli in Drosophila melanogaster. Autophagy is induced in response to starvation in the larval fat body to promote cell survival. Atg mutants were shown to have a shorter life span compared to wild type animals when they were under starvation.6, 7 Mutations in Atg genes were shown to cause cell death defects, including incomplete degradation of larval salivary glands, reduced DNA fragmentation in larval midgut and mid-stage  A version of this chapter will be submitted for publication. Hou YC, Moradian A, Morin G, Gorski SM. 2009. Effector caspase Dcp-1 catalytically regulates starvation-induced autophagy.  98  egg chambers, and suppression of amnioserosa cell death, suggesting autophagy can function as a cell death effector.7-10 One possibility that explains the mechanistic role of autophagy in cell death is that autophagy facilitates the degradation of specific cell survival factors. Such an example exists in mammalian cells where it was shown that autophagy assists in the degradation of the reactive oxygen species (ROS) scavenger catalase and triggers cell death.11 Cell death caused by autophagy mediated degradation of survival promoting proteins has not been demonstrated in Drosophila melanogaster. An alternative explanation for the autophagy-related death promoting effects is that high levels of autophagy may deplete cellular components and organelles leading to a metabolic crisis and cell death. For example, overexpression of Drosophila melanogaster Atg1 was shown to lead to reduced cell growth and caspase-dependent apoptotic death in the larval fat body. We previously demonstrated that six cell death genes, Dcp-1, hid, Bruce, Buffy, debcl, and p53 as well as Ras/Raf/MAPK signaling pathway components have a role in autophagy regulation in Drosophila cultured cells. We showed that Dcp-1 is required for cell death in germaria, and is also necessary for starvation-induced autophagy in both germaria and mid-stage egg chambers. Further, overexpression of Dcp-1 was sufficient to induce autophagy at these two stages even under well fed conditions. Loss-of-function mutations in Bruce resulted in ectopic autophagy and cell death in both stages, regardless of nutrient status, indicating that Bruce acts normally to suppress both autophagy and cell death during Drosophila oogenesis.9 Bruce is one of the inhibitor of apoptosis (IAP) family members, and IAPs are classified by the presence of baculoviral-IAP-repeat (BIR) domains that mediate protein-protein interactions and block the access of the catalytic  99  residue cysteine of caspases to their substrates.12, 13 Thus, the presence of a BIR domain in Bruce suggests that Bruce may suppress Dcp-1 activity and epistasis analyses will be required to prove or disprove this hypothesis. Dcp-1 is one of four effector caspases in Drosophila melanogaster and its role in apoptosis has been described. During Drosophila melanogaster development, apoptotic cell death is mostly mediated by the apical caspase Dronc that cleaves and activates downstream effector caspases Dcp-1 and drICE.14-16  Dcp-1 and drICE share high  sequence similarity with each other and both of them share sequence similarity with mammalian effector caspase-3.17 However, genetic studies in flies indicate differences between the roles of the two prominent effector caspases Dcp-1 and drICE in apoptosis. Flies carrying mutations in drICE are pupal lethal and have reduced cell death in the embryonic nervous system, pupal retina, adult wing, and in response to stresses, including irradiation and the inhibition of protein synthesis.18, 19 In contrast, null mutants of Dcp-1 are viable and only show defects in starvation-induced cell death in germaria and mid-stage egg chambers.9, 20 Double mutants of drICE and Dcp-1 show more severe phenotypes in several Drosophila melanogaster tissues compared to those in flies lacking only drICE, indicating that Dcp-1 probably plays a partly redundant role with drICE and/or is able to compensate for its depletion.19 Biochemically, drICE and Dcp-1 have slightly different enzymatic specificities, since only Dcp-1 but not drICE was shown to be able to cleave human lamins.21 In addition, Dcp-1 can cleave itself or drICE in vitro but drICE could not cleave itself.21 These findings suggest that the mechanistic roles of Dcp-1 and drICE in developmental cell death might be different, and these two prominent  100  effector caspases may have at least some distinct enzymatic properties despite the fact that they share high sequence similarity with each other. Numerous studies have focused on characterizing the mechanistic roles of caspases in apoptosis but the upstream regulatory pathways and substrates of caspases in non-apoptotic processes have been less well studied. Here I investigate the regulation of Dcp-1 and identify its candidate substrates during the process of autophagy. Since the presence of a BIR domain in Bruce suggests that Bruce may be a negative regulator of Dcp-1 mediated autophagy, I created Dcp-1 Prev; Bruce E81 double mutants to examine the genetic relationship between Bruce and Dcp-1. In addition, I determined the roles of Bruce and Dcp-1 in starvation-induced autophagy in the larval fat body and midgut where the function of autophagy is to promote survival instead of death. I also examined the activation status of Dcp-1 and drICE, and requirement of Dcp-1 or drICE mediated proteolytic events in cells undergoing starvation-induced autophagy. Finally, to gain a better understanding of the molecular mechanisms of Dcp-1 in the autophagy process, I performed an immuno-affinity purification (IP) and tandem mass spectrometry (MS/MS) fragmentation based assay to identify potential substrates of Dcp-1 in autophagy inducing conditions.  3.2 Materials and methods 3.2.1 Cell culture and transfection Drosophila l(2)mbn cells (provided by A. Dorn) were maintained in Schneider’s Drosophila medium (Invitrogen) supplemented with 10% FBS (nutrient full medium) in 25-cm2 suspension flasks (Sarstedt) at 25oC. All experiments were performed 3 days  101  after passage, and cells were discarded after 25 passages. For cell transfection, 3μg of plasmid DNA and 18μL of Cellfectin (Invitrogen) were combined in 200μL Grace serum-free medium (Invitrogen), incubated at room temperature for 30 minutes and then added to 3x106 cells in 800μL Grace medium. Cells were incubated overnight (16 hours) at 25oC in one well of a 24 well suspension plate (Sarstedt). After incubation, cells were split and 2mL of 10% FBS Schneider’s medium were added. 3.2.2 Immunofluorescence Transient-transfected cells were resuspended and 100μL of the suspension was added to each well of an 8-well CC2 coated chamber slide (Nunc) and incubated for an hour at room temperature. Nutrient-full medium was replaced with 2mg/mL glucose/PBS for 2 hours. Cells were fixed with 4% paraformaldehyde for 30 minutes, washed with PBS three times, permeablized with 0.2% Triton X-100 for 5 minutes and blocked with 1% BSA for 30 minutes.  Anti-V5 (1:1000, Sigma) and Anti-LC3 (1:100, NanoTools)  primary antibodies were incubated overnight at 4oC. Anti-mouse IgG conjugated to Alexa 488 (1:1000, Invitrogen) and anti-rabbit IgG conjugated to Cy3 (1:1000, Jackson Laboratories) secondary antibodies were incubated for 1 hour at room temperature. After immunostaining, cells were mounted with Slowfade Gold with DAPI (Invitrogen). Images were obtained using a 40X objective on a microscope (Axioplan2, Carl Zeiss, Inc.) and captured with a cooled mono 12-bit camera (QImaging) and Northern Eclipse image analysis software (Empix Imaging, Inc.). GFP-LC3 punctate dots were counted in cells expressing plasmids which were identified by positive V5 immuno-staining. A minimum of 50 cells per sample was counted for triplicate samples per condition.  102  3.2.3 LysoTracker Red (LTR) and DAPI staining For larval fat body and midgut LTR analyses, approximately twenty second instar larvae 48hr after hatching were transferred to a cornmeal/dextrose fly food agar plate supplemented with yeast paste. 24hr later, larvae (fed) were immediately dissected or larvae (starved) were placed on a plate containing 20% sucrose for 1-4 hours prior to dissection. For LTR staining in ovaries, flies were conditioned on yeast paste for 2 days (fed) or placed in a dry vial with access to a 10% sucrose solution for 4-5 days. Tissues, including the larval fat body, midgut, and ovary, were dissected in PBS and immediately transferred into PBS containing 0.8μM LTR (Invitrogen) for 2-5 minutes at room temperature in the dark. Tissues were then stained with 0.1mg/mL DAPI for 30 seconds, washed three times with PBS, and mounted with SlowFade (Invitrogen) at room temperature. Images were obtained with a 20 or 40X objective (Carl Zeiss Inc.) on an Axioplan 2 microscope and captured with a cooled mono 12-bit camera and Northern Eclipse image analysis software. 3.2.4 Immunoprecipitation (IP) and MS/MS analysis For large-scale IP experiments, 96mL of V5-Dcp-1C196A or V5-vector control transfected l(2)mbn cell culture were combined into two 50mL polypropylene tubes and centrifuged at 800 rpm for 10 minutes. Nutrient full medium was replaced with either new 10% FBS/Schneider medium (Fed) or 2mg/mL glucose/PBS for 2 hour starvation treatment. Cells were centrifuged at 800 rpm for 10 minutes and crosslinked with 0.25% paraformaldehyde at 25oC for 40 minutes. 1.25M glycine (final 0.125M) was added and incubated for 5 minutes at room temperature to stop the cross-linking reaction. Samples  103  were then centrifuged at 800 rpm for 10 minutes. 10mL of lysis buffer (20mM Tris pH 7.5, 150mM NaCl, 1mM EDTA, 1% NP-40, 10mM β-glycerophosphate, 2mM sodium orthovanadate, 1mM AEBSF, 10μg/mL pepstatin A, 10μg/mL Leupeptin and 10μg/mL aprotinin) was added to samples. Cells were disrupted by passing through a 21G syringe five times and lysates were incubated at 4oC with agitation for 30 minutes. Cell extracts were centrifuged at 20,000g for 30 minutes and supernatants were stored at -80oC overnight. Supernatants were incubated with 100μL of a 50% slurry of Sepharose 4B (Sigma) and agitated gently for 1 hour at 4oC.  Sepharose 4B was removed by  centrifugation and supernatants were incubated with 40μL of a 50% anti-V5 affinity agarose resin for three hours at 4oC.  Anti-V5 resins were recovered by centrifugation  and washed 5X with cold lysis buffer and 2X lysis buffer with 500mM NaCl. Bound proteins were eluted by re-suspension in 0.5M formic acid for 30 minutes at 4oC. Eluates were boiled for 20 minutes at 95oC to reverse the formaldehyde cross-links. Eluates were then vacuum dried, re-suspended in protein sample buffer (Invitrogen) and separated by SDS-PAGE using a 10% NuPAGE gel (Invitrogen) and 1X MES buffer. Protein bands were visualized with the colloidal coomassie stain and each lane was cut into 16 equal sections.  Gel slices were transferred into a 96 well plate, reduced with 10mM  dithiothreitol (DTT), S-alkylated with 100mM iodoacetamide and then subjected to in-gel trypsin digestion with 20μL of 20ng/uL trypsin per well overnight at 37 oC. Peptide mixtures were subjected to LC-MS/MS analysis on a Finnigan LCQ (PTRL-West) or a 4000QTRAP (Applied Biosystems) ion trap mass spectrometer via reversed phase HPLC nano-electrospray ionization.  All MS-MS spectra were queried against Drosophila  Ensembl sequence databases using Mascot (Matrix Sciences, London, UK) or X!tandem  104  algorithms. An in-house web-based database, SpecterWeb (Sun M, Kuzyk M, and Morin GM, unpublished), was employed to process raw mass spectrometric protein identifications. Non-specifically binding proteins that were identified in the IPs from the negative control (V5-vector only) were subtracted from identified proteins in the IPs from cells transfected with V5-Dcp-1C196A using SpecterWeb for each condition (fed or 2 hr starvation). 3.2.5 Western Blot Drosophila l(2)mbn cells were subjected to different time periods of starvation treatment (2mg/mL glucose/PBS), ranging from 30 minutes to 8 hours. Protein lysates were loaded onto a 10% NuPAGE SDS-PAGE gel (Invitrogen) and separated with 1X MES buffer (Invitrogen).  Proteins were transferred to a PVDF or nitrocellulose membrane  (Invitrogen) and processed according to the standard protocol of Li-COR system. AntiDcp-1 (1:1000)22 or anti-drICE (1:1000)22 with anti-Actin JLA20 (1:1000; DSHB) were incubated overnight at 4oC. Anti-mouse IR800 (Rockland Immunochemicals) and antiguinea pig IR700 (Rockland Immunochemicals) were incubated for 1 hour at room temperature. Membranes were imaged using an Odyssey Infrared imaging System (LiCOR Biosciences). Odyssey software v.3.1 was employed to quantify the integrated intensity of each band. Anti-Actin antibody was used for evaluating loading controls. 3.2.6 Computational analyses of protein sequences Protein sequences, corresponding to identified peptides, were obtained in FASTA format from Flybase. A subset of high confidence candidates (unique peptides >2 and log(E)  105  <-15) from both fed and 2 hour starvation was selected for caspase cleavage site analyses. Prediction of caspase cleavage sites was performed using the CasPredictor program.  23  We selected our thresholds (score>=0.3; statistical>0.75) based on scores of ten known mammalian caspase substrates predicted by the CasPredictor program.23 Mammalian orthologues of identified Drosophila proteins were obtained using the InParanoid database.24  3.3 Results 3.3.1 Effector caspase Dcp-1 genetically interacts with IAP protein Bruce and functions downstream of Bruce IAP family members have been shown to inhibit caspase activity by binding via a baculoviral IAP repeat (BIR) domain, and the presence of a BIR domain in Bruce suggests that it may have caspase inhibitory activity. Since Bruce and Dcp-1 loss-offunction mutants have opposite phenotypes in the Drosophila melanogaster ovary, it is possible that they interact genetically with each other. To test this possibility, we created Dcp-1 Prev/Dcp-1 Prev; Bruce E81/Bruce E81 (Dcp-1 Prev; Bruce E81 ) double mutants. In the Dcp-1 Prev; Bruce E81 double mutants, the ovarian atrophy phenotype of Bruce E81 loss-offunction mutants was rescued (Figure 3.1A). The size of the ovaries in the Dcp-1 Bruce  E81  E81  double mutants was larger compared to those in the Bruce  Prev  ;  mutants (Figure  3.1A) suggesting that loss of Dcp-1 function might be able to rescue the Bruce phenotype. To understand if the rescue of the ovarian atrophy phenotype is due to suppression of cell death and/or autophagy, we placed Dcp-1  Prev  ; Bruce  E81  double  mutants under well fed and nutrient deprivation conditions. Degenerating mid-stage egg  106  chambers in the well-fed Dcp-1Prev/Cyo, BruceE81/Bruce  E81  mutants had condensed  nuclei (Figure 3.1 B), as expected for the BruceE81 mutant phenotype. In contrast, in the well-fed Dcp-1  Prev  /Dcp-1  Prev  ; Bruce  E81  /Bruce  E81  double mutants, we observed no  degenerating mid-stage egg chambers. Degenerating mid-stage egg chambers in the starved Dcp-1  Prev  /Dcp-1  Prev  ; Bruce  E81  /Bruce  E81  double mutants had a persistent nurse  cell nuclei phenotype which was reported previously in the starved Dcp-1Prev mutants (Figure 3.1C).20 In addition, starved Dcp-1 Dcp-1  Prev  Prev  ; Bruce  E81  double mutants showed the  mutant phenotype of no or low LTR staining in degenerating stage 8 egg  chambers (Figure 3.1D).9 These findings place Dcp-1 downstream of Bruce, suggesting that Bruce might suppress cell death and autophagy in the ovary through restraining Dcp1 activity. 3.3.2 IAP protein Bruce delays starvation induced autophagic responses in the larval fat body and midgut Previously, I showed that loss of Bruce protein is sufficient to trigger starvationinduced autophagy using LysoTracker Red (LTR) staining in both germaria and degenerating midstage egg chambers during Drosophila melanogaster oogenesis regardless of the nutrient availability status.  To further investigate whether Bruce  regulates starvation-induced autophagy in other tissues in Drosophila melanogaster, I employed LTR staining in fed Bruce loss-of-function mutants.  The larval fat body  serves as a nutrient source and undergoes autophagy to provide energy for developing imaginal tissues during metamorphosis and facilitates survival in response to starvation.6 Studies done by Scott et al. showed that the fat body from third instar fed larvae displayed diffuse or faint LTR staining.6 In contrast, the fat body from nutrient deprived  107  third instar larvae displayed intense, punctate LTR staining and reached a maximal level of LTR staining after 4hr starvation.6 The fat body of fed 3rd instar larvae from two Bruce deletion mutants (BruceE81 and BruceE16) had faint LTR staining similar to control animals (Figure 3.2 A-C). However, the fat body from both Bruce mutants reached intense LTR punctate staining in each cell after just 1hr starvation, in contrast to control larvae (w1118) under the same conditions (Figure 3.2 D-F). These data suggest that loss of Bruce function accelerates the autophagic response to nutrient deprived conditions in the larval fat body. Further, I analysed the autophagic response in the midgut of third instar larvae. The larval midgut dissected from well fed control animals had low or no LTR staining (Figure 3.2 G). When control larvae were subjected to 4 hours starvation, there was intense LTR punctate staining in each cell of the third instar larval midgut, suggesting that the larval midgut also has a robust autophagic response to nutrient deprivation (Figure 3.2 H). Similar to the fat body, I found the midgut from Bruce mutants reached a maximal level of LTR staining after 1hr starvation in contrast to control animals that did not reach a comparable level until starved for at least 3-4 hours (Figure 3.2 I-J). Unlike in the ovary, loss of Bruce function is not sufficient to induce autophagy in the larval fat body and midgut but its absence results in an accelerated LTR response to starvation in these larval tissues. 3.3.3 The effector caspase Dcp-1 accelerates the starvation-induced autophagic response but is not required for autophagy in the larval fat body Next I investigated the role of the effector caspase Dcp-1 in starvation induced autophagy in the larval fat body. Under nutrient rich conditions, I observed low or no LTR staining in the fat body cells that expressed full length Dcp-1 under the control of a  108  fat body GAL4 driver (CG-GAL4) (Figure 3.3 A). When CG-GAL4/UAS-fl-Dcp-1 larvae were transferred to 1 hour starvation, I observed an intense level of LTR staining in the fat body cells in contrast to control animals which still had faint LTR staining (Figure 3.3 B). Similar to Bruce loss-of-function mutants, expression of Dcp-1 was not sufficient to induce an autophagic response but instead accelerated the autophagic response to nutrient withdrawal in the larval fat body. To determine whether Dcp-1 is required for starvation induced autophagy in the larval fat body, I employed LTR staining in Dcp-1 loss-offunction mutants. After four hours of nutrient deprivation, I still observed an abundance of LTR puncta in the larval fat body of Dcp-1Prev mutants indicating that Dcp-1 is not required for starvation-induced autophagy in the larval fat body (Figure 3.3 C and D). Similar results were observed in the larval midgut, in that after four hours of starvation, the larval midgut of Dcp-1Prev mutants had numerous LTR puncta similar to wild type (Figure 3.3 E and F). 3.3.4 Starvation has a rapid effect on activation of Dcp-1 but not drICE. Next, I wanted to examine whether starvation had differential effects on activation of two effector caspases, Dcp-1 and drICE.  To investigate this question, I starved  Drosophila l(2)mbn cells in amino acid deprived medium (glucose/PBS) for different time periods, ranging from 30 minutes to 16 hours. Previous studies of Rpr-mediated apoptosis showed that Dcp-1 was cleaved at the inter-domain activation cleavage site Asp33 and Asp215, and drICE was cleaved at Asp28 and Asp230.22  Immunoblot  analysis of endogenous Dcp-1 using an anti-Dcp-1 antibody that specifically recognizes the completely processed p20 subunit and full-length Dcp-1 showed that the inter-domain cleavage of Dcp-1 occurred even under the fed condition in l(2)mbn cells. Following 30  109  minutes of nutrient deprivation, I observed increased levels of p20 subunits of Dcp-1 (3 fold increase in expression compared to fed condition). Levels of p20 subunits of Dcp-1 continued to increase after 1 hour of starvation (6 fold increase in expression), and showed no differential expression compared to fed condition after 2 to 16 hours of starvation. In contrast, after 16 hours of starvation, inter-domain cleavage of drICE was not detectable as shown in the immunoblot analysis with an anti-drICE antibody that has been shown to recognize both full length and p20 subunit of drICE (Figure 3.4).22 These results demonstrate that nutrient deprivation has a rapid effect on proteolysis of Dcp-1 but not drICE. 3.3.5 The catalytic activity of Dcp-1 but not drICE is required for the induction of starvation-induced autophagy To determine whether Dcp-1-mediated proteolytic events are required for the induction of starvation induced autophagy, a catalytically inactive Dcp-1 construct harboring a mutation of Cys to Ala was employed (Dcp-1C196A).22 A fusion of the wildtype Dcp-1 with a V5 tag was expressed in l(2)mbn cells stably transfected with a marker for autophagy, mammalian LC3 (Atg8 homologue) fused to GFP protein, and showed an increase in GFP-LC3 puncta in starved cells compared to cells transfected with the vector control (Figure 3.5 A and B). In contrast, expression of catalytically inactive Dcp-1C196A failed to show a relative increase in GFP-LC3 puncta under the same conditions, indicating Dcp-1-mediated proteolytic events are required to regulate autophagy (Figure 3.5 A and B). Further I tested whether drICE mediated proteolytic events are required for autophagy. Expression of wild-type drICE or catalytically inactive drICEC211A did not show an increase in GFP-LC3 puncta compared to the negative control (vector only)  110  (Figure 3.5 B). These results rule out the possibility that cellular stress due to expression of the effector caspase drICE results in the induction of autophagy.  Instead, these  findings indicate that Dcp-1 mediated proteolytic events are essential for the induction of autophagy, suggesting that Dcp-1 cleaved substrates might play a role in autophagy regulation. 3.3.6 Potential regulators or substrates of Dcp-1 in cells undergoing autophagy To identify potential substrates of Dcp-1 in autophagy inducing conditions, we performed an immuno-affinity purification (IP) and tandem mass spectrometry (MS/MS) fragmentation based assay. A study done by Kamada et al. used the yeast two-hybrid system and a modified caspase construct as the bait. They introduced a point mutation into caspase 3 which substituted serine for the active site cysteine and prevented proteolytic cleavage of substrates.25  They successfully identified gelsolin, an anti-  apoptotic gene, as a substrate of caspase-3.25 We employed a similar modified strategy by expressing the N-terminally V5 tagged Dcp-1C196A as an IP bait protein in Drosophila l(2)mbn cells to prevent cleavage of substrates. In addition, we used the cross-linker, formaldehyde, to strengthen the transient association of Dcp-1 with potential substrates or interaction partners and then removed non-specifically bound proteins by subjecting complexes to extensive washing.  V5 tagged Dcp-1C196A under the control of the actin  promoter or a V5 vector-only negative control was transfected into l(2)mbn cells and then subjected to full medium (fed) or 2 hours starvation.  Cell lysates were  immunoprecipitated with the anti-V5 antibody and immunoprecipitates were analyzed by LC-MS/MS for protein detection and identification.  A subset of high confidence  candidates (unique peptides >2 and log(E) <-15) from both fed and 2 hour starved  111  conditions, and that were not detected in the V5 vector-only control, was selected for analyses of caspase cleavage site prediction (Table 3.1). We chose the CaSPredictor program which combines a PEST-like index and position-dependent amino acid matrices for prediction of potential caspase cleavage sites.23  Sixteen out of our twenty-one  candidates that met the selection threshold contain potential caspase cleavage sites. We used the Inparanoid eukaryotic ortholog database24 to search for mammalian orthologs of our twenty-one Drosophila melanogaster candidate proteins. We then searched these mammalian orthologs to determine whether they are known caspase substrates based on data from two recent studies26,  27  and the CASBAH database.28  The mammalian  orthologs of seven of our candidate proteins are known caspase substrates, further suggesting that the Drosophila melanogaster counterparts of these seven proteins may also be caspase substrates. Overall, our IP-MS studies provide preliminary data that can be used to build a working model to help understand the molecular mechanism of Dcp-1 in autophagy regulation.  3.4 Discussion The genetic relationship between Dcp-1 and Bruce was unknown previously, and we addressed this question by generating Dcp-1  Prev  ; Bruce  E81  double mutants. Our  findings showed that loss-of-function of Dcp-1 could rescue the ovary atropy phenotype of Bruce in the fed double mutants. In the starved Dcp-1  Prev  ; Bruce E81 double mutants,  degenerating mid-stage egg chambers had persistent cell nuclei and faint LTR staining which are phenotypes of Dcp-1Prev mutants. Thus, the rescue of the ovary atrophy phenotype in Dcp-1 Prev; Bruce E81 double mutants appears to result from the suppression of both cell death and autophagy. Our data suggest that Bruce acts to restrain Dcp-1  112  activity and suppress Dcp-1 mediated-cell death and autophagy in the ovary. We cannot rule out the possibility that other IAP proteins, such as DIAP1, play a similar function and also limit Dcp-1 activity during cell death or autophagy. In addition, it is possible that Bruce might suppress other effector caspases, such as drICE, to aid in the prevention of cell death and autophagy in the ovary. Caspases are a family of proteases that are known to mediate the execution of apoptosis by cleaving cellular substrates. In addition to apoptosis, caspases function in non-apoptotic processes, including immunity, cell fate determination and compensatory proliferation.29,  30  The molecular mechanisms by which Dcp-1 regulates starvation-  induced autophagy are still unclear. Local activation or compartmentalization of active caspases is one regulatory mechanism by which high levels of active caspases could regulate non-apoptotic functions without triggering apoptosis.29,  30  For example,  immuno-reactivity for caspase activity using caspase 3 antibody is detected only in a cytoskeletal membrane complex, termed the individualization complex (IC), during the non-apoptotic process of Drosophila melanogaster spermatid individualization.31,  32  During Drosophila metamorphosis, the larval brain undergoes a massive elimination of dendrites and axons without triggering cell death of the entire tissue, perhaps through localizing active caspases to dendrites only.  33  Therefore, it is possible that Dcp-1 is  localized to specific cellular organelles (e.g., autophagosomes) during its role in autophagy. However, I think that this might not be the case, since Dcp-1 immunostaining did not seem to localize to specific cellular organelles but instead appeared to be distributed throughout the cytosol. In addition, my results showed that the catalytic activity of Dcp-1 but not drICE is essential for starvation-induced autophagy.  This  113  observation suggests that expression of effector caspases do not in general induce cellular stress which in turn triggers an autophagy response indiscriminately. Instead, our results suggest that Dcp-1 might recognize and cleave substrates which mediate autophagy regulation distinctively. Caspases recognize at least four contiguous amino acids (P4-P3P2-P1) in their substrates, and cleave after P1 which is usually an Asp residue.34 Dcp-1 has substrate specificity that is similar to mammalian caspase 3 and CED-3, the effector caspase in C.elegans.21 Using positional scanning peptide libraries, it was shown that Dcp-1 has an absolute requirement for Asp in P4, a strong preference for Glu in P3 and is tolerant of several substitutions in P2.21 Thus, Dcp-1 has an optimal recognition motif of DEVD, which is the same as other caspases including caspase 2, 3, and 7 and CED-3.21 A scanning peptide library study has not been performed on drICE but it also has a clear preference for Asp in P1.35 A study from Song et al. showed that only Dcp-1 but not drICE was able to cleave human lamins, indicating that the specificities of Dcp-1 and drICE are not identical.21  Based on our findings and these biochemical studies of  substrate specificity, we speculate that the two prominent Drosophila effector caspases, Dcp-1 and drICE, might recognize different sets of substrates mediating apoptosis and autophagy independently or co-ordinately. Caspases are synthesized as single-chain zymogens and exist as homo-dimers constitutively. After cleavage in the inter-domain cleavage site, the catalytic activity of caspases is increased significantly.36 The inter-domain cleavage causes conformation changes which allow caspases to expose the catalytic cysteine and allow subsequent substrate binding. Our data showed that cleavage of endogenous Dcp-1 in the interdomain site already occurred in nutrient full medium and cleaved p20 subunits of Dcp-1  114  were increased following 30 minutes of starvation treatment. These data suggest that nutrient deprivation might facilitate the inter-domain cleavage of Dcp-1 and thus help the subsequent binding/cleaving of Dcp-1 substrates for the execution of autophagy. It is possible that the timing of Dcp-1 catalytic activation could determine the sensitivity thresholds of autophagic and apoptotic responses. Nutrient deprivation might enhance Dcp-1 catalytic activities which promotes autophagy for cell survival, giving the cells a chance to recover. Prolonged starvation might result in activation of drICE that triggers apoptosis. The discrepancies of Bruce and Dcp-1 mediated autophagic responses between larval tissues (fat body and midgut) and the ovary remain to be resolved. In the ovary, loss-of-function of Bruce is sufficient to trigger autophagy and cell death, and loss-offunction of Dcp-1 suppresses both processes.9 However, loss-of-function of Bruce or ectopic expression of Dcp-1 resulted in an acceleration of the autophagic response but was not sufficient to trigger autophagy in the absence of nutrition deprivation in the larval fat body and midgut. In addition, we were unable to identify an essential role for Dcp-1 in starvation-induced autophagy in both larval tissues. I speculate that insulin/PI3K/Akt signaling may play a role in determining the differences of Bruce and Dcp-1 mediated autophagic responses in these tissues. The larval fat body and midgut are terminally differentiated endoreplicating tissues (ERTs) that provide nutrients for undifferentiated imaginal cells during the larval stage.37 Cells in the ERTs do not directly respond to changes in dietary protein but instead use secondary humoral signals, most likely insulins, to continue cell growth before cells are severely depleted of amino acids.37 Protein starvation causes loss of PI3K activity in ERTs.37 I observed a low expression of  115  Bruce in the fed larval fat body and an increase in Bruce expression following just one hour of starvation, suggesting that activity of Bruce is also modulated in response to starvation (Figure 3.7). On the other hand, Akt kinase, which is activated by PI3K was shown to be expressed at a relatively low level in nurse cells of mid-stage egg chambers.38 Cell death in mid-stage egg chambers and salivary gland cells shares many similarities, and autophagy is induced following down-regulation of PI3K expression in salivary glands.8,  39  Based on these previous results, clearly there are differences in  PI3K/Akt levels between tissues such as the fat body, where autophagy is important for survival in response to starvation, and tissues such as salivary glands and the ovary where autophagy plays a role in the cell death process. Thus, the delay in response to loss of Bruce or ectopic Dcp-1 expression in the larval fat body and midgut might be due to the time required for the generation of secondary humoral signals and the loss of PI3K activity. However, the reason why we did not observe an essential role for Dcp-1 in starvation induced autophagy in the larval fat body and midgut remains an outstanding question. Similarly, overexpression of a pro-apoptotic gene Hid has been shown to induce autophagy but is not essential for starvation induced autophagy in the fat body. It is possible that a redundant pathway (e.g., ecdysone) can compensate for autophagy regulation in the larval fat body of Dcp-1 and Hid mutants. Our IP-MS studies provide a preliminary set of potential substrates and interacting proteins of Dcp-1 which can be used to generate a working model for how Dcp-1 executes autophagy (Table 3.1 and Figure 3.6). The clathrin-uncoating ATPase Hsc70-4 not only is a TOR interacting protein but is also involved in autophagy and endocytosis in the larval fat body.40 Loss-of-function Hsc70-4 mutants have decreased  116  TOR, Akt and phospho-Akt levels.40 Based on the CasPredictor program,23 we found a potential caspase cleavage site in the Hsc70-4 protein sequence and its mammalian homologue, HSP7C, is a known caspase substrate.24,  26  Inhibition of Hsp90, the  mammalian homologue of Drosophila melanogaster Hsp83, suppresses the Akt/TOR pathway and induces autophagy in murine embryonic fibroblasts.41 We also found a potential caspase cleavage site in the Drosophila melanogaster Hsp83 protein sequence. Therefore, we hypothesize that Dcp-1 might cleave Hsc70-4 and/or Hsp83 and result in decreased Akt/TOR activity that then triggers autophagy. We identified one of the Drosophila peroxiredoxin family members, Jafrac1, as a potential Dcp-1 substrate.42 Purified recombinant Jafrac1 proteins are shown to reduce H2O2, and expression of Jafrac1 increases cell survival following H2O2 treatment in Drosophila S2 cells.42 Starvation has been shown to trigger production of reactive oxygen species (ROS) such as H2O2, and HsAtg4, the mammalian homologue of yeast Atg4, is a direct target for oxidation by H2O2.43 Oxidation of HsAtg4 leads to induction of the autophagy process.43 Thus, it is possible that Dcp-1 cleaves Jafrac1, resulting in increased levels of H2O2 that could oxidize DmAtg4 and trigger autophagy.  Our data  demonstrated that prolonged starvation leads to increased Dcp-1 activity which could result in much higher levels of H2O2 leading to cell death. This speculative model is consistent with a previous study that showed autophagy degrades the ROS scavenger catalase, resulting in cell death in mammalian cells.11 We generated a putative model of Dcp-1 mediated autophagy based on some of our IP-MS data and previous observations (Figure 3.6). The important next steps will be to validate our interaction data, verify if candidate proteins with potential caspase cleavage sites are Dcp-1 substrates using  117  biochemical assays, and determine if the identified substrates have a functional significance in mediating autophagy. However, we cannot rule out the possibility that these candidate proteins might also function in apoptosis. Future studies that elucidate the roles of the candidate proteins in autophagy and/or apoptosis will help to establish the regulatory mechanisms of Dcp-1 governing the crosstalk between these two processes.  118  Figure 3.1 Ovarian atrophy phenotype resulting from Bruce mutations is rescued by Dcp-1 (A)The ovaries of BruceE81 mutants are poorly developed and atrophied even under wellfed conditions (left). In contrast, the ovaries of Dcp-1 Prev/Dcp-1 Prev; Bruce E81/Bruce E81 (Dcp-1 Prev; Bruce E81 ) double mutants (right) are better developed compared to those of Bruce single mutants. (B) In well-fed Dcp-1Prev/Cyo, BruceE81/Bruce E81flies, ovaries had numerous dying stage 8 egg chambers (diamond arrows). (C) In nutrient-deprived Dcp1Prev; BruceE81 double mutants, ovaries had degenerating mid stage egg chambers (arrows) with a phenotype similar to Dcp-1Prev mutants, consisting of the persistent nurse cell nuclei and disappearance of follicle cells. n=3 animals. Scale bar: 100μm. (D) Degenerating mid-stage egg chambers (arrow) in nutrient-deprived Dcp-1Prev; BruceE81 mutants showed no or low LTR staining that is characteristic of the Dcp-1Prev mutant phenotype. n=45 degenerating mid-stage egg chambers scored in three different animals. Percentage of LTR positive mid-stage egg chambers is 15.6%. (E) DAPI staining of the same degenerating mid-stage egg chamber shown in (D) in nutrient-deprived Dcp-1Prev; BruceE81 mutants. Scale bar: 50μm. DAPI staining of nuclei is shown in white.  119  Figure 3.1  120  Figure 3.2 Bruce postpones starvation-induced autophagy in the larval fat body and midgut. (A-C) The larval fat body from fed control w1118, BruceE81 and BruceE16 mutants showed low levels of LTR (red) staining. (D-F) Following 1hr starvation, fat body cells from control w1118 animals showed low levels of LTR (red) staining, whereas the fat body cells from two different loss-of-function Bruce mutants displayed intense, punctate LTR staining. (G) The larval midgut dissected from fed control w1118 animals displayed faint LTR (white) staining. (H) In contrast, the midgut from 4hr starved control animals displayed intense LTR dots (white) in all cells. (I-J) Following 1hr starvation, the midgut from control animals showed low LTR (red) staining, whereas the midgut from Bruce mutants displayed numerous LTR (red) puncta. Scale bar: 100μm. At least six different animals were examined for (A-F) and at least three different animals were examined for (G-J). DAPI staining of nuclei is shown in blue.  121  Figure 3.2  122  Figure 3.2 continued  123  Figure 3.3 Expression of Dcp-1 accelerates autophagy in response to nutrient withdrawal but is not essential for autophagy in the fat body and midgut (A) Following 1hr starvation, fat body cells from control animals (CG-GAL4) display faint LTR (white) staining. (B) In contrast, expression of full length Dcp-1 in the fat body (CG-GAL4/UAS-fl-Dcp-1) resulted in intense LTR staining following 1hr starvation. Scale bar: 100μm. (C-D) Following 4hr starvation treatment, fat body cells from control w1118 and Dcp-1Prev mutants both displayed abundant LTR puncta. Scale bar: 20 μm. (E-F) Following 4hr starvation treatment, midgut cells from control w1118 and Dcp-1Prev mutants displayed intense LTR staining. Scale bar: 100 μm. At least ten animals from each strain were examined in each condition.  124  Figure 3.3  125  Figure 3.4 Time course analyses of Dcp-1 and drICE cleavage at the inter-domain site during nutrient deprivation. Drosophila l(2)mbn cells were subjected to different time periods of starvation treatment, ranging from 30 minutes to 16 hours. Cont = Control cells not subjected to starvation. Immunoblot analyses using anti-Dcp-1 and anti-drICE antibodies that specifically recognize the full length and the processed p20 subunit of Dcp-1 and drICE showed an increase in processed p20 subunits of Dcp-1 following 30 minutes starvation treatment. Inter-domain cleavage of drICE was not detectable even after 16 hours of starvation treatment. Actin was used as a loading control. The blot shown is representative of three independent experiments. f.l Dcp-1= full length Dcp-1; ΔDcp-1= prodomain removed Dcp-1; Dcp-1_p20=processed p20 subunit of Dcp-1; fldrICE=full length drICE.  126  Figure 3.4  127  Figure 3.5 The catalytic activity of Dcp-1 but not drICE is required for starvationinduced autophagy (A) Representative images of GFP-LC3 staining in l(2)mbn-GFP-LC3 cells transfected with V5-Dcp-1and V5-Dcp-1C196A following 2hr starvation treatment. Arrows denote representative cells expressing indicated plasmid. (B) Fluorescent microscopic quantification of GFP-LC3 puncta in l(2)mbn-GFP-LC3 cells transfected with indicated plasmid following 2hr starvation treatment. Results shown represent mean ± SEM for combined data from three independent experiments. At least fifty V5 positive cells were examined in each of three independent experiments for each condition. Scale bar: 20μm.  128  Figure 3.5 A  B  129  Figure 3.6 Putative model of Dcp-1 mediated autophagy  In this model, the substrates of effector Dcp-1 are involved in the regulation of starvation induced autophagy in Drosophila. Dcp-1 mediates the cleavage of Hsc70-4 and Hsp83 which results in the loss of Akt/TOR activity and the induction of autophagy. Dcp-1 mediated cleavage of Jafrac1, a ROS scavenger, might result in increased levels of H2O2 that leads to DmAtg4 oxidation and autophagy induction.  130  Figure 3.7 Bruce expression increases in response to nutrient withdrawal The larval fat body from a wild type (w1118) animal grown under well fed (top) or 1hr starvation (bottom) conditions was stained with anti-Bruce antibody. An increase in Bruce expression is evident following 1 hour starvation compared to the well fed condition. Scale bar=100μm. At least six animals were examined for each condition. DAPI staining of nuclei is shown in blue.  131  Figure 3.7  132  Table 3.1 Potential Dcp-1 substrates Name Hsc70-4 Uba1  Site IEIDS  Act42A  ELPDG  TER94 Socs16D Ef1beta  DEIDA DLLDE DDVDL  GO Molecular Function ATPase activity ubiquitin activating enzyme activity structural constituent of cytoskeleton syntaxin-1 binding thioredoxin peroxidase activity ATPase activity translation elongation factor activity ATP:ADP antiporter activity DAG-activated phospholipiddependent PKC inhibitor transcription regulator activity structural constituent of cytoskeleton; RNA binding malate dehydrogenase activity aminoacyl-tRNA ligase activity UDP-glucose 6-dehydrogenase activity ATP binding; chaperone binding hydrogen-exporting ATPase activity, structural constituent of cytoskeleton ATPase activity; serine-type endopeptidase activity protein binding translation elongation factor activity  alphaTub84B tomosyn Jafrac1 Hsp83 Ef1alpha48D sesB 14-3-3 epsilon spen  DSGDG NKLDG CSTDS DEADD DALDA  mask disco-r CG34422  DEPDS DEIDS DSTDS  CG8184  SSVDS  ubiquitin-protein ligase E3 activity  DEADS  sgl Hsc70Cb blw  UniProt HSP7C_HUMAN* UBE1_HUMAN TBA1A_HUMAN* STXB5_HUMAN PRDX2_HUMAN HS90A_HUMAN* EF1A1_HUMAN ADT2_HUMAN 1433E_HUMAN MINT_HUMAN* 4EBP3_HUMAN BNC2_HUMAN NA UGDH_HUMAN HS74L_HUMAN ATPA_HUMAN ACTB_HUMAN* TERA_HUMAN SOCS2_HUMAN EF1B_HUMAN* HUWE1_HUMAN*  Gene symbols and GO molecular functions are from Flybase. Predictions of caspase cleavage sites of identified proteins were determined by the CaSPredictor program and are listed in column 2. Mammalian orthologs of identified proteins are listed in column 4. An asterisk adjacent to the UniProt identifier indicates that the mammalian ortholog of our identified Drosophila protein is a known caspase substrate.  133  3.5 References 1.  Kissova I, Salin B, Schaeffer J, Bhatia S, Manon S, Camougrand N. Selective and  non-selective autophagic degradation of mitochondria in yeast. Autophagy 2007; 3:32936. 2.  Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima  R, Yokoyama M, Mishima K, Saito I, Okano H, Mizushima N. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 2006; 441:885-9. 3.  Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, Ueno T, Koike M,  Uchiyama Y, Kominami E, Tanaka K. Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 2006; 441:880-4. 4.  Ravikumar B, Duden R, Rubinsztein DC. Aggregate-prone proteins with  polyglutamine and polyalanine expansions are degraded by autophagy. Human molecular genetics 2002; 11:1107-17. 5.  Klionsky DJ. Autophagy. Georgetown: Landes Bioscience, 2004.  6.  Scott RC, Schuldiner O, Neufeld TP. Role and regulation of starvation-induced  autophagy in the Drosophila fat body. Dev Cell 2004; 7:167-78. 7.  Juhasz G, Erdi B, Sass M, Neufeld TP. Atg7-dependent autophagy promotes  neuronal health, stress tolerance, and longevity but is dispensable for metamorphosis in Drosophila. Genes Dev 2007; 21:3061-6. 8.  Berry DL, Baehrecke EH. Growth arrest and autophagy are required for salivary  gland cell degradation in Drosophila. Cell 2007; 131:1137-48.  134  9.  Hou YC, Chittaranjan S, Barbosa SG, McCall K, Gorski SM. Effector caspase  Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis. J Cell Biol 2008; 182:1127-39. 10.  Mohseni N, McMillan SC, Chaudhary R, Mok J, Reed BH. Autophagy promotes  caspase-dependent cell death during Drosophila development. Autophagy 2009; 5:32938. 11.  Yu L, Wan F, Dutta S, Welsh S, Liu Z, Freundt E, Baehrecke EH, Lenardo M.  Autophagic programmed cell death by selective catalase degradation. Proc Natl Acad Sci U S A 2006; 103:4952-7. 12.  Vaux DL, Silke J. IAPs, RINGs and ubiquitylation. Nat Rev Mol Cell Biol 2005;  6:287-97. 13.  Srinivasula SM, Ashwell JD. IAPs: what's in a name? Mol Cell 2008; 30:123-35.  14.  Hawkins CJ, Yoo SJ, Peterson EP, Wang SL, Vernooy SY, Hay BA. The  Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID, and GRIM. J Biol Chem 2000; 275:27084-93. 15.  Meier P, Silke J, Leevers SJ, Evan GI. The Drosophila caspase DRONC is  regulated by DIAP1. Embo J 2000; 19:598-611. 16.  Xu D, Li Y, Arcaro M, Lackey M, Bergmann A. The CARD-carrying caspase  Dronc is essential for most, but not all, developmental cell death in Drosophila. Development 2005; 132:2125-34. 17.  Hay BA, Guo M. Caspase-dependent cell death in Drosophila. Annual review of  cell and developmental biology 2006; 22:623-50.  135  18.  Muro I, Berry DL, Huh JR, Chen CH, Huang H, Yoo SJ, Guo M, Baehrecke EH,  Hay BA. The Drosophila caspase Ice is important for many apoptotic cell deaths and for spermatid individualization, a nonapoptotic process. Development 2006; 133:3305-15. 19.  Xu D, Wang Y, Willecke R, Chen Z, Ding T, Bergmann A. The effector caspases  drICE and dcp-1 have partially overlapping functions in the apoptotic pathway in Drosophila. Cell Death Differ 2006; 13:1697-706. 20.  Laundrie B, Peterson JS, Baum JS, Chang JC, Fileppo D, Thompson SR, McCall  K. Germline cell death is inhibited by P-element insertions disrupting the dcp-1/pita nested gene pair in Drosophila. Genetics 2003; 165:1881-8. 21.  Song Z, Guan B, Bergman A, Nicholson DW, Thornberry NA, Peterson EP,  Steller H. Biochemical and genetic interactions between Drosophila caspases and the proapoptotic genes rpr, hid, and grim. Mol Cell Biol 2000; 20:2907-14. 22.  Tenev T, Zachariou A, Wilson R, Ditzel M, Meier P. IAPs are functionally non-  equivalent and regulate effector caspases through distinct mechanisms. Nat Cell Biol 2005; 7:70-7. 23.  Garay-Malpartida HM, Occhiucci JM, Alves J, Belizario JE. CaSPredictor: a new  computer-based tool for caspase substrate prediction. Bioinformatics (Oxford, England) 2005; 21 Suppl 1:i169-76. 24.  O'Brien KP, Remm M, Sonnhammer EL. Inparanoid: a comprehensive database  of eukaryotic orthologs. Nucleic acids research 2005; 33:D476-80. 25.  Kamada S, Kusano H, Fujita H, Ohtsu M, Koya RC, Kuzumaki N, Tsujimoto Y.  A cloning method for caspase substrates that uses the yeast two-hybrid system: cloning of the antiapoptotic gene gelsolin. Proc Natl Acad Sci U S A 1998; 95:8532-7.  136  26.  Mahrus S, Trinidad JC, Barkan DT, Sali A, Burlingame AL, Wells JA. Global  sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini. Cell 2008; 134:866-76. 27.  Dix MM, Simon GM, Cravatt BF. Global mapping of the topography and  magnitude of proteolytic events in apoptosis. Cell 2008; 134:679-91. 28.  Luthi AU, Martin SJ. The CASBAH: a searchable database of caspase substrates.  Cell Death Differ 2007; 14:641-50. 29.  Kuranaga E, Miura M. Nonapoptotic functions of caspases: caspases as regulatory  molecules for immunity and cell-fate determination. Trends Cell Biol 2007; 17:135-44. 30.  Yi CH, Yuan J. The Jekyll and Hyde functions of caspases. Dev Cell 2009; 16:21-  34. 31.  Kuo CT, Zhu S, Younger S, Jan LY, Jan YN. Identification of E2/E3  ubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron dendrite pruning. Neuron 2006; 51:283-90. 32.  Williams DW, Kondo S, Krzyzanowska A, Hiromi Y, Truman JW. Local caspase  activity directs engulfment of dendrites during pruning. Nature neuroscience 2006; 9:1234-6. 33.  Huh JR, Vernooy SY, Yu H, Yan N, Shi Y, Guo M, Hay BA. Multiple apoptotic  caspase cascades are required in nonapoptotic roles for Drosophila spermatid individualization. PLoS biology 2004; 2:E15. 34.  Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, Garcia-Calvo M,  Houtzager VM, Nordstrom PA, Roy S, Vaillancourt JP, Chapman KT, Nicholson DW. A combinatorial approach defines specificities of members of the caspase family and  137  granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem 1997; 272:17907-11. 35.  Snipas SJ, Drag M, Stennicke HR, Salvesen GS. Activation mechanism and  substrate specificity of the Drosophila initiator caspase DRONC. Cell Death Differ 2008; 15:938-45. 36.  Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis.  Nat Rev Mol Cell Biol 2004; 5:897-907. 37.  Britton JS, Lockwood WK, Li L, Cohen SM, Edgar BA. Drosophila's insulin/PI3-  kinase pathway coordinates cellular metabolism with nutritional conditions. Dev Cell 2002; 2:239-49. 38.  Cavaliere V, Donati A, Hsouna A, Hsu T, Gargiulo G. dAkt kinase controls  follicle cell size during Drosophila oogenesis. Dev Dyn 2005; 232:845-54. 39.  McCall K. Eggs over easy: cell death in the Drosophila ovary. Dev Biol 2004;  274:3-14. 40.  Hennig KM, Colombani J, Neufeld TP. TOR coordinates bulk and targeted  endocytosis in the Drosophila melanogaster fat body to regulate cell growth. J Cell Biol 2006; 173:963-74. 41.  Kundu M. LT, Yang CY., McCastlain K., Hennessy K., Wu J., Ney P., Thompson  C. Hsp90 Regulates Ulk1-Mediated Autophagic Clearance of Mitochondria Keystone symposia on cell death pathway. Whistler BC, 2009. 42.  Radyuk SN, Klichko VI, Spinola B, Sohal RS, Orr WC. The peroxiredoxin gene  family in Drosophila melanogaster. Free radical biology & medicine 2001; 31:1090-100.  138  43.  Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z. Reactive oxygen  species are essential for autophagy and specifically regulate the activity of Atg4. Embo J 2007; 26:1749-60.  139  Chapter 4 Conclusions and future research 4.1 Overall summary and significance of the thesis research Macroautophagy (autophagy hereafter) is a lysosome-mediated catabolic process involved in the degradation and recycling of intracellular components. The association of autophagy with cell death has attracted considerable attention and raised many unanswered questions. To contribute to a better understanding in this area, our approach was to conduct a systematic RNAi-based screen of Drosophila apoptosis-related genes to identify  potential  apoptosis-related  modifiers  of  starvation-induced  autophagy.  Starvation or nutrient deprivation is a well characterized inducer of autophagy in Drosophila1 and many other organisms.2,  3  We developed an efficient flow cytometer  based Lysotracker Green (LTG) assay as a primary screen and coupled that to a secondary GFP-LC3 redistribution assay, both in Drosophila l(2)mbn cells, to provide readouts representing late and early stages of autophagy, respectively. As an initial validation of our screening strategy, we designed dsRNAs corresponding to autophagy genes and known autophagy regulators. Our findings showed that dsRNAs corresponding to 11 Drosophila Atg homologues were able to reduce LTG levels in starved l(2)mbn cells. Knockdown of known positive and negative autophagy regulators using RNAi also produced expected alterations in LTG and GFP-LC3. We next screened twenty apoptosis-  A version of this chapter has been published. Hou Y-C. C, Hannigan A.M, Gorski S.M. An executioner caspase regulates autophagy. Autophagy 2009; 5: 530-33. (Invited by the editorial board)  140  related and Ras pathway-related genes and found 9 that modified LTG and GFP-LC3 levels significantly. Knockdown of Dcp-1, hid, debcl, buffy and p53 suppressed LTG and GFP-LC3 levels in starved cells, identifying these genes as positive regulators of autophagy. RNAi-mediated knockdown of Bruce, Ras, Raf, and MAPK enhanced LTG and GFP-LC3 levels in starved cells, identifying these genes as negative regulators of autophagy. How might the identified gene products act to regulate or modulate the autophagic response in nutrient-deprived cells? Our data showed that the pro-apoptotic gene, hid, but not rpr, grim, or skl, acts to regulate starvation-induced autophagy in Drosophila l(2)mbn cells. Consistent with our findings, overexpression of hid was shown previously to induce autophagy in various Drosophila tissues including fat body, midgut, and salivary glands.4 Survival Ras/Raf/MAPK signaling has been shown to specifically inhibit the pro-apoptotic activity of Hid,5 and our observations indicate that the Ras/Raf/MAPK pathway also plays an inhibitory role in starvation-induced autophagy. The Hid protein contains five MAPK phosphorylation consensus sites;5 thus it is possible that survival signals regulate the crosstalk between autophagy and apoptosis through different threshold levels of MAPK-mediated phosphorylation on Hid. In addition, Hid is known to promote polyubiquitination of DIAP1 and antagonize its anti-apoptotic activity through proteosomal-dependent degradation.6 Surprisingly, we found that another IAP protein, Bruce, but not DIAP1 acts as a suppressor of autophagy suggesting that Bruce, instead of DIAP1, might be the downstream target of Hid and act to antagonize Hidmediated autophagy. Bruce and its mammalian homologue, Apollon, share sequence conservation in the BIR (baculoviral-IAP-repeat) and UBC (ubiquitin-conjugating  141  enzyme) domains.7 Apollon has been shown to ubiquitinate and promote degradation of SMAC, the mammalian homologue of Hid.7 Perhaps Drosophila Bruce has a similar molecular function as Apollon and promotes degradation of Hid through ubiquitination, thereby acting to negatively regulate autophagy. However, Bruce is a large protein (530 kDa) and it is plausible that it could regulate autophagy through protein interactions with one of its other protein regions. Another candidate Bruce-interacting protein that we identified in our screen is the effector caspase Dcp-1. IAP family members can bind directly to caspases, and inhibit their activity.8 Thus, it is possible that Bruce suppresses Dcp-1 activity or promotes Dcp-1 degradation through its BIR and/or UBC domains. Such an interaction would be consistent with our identification of Dcp-1 as a positive regulator of autophagy. Based on our recent observations and these previous findings, we propose a hypothetical pathway for the regulation of starvation-induced autophagy in Drosophila (Figure 4.1). Clearly, epistasis analyses and protein interaction studies are required to prove or disprove this model, and understand how it integrates with other components (eg. Tor) already known to control the autophagic response to starvation. To validate the autophagy modulating effects of some of the identified cell deathrelated genes in vivo, we used Drosophila melanogaster oogenesis as a model system. Nutrient deprivation triggers germline cell death at two specific stages during oogenesis, the germarium and mid-stage oogenesis.9 Using a GFP-LC3 transgenic Drosophila line10 as well as Lystotracker Red staining, we found that autophagy also occurs in response to nutrient deprivation at these two stages in oogenesis. An earlier study in Drosophila virilis similarly reported the presence of autophagic structures, as observed by TEM, in mid-stage (as well as late-stage) oogenesis.11 Dcp-1 was shown previously to be required  142  for mid-stage egg chamber cell death.12 We further demonstrated that Dcp-1 is required for cell death in germaria, and is also necessary for starvation-induced autophagy in both germaria and mid-stage egg chambers. Further, overexpression of Dcp-1 was sufficient to induce autophagy at these two stages even under well fed conditions (Figure 4. 2). Lossof-function mutations in Bruce resulted in ectopic autophagy and cell death in both stages, regardless of nutrient status, indicating that Bruce acts normally to suppress both autophagy and cell death during Drosophila oogenesis. Thus, our observations using RNAi targeting Dcp-1 and Bruce in the l(2)mbn cell line were confirmed in vivo during Drosophila melanogaster oogenesis. However, we observed that loss-of-function of Bruce or overexpression of Dcp-1 in the larval fat body or midgut resulted in a starvation-dependent acceleration of the autophagic response following starvation, rather than ectopic autophagy. Therefore, Bruce and Dcp-1-induced autophagy responses were different between the larval fat body, midgut and the ovary. What are the factors that contribute to the discrepancies of Bruce and Dcp-1 mediated autophagic responses between larval tissues (fat body and midgut) and the ovary? Several reports have demonstrated that upstream signaling pathways such as TOR and PI3K can affect the mechanistic role of autophagy in cell death, cell growth and cell survival. In the larval fat body, an inverse relationship exists between autophagy and cell growth, and autophagy-defective cells had a growth advantage under physiological conditions.13 However, in cells which had defective TOR signaling, autophagy did not have an inhibitory effect on cell growth.1,  13  In the salivary gland, growth arrest and  autophagy occur concurrently and autophagy contributes to cell death in the presence of apoptotic factors, suggesting that the levels of cell growth signaling might affect the  143  mechanistic role of autophagy.14 A recent report by Lu et al. showed that controlled expression of the tumor suppressor gene aplasia Ras homolog member I (ARHI) resulted in autophagic cell death in human ovarian cancer cells.15 However, multiple factors, including growth factors, inflammatory cytokines, and extracellular matrix proteins within xenograft tumors in mice switched ARHI-induced autophagy to promote tumor cell survival instead of cell death suggesting that levels of PI3K signaling might differentiate the role of autophagy in cell survival and cell death.15 In Drosophila, a relatively high level of PI3K signaling was observed in the larval fat body, and protein starvation resulted in loss of PI3K activity.16 In contrast, a relatively low level of expression of Akt expression, a PI3K substrate, was observed in the nurse cells of midstage egg chambers but not in the nurse cells of egg chambers of other stages.17 Thus, I speculate that upstream signaling pathways, such as the PI3K/Akt pathway, might be the factors that contribute to the discrepancies of Bruce and Dcp-1-mediated autophagic responses in different tissues. It will be important to determine the mechanistic roles of Bruce and Dcp-1-induced autophagy in cell death, growth and cell survival under conditions of competent or interrupted PI3K and TOR signaling in the future. If an effector caspase is required for autophagy and apoptosis, what determines the balance between these two processes and what is the final cellular outcome? In the Drosophila ovary, the two cellular stress responses occurred together and it is possible that autophagy is part of the apoptotic response itself, an idea put forward already by Thorburn.18 Several cell death regulators have functions that are involved in the adaptation to stress.19 For example, EGL-1, a BH3-only protein, is required for metabolic stress,20 and AIF plays a role in redox stress.21 Hence, an alternative idea is that some  144  proteins involved in stress responses, such as autophagy, also evolved roles as cell death effectors. A previous biochemical study showed that the effector caspase Dcp-1 was able to auto-cleave/auto-activate and also cleave another effector caspase drICE.22 In contrast, drICE did not act to cleave itself.22 Our data showed that nutrient deprivation had different effects on Dcp-1 and drICE inter-domain cleavage, an indication of increased catalytic activity. Following 30 minutes starvation, we observed increased levels of p20 subunits of endogenous Dcp-1, suggesting that catalytic activity of Dcp-1 was increased shortly after starvation. In contrast, we did not observe p20 subunits of endogenous drICE even following 16 hours of starvation. It is possible that the timing of Dcp-1 activation could determine the sensitivity thresholds of autophagic and apoptotic responses. Starvation signals might initially induce Dcp-1 activation which promotes autophagy for cell survival, giving the cells a chance to recover and allow continued development.  Prolonged starvation signals might result in activation of drICE and  triggers apoptosis. If activation of Dcp-1 determines the sensitivity thresholds of autophagic and apoptotic responses, what is the mechanism that regulates Dcp-1 activity?  We  discovered a genetic interaction between Dcp-1 and Bruce, where a mutation in Dcp-1 blocked the ectopic autophagy and cell death observed in the ovaries of Bruce mutants. Thus, our genetic data showed that IAP protein Bruce might restrain Dcp-1 activity. Other IAP protein family member such as DIAP1 could also play a role in the regulation of Dcp-1 activation.  In mammalian cells, Cytochrome c (Cyt c) is released from  mitochondria to the cytosol where it binds to Apaf-1 leading to caspase activation during an apoptotic stimulus.23 The release of Cyt c is negatively regulated by the anti-apoptotic  145  Bcl-2 family members such as Bcl-2 and Bcl-XL; thus activation of mammalian caspases is held in check by Bcl-2 proteins.24 Several mammalian Bcl-2 family proteins have been shown to also play an important role in autophagy regulation. Particularly, anti-apoptotic Bcl-2 negatively regulates autophagy through its interaction with autophagy protein Beclin 1 indicating that a threshold of autophagic response is also held in check by Bcl-2 proteins.25 Could activation of Dcp-1 be regulated by Bcl-2 family members or factors released from mitochondria? In Drosophila, the role of mitochondria in cell death has been a subject of debate. Considerable evidence suggests that Cyt c does not play a clear role in Drosophila apoptosis.26-28 The roles of two Bcl-2 family proteins, Debcl and Buffy, in apoptotic regulation have been studied in Drosophila, and data clearly showed that Debcl has a pro-apoptotic function.29 The role of Buffy still appears to be somewhat elusive but most data suggest that it is anti-apoptotic.30, 31 The role of the Drosophila Bcl-2 proteins in the regulation of mitochondrial changes (i.e. the release of Cyt c or other mitochondria factors) during apoptosis is still unclear. Our RNAi screen showed that both Debcl and Buffy have a pro-autophagic function. Unlike mammalian cells, Bcl2 family proteins do not seem to play a role in the regulation of caspase activation, and the autophagic response may not be held in check by Bcl-2 proteins in Drosophila. However, we cannot rule out the possibility that additional Bcl-2 family members still await to be discovered in Drosophila. Caspases are known to mediate the execution of apoptosis by cleaving cellular substrates. Numerous studies have been conducted to identify specific substrates of caspases in apoptosis; however, substrates of caspases in non-apoptotic processes are less characterized. Our results demonstrated that catalytic activity of Dcp-1, but not drICE, is  146  required for starvation-induced autophagy suggesting that substrates of Dcp-1 mediate the execution of autophagy and that substrates of Dcp-1 in autophagy could be distinct from its substrates in apoptosis. An immunoprecipitation and mass spectrometry (IP-MS) based strategy was employed to identify substrates of Dcp-1 in cells undergoing autophagy. Future work is required to verify if identified proteins from our IP-MS studies are Dcp-1 substrates, and to determine if identified proteins have a functional significance in mediating autophagy. However, our initial IP-MS studies did provide a preliminary list of promising substrates which can be used to generate a working model for how Dcp-1 might act to regulate autophagy. We identified Hsc70-4, which genetically interacts with the nutrient sensor TOR32, as a potential substrate of Dcp-1. Loss-offunction Hsc70-4 larvae showed decreased levels of Akt, phospho-Akt and TOR32; thus our data are consistent with the possibility that Dcp-1 mediated-cleavage of Hsc70-4 leads to suppression of TOR activity and the subsequent induction of autophagy. Our data are also consistent with the possibility that Dcp-1 might mediate autophagy by regulating levels of reactive oxygen species (ROS), shown previously to cause oxidation of Atg4 and subsequent autophagy induction in mammals.33 We identified one of the Drosophila peroxiredoxin family members, Jafrac1, as a potential Dcp-1 substrate.  Jafrac1  scavenges ROS and peroxides (H2O2), and expression of Jafrac1 promotes cell survival of S2 cells following H2O2 treatment.34 Dcp-1 might cleave Jafrac-1 resulting in increased levels of H2O2 that oxidize DmAtg4 and trigger autophagy. The role of autophagy in cell death is still not well understood and appears to be context dependent. During developmental cell death, such as embryogenesis and insect metamorphosis, it has been proposed that autophagy acts to assist dead cell clearance  147  when insufficient phagocytes are available for corpse removal.35, 36 Three recent studies have demonstrated that autophagy is involved in developmental cell death processes.14, 37, 38  In a mouse embryoid body cavitation model37 and in a mouse neuroepithelium model,38  autophagy was shown to be essential for the clearance of dying cells by generating engulfment signals, including lysophosphatidylcholine secretion (come-get-me signal) and phosphatidylserine exposure (eat-me signal). During Drosophila metamorphosis, autophagy genes were demonstrated to be required for complete salivary gland cell degradation.14 In the Drosophila ovary, nutrient deprivation signals trigger germarium (region 2A) and mid-oogenesis cell death to remove defective egg chambers before the investment of energy into them,9, 39 and our results showed that nutrient deprivation also triggers autophagy at these two stages. To investigate the role of autophagy during germarium and mid-oogenesis cell death, we analyzed the phenotype of DmAtg1 germline clones and DmAtg7 mutant ovaries. Although chromatin condensation appeared normal, TUNEL staining, an indicator of DNA fragmentation, appeared reduced in germarium cells and degenerating mid-stage egg chambers in DmAtg1 and DmAtg7 mutants. How might autophagy be involved in DNA degradation during Drosophila germarium and mid-stage nurse cell death? DNA degradation is mediated by multiple nucleases40, including cell-autonomous and waste-management nucleases. Most cell autonomous nucleases generate TUNELreactive DNA fragments.41 Autophagy might positively regulate the activity of cellautonomous nucleases and thus be involved directly in the regulation of DNA fragmentation. Of the numerous cell-autonomous nucleases identified so far, apoptosisinducing factor (AIF) might be a promising target of autophagy. Following an apoptotic  148  stimulus, AIF is released from the mitochondrial inter membrane space and translocates to the nucleus resulting in DNA fragmentation.42 Recent publications show that defective mitochondria are selectively targeted by autophagy for degradation.43-45 Inhibition of autophagy might prevent the release of AIF from mitochondria resulting in decreased DNA fragmentation.  Alternatively, autophagy could modulate the activity of the  lysosomal nuclease, DNAseII, which generates 5’-hydroxyl and 3-phosphate ends that are unrecognizable substrates for TdT of the TUNEL assay. In this scenario, autophagy might act normally to delay or suppress DNAse II-mediated DNA degradation,41 and thus autophagy inhibition would result in accelerated DNAseII activity and a concomitant decrease in TUNEL positive DNA. Electron microscopy analyses have shown that nurse cell debris is engulfed by surrounding follicle cells in dying mid-stage egg chambers.46 Therefore, the TUNEL negative nurse cell nuclei in the DmAtg1 and DmAtg7 mutants could also be accumulated cell corpse DNA which could not be recognized by engulfing cells because they failed to display engulfment signals. However, in mouse embryoid body and retinal neuroepithelium models, dying cells in Atg gene mutants failed to display engulfment signals but still showed TUNEL staining,37,  38  a result that differs  from our observations in the Drosophila ovary. The possible role(s) of autophagy in DNA degradation, as illustrated in Figure 4.3, remain to be further investigated. It is interesting to note that cell-autonomous DNA degradation is not essential for cell death but appears to affect the efficiency or extent of death at least in some systems. In contrast, removal of dead cell debris can be required for sustained organism survival.40 Thus, we propose that at least one of the functions of autophagy in the Drosophila ovary is to  149  enhance or suppress the efficiency of cell degradation and/or promote corpse clearance associated with cell death.  4.2 Current limitations and summary of future research directions Our RNAi screen identified nine cell death related genes that are required for starvation-induced autophagy. Additional cell death genes might function in autophagy regulation but were either not tested in our screen or not detected because of insufficient knockdown by RNAi, a long half-life of the corresponding proteins, and/or functional redundancy. Future validation of the remaining identified genes in relation to autophagy during Drosophila oogenesis or other in vivo systems will be valuable in the future. In addition, epistasis analyses of these genes can be achieved either by RNAi or Drosophila genetic approaches and can be used to establish the pathway of starvation-induced autophagy in Drosophila. The role of autophagy in oogenesis cell death, especially DNA degradation, remains an outstanding question to be investigated. Our data suggest that discrepancies exist for Bruce and Dcp-1 mediated autophagic responses between larval tissues (fat body and midgut) and the ovary. Increased numbers of animals are required to validate the results observed in larval fat body and midgut. Further, immunostaining analyses of PI3K or Akt expression levels may provide insights to resolve the discrepancies. Our initial IP-MS studies provide a preliminary list of potential Dcp-1 substrates that may mediate autophagy and this data will be strengthened by performing biological replicates. A weakness of our approach is that unlike other methods that directly analyze cleavage events of caspase, our method utilized a binding approach that is indirect. Thus, our approach could not distinguish the actual substrates of Dcp-1 from interacting proteins that bind to Dcp-1. An in vitro cleavage assay need to be employed  150  to carefully examine whether identified proteins are cleaved by Dcp-1. The most important next step will be to verify and determine if these putative Dcp-1 substrates have any functional significance in mediating the execution of autophagy, and our RNAi screen is one method that can be employed to test for their involvement in autophagy.  4.3 Potential applications of the research findings Mammalian homologues of Drosophila Bruce, Hid and Dcp-1 are Apollon, Smac and Caspase-3, respectively. It remains to be tested whether the autophagy regulating functions of Bruce, Hid and Dcp-1 are conserved in these mammalian counterparts. Interestingly, overexpression of Apollon can suppress apoptosis, and recent evidence suggests that this occurs indirectly via p53.47 As noted above, Apollon can also ubiquitinate the pro-apoptotic protein Smac, as well as Caspase-9.7 Smac/DIABLO is released from the mitochondria to antagonize IAPs, namely XIAP, cIAP-1 and -2, survivin and Apollon.8 In this way, Smac promotes the activation of Caspase-3 and is pro-apoptotic. Perhaps it has a similar pro-autophagy mode of action. Low levels of Smac48 and Caspase-349 have been associated with chemotherapy resistance, and based on our model in Figure 4.1, low level activation would be consistent with induction of autophagy. While genetic studies showed that autophagy may act as a tumor suppressor mechanism,50, 51 it has been demonstrated also that autophagy can play a protective role during chemotherapy and radiation treatment.52-56 Since Smac mimetics and suppression of IAP proteins are under active investigation as anti-cancer treatments,57 it may be worthwhile to investigate a therapeutic strategy that combines Smac mimetics with autophagy inhibition. If Smac, Apollon and Caspase-3 do function in autophagy regulation, it will be important to understand their effects in both normal and cancer cells.  151  And given the complexity of apoptotic signaling pathways, it is likely that additional cell death-related genes with a link to autophagy will be discovered.  152  Figure 4.1 A hypothetical pathway for the regulation of sensitivity thresholds leading to autophagy or apoptosis. Based on the known apoptosis-related interactions of the gene products identified in our study, we propose a putative pathway involved in the regulation of starvationinduced autophagy in Drosophila. In this model, the effector caspase Dcp-1 plays a key role in defining the balance between autophagic and apoptotic responses. Starvation facilitates activation of Dcp-1 that leads to induction of autophagy resulting in cell survival or cell death depending on cellular context. Dcp-1 activation perhaps leads to subsequent cleavage of drICE and eventually cell death.  153  Figure 4.1  154  Figure 4.2 The effector caspase Dcp-1 is sufficient for the induction of autophagy during Drosophila oogenesis. Expression of activated Dcp-1  in the germline (UASp-GFP-LC3/+; nanos-  GAL4/nanos-GAL4 UASp-tDcp-1)58 resulted in nurse cell death during mid-oogenesis (arrow) and dying nurse cells had numerous GFP-LC3 puncta (green). DAPI staining of nuclei is shown in white. Scale Bar: 50μm  155  Figure 4.2  156  Figure 4.3 Possible relationships between autophagy and DNA degradation in Drosophila oogenesis This diagram depicts how autophagy might play a role in the DNA degradation process based on the reduced TUNEL staining phenotype observed in dying mid-stage egg chambers of DmAtg1 and DmAtg7 mutants. Autophagy could positively regulate the activities or sub-cellular localization of cell-autonomous nucleases (that generate TUNEL-reactive fragments) and thereby enhance DNA degradation. Alternatively, autophagy may negatively regulate the activity of lysosomal nuclease DNAseII (that generates TUNEL non-reactive DNA ends), and thereby suppress or delay DNAse-II mediated DNA degradation . Finally, autophagy could sustain the high ATP levels that are required for display of engulfment signals, including lysophosphatidylcholine (comeget-me) and/or phosphatidylserine (eat-me).  157  Figure 4.3  158  4.4 References 1.  Scott RC, Schuldiner O, Neufeld TP. 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Dev Biol 2003; 260:113-23.  165  Appendices Appendix A: abbreviations list CT, cycle threshold dsRNA, double stranded ribonucleic acid l(2)mbn, lethal (2) malignant blood neoplasm LTG, lysotracker green LTR, lysotracker red IP, immuno-affinity purification MS, mass spectrometry PBS, phosphate buffer saline QRT-PCR, quantitative reverse transcription PCR UAS, Upstream Activating Sequence CMA, Chaperone-mediated autophagy ER, endoplasmic reticulum CARD caspase recruitment domains DED death effector domains MEFs, mouse embryonic fibroblasts zVAD, pan caspase inhibitor ROS, reactive oxygen species TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling FBS, fetal bovine serum 3MA, 3 methyladenine Baf, Bafilomycin  166  PI, propidium iodide DAPI, 4',6-diamidino-2-phenylindole BIR, baculoviral IAP repeat GLCs, germline clones IC, individualization complex ERTs, endoreplicating tissues UBC, ubiquitin-conjugating TEM, transmission electron microscopy Atg, autophagy-related AIF, Apoptosis-inducing factor TRAIL, TNF-related apoptosis-inducing ligand FADD, Fas-associated protein with death domain DAPK, death-associated protein kinase TOR, target of rapamycin Nc, Dronc (initiator caspase) Dcp-1, death caspase-1 (effector caspase) Ice, drICE (effector caspase) rpr, Reaper (core cell death effector) Skl, Sickle (core cell death effector) DIAP1 (th), Drosophila inhibitor of apoptosis protein-1 Raf (phl), MAP kinase kinase kinase MAPK (rl), (mitogen activated protein kinase) SMAC, Second mitochondria-derived activator of caspase  167  XIAP, X-linked inhibitor of apoptosis cIAP-1 (BIR2), Baculoviral IAP repeat-containing protein 2 cIAP2 (BIR3), Baculoviral IAP repeat-containing protein 3 DFCP1, double FYVE domain containing protein Lamp2a, lysosome associated membrane protein type 2a Class III PI3K, Class III phosphatidylinositol 3 kinase PI3P, phosphatidylinositol 3-phosphate UVRAG, Ultroviolet irradiation resistance associated tumour suppressor gene PE, phosphatidylethanolamine MAP1LC3B or LC3, microtubule associated protein 1 light chain 3B  168  

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