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Characterization of Lamp1 in Drosophila Melanogaster

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Characterization of Lamp1 in Drosophila Melanogaster Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2019 Characterization of Lamp1 in Drosophila melanogaster Norin Yasin Chaudhry Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry Commons, and the Molecular Biology Commons Recommended Citation Chaudhry, Norin Yasin, "Characterization of Lamp1 in Drosophila melanogaster" (2019). Graduate Theses and Dissertations. 17422. https://lib.dr.iastate.edu/etd/17422 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Characterization of Lamp1 in Drosophila melanogaster by Norin Yasin Chaudhry A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Molecular, Cellular and Developmental Biology Program of Study Committee: Gustavo MacIntosh, Major Professor Linda Ambrosio Hua Bai The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this thesis. The Graduate College will ensure this thesis is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2019 Copyright © Norin Yasin Chaudhry, 2019. All rights reserved. ii TABLE OF CONTENTS Page LIST OF FIGURES iii LIST OF TABLES iiv ACKNOWLEDGMENTS v ABSTRACT vii CHAPTER 1. GENERAL INTRODUCTION 1 Cellular Degradation Machinery 1 The Family of Lysosome Associated Membrane Proteins (LAMPs) in Eukaryotes 2 Structural Characterization of LAMPs 3 LAMPs and Autophagy 5 Other Biological Functions for LAMP Proteins 14 Summary 18 References 19 CHAPTER 2. CHARACTERIZATION OF LYSOSOME-ASSOCIATED MEMBRANE PROTEIN, LAMP-1, IN DROSOPHILA MELANOGASTER 29 Abstract 29 Introduction 29 Materials and Methods 32 Results 35 Discussion 52 Conclusion 58 References 59 CHAPTER 3. GENERAL CONCLUSION 68 Conclusion 68 Future Directions 71 References 73 APPENDIX. LIST OF PRIMERS AND DATABASES USED IN THE STUDY 82 iii LIST OF FIGURES Page Figure 1. (A) 3D representation of mouse LAMP-2 (PDB ID: 5GV3) depicting the secondary structure, transmembrane helix and disulphide bridges of the protein. .................................................................................................................4 Figure 2. (previous page) Initiation of phagophore formation involving 3 multiprotein complexes: ULK1 complex, Vps34 complex and Atg16/Atg5/Atg12 complex. ...............................................................................................................9 Figure 3: Total RNA extracted from 3rd IL of Lamp1e00879 and w1118 was subjected to RT-PCR using Lamp-1 primers. ......................................................................... 35 Figure 4: The line across the body of the female and male flies indicates how the body measurements were calculated. ........................................................................... 36 Figure 5: Representative data from the larval crawling assay using Lamp1e00879 and w1118 3rd instar larvae at room conditions. ......................................................... 37 Figure 6: (A) Phase-contrast microscopy was conducted on the fat-bodies of 3rd instar larvae.................................................................................................................. 39 Figure 7: (A) Phase-contrast microscopy was conducted on the fat-bodies of 3rd instar larvae.................................................................................................................. 40 Figure 8: (A) Phase-contrast microscopy was conducted on the fat-bodies of 3rd instar larvae.................................................................................................................. 41 Figure 9: (A) Confocal microscopy was used to observe the LTR-positive puncta in the fat-bodies of adult flies ....................................................................................... 43 Figure 10: (A) Electron microscopy of fat body cells of Lamp1e00879 and w1118 3rd IL .......... 45 Figure 11: (A) Electron microscopy of fat body cells of Lamp1e00879 and w1118 adult flies .................................................................................................................... 46 Figure 12: Hierarchical arrangement of organisms in the tree of life. ..................................... 48 Figure 13: The consensus sequence for the putative domain of Lamp-1 protein [(A) & (B)] and the transmembrane helix [(C)] based on sequence alignment data from the listed organisms. ................................................................................... 50 iv LIST OF TABLES Table A1: List of primers used. 81 Table A2: Databases used for phylogenetic analysis 81 v ACKNOWLEDGMENTS I would like to thank my research advisor, Gustavo MacIntosh for his constant guidance and insight about my research as well as professional goals. He has been a constant source of support and taught me how to develop better research questions and research methodologies. I would also like to extend gratitude to my Program of Study Committee (POSC) members, Dr. Linda Ambrosio and Dr. Hua Bai for their invaluable input and constant feedback on my research. I am grateful to Dr. Ambrosio for training me how to work with flies and be organized in my lab work. I am indebted to Dr. Bai for keeping all his microscopy and molecular biology equipment and resources at our disposal and all his lab members, especially Ping Kang and Mark Bouska, for training me on how to work with the technology and optimizing my results. I would not have been able to achieve without both of their guidance. I am also thankful to all the lab members of the MacIntosh lab including Ang-Yu Li, fellow graduate student, for his constant insight and inquisitiveness about my results that enabled me to read more and learn more every day. I would also like to acknowledge Adem Selimovic for being a hard-working undergraduate researcher in our lab and helping in conducting molecular biology experiments and being a constant source of support as well as asking hard questions to test both of our knowledge and understanding of our research. I am also grateful to Nyamal Diew for working on Drosophila behavioral analysis with me. I would also like to acknowledge the support and love of my entire family from thousands of miles away, for extending support and motivation to keep working hard and achieve my goals. I am also grateful to the Fulbright Foreign Students Program funded by the United States Department of State, Bureau of Education and Cultural Affairs for providing me vi with the opportunity to pursue my Master’s degree at Iowa State University and enabling me to play a part in cultural exchange between United States and Pakistan with pride. vii ABSTRACT Lysosome Associated Membrane Proteins (LAMPs) are the most abundant proteins in the lysosomal membrane and are important for maintaining the structural integrity of the lysosomes and separating the hydrolytic enzymes inside the lysosomal lumen from the cell cytoplasm. Additional functions of Lamp proteins are continued to be discovered and they have been recently characterized for their involvement in autophagy in different mammalian systems. LAMP-1 and LAMP-2 are ubiquitously expressed in mammals. LAMP-2 has 3 splice variants and mutations in LAMP-2B have been shown to cause Danon disease in humans, characterized by the accumulation of autophagic vacuoles in the heart and skeletal muscle. In mice, the knockdown of both LAMP-1 and LAMP-2 leads to embryonic lethality with similar accumulation of autophagic vacuoles in their heart and skeletal muscle tissues. In Drosophila melanogaster, there is only 1 ortholog of LAMP proteins, Lamp-1, that has been solely used as a lysosomal marker with its basic function remaining unknown. We hypothesize that Drosophila can be a good model to study lysosomal defects and Danon disease. Our lab had previously characterized a loss of function (LOF) mutant for Lamp-1 and unlike the deletion of LAMP-1/LAMP-2 in vertebrate, the LOF flies are viable and fertile with no morphological abnormalities. However, the analysis of their cellular phenotype showed accumulation of Lysotracker- red (LTR) positive puncta suggesting an increase in the accumulation or decreased degradation of autophagic vacuoles (autophagosomes, lysosomes or autophagolysosomes) in fat body tissues. In preliminary evaluation of larval crawling ability of the both wild-type and mutant 3rd instar larvae, we found that the mutant larvae also present with decreased crawling ability. This behavioral phenotype together with the observed cellular viii phenotype suggest important functions for Lamp-1, but more research is needed to obtain a deeper understanding of the role of Lamp-1 in the lysosomes of Drosophila melanogaster. 1 CHAPTER 1. GENERAL INTRODUCTION Cellular Degradation Machinery There are two main degradation processes inside the cell, the lysosomal network and the ubiquitin proteasome system. Lysosomes are an essential component of the cells for a multitude of reasons that are continually being discovered. They play crucial roles in the recycling and degradation of endocytic,
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