Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations

2019

Characterization of Lamp1 in Drosophila melanogaster

Norin Yasin Chaudhry Iowa State University

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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.

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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 Associated Membrane (LAMPs) in Eukaryotes 2 Structural Characterization of LAMPs 3 LAMPs and 5 Other Biological Functions for LAMP Proteins 14 Summary 18 References 19

CHAPTER 2. CHARACTERIZATION OF LYSOSOME-ASSOCIATED MEMBRANE , 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

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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

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LIST OF TABLES

Table A1: List of primers used. 81

Table A2: Databases used for phylogenetic analysis 81

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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.

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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 and separating the hydrolytic enzymes inside the lysosomal lumen from the cell . 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 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 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, autophagic and secretory molecules and therefore are responsible for regulating cellular homeostasis (Mullins & Bonifacino, 2001). These organelles are acidic compartments bound by a single membrane, and contain a variety of hydrolases. These enzymes include , peptidases, phosphatases, nucleases, glycosidases, sulfatases, RNases and lipases; with each hydrolase targeting its specific substrate and collectively are capable of degrading all macromolecules (Bainton, 1981; Futai,

Miyata, & Mizuno, 1969; Pisoni, 1996; Saha, Graham, & Schlessinger, 1979). Among the proteases, the best-known enzymes are the family of cathepsins which are distinguished based on the amino acid found in their active site; serine, cysteine or aspartic acid. The lysosomal membrane, functioning to separate the hydrolytic enzymes present inside the lumen of the lysosomes from the rest of the cytoplasm is composed of cholesterol and phospholipids, as well as present in the form of (Bleistein,

Heidrich, & Debuch, 1980). The lysosomal membrane in mammals contains approximately

25 transmembrane proteins that have been identified to date (Lübke, Lobel, & Sleat, 2009;

Sleat, Della Valle, Zheng, Moore, & Lobel, 2008; Sleat, Jadot, & Lobel, 2007).

The hydrolytic enzymes contained within the lysosomes are synthesized in the endoplasmic reticulum and are transported to the lysosomes with the help of mannose-6- phosphate receptors (MPRs) (Jadot, Dubois, Wattiaux-De Contnck, & Wattiaux, 1997). 2

There are two different kinds of MPRs in mammalian cells, cation-independent and cation- dependent, and both are equally important for retaining the lysosomal enzymes inside the cells (Hille-Rehfeld, 1995). These receptors are present in the trans-Golgi network (TGN) where they bind with the hydrolases and transport them in the form of clathrin-coated vesicles to the early or late endosomes (Eskelinen et al., 2002). Early endosomes mature into late endosomes by acquiring lysosomal proteins and acidic pH environment. These endosomes can be distinguished from the lysosomes because the lysosomes lack MPRs. Due to the acidic environment within the lysosomes, the receptors dissociate from the enzymes and recycle back to the TGN, binding with more hydrolytic enzymes and continuing their transport to the endosomes (Eskelinen, Tanaka, & Saftig, 2003; Kornfeld & Mellman, 1989).

Besides the separation of the hydrolytic enzymes, the lysosomal membrane is also important for maintaining the acidic pH of 4.8 of the interior and for the eventual transportation of the degraded products from the lumen of the lysosomes back into the cytoplasm (Carlsson &

Fukuda, 1989; Eskelinen et al., 2002; Lloyd & Forster, 1986; Peters & von Figura, 1994).

The Family of Lysosome Associated Membrane Proteins (LAMPs) in Eukaryotes

The lysosomal membrane contains multiple glycosylated proteins, among which some of the most abundant include the lysosome associated membrane proteins (LAMPs). In humans, there are 5 proteins in the LAMP family; LAMP-1, LAMP-2, LAMP-3 (also referred to as DC-LAMP), BAD-LAMP and macrosialin (CD68); however, in Drosophila melanogaster, there is only one ortholog , Lamp-1. The thickness of the dense coat formed on the inside of the lysosomal membrane by the heavily glycosylated luminal domains of the integral lysosomal membrane proteins, including but not limited to LAMPs, ranges from 5 to 12 nm (Johnson, Warner, Yancey, & Rothblat, 1996; Neiss, 1984). All the 3 lysosomal membrane proteins contain sorting signals within their cytosolic domains (Huotari

& Helenius, 2011).

LAMP-1 and LAMP-2 are the major components of the lysosomal membrane and constitute 0.1% to 0.2% of the total cell protein and are extensively used as markers for lysosomes (Chen, Murphy, Willingham, Pastan, & August, 1985). These two proteins have been found to be evolutionary related while also being structurally similar (Eskelinen et al.,

2002). They are ubiquitously expressed in human tissues and cell types, with increased expression in metabolically active cells (Aumüller, Renneberg, & Hasilik, 1997; Konecki,

Foetisch, Zimmer, Schlotter, & Konecki, 1995).

Structural Characterization of LAMPs

LAMP proteins are characterized as type I transmembrane proteins as they consist of a largely conserved and heavily N-glycosylated luminal domain, O-glycosylated and proline- rich hinge region, a transmembrane domain and a short 11-amino acid long C-terminal cytoplasmic tail that contains lysosomal/endosomal targeting signature (Winchester, 2001)

(Figure 1). Although the luminal domain and transmembrane domains are largely conserved between both LAMP-1 and LAMP-2 (Hunziker & Geuze, 1996), their cytoplasmic tail is more variable. LAMP-2 has 3 splice variants in humans, LAMP-2A, LAMP-2B and LAMP-

2C, and the differences in their cytoplasmic tail can distinguish between the three isoforms and their involvement in different cellular pathways (Carlsson & Fukuda, 1989; Gough &

Fambrough, 1997). All splice variants are involved in some of the same processes including antigen processing, cholesterol trafficking, lysosome biogenesis and phagocytosis (Eskelinen et al., 2002; Huynh et al., 2007; Schneede et al., 2011). However, the subtle difference in their cytosolic tail also enables them to take on unique roles. For example, LAMP-2A is involved in chaperon-mediated autophagy (CMA), LAMP-2B is involved in stabilizing the 4 lysosomal polypeptide transporter, Transporter Associated with antigen Processing Like

(TAPL), within the lysosomal membrane and LAMP-2C is being recognized to play a crucial role in RNA/DNAautophagy (Ana Maria Cuervo & Dice, 1996; Demirel et al., 2012;

Fujiwara, Furuta, et al., 2013; Fujiwara, Kikuchi, et al., 2013; Hubert et al., 2016).

Figure 1. (A) 3D representation of mouse LAMP-2 (PDB ID: 5GV3) depicting the secondary structure, transmembrane helix and disulphide bridges of the protein. The second 3D image is rotated by approximately 45 degrees. (B) The protein structure is composed of two internal homologous lysosome-luminal domains separated by a proline-rich hinge region. The C-terminal extremity is spanned by a transmembrane helix, followed by a short cytoplasmic tail.

Amidst their structural similarity, LAMP-1 and LAMP-2 only share 37% amino acid and are located on different , representative of their early 5 evolutionary divergence (Eskelinen, 2006). As mentioned earlier, the luminal region of the proteins consist of two N-glycosylated LAMP domains, separated by a proline-rich and O– glycosylated hinge region and two bridges contained in each luminal domain in mammals (Carlsson & Fukuda, 1989). The N-linked have been suggested to play a vital role towards protecting the LAMP proteins against degradation during their synthesis

(Kundra & Kornfeld, 1999). The luminal domain directly adjacent to the transmembrane helix, commonly referred to as the ‘LAMP domain’, is conserved amongst all 5 human

LAMPs (Wilke, Krausze, & Büssow, 2012). Furthermore, the solved crystal structure for the

DC-LAMP (LAMP-3) also shows that the conserved disulphide bridges give the domain a distinct spatial alignment conducive to its functionality and allow forit to have multiple molecular interactions with neighboring membrane proteins. The first disulphide bridge stabilizes the β -prism fold by linking the two β-sheets formed by the domain and the second disulphide bridge connects the C-terminal transmembrane helix to the center of the front β- sheet. The two domains are connected by a proline-rich linker region, ‘hinge’, that in human

LAMP-1 consists of 23 residues including 11 prolines and 6 O- sites (Wilke et al., 2012). The C-terminal tail of LAMP-1 and LAMP-2 contains a specific lysosomal targeting motif that is has been determined to be a tyrosine residue, preceded by glycine and four amino acids away from a hydrophobic C-terminal residue (Tabuchi, Akasaki, & Tsuji,

2000).

LAMPs and Autophagy

In mammals, there are three types of autophagic mechanisms, macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). LAMP proteins are an integral part of the autophagy machinery inside the cells and all digestive bodies formed through autophagy eventually fuse with the lysosomes for final degradation. In mammals, the process 6 of autophagy is nutrient-dependent and therefore the level of amino acids and insulin primarily regulate the main regulator of nutrient signaling, mTOR, that in turn mediates autophagy (Mizushima, 2007). In Drosophila melanogaster, the PI3K/Akt/p70S6K signaling is conserved and the homolog, dTOR, is equally crucial for the normal organismal cell growth. Fruit fly’s larvae are composed of endoreplicating tissues (ERTs) including fat body that is most sensitive to the nutrients and levels of amino acids. And thus, the regulation of dTOR signaling is also nutrient-dependent as in mammals (Bradley & Leevers, 2003).

The process of autophagy begins at the endoplasmic reticulum with the formation of phagophores (open-ended ) that either engulf cytoplasmic cargo and organelles or incorporate them through a cargo-specific loading process (Figure 2). The initiation of phagophore formation is regulated through 3 multiprotein complexes including ULK1 complex (ULK1, Atg13, Atg101 and FIP200), the Vacuolar protein sorting (Vps) 34 complex (Beclin1, Atg14L and Vps34) and the Atg16/Atg5/Atg12 complex. Following initiation, Atg3 and LC3 (Atg8) are recruited for development of the phagophore. After the membrane of the phagophore closes, producing a vesicle with a double membrane, LC3 persists and the phagophore matures into autophagosomes (Nakatogawa, Ichimura, &

Ohsumi, 2007). At this time, all the Atg proteins and the initiation multiprotein complexes dissociate from the mature autophagosomes (Krämer, 2013). These mature autophagosomes next fuse to the lysosomes because of protein interactions on their surfaces. Formation of autolysosomes requires Rab7 effector Oxysterol-binding Protein Homologue (ORP1L) and

Rab Interacting Lysosomal Protein (RILP) present on the lysosome, to bind to the homotypic fusion and protein sorting (HOPS) complex, consisting of 6 Vps subunits (Ganley, Wong,

Gammoh, & Jiang, 2011; Jäger et al., 2004; Johansson, 2003; Kimura, Noda, & Yoshimori, 7

2007). These associations cause the autophagosomes to move along the microtubules by recruiting dynein-dynactin motor proteins. HOPS complex attaches the autophagosomes to the lysosomes by cross-linking Rab7 on the lysosome to Qa-SNARE syntaxin 17 (STX17) on autophagosomes to R-SNARE VAMP8 on the lysosomal membrane in the presence of Qbc-

SNARE SNAP-29 (that can be present in the form of a SNAP-29/Stx6 SNARE complex), forming a tetrameric trans-SNARE complex (Ganley et al., 2011; Itakura, Kishi-Itakura, &

Mizushima, 2012; Jiang et al., 2014; kleine Balderhaar & Ungermann, 2013; Wong et al.,

1999). This cross-linking is mediated by the dimeric form of Atg14 that essentially stabilizes the STX17/SNAP-29 complex (Diao et al., 2015). STX17 is believed to be first recruited to the outer membrane of the autophagosomes and it recruits SNAP-29 from there, followed by the binding to VAMP8 which is present on the lysosomal membrane (Hegedűs, Takáts,

Kovács, & Juhász, 2013). After the autophagosomes have fused with the lysosomes, the cis-

SNARE complex then disassembles from the membrane with the help from NSF (N- ethylmaleimide-sensitive factor) and NAPA (NSF attachment protein, α, also known as α-

SNAP) (Hong, 2005).

In the deficiency of LAMP-2, the expression of STX17 on the autophagosomes is drastically reduced, suggesting that LAMP-2 is integral at this step of autophagolysosome biogenesis; although the mechanism is unclear, increasing evidence suggests its importance resides with correct translocation of STX17 to autophagosomes (Hubert et al., 2016). The rescue with LAMP-2A partially corrects for the loss of STX17 expression, establishing a functional relationship between STX17 and LAMP-2A (Hubert et al., 2016). LAMP2 deficiency can also have implications on the degradations of long-lived proteins including inhibitors of T-cell receptor signaling that can therefore be downregulated in the absence of 8 the splice variant LAMP-2A (Valdor et al., 2014). Cholesterol export can also be disrupted, which can lead to the lowering in the concentration of cholesterol in the lysosomes and the endoplasmic reticulum and therefore alter the recruitment pathway for Stx6 (Schneede et al.,

2011). Deficiencies in the recruitment of Stx6 can affect the interaction between SNAP-29 and STX17 that can in turn affect the fusion of autophagosomes with lysosomes. Levels of cholesterol also regulate ORP1L activity and lower levels of cholesterol can lead to an endoplasmic reticulum protein, VAP-A, binding to ORP1L that functions to prevent the tethering of autophagosomes with lysosomes (Wijdeven et al., 2016). Therefore, the autophagosomes are unable to mature in the absence of any one of STX17, SNAP-29 or

VAMP8, which all function as part of the same SNARE complex in the deficiency of LAMP-

2 because of its regulation at multiple levels of the process as discussed here. Therefore, research shows the accumulation of double-membrane autophagosomes and impaired fusion with lysosomes in the deficiency of either of these proteins (Takáts et al., 2013).

Therefore, LAMP-2 has been recognized as an important part of the autophagy machinery with each of its splice variants either characterized for their unique roles or are still understudied. Among the three splice variants of LAMP-2, LAMP-2A, LAMP-2B and

LAMP-2C, the luminal domains are largely conserved, however, the cytosolic tails are different and determine their specificity in autophagy.

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Figure 2. (previous page) Initiation of phagophore formation involving 3 multiprotein complexes: ULK1 complex, Vps34 complex and Atg16/Atg5/Atg12 complex. After the formation and elongation of phagophore, it matures into autophagosomes which then fuses with lysosomes to form autophagolysosomes (see text for details).

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LAMP-2A and Chaperone-Mediated Autophagy (CMA)

LAMP-2A is an integral component of chaperone-mediated autophagy (CMA) and

LAMP-2A’s downregulation is characteristic of aging cells and tissues (Ana Maria Cuervo

& Dice, 2000b; Dice, 1982). This form of autophagy differs from other modes of autophagy both in how the proteins are specifically targeted and how these proteins are translocated to the lysosomal lumen for degradation by the hydrolases (Ana Maria Cuervo, 2010; Dice,

2007).

All substrate proteins directed towards CMA for degradation contain a Lys-Phe-Glu-

Arg-Gln (KFERQ)-like motif in their amino acid sequence that is recognized by a chaperone protein, the heat shock cognate protein of 70 kDa (Hsc70) that then transports the cargo protein to the lysosomal surface in the presence of its co-chaperones, the Bcl2-associated athanogene 1 protein (Bag1), the Hsp70-interacting protein (Hip), the Hsp70-Hsp90 organizing protein (Hop) and Hsp40 (Chiang, Plant, & Dice, 1989; Dice, 1990).

Approximately 30% of the total cytosolic proteins are substrates for CMA as they contain the necessary KFERQ-like motif in their sequence as studied by protein sequence analysis

(Wing, Chiang, Goldberg, & Dice, 1991). Before it can be translocated across the lysosomal membrane, the substrate protein is unfolded and the substrate protein-chaperone complex next interacts with the free cytosolic tail of LAMP-2A that functions as a receptor of CMA and helps to dock the complex onto the lysosomal membrane (Ana Maria Cuervo & Dice,

1996; Salvador, Aguado, Horst, & Knecht, 2000). LAMP-2A has been shown to exist as a trimer (three transmembrane α-helices bound tightly to form a coiled coil trimer) across the lysosomal membrane in the DPC micelles using NMR spectroscopy (Rout, Strub, Piszczek,

& Tjandra, 2014). A previous study was also able to show that in the membrane of the lysosomes isolated from rat livers, the translocation of the substrate across the membrane 11 does require a higher order LAMP-2A oligomer complex of 700 kDA to form

(Bandyopadhyay, Kaushik, Varticovski, & Cuervo, 2008). Moreover, the substrate protein and chaperones have been shown to bind through a bimodal or simultaneous interaction where the binding of the substrate and its targeting to the lysosomal membrane are coupled

(Rout et al., 2014). It had been previously speculated that the binding of this complex to the cytosolic tail of LAMP-2A induces a conformational change to assist in the translocation of substrate protein inside the lysosomal lumen by the translocation complex composed of the luminal form of Hsc70. However it has been shown through biophysical analysis that the simultaneous binding of the chaperone and the substrate protein with the free cytosolic tail of

LAMP-2A does not induce any conformational change in its transmembrane helix or the luminal domain (Agarraberes & Dice, 2001; Agarraberes, Terlecky, & Dice, 1997; Ana

Maria Cuervo, Dice, & Knecht, 1997; Rout et al., 2014). Therefore, the formation of the oligomeric form of LAMP-2A and successful translocation of the substrate protein seems to be independent of its own structural features (Rout et al., 2014). More research is required to fully understand the structural arrangement of LAMP-2A in the lysosomal membrane and their role in cargo translocation. After the translocation of the substrate protein, the substrate protein is degraded by the hydrolases present in the lysosome (Agarraberes et al., 1997; Ana

Maria Cuervo et al., 1997), however, it remains to be concluded how LAMP-2A assists the heat shock family of 70 kDa (Hsc70) present on the inside of the lysosome for subjecting the substrates to the proteolytic pathway .

The expression levels of LAMP-2A directly impact the CMA activity, as the binding of the substrate protein to its cytosolic tail is a limiting step (Ana Maria Cuervo & Dice,

2000b). Changes in its expression level does not require de novo synthesis or transcriptional 12 induction (except under specific conditions e.g. oxidative stress). LAMP-2A degradation at the membrane of the lysosome is controlled by the cleavage activity of cathepsin A and an unknown metalloprotease. The cleaved LAMP-2A is degraded like other proteins in the lysosomal lumen after it is released from the membrane (Ana Maria Cuervo & Dice, 2000a;

Ana Maria Cuervo, Mann, Bonten, d’Azzo, & Dice, 2003). The rate of degradation decreases in response to activation or upregulation of CMA.

LAMP-2C and RN/DNautophagy

LAMP-2C has been shown to interact with RNA and possibly assist its degradation inside the lysosomes through a new pathway called RNautophagy (Aizawa et al., 2016).

Lysosomes have been known to harbor acidic RNases for the degradation and recycling RNA inside the cell to contribute to overall cellular homeostasis (Campomenosi et al., 2006; Futai et al., 1969; Haud et al., 2011; Pisoni, 1996; Saha et al., 1979). The cytosolic sequence of

LAMP-2C interacts with SIDT2, the ortholog of Caenorrhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), that localizes to the lysosomal membrane and translocates mRNAs and rRNAs across the membrane in an ATP-dependent manner in vitro. It remains to be investigated if SIDT2 can also transport hairpin RNA containing double and single-stranded regions and dsRNA as its homolog SID-1 (Aizawa et al., 2016). The knockout of Sidt2 leads to corresponding reduction of total RNA degradation in wild-type MEFs. Once inside the lysosomes, RNA is effectively degraded by the acidic

RNases present inside. Moreover, the overexpression of LAMP-2C alone contributes to the increase in RNautophagy, suggesting its unique role in autophagy. The LAMP-2 KO mice showed great reduction in the level of RNautophagy and RNA accumulated inside their brain tissues. The mechanism by which SIDT2 translocates RNA remains unknown. However

Aizawa et al., (2016) suggests that it could be either that the SIDT2 functions as an RNA 13 transporter/channel or that it causes the lysosomal membrane to deform to ingest RNA in a microautophagy-like process (Aizawa et al., 2016). The N-terminus of SIDT2 extends into the lysosomal lumen with the rest of 9 predicted transmembrane domains present in the lysosomal membrane. The C-terminus in the cytosol has been shown to form a tetrameric structure that is stable, however it remains to be seen how it interacts with RNA (Aizawa et al., 2016). Moreover, SIDT2 has been reported to be a hydrolase, with conservation of its serine residue in position 564 amongst all the member proteins of transmembrane hydrolase superfamily and could have important implications for the role of the transmembrane protein in the translocation of RNA across the membrane (Pei, Millay, Olson, & Grishin, 2011). The specificity of LAMP-2C to bind with RNA and DNA substrates has been shown to be specific for sequences that have more than 3 nucleotides of the type poly-G/dG. The sequences that are poly-A/dA, poly-C/dC, poly-T/dT or poly-U/dU as in RNA’s case were not bound by LAMP-2C and were not degraded inside the lysosomes as poly-G/dG sequences were. The sequences with 3 or less than 3 G nucleotides were also not sufficient for binding with LAMP-2C (Hase et al., 2015). Moreover, the RNA/DNA binding with the cytosolic domain of LAMP-2C has been shown to take place because of a unique arginine- rich motif (ARM) and conserved lysine residues (Fujiwara, Hase, Wada, & Kabuta, 2015).

Any substitution in either the ARM or the lysine residue was able to abolish the binding of

RNA or DNA with LAMP-2C. A glutamine residue in the tail may also assist in binding with

DNA as its substitution leads to reduced binding of the cytosolic tail to purified DNA

(Fujiwara et al., 2015). The cytosolic sequence of Drosophila’s Lamp-1 shows strongest homology with human LAMP-2C suggesting that it may also be part of a similar

RNautophagy pathway in the fly system that still needs to be investigated. 14

Other Biological Functions for LAMP Proteins

Much of the earlier research on lysosome associated membrane proteins revolved on the basic idea that their main purpose was to maintain the integrity of the lysosomal membrane and separate the hydrolytic enzymes presents in its lumen from rest of the cytoplasm. However, with time and greater research being conducted, their roles evolved from just being a protective barrier to a role mediating cellular homeostasis through autophagy and other associated processes.

Major part of our knowledge about the biological function of LAMP proteins is derived from the studies conducted on mice models with targeted disruptions in LAMP-1 and

LAMP-2. Mice deficient in LAMP-1 are viable and present mild in the brain

(mild astrogliosis in the dorsal cortex) with all the other tissues being normal. They also show an upregulation of LAMP-2 expression in the kidney, spleen and heart tissues, possibly because of a compensatory mechanism (Andrejewski et al., 1999). However, the deletion of

LAMP-2 causes a more severe phenotype with half of the mice dying between three and six weeks of birth (Tanaka et al., 2000), accumulation of large autophagic vacuoles in multiple tissues including skeletal muscle and heart, enlarged heart, and severe reduction of the contractile function of the papillary muscles, as compared to control mice (Stypmann et al.,

2006; Tanaka et al., 2000). With age, the accumulation of autophagosomes becomes more pronounced in both the heart and skeletal muscle tissues of the mice that do survive (Tanaka et al., 2000). Moreover, LAMP-1 does not compensate for the deficiency of LAMP-2 suggesting that there are specific and different functions that the two similar proteins are responsible for. Their expressions are also different in human tissues with LAMP-2 being more abundant in the brain tissue than LAMP-1 (Furuta, Yang, Chen, Hamilton, & August,

1999). On the other hand, the LAMP-1/LAMP-2 double deficiency is embryonic lethal in 15 mice, and the microscopic evaluation of the organs in fetuses shows a higher accumulation of cytoplasmic vacuoles. The number of autophagic vacuoles was also found to be higher in double deficient fibroblast cell lines derived from LAMP-double deficient embryos

(Eskelinen, 2006). In humans, the LAMP-2 deficiency causes an X-linked vacuolar cardiomyopathy and myopathy known as Danon disease (DD) that predominantly shows severe symptoms in males, with affected females presenting milder cardiac symptoms later in their lives (Saftig, Von Figura, Tanaka, & Lüllmann-Rauch, 2001). The fusion of late endosomes/lysosomes with autophagosomes was found to be impaired in LAMP-double deficient fibroblasts (González-Polo et al., 2005) as well as the recruitment of small GTPase

Rab7 to the autophagosomes for the formation of autolysosomes (Jäger et al., 2004). These studies set the basis to all the future studies to prove that lysosomal membrane proteins,

LAMP-1 and LAMP-2, were involved in more important process than just maintaining the structural integrity of the lysosomal membrane and could be important for stabilizing the autophagosomal pathway.

Recent research has also shown that LAMPs are important for the functionality of exocrine pancreas. The induction of experimental pancreatitis leads to marked decrease in the expression level of LAMP-1 and LAMP-2 (Mareninova et al., 2015). LAMP-2 null mice develop pathologies of pancreatitis characterized by acinar cell vacuolization. It was discovered that these vacuoles are autolysosomes, and their accumulation was confirmed to be due to impairment of autophagic influx. It was also reported that this accumulation is accompanied by an increase in the expression of a protein that is localized to the autophagosomal membrane (microtubule-associated protein 1 chain 3-II [LC3-II]) and a protein that is degraded through autophagy (p62/SQSTM-1) (Fortunato et al., 2009; 16

Gukovskaya & Gukovsky, 2012; Gukovsky, Li, Todoric, Gukovskaya, & Karin, 2013;

Mareninova et al., 2009). The experimental pancreatitis pathologies were also accompanied by trypsinogen activation, -driven inflammation and acinar cell death.

Furthermore, the pancreatitis-induced decreases in the expression levels of LAMP proteins were only observed for LAMP isoforms mediating macroautophagy and not for any other lysosomal membrane proteins, for example, LIMP-2. In vitro analysis showed that the degradation of LAMP-2 is mediated by CatB since the C-terminal region of LAMP-2 has

CatB cleavage sites within the luminal domain of the protein, closer to the transmembrane helix (Mareninova et al., 2015). The results are consistent with the degradation of LAMP-2A isoform by CatA previously shown elsewhere (Ana Maria Cuervo et al., 2003).

Coxiella burnetti is an obligate intracellular bacterial pathogen that induces the formation of specific vacuoles where it can escape host defenses. LAMP-1 and LAMP-2 have been found to be important for causing maturation delay of parasitophorous Coxiella- containing vacuoles (PV) and their fusion with endosomes, lysosomes and autophagic vesicles (Schulze-Luehrmann et al., 2016). The bacteria primarily targets and but has also been found to be infectious to fibroblast or epithelial cell lines

(Maurin, Raoult, Location, & Cycle, 1999; Voth & Heinzen, 2007). After the mammalian cells take up the bacteria, C. burnetti remains inside a membrane-bound vacuole that initially matures in a way similar to phagosomes by undergoing fusion with the endosomes and lysosomes to form a phagolysosomal compartment that has been found to be labelled with

Rab7 (late endosomal marker), vATPase and cathepsin D. LAMP-1/2 are not required for the uptake of C. burnetti, however, they do restrict replicative ability or multiplication of the bacteria measured by the amount of intracellular bacteria present in the KO cell line as 17 compared to the wild-type control at 48 and 72 hours post-infection (when they are fast replicating). The lysosomal membrane proteins instead play an important role in the establishment of the spacious but fragile PVs in wild-type mouse embryonic fibroblasts

(MEF). The change in the replicative ability of the bacteria was not found to be caused by the defect in the acidification or degradative ability of the vacuole in the infected MEFs but instead because of the lack of LAMP-1 and LAMP-2. These MEF cells had significantly smaller PVs in the LAMP-1/2 KO as compared to wild-type MEFs after 24 hours of infection. Therefore, any further maturation of these vacuoles has been discovered to be delayed by the presence of lysosome associated membrane proteins, LAMP-1 and LAMP-2 that do not have to bind with the membrane of the PV to cause this delay. Wild-type cells have spacious PVs with smaller number of bacteria, however, the LAMP-1/2 KO cell lines have smaller but tightly packed PVs indicating their fast maturation process. The ectopic expression of LAMP-1 partially delayed the maturation in KO MEFs, while the ectopic expression of LAMP-2A was sufficient to cause the complete maturation delay of the PV

(Schulze-Luehrmann et al., 2016). Moreover, PVs in wild-type MEFs have reduced fusion with lysosomes, endosomes and phagosomes as compared to LAMP-1/2 KO MEFs which suggests an additional role for the LAMP proteins of controlling the fusion rate of the PV

(Schulze-Luehrmann et al., 2016).

Overexpression of LAMP2 has also been described for certain cells, especially prostate and thyroid (Morell et al., 2016). In epithelium, LAMP-1 and

LAMP-2 have enhanced expression suggesting their involvement in neoplastic progression; however, no direct association between the overexpression and cell proliferation has been shown (Furuta et al., 2001). In neuroendocrine (NE) cells, LAMP-2 has been found to serve 18 as a potential biomarker and target for NE prostate cancer as its overexpression has been found to assist NE differentiation of serum-starved LNCαP cells and facilitates autophagy activity, possibly by increasing the fusion of autophagosomes with lysosomes, to attain NE phenotype and cell survival (Morell et al., 2016). On the other hand, the knockdown of

LAMP-2 hindered the differentiation of NE cells. Previous studies have also conclusively shown the elevated level of LAMP-2A expression and CMA in over 40 different types of human tumors when compared to the expression level in the normal tissue surrounding the tumor (Kon et al., 2011).

In another recent report, ectopic expression of human LAMP-2A has been shown to induce autophagic influx and serve as a neuroprotective agent in Drosophila brain (Issa et al.,

2018). The pan-neuronal expression of LAMP2A led to the development of resistance against the stresses being induced by nutrient starvation, accumulation of SNCA and paraquat exposure that all collectively lead to an increase in the accumulation of reactive oxygen species (ROS) in Drosophila nervous system. Therefore, LAMP2A was able to induce the stimulation of autophagic mechanism in this organism. Furthermore, this protective behavior also lead to improvement in the healthspan of the adult flies, reflected from the improved climbing ability (SING), but had no impact on the lifespan.

Summary

Lysosome associated membrane proteins (LAMPs) had long been believed to have roles in maintaining the integrity of the lysosomal membrane and serve as a barrier to protect the cytoplasm from hydrolytic enzymes present in its lumen. However, continued research on these membrane proteins has been able to suggest their unique roles and functions in maintaining cellular homeostasis by having unique roles in the processes of chaperone mediated autophagy (LAMP-2A) and RN/DNautophagy (LAMP-2C). The sequence analysis 19 of the luminal domain, transmembrane domain and a short cytosolic tail of LAMPs shows these structural features to be conserved in multiple organisms to a large extent. In

Drosophila melanogaster, there is only 1 homolog gene, Lamp-1, and it is closest in homology to human LAMP-2, specifically LAMP-2C in humans with 50% amino acid sequence homology of the cytosolic sequence that has been discovered to interact with DNA and RNA. Due to the availability of advanced genetic tools in Drosophila, it can be an excellent model to dissect the putative function of this protein as it related to autophagy as well as its possible involvement in a similar RNA/DNA autophagy pathway as LAMP-2C.

Therefore, the focus of this study has been to characterize a knockout mutant of

Lamp-1 for its cellular phenotype in the fruit fly system and draw parallels to its known functionality and disease phenotype in human and mouse systems. In mice, the knockout of

Lamp proteins (LAMP-1 and LAMP-2) leads to embryonic lethality and accumulation of autophagic vacuoles in the cardiac and skeletal muscle cells. In humans, the knockout of

Lamp2 leads to Danon disease which is also characterized by similar accumulation of autophagic vacuoles in the brain, cardiac and skeletal muscles tissues. The patients develop cardiomyopathy, myopathy and mental retardation. The establishment of a fruit fly model could therefore advance our understanding of human pathologies associated with LAMP deficiencies. Additionally, we sought to characterize the role of Lamp-1 in the overall autophagic pathway and hence explore the conservation of protein’s function as observed in mammalian systems.

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CHAPTER 2. CHARACTERIZATION OF LYSOSOME-ASSOCIATED MEMBRANE PROTEIN, LAMP-1, IN DROSOPHILA MELANOGASTER

Norin Yasin Chaudhry1, Adem Selimovic1, Linda Ambrosio1 and Gustavo MacIntosh1

1Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State

University, Ames, Iowa, United States of America

Modified from a manuscript to be submitted to Molecular Genetics and Genomics

Abstract

Lysosome Associated Membrane Proteins (LAMPs) have long been believed to have important roles in maintaining the structural integrity of the lysosomes and separating the hydrolytic enzymes inside the lysosomal lumen from the cell cytoplasm only. Recently, however, Lamp proteins have been characterized for their involvement in autophagy in different mammalian systems. Humans have 5 Lamp proteins but only LAMP-1 and LAMP-

2 are ubiquitously expressed. LAMP-2 has 3 splice variants and mutations in LAMP-2B have been shown to cause Danon disease, characterized by the accumulation of autophagic vacuoles in the heart and skeletal muscle and present as cardiomyopathy, myopathy and mental retardation in some patients. 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 predominantly used as a lysosomal marker, but its basic function remains 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 develop normally to the adult stage. Although the mutant flies have no morphological abnormalities, the analysis of their cellular phenotype showed accumulation 29 of Lysotracker- red (LTR) positive puncta and vesicle aggregates present inside enlarged lysosomes that could be attributed to increase in accumulation or decreased degradation of autophagic vacuoles in fat body tissues. The mutant larvae also present with decreased crawling ability suggesting a behavioral phenotype that together with cellular phenotype results require deeper understanding of the putative role of Lamp-1 in the lysosomes of

Drosophila melanogaster.

Introduction

Lysosomes are intracellular organelles in eukaryotes that are responsible for degrading endocytic, autophagic and secretory molecules (Mullins & Bonifacino, 2001).

They contain approximately 60 types of hydrolytic enzymes that target a variety of substrates and are separated from the cellular cytoplasm by a single phospholipid bilayer that is 7 –

10nm thick (Bainton, 1981; Saftig, Schröder, & Blanz, 2010). The lysosomal membrane in mammals contains multiple proteins that are heavily glycosylated and is known to be spanned by at least 25 transmembrane proteins, with lysosome associated membrane proteins

(LAMPs) being the most abundant proteins in the lysosomal membrane (Lübke et al., 2009;

Sleat et al., 2008, 2007).

Humans have five LAMP proteins that are either ubiquitously expressed (LAMP-1 and LAMP-2) or have tissue-specific expression (DC-LAMP, CD68 and macrosialin)

(Aumüller et al., 1997; Konecki et al., 1995). LAMP-1 and LAMP-2 are major components of the lysosomal membrane and have been extensively used as a biomarker for lysosomes

(Chen et al., 1985). They have been characterized as type I transmembrane proteins as they are composed of a luminal domain, a transmembrane helix and a short 11- amino acid long

C-terminal tail present inside the cytoplasm. The luminal domain of the LAMP proteins consists of 2 largely conserved N-glycosylated LAMP domains that are separated by a 30 proline-rich and O-glycosylated hinge region in mammals (Carlsson & Fukuda, 1989). There are two conserved disulphide bridges present in each of the LAMP domains and have been known to give this domain a distinct spatial arrangement important for its functionality. The unique arrangement of the protein allows it to have multiple molecular interactions with neighboring membrane proteins (Wilke et al., 2012). The LAMP domain directly adjacent to the transmembrane helix is conserved across all 5 human LAMPs (Wilke et al., 2012). The cytoplasmic tail of all LAMP proteins contains unique lysosomal/endosomal targeting signature (Winchester, 2001). This unique motif has been found to be a tyrosine residue, preceded by glycine and four amino acids away from a hydrophobic C-terminal residue

(Tabuchi et al., 2000).

LAMP-2 in mammals has 3 splice variants; LAMP-2A, LAMP-2B and LAMP-2C, and each of the three splice variants have unique roles and participate in difference cellular pathways, defined by the variability in their cytoplasmic tail’s amino acid motifs (Carlsson &

Fukuda, 1989; Gough & Fambrough, 1997). All LAMP proteins have been found to be involved in antigen processing, trafficking of cholesterol, biogenesis of lysosomes and phagocytosis (Eskelinen et al., 2002; Huynh et al., 2007; Schneede et al., 2011), however, the subtle differences between their cytoplasmic tails have enabled them to adopt unique roles as well. They have been increasingly found to be associated with autophagy and therefore integral to maintaining cellular homeostasis. LAMP-2A is involved in chaperone mediated autophagy (CMA) and downregulation of its expression is characteristic of aging cells (A M

Cuervo & Dice, 2000; Dice, 1982). LAMP-2B assists in stabilizing the lysosomal polypeptide transporter, Transporter Associated with antigen Processing Like (TAPL) within the lysosomal membrane (Demirel et al., 2012). Mutations in this splice variant leads to 31

Danon disease in humans characterized by cardiomyopathy, myopathy and mental retardation (Nishino et al., 2000). Lastly, the role of LAMP-2C is being investigated in relation to DN/RNautophagy (Fujiwara, Furuta, et al., 2013; Fujiwara, Kikuchi, et al., 2013).

Mus musculus has 2 LAMP proteins. Earlier studies in mice models have found the

Lamp1/Lamp2 double knockout mice to be embryonic lethal and show accumulation of autophagic vacuoles in the cardiac and skeletal muscle cells, like the human patients of

Danon disease. In this study, we used Drosophila melanogaster as our animal model of choice because there is only one ortholog of LAMP-1 and LAMP-2, Lamp1, and its loss of function mutants (Lamp1-KO) are viable and fertile. Therefore, we characterized phenotypic and cellular characteristics for our Lamp1-KO 3rd instar larvae and adult flies to study the putative role of Lamp1 in Drosophila melanogaster.

Materials and Methods

Drosophila melanogaster Stocks

All fly stocks were maintained at 25 °C, 80% humidity and 12-hour light/dark conditions and adults were reared on standard cornmeal media unless otherwise indicated.

The Drosophila melanogaster strain w1118 was obtained from the Bloomington Drosophila

Stock Center at Indiana University (Bloomington#5905 and #3605) and Lamp1e00879

(PBac{RB}Lamp1e00879) was obtained from Exelixis at Harvard Medical School

(https://Drosophila.med.harvard.edu/). w1118 were used as wild-type flies in all experiments.

Age-Synchronized Larval and Adult Flies Collection

For age-synchronized larval collection, 150 females and 50 males were placed in collection bottles (15mm x 60mm Bloomington’s Drosophila Stock Center molasses-agar media plates dusted with Baker’s yeast). Eggs were collected and 43 eggs were transferred to each Bloomington’s Drosophila Stock Center yeast soft-food plate for growing 3rd instar 32 larvae at at 25 °C, 80% humidity and 12-hour light/dark conditions. The 3rd IL crawling on the lids of the petri dishes were collected for all experiments. For adults, flies were left to mate and lay eggs for 24 hours in glass bottles with standard cornmeal media. After 24 hours, the parents were removed, and the bottles were incubated at standard incubation conditions for 10 days until new adults eclosed.

Starvation Treatment

Age-synchronized eggs were collected in the same way as described and the 3rd instar larvae developing from the soft-food plates were placed onto Whatman I filter paper saturated with 1X PBS at 25 °C for 4 hours. After 4 hours, the larvae were either immersed in TRIzol for dispersion using QIAGEN’s Tissue Lyser II and stored at -80 °C for molecular analyses, or were dissected in 1X PBS for fluorescent microscopy. For control, well-fed 3rd instar larvae were also collected from the same set of plates/bottles for all corresponding experiments.

Fat Body Tissue Staining and Fluorescence Microscopy

Larval and adult flies fat body tissues were dissected in 1X PBS, and then incubated for 3 to 5 min in 100 µM Lysotracker- Red DND-99 (Life Technologies, Carlsbad, CA,

USA) for staining lysosomes and autolysosomes, and 1 µM Höechst 33342 (Thermo Fisher

Scientific Inc, Rockford, IL, USA) for staining DNA in PBS as described previously (Scott,

Schuldiner, & Neufeld, 2004). At the end of incubation, fat body tissues were transferred to

PBS on glass slides and immediately imaged using an Olympus BX51WI upright fluorescence microscope.

Quantitative RT-PCR

Total RNA was extracted using TriZol reagent (Thermo Fisher Scientific, Waltham,

MA, USA) from 25~30 synchronously staged larvae or whole adult flies. Extracted RNA 33 was DNase-treated using Turbo DNA-free (Ambion) , and approximately 500 ng total

RNA was reverse transcribed using SuperScript III First Strand (Invitrogen). qPCR was performed in triplicates for each experimental condition with an ABI prism 7300 Sequence

Detection System (Thermo Fischer Scientific, Waltham, MA, USA) using Absolut qPCR with SYBR Green + Rox kit (Thermo Fischer Scientific). mRNA abundance of each gene was compared with the transcript of ribosomal protein L32 gene (RPL32) as the control using

DDCT method (Rao, Huang, Zhou, & Lin, 2013). All experiments were conducted according to the manufacturer’s instructions. The primer sequences used for qPCR are presented in

Table 1 in Appendix A.

Phenotypic Analysis

10 adult females and males were placed in each vial with standard cornmeal media for mating and laying eggs for 3 hours. The parents were then removed, and the vials were incubated at standard conditions for 10 days till the new adults eclosed. The flies were left to mature for 3-4 days before their body and wing lengths were measured. The experiment was conducted in triplicate at both 18°C and 25°C.

Statistical Analysis

GraphPad Prism 8 (GraphPad Software, La Jolla, CA) was used for statistical analysis.

Two-tailed t-test was used for comparing the mean expression of Lamp-1 and Atg5 mRNA normalized against an internal standard control, ribosomal protein L32 (RpL32), in

Lamp1e00879 with that of w1118 in both normal conditions. The same analysis was also used to compare the percentage of fat body cells that contained more than 5 LTR-positive puncta in

3rd IL under normal and starvation conditions. The percentage of cells containing any LTR- 34 positive puncta in adult flies were only compared under normal conditions. The corresponding p values are represented as asterisks; **** (< 0.0001), ** (< 0.001) and * (< 0.05).

Behavioral Assays

In order to assay Drosophila behavior, larval crawling assay (referred to as LCA) and

Rapid Iterative Negative Geotaxis (RING) assay were conducted as described (Nichols, Becnel

& Pandey, 2012).

Database Searches and Sequence Identifications For Protein Sequence Analysis

Identification of Lamp genes was done using BLASTP (Altschul, Gish, Miller, Myers,

& Lipman, 1990) across eukaryotes genomes representing different phylogenetic branches, using Drosophila melanogaster Lamp1 protein as the query. The Lamp proteins used for further analyses are listed in Table 2. The BLAST hits with the lowest Expect value (E) were selected and these sequences were cross-checked using their annotated genome databases

(links are also provided in Table X2). The protein sequences of the 20 identified organisms were then aligned in CLC Sequence Viewer 8 (QIAGEN Bioinformatics). Amino acids were color-coded in CLC using RASMOL biomolecular graphics tool (Sayle & Milner-White,

1995) (https://www.umass.edu/microbio/rasmol/distrib/rasman.htm). The prediction of signal peptides and transmembrane helix were carried out using SignalP (Bendtsen, Nielsen, von

Heijne, & Brunak, 2004) and TMHMM Server v. 2.0 (Server, 2015) respectively.

Results

Previous work from our lab characterized a viable and fertile loss-of-function mutant for Lamp1 (hereon referred to as Lamp1e00879), and showed that Lamp1 is constitutively expressed at all developmental stages of Drosophila (Riaz, 2014). We continued the work on the same mutant to dissect its cellular phenotype as well as behavioral traits that could help to 35 discover a functional relationship of Lamp1 with other participants of autophagy in

Drosophila and a biochemical understanding of Danon disease in humans. We confirmed the lack of Lamp1 through qPCR gene expression analysis in Lamp1e00879 3rd instar larvae and adult flies and the results successfully showed lack of Lamp1 expression in all samples (Figure 3). Therefore, we proceeded to use this mutant for our subsequent experiments.

Figure 3: Total RNA extracted from 3rd IL of Lamp1e00879 and w1118 was subjected to RT- PCR using Lamp-1 primers. No amplification was observed in Lamp1e00879 indicating loss of activity of Lamp-1 in the mutant. Ribosomal protein L32 (RpL32) was used as an internal standard for normalization.

Lamp1e00879 Adult Flies Have No Observable Developmental Defects.

We were initially interested in conducting a phenotypic analysis of the flies to study any obvious developmental defects in the KO flies because the LAMP1/LAMP2 double KO mice present with craniofacial abnormalities (Eskelinen, 2006). Lamp1e00879 larvae develop to the adult stage without any physical abnormalities or developmental delay. Moreover, we measured the body lengths of the adult flies according to the model shown in Figure 4. Male

Lamp1e00879 and w1118 adult flies consistently had a body length of 2.5 (± 0.0) mm and the value was 3.0 (± 0.0) mm for females, without any observable difference between mutant and wild-type. 36

Figure 4: The line across the body of the female and male flies indicates how the body measurements were calculated. For the male flies, the reading was consistently found to be 2.5 mm and for females, it was conserved at 3.0 mm for both Lamp1e00879 adult flies and w1118 with no statistical difference between them.

The ratio of body weight of Lamp1e00879 adult flies to w1118 was 0.91 suggesting no difference in the composition of the body mass between both the fly lines. Therefore, the mutant flies do not have any detectable developmental defect that could be associated with the lack of an abundant cellular protein and do not have any observable phenotypic abnormalities as represented in our results.

Lamp1e00879 3rd IL Present With Deteriorated Crawling.

Earlier studies conducted with Danon disease patients observed that the male patients with skeletal myopathy had muscle weakness and was presented in their difficulty to walk. (D’souza et al., 2014). Therefore, we wanted to observe if the flies with the knockout of Lamp1 portrayed any similar decrease in their crawling or climbing abilities. Larval Crawling Assay (LCA) and

Rapid Iterative Negative Geotaxis (RING) protocols are well-established methods to measure the neurological effects of mutation on the behavior of D. melanogaster (Nichols, Becnel, &

Pandey, 2012), and thus were selected to evaluate mutant mobility. 37

Our preliminary results suggest that the 3rd instar larvae with Lamp1 knocked out moved slower as compared to wild-type larvae and the results were not always consistent

(Figure 5). Their speed was measured by the number of gridlines crossed by the 3rd IL in one minute while placed on a 2% agarose gel. They often also showed a tendency to turn around more than the wild-type 3rd IL, although this observation was not quantified.

Figure 5: Representative data from the larval crawling assay using Lamp1e00879 and w1118 3rd instar larvae at room conditions. Quantification was conducted using 5 data sets; counting the number of gridlines crossed by larvae per minute under room temperature conditions and using t-test to calculate p-value (p< 0.01). Visualizing The Accumulation Of Lysotracker- Red (LTR)-Positive Puncta In Fat Body Cells Under Starvation Conditions In W1118 3rd Instar Larvae

Next, we wanted to observe if these behavioral traits were also translated into any cellular phenotype in the fat body tissue of the fly. Fat body tissues have been predominantly analyzed to observe cellular phenotypes associated with autophagy.

To this end, age synchronous eggs were collected on molasses plates and incubated at standard conditions (25°C) on soft-food yeast plates until 3rd instar larvae (IL) were found to be crawling on the lids of the petri dishes as described in materials and methods. The larvae were then dissected to obtain the fat body cells, which were in turn incubated with 38

Lysotracker Red DND-99 for staining all of the acidic compartments inside the cells and

Hoechst for staining the nucleus and analyzed by phase-contrast microscopy. The fat body cells with more than 5 LTR-positive puncta (chosen arbitrarily to discount cells with basal autophagy) were counted towards the total number of cells with elevated signal for

Lysotracker- red in both the samples.

In order to check for the efficiency of the Lysotracker- red, the fat bodies of the wild- type 3rd IL were dissected from flies in well-fed and starvation conditions. As nutrient starvation leads to accumulation of LTR-positive puncta in the fat body cells of the 3rd IL

(Scott et al., 2004), we were interested in observing if our experimental conditions also led to similar accumulation of LTR-positive puncta in the fat bodies and could be visualized using

Lysotracker- red. As expected, the wild-type fat bodies showed accumulation of LTR- positive puncta in their cells due to nutrient starvation and upregulation of autophagy after being starved for 4 hours (Figure 6A). The number of cells that contained more than 5 LTR- positive puncta were counted for both normal and starvation conditions. We observed a 2- fold increase in the accumulation of LTR-positive puncta in the fat body cells between well- fed and starvation conditions (p< 0.0001) (Figure 6B). 39

Figure 6: (A) Phase-contrast microscopy was conducted on the fat-bodies of 3rd instar larvae, dissected in PBS; incubated for 3-5 minutes with Lysotracker and DAPI. Images are representative of the results obtained. (B) Quantification was conducted using all the microscopy data; counting the number of cells in w1118 that contained more than 5 LTR- positive puncta under normal and starvation conditions and using t-test to calculate p-value (Scale bar = 20 µm and p< 0.0001). Lamp1e00879 3rd Instar Larvae Depict The Accumulation Of Lysotracker- Red (LTR)- Positive Puncta In Fat Body Cells Under Normal and Starvation Conditions

We repeated the staining and microscopy experiment as described in the previous result, using the fat body cells of both w1118 and Lamp1e00879 3rd IL under well-fed and starvation conditions next. Our microscopy analysis showed that the mutant fat body cells have accumulation of LTR-positive puncta in 83.9 ± 3.2% of their cells under well-fed 40 conditions while only 24.7 ± 4.8% of the w1118 cells show accumulation of LTR-positive puncta (p< 0.0001) (Figure 7).

Figure 7: (A) Phase-contrast microscopy was conducted on the fat-bodies of 3rd instar larvae, dissected in PBS; incubated for 3-5 minutes with Lysotracker and DAPI under normal, well- fed conditions. Images are representative of the results obtained. (B) Quantification was conducted using all the microscopy data; counting the number of cells in w1118 and Lamp1e00879 that contained more than 5 LTR-positive puncta and using t-test to calculate p- value. (Scale bar = 20 µm, p<0.0001) When these 3rd IL were next starved for 4 hours there was a further increase in the accumulation of LTR-positive puncta in both genotypes (Figure 8A). The quantification of 41 the staining observed yielded a statistically significant difference in the appearance of the

LTR-positive puncta between the Lamp1e00879 and w1118 3rd IL (p< 0.001) (Figure 8B).

Figure 8: (A) Phase-contrast microscopy was conducted on the fat-bodies of 3rd instar larvae, dissected in PBS; incubated for 3-5 minutes with Lysotracker and DAPI under starvation conditions. Images are representative of the results obtained. (B) Quantification was conducted using all the microscopy data; counting the number of cells in w1118 and Lamp1e00879 that contained more than 5 LTR-positive puncta and using t-test to calculate p- value. (Scale bar = 20 µm, p<0.001)

Therefore, these results together suggest that the Lamp1e00879 3rd IL have elevated number of vesicles that can be stained with Lysotracker- red in their fat body cells. Since 42

Lysotracker- red marks all the acidic compartments within the fat body cells, these results could indicate an elevated basal level of autophagosomes, autophagolysosomes or lysosomes.

Further inspection using anti-Atg8a or Lamp1-GFP could help in characterizing these puncta.

Lamp1e00879 Male Adult Flies Depict Similar Accumulation Of Lysotracker- Red (LTR)- Positive Puncta In Their Fat Body Cells Under Normal, Well-Fed Conditions

A potential explanation for the accumulation of LTR-positive puncta observed in the mutant

3rd IL fat bodies could be changes in the timing of developmental autophagy of the larva.

Therefore we repeated the experiment in the fat bodies of the adult flies under well-fed conditions. The fat bodies of the adult Lamp1e00879 also showed an increase in the accumulation of LTR-positive puncta, reaching 65.7 ± 7.8% of the total cells in the mutant as compared to 9.1 ± 1.9% of the total fat body cells in w1118 (Figure 9). Confocal microscopy was used to observe these LTR-positive puncta, however, phase-contrast microscopy was used to complete the quantification of the cells containing these puncta for both samples.

Therefore, statistically, there is a very significant difference in the accumulation of LTR- positive puncta in mutant cells which is highly unlikely to result from developmental autophagy (p< 0.0001) alone. 43

Figure 9: (A) Confocal microscopy was used to observe the LTR-positive puncta in the fat- bodies of adult flies, dissected in PBS; incubated for 3-5 minutes with Lysotracker and DAPI under normal, well-fed conditions. Images are representative of the results obtained. (B) Quantification was conducted using all the phase-contrast microscopy data; counting the number of cells in w1118 and Lamp1e00879 that contained any LTR-positive puncta and using t-test to calculate p-value (p<0.0001).

e00879 Lysosomes of Lamp1 Present Morphological Changes and Seem To Contain Vesicle Aggregates

We were interested in conducting ultrastructural analysis of the lysosomes in the mutant fat body cells for any observable changes. Our preliminary results indicate vesicle aggregates 44 that are not normally present in the wild-type in both 3rd IL (Figure 10A) as well as adult flies (Figure 11A). These aggregates are more pronounced in the 3rd instar larvae and could either be lysosomes fused together or enlarged lysosomes with the undigested cargo present inside them. Average diameter of a lysosome is between 0.5 – 1 µm (Alberts et al., 1994), however, the mutant lysosomes seem to be approximately double in diameter than the wild- type lysosomes in 3rd IL (Figure 10B) with the difference being statistically significant (p <

0.0001). In adults, there was no difference in the diameter of the lysosomes (Figure 11B) however lysosomes from mutant flies seem to be more electron dense than the wild-type lysosomes suggesting undigested cargo present in them. 45

Figure 10: (A) Electron microscopy of fat body cells of Lamp1e00879 and w1118 3rd IL depicting vesicle aggregates in the enlarged lysosomes (marked by the black arrow) in the mutant fat body as opposed to smaller lysosomes in the wild-type fat body cells (Scale bar = 2 µm). (B) Average diameter of the lysosomes in Lamp1e00879 is approximately double that of the lysosomes in w1118 (p<0.0001). 46

Figure 11: (A) Electron microscopy of fat body cells of Lamp1e00879 and w1118 adult flies depicting vesicle aggregates in the amorphic and denser lysosomes (marked by the black arrow) in the mutant fat body as opposed to circular lysosome in the wild-type fat body cells (Scale bar = 2 µm). (B) Measurement of the average diameter of the lysosomes in Lamp1e00879 and w1118 did not reveal any significant difference between them. The Ancestral Form of The Lysosome Associated Membrane Protein Has 1 Conserved Luminal/LAMP Domain and 1 Transmembrane Domain

Many mammals have multiple LAMP genes that are either expressed in a tissue- specific or ubiquitous manner, and each of them has evolved to adopt specific functions.

Drosophila melanogaster, an arthropod, has only 1 Lamp ortholog gene, which presents the highest degree of homology to LAMP-2C among human and mouse LAMP proteins 47

(Fujiwara, Furuta, et al., 2013). Therefore, we were interested in exploring whether the presence of only 1 ortholog is a common feature outside of mammals, how its function may have evolved over time, and the evolutionary relationship of flies Lamp-1 to other orthologs in additional metazoan species. We hypothesized that ancestrally, there could have been multiple Lamp genes with diverse functions, but they were lost in the Drosophila’s genome due to rapid sequence evolution or related gene loss events. As a result, other genes may have evolved to inherit the additional functions of its other orthologs in mammalian systems or

Lamp-1 acquired all the functions carried out by mammalian LAMP-1 and LAMP-2.

We first analyzed full genomes from organisms from different metazoan classes to note how many Lamp genes were present in organisms from Bilateria, Myxozoa, Cnidaria,

Ctenophora, Placozoa and Porifera. Figure 12 shows the hierarchy of these organisms and animal groups as they are represented in the tree of life, based on their phylogenetic relationships. The organisms shaded in yellow have only one ortholog of the Lamp-1 gene in their genomes and the ones in green have more than one Lamp-1 gene. The Myxozoa,

Ctenophora and Placozoa organisms analyzed, highlighted in grey, do not contain orthologous Lamp-1 genes in their genomes. 48

Figure 12: Hierarchical arrangement of organisms in the tree of life. The species corresponding to classes in the grey boxes did not contain any protein with LAMP domain/sub-family. Yellow boxes indicate species representative of the clade that have only one Lamp in their genome, and green represent species with more than one. After selecting twelve organisms from these six classes, we aligned the protein sequences of their Lamp genes to identify the regions of greatest conservation and specific features that may have been ancestrally inherited, rather than adopted more recently within each organism. The alignment yielded the highly conserved LAMP luminal domain and the transmembrane helix as shown in Figure 13. Even though human and mice LAMPs have two

LAMP domains, we found that only one domain is conserved across Lamp genes in all genomes. This luminal domain is marked by 4 conserved cysteine resides (highlighted in yellow) that participate in creating 2 disulphide bridges except for in Hydra vulgaris and

Amphimedon queenslandica which only have the latter 2 cysteine residues, creating 1 disulphide bridge. The consensus sequence for the alignments of the luminal domain is also shown in red. 49

Furthermore, the alignment of the protein sequences also yielded conservation of the transmembrane helix. All human LAMPs have been previously shown to have 2 conserved glycine residues and 1 conserved proline residue in the transmembrane helix (Wilke et al.,

2012). In our alignment of the transmembrane helices of the 12 organisms, similar conservation of the glycine residue (in the 7th position) and the proline residue (in the 3rd position) have been found. However, the 2nd glycine conservation has been found to be either substituted with Phenylalanine (in the 12th position) or Tyrosine/Alanine (in the 24th position) amino acids (Figure 12C). The amino acids are also color coded according to RASMOL which colors them based on their localization on the outside or the inside of the protein

(Sayle & Milner-White, 1995). Specifically, the colors green refers to hydrophobic amino acids, orange refers to proline and blue refers to charged amino acids. We further analyzed all the protein sequences in TMHMM to confirm for the presence and localization of the transmembrane helix and the retrieved data coincided with the alignment results (data not shown). Furthermore, consistent with the basic structural features of the membrane proteins, all LAMP proteins of the 12 organisms contained signal peptide sequences as verified through SignalP software (data not shown). 50

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. The cysteines (highlighted in yellow) at the start and end of the sequences form disulphide bridges that are conserved in each of the duplicated homologous luminal LAMP domains. They are followed by a transmembrane helix and a short cytoplasmic tail (not shown). The colors green refers to hydrophobic amino acids, orange refers to proline and blue refers to charged amino acids. 51

Discussion

Lysosome associated membrane protein (LAMPs) have been recognized in recent years to hold important functions with regards to assisting in the recognition and translocation of cargo that needs to be degraded as well as help keep the hydrolytic enzymes physically separated from rest of the cytoplasm. In mammals, there are two main orthologues that are ubiquitously expressed, LAMP-1 and LAMP-2 (Aumüller, Renneberg, & Hasilik,

1997; Konecki, Foetisch, Zimmer, Schlotter, & Konecki, 1995; Zot & Fambrough, 1990).

These proteins have been known to participate in the broader context of autophagy through studies in mice and human disease models. In mice, the knockout of Lamp proteins (LAMP-1 and LAMP-2) leads to embryonic lethality and accumulation of autophagic vacuoles in the cardiac and skeletal muscle cells. The deletion of LAMP-1 alone does not lead to lethality and only results in mice having mild astrogliosis, and the lack of its expression is compensated by upregulation of LAMP-2 (Andrejewski et al., 1999; Eskelinen, 2006;

Lacoste-Collin et al., 2002; Nishino et al., 2000; Saftig et al., 2001). In humans, the mutation in LAMP-2 leads to Danon disease which is also characterized by similar accumulation of autophagic vacuoles in the brain, cardiac and skeletal muscles tissues. It is X-linked as

LAMP-2 gene is present on it and affects males predominantly and affected mothers only have milder and later-onset of cardiomyopathy. The patients develop cardiomyopathy and myopathy, leading to the enlargement of the heart muscle. Mental retardation has only been described in a small proportion of the patients, however, it affected 70% of the total males studied and only 6% of the total females in cases where it has been observed (Nishino et al.,

2000; Saftig et al., 2001; Sugie et al., 2002). Moreover, the depletion of LAMP-2 in Danon disease patients has been known to lead to skeletal myopathy characterized by muscle weakness and the development of mild issues with walking and sitting with the passage of 52 time (D’souza et al., 2014). Since the mice with the LAMP-1/2 both knocked out are embryonic lethal, we needed to explore another animal model that can draw parallel between the cellular phenotype of the Danon disease patients and mice while being viable and fertile.

Understanding the biochemistry of Drosophila cells with the deficiency of Lamp-1 can not only help our understanding of the disease but also fill the broader gaps in the realm of its role in autophagy.

In this study, we used a Drosophila melanogaster Lamp-1 loss-of-function (LOF) mutant, Lamp1e00879, that had been characterized previously to study the cellular phenotype and elucidate the role of Lamp-1 since it is both viable and fertile (Riaz, 2014). D. melanogaster has only 1 ortholog of Lamp-1 gene and previous research in our lab has shown that it is expressed at all developmental stages of the fly (Riaz, 2014). Moreover, we had also found previously that upon the induction of autophagy, Lamp-1 expression is also upregulated, suggesting its possible role in autophagy.

We hypothesized that the deficiency of Lamp-1 can lead to the upregulation of basal autophagy in the mutant flies as well as cause behavioral defects in the animal with regards to its crawling and climbing abilities based on the phenotypic implications in humans and mice with the corresponding deleterious mutation. As shown in the preliminary results, the

3rd IL had reduced crawling ability when compared to the wild-type 3rd IL, possibly as a side- effect of the knockout of Lamp1. The deteriorated crawling ability could suggest the reduced function of the muscle tissue in the 3rd IL, however, our preliminary results weren’t consistent to strongly suggest so. In the latter case, the 3rd IL may have restricted locomotion based on increased sensitivity to the light. Even though studies in humans showed that the symptoms of skeletal myopathy were more pronounced in males (Sugie et al., 2002), we used 53

3rd IL of both sexes to characterize the crawling ability. In future, there could be a gender- specific analysis to shed light on the differences that may be present in these LOF 3rd larvae in line with the different mutational effects on specific gender in humans.

We also expected that the knock out of a protein that is constitutively expressed in D. melanogaster would lead to delays in the developmental profile of the fly or manifestation of developmental defects including reduced body length or decreased body mass. However, no such development defects were observed in the Lamp1e00879 mutant adult flies. The mice with double KO did present with craniofacial abnormalities and the humans had ophthalmic abnormalities (peripheral pigmentary retinopathy, lens changes, myopia, abnormal electroretinogram and visual fields) which were not evaluated as part of this study but would be interesting to look at in the future to explore any manifestation of similar development defects in our fly mutant (Eskelinen, 2006; Prall et al., 2006).

Cellularly, the Lamp-1 LOF mutant 3rd IL presented with the accumulation of

Lysotracker-red (LTR)- positive puncta under well-fed as well as starvation conditions in the fat body cells. Previous research has shown that the effects of upregulated autophagy or starvation-induced autophagy can be observed well in the fat body cells of D. melanogaster and therefore it was the tissue of choice in our study (Scott et al., 2004). These LTR-positive puncta can be either mature autophagosomes, autophagolysosomes or lysosomes since

Lysotracker- red stains all the acidic compartments inside the cell. The accumulation of these puncta raises an important question of whether the greater number of puncta in the fat body cells are due to an increased rate of autophagosome/autophagolysosome production or decreased rate of their degradation in the cells of the mutant. The results from the study conducted by Hubert et al., (2016) were able to answer whether the deficiency of LAMP-2 54 affected the maturation of autophagosomes or their degradation by fusion with lysosomes through their research on LAMP-2 deficient MEFs. Using dual-tagged tfLC3 construct which keeps fluorescing after the cargo has been delivered to the lysosomes and not quench after incorporated into the autophagosomes, the researchers were able to demonstrate that the autophagosomal degradation was severely inhibited in the absence of LAMP-2 and it was equally severe in LAMP-2-single deficient cells and strongly suggested that LAMP-2 was responsible. This result is supported by the fact that the defect was reversed by complementation with LAMP-2A at levels that also restored CMA. It would be interesting to use a similar construct in our LOF mutants to observe whether in our fat body cells there is also impaired autophagosomal degradation. Moreover, LysoSensor™ Yellow/Blue DND-160 can also be used to measure the pH of the autophagosomes/autophagolysosomes which have been successfully used to also measure the changes and fluctuation in the pH of the vacuoles before and after fusing with the lysosomes (Schulze-Luehrmann et al., 2016). Moreover, with regards to the identity of these puncta, research has shown that the autophagosomes are also mildly acidic in nature (5.8 to 6.2) (Maulucci et al., 2015) and therefore these compartments cannot be characterized as any specific organelle with just the Lysotracker- red dye.

Therefore, if Lamp-1 deletion is associated with autophagy, it would be interesting to be able to identify these puncta to elucidate where in the path of degradation by the autophagic pathway is LAMP-1 involved and can lead to potential blockage owing to its knockout.

Additionally, it will be insightful to also measure the pH of these puncta for more information.

We also observed the lysosomal morphology through electron microscopy. The preliminary imaging of the fat body cells in the 3rd IL and adult flies of the Lamp1e00879 as 55 compared to the wild-type presented with somewhat enlarged lysosomes (double in the diameter in 3rd IL) that had vesicle aggregates inside them that had not been degraded. These aggregates were more pronounced in the 3rd IL than in the adult fly. Further quantification is required to evaluate the consistency of this phenotype and measure the average size of the lysosomes in the mutant that could be the results of Lamp-1 depletion.

Moreover, we were interested in understanding the evolution of Lamp-1 gene along the metazoan organisms. We hypothesized that there were multiple Lamp genes ancestrally, but they were lost in D. melanogaster due to gene loss events. Therefore, it could be that other genes may have evolved to inherit the additional functions of its other orthologs in mammals. Based on our analysis of full genomes and protein sequence alignment results, we observed the conservation of one LAMP luminal domain and the transmembrane helix across the twelve selected organisms in different phyla of the metazoan classification. The animal groups of Myxozoa, Ctenophora and Placozoa contain no Lamp gene. While the organisms in the Deuterostomia phylum have multiple orthologs of Lamp-1, it can be suggested that since these organisms underwent genome duplication events, they may have acquired multiple copies of the gene and therefore have more orthologs (Friedman & Hughes, 2001).

However, most phyla contain only one Lamp gene in their genome, suggesting that the last common ancestor had only one Lamp protein.

An interesting case can be observed in the phylum Nematoda where the two representative organisms, Caenorhabditis elegans and Brugia malayi, have difference in the number of Lamp genes present. Nematodes have undergone genetic evolution through both the events of duplication and genome loss (Woollard, 2005). In the taxonomic classifications of the nematodes based on protein sequence evolution, all the nematodes have been classified 56 into five clades. B. malayi belongs to clade III whereas Caenorhabditis elegans is in clade V which has appeared most recently (Blaxter et al., 1998). Therefore, B. malayi may represent the ancestral form of the Lamp-1 protein as well and suggest that ancestrally there was only one Lamp-1 gene that may have evolved in more recent nematode species due to gene duplication. With more molecular evaluation, this hypothesis can be tested to shed more light on their functional divergence as it is being used to study the evolutionary divergence of levamisole-sensitive acetylcholine receptors in nematodes (Duguet et al., 2016). If the alternate hypothesis that ancestrally there was only one Lamp gene and it multiplied into more orthologs due to gene duplication events can be considered, it can be suggested that these events led to sub-functionalization and/or neo-functionalization of the protein and therefore formed more orthologs which later adopted additional functionalities, specifically the unique roles of the splice variants of LAMP-2 in humans and mice that may have been carried out by one protein in an ancestral organism. It can be argued that LAMP-2 adapted through evolution and adopted new functionality and therefore became more useful in its molecular context (either in the process of CMA or DN/RNautophagy) than the original

Lamp gene. This could also explain why the knockout of LAMP-1 in mice is dispensable to the animals as compared to the knockout of LAMP-2 that leads to embryonic lethality in mice and Danon disease in humans. It would also explain why in D. melanogaster, the knockout of a protein like Lamp-1 does not lead to lethality and the flies are both viable and fertile. The hypothesis therefore can be extended to further state that the ancestral form of the protein contained a highly conserved luminal/LAMP domain, as opposed to two domains in mammals, and a transmembrane domain. Nevertheless, a molecular framework is required to 57 study these observations further and prove the phylogenetic evolution of LAMP genes through the metazoan phyla.

Conclusion

In this study, we investigated the phenotypic and cellular features of our previously characterized LOF mutant of Lamp-1 in Drosophila melanogaster, Lamp1e00879 to dissect the function of Lamp-1 in this animal model and study its correlation with autophagy. We found that there were no developmental defects in our mutant adult flies with regards to their body size or body mass. However, the 3rd IL have shown deteriorated crawling ability in our initial evaluation which may be reflective of the skeletal myopathy observed in Danon disease patients with Lamp-2 deleterious mutations. Moreover, the fat body cells of these LOF mutants shows an accumulation of LTR-positive puncta under well-fed as well as starvation conditions. These elevated LTR-positive puncta may refer to an accumulation of autophagosomes, autophagolysosomes or lysosomes and can also be suggestive of impaired degradation of these autophagic vacuoles in the deficiency of Lamp-1 as observed in LAMP-

2 deficient MEFs. Furthermore, the electron microscopy analysis revealed that the

Lamp1e00879 3rd IL lysosomes are approximately double in diameter and contain vesicle aggregates. The adult flies, on the other hand, also reveal the lysosomes to be more electron dense suggesting that they may contain undigested cargo and sometimes may appear to be amorphic in shape. We also studied the phylogenetic evolution of Lamp-1 gene to find the organisms in Arthropod, Annelida and Mollusca phylum contain only 1 ortholog.

Deuterostomes on the other hand also present with multiple orthologs which can be correlated with the whole genome duplication events prevalent in their classes.

58

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CHAPTER 3. GENERAL CONCLUSION

Conclusion

There are two main cellular degradation machineries operating inside a cell, the lysosomes and the ubiquitin proteasome system. Lysosomes are an integral component of the cell primarily for maintaining cellular homeostasis, recycling and degrading autophagic, endocytic and secretory molecules (Mullins & Bonifacino, 2001). They are acidic organelles enclosed by a single membrane and contain a multitude of hydrolytic enzymes in their lumen, varying from proteases, phosphatases, RNases and lipases (Bainton, 1981; Futai et al.,

1969; Pisoni, 1996; Saha et al., 1979). Therefore, they can degrade all macromolecules and are central to maintaining the normal functioning of the cell. The lysosomal membrane is composed of glycoproteins, phospholipids and cholesterol and contains up to 25 transmembrane proteins in mammals (Bleistein et al., 1980; Lübke et al., 2009; Sleat et al.,

2008, 2007). Among all of the glycosylated protein families present in the lysosomal membrane, the most abundant family is that of lysosome associated membrane proteins

(LAMPs) that are also the most abundant cellular proteins and heavily glycosylated (Carlsson

& Fukuda, 1989; Chen et al., 1985; Saftig, 2005).

In humans, there are five LAMP proteins including LAMP-1 and LAMP-2 that are ubiquitously expressed (Aumüller et al., 1997; Konecki et al., 1995). They are structurally similar (Eskelinen et al., 2002). Mus musculus has 2 orthologs, LAMP-1 and LAMP-2, whereas, Drosophila melanogaster only has one ortholog, Lamp-1. They are type I transmembrane protein and structurally are composed of a conserved luminal domain, a transmembrane helix and a short cytoplasmic tail that is more variable (Winchester, 2001). In humans, there are three splice variants of LAMP-2, LAMP-2A, LAMP-2B and LAMP-2C. 68

They each are distinguished from each other on the basis of their cytoplasmic tail that also in turn dictates their involvement in different cellular pathways (Carlsson & Fukuda, 1989;

Gough & Fambrough, 1997). While they are all collectively important for antigen processing, cholesterol trafficking, lysosome biogenesis and phagocytosis (Eskelinen et al.,

2002; Huynh et al., 2007; Schneede et al., 2011), they each also have adopted unique roles in chaperone mediated autophagy (LAMP-2A), stabilizing TAPL in the lysosomal membrane

(LAMP-2B) and RN/DNautophagy (LAMP-2C) (Ana Maria Cuervo & Dice, 1996; Demirel et al., 2012; Fujiwara, Furuta, et al., 2013; Fujiwara, Kikuchi, et al., 2013; Hubert et al.,

2016).

In this study, we explored the putative role of Drosophila melanogaster Lamp-1, that has been shown to be closest in homology to human LAMP-2C for interacting with RNA and

DNA as well as share conserved luminal domain with the other splice variants of LAMP-2

(Fujiwara, Furuta, et al., 2013). The availability of advanced genetic and molecular tools in

D. melanogaster make this organism an excellent model to study the relation of this protein with basal autophagy as well as RN/DNautophagy. We used a previously characterized loss- of-function (LOF) mutant of Lamp-1, Lamp1e00879, in D. melanogaster for observing its morphological and cellular phenotype (Riaz, 2014). In mice, the double knockout of LAMP-

1 and LAMP-2 is embryonic lethal and presents with accumulation of autophagic vacuoles in the cardiac, skeletal muscle and brain cells (Eskelinen, 2006). In humans, the deleterious mutations in LAMP-2 leads to the development of Danon disease characterized by cardiomyopathy, myopathy and mental retardation, that are more pronounced in males than females (Saftig et al., 2001; Sugie et al., 2002). 69

Our results collectively showed that there are no apparent development defects or physical abnormalities in our LOF mutant larvae and adult flies (no significant changes in their body lengths or body mass). When observed for their behavioral traits, we found that the LOF mutant 3rd instar larvae represented deteriorated crawling ability as compared to the wild-type 3rd instar larvae in our initial evaluation. The deteriorated crawling ability of the larvae may draw a parallel with the skeletal myopathy observed in Danon disease patients who also show muscle weakness with the passage of time (Sugie et al., 2002). However, the larval crawling assay results were not always consistent and therefore require further investigation. Furthermore, we observed accumulation of Lysotracker-red (LTR) positive puncta in the fat body cells of the 3rd instar larvae under normal and starvation conditions.

Similar observations were recorded for adult flies under well-fed conditions. These LTR- positive puncta may refer to lysosomes since Lysotracker-red is preferentially used to stain for this organelle. However, it could also revel other intermediate autophagic vacuoles as the dye does marks for the acidic compartments inside the cell. The accumulation of these puncta can be associated with either impaired degradation of the autophagic vacuoles or increase in their rate of production and therefore will be an interesting question to pursue in the future.

Lastly, we were also interested in studying the phylogenetic evolution of the Lamp-1 gene in the metazoan organisms and understand if the gene duplication or rapid sequence evolutionary events led to gene loss in D. melanogaster or replication in mammals. While the organisms present in Arthropod, Annelida and Mollusca phyla contain only 1 ortholog,

Deuterostomia organisms all have multiple orthologs which can be correlated with the whole genome duplication events prevalent in their classes and it would be interesting to establish a molecular framework to further study the evolution of this gene and to investigate if in D. 70 melanogaster, other genes have evolved along the way to adopt its additional functions represented in the other multiple orthologs of Lamp-1 in mammals.

Future Directions

From this study, we were able to understand the cellular phenotype of the fat body cells under

Lamp-1 deficient conditions. These fat body cells presented with accumulation of LTR-positive puncta that can be autophagosomes, autophagolysosomes or lysosomes. Therefore, it will be interesting to identify these puncta. This identification will help in shedding more light on where in the autophagic pathway is Lamp-1 involved and verify if, similar to the LAMP-2 deficient MEFs, these puncta are also accumulating as a result of impaired degradation of the autophagosomes (Hubert et al., 2016). The identity of these puncta can be molecularly explored using similar constructs such as the LC3 tandem fluorescent tag created by Kimura et al., (2007) that continues to fluoresce even after the cargo has been transferred to the lysosome for degradation starting from autophagosomes and therefore has been known to distinguish between the autophagosomes and lysosomes.

Alternatively, LysoSensor™ Yellow/Blue DND-160 can be used to track the pH of the autophagic vacuoles and therefore study the transitions as observed through the all of the intermediates including autophagosomes, autophagolysosomes and lysosomes as suggested in an earlier research study

(Schulze-Luehrmann et al., 2016). This can also help in understanding if under the conditions of

Lamp-1 deficiency, the fusion of autophagosomes with lysosomes is impaired and therefore leads to the accumulation of LTR-positive puncta as observed in our results. Moreover, the lysosomes can be isolated and studied outside the cellular system and observe their degradation of the cargo in vitro by tagging them appropriately. Tagged Atg8a autophagosomes can also be added to this system to observe the fusion of autophagosomes with lysosomes in the presence and absence of Lamp-1.

Moreover, it will be interesting to study the molecular partners of Lamp-1 as they have been observed in other mammalian systems for both of its orthologs, LAMP-1 and LAMP-2. Currently, our understanding of Lamp-1-mediated degradation processes is limited in D. melanogaster and 71 therefore, it will be insightful to observe if the known Lamp-1 protein partners in mammals

(including SNARE proteins, SidT2 and Stx17) interact directly with Lamp-1 or not to mediate the fusion of lysosomes and autophagosomes and assist in the translocation of cargo inside the lysosomes for degradation. LAMP-2C, a splice variant of mammalian LAMP-2, has been implicated in

RN/DNautophagy and has been shown to interact directly with RNA/DNA in the cytoplasm. SidT2 helps in the translocation and delivery of the RNA inside the lysosomes where it is recycled. D. melanogaster’s Lamp-1 shares the most homology with LAMP-2C and therefore we hypothesize that it may also participate in the recognition and ultimate degradation/recycling of RNA in the fruit-fly.

Therefore, it will be important to shed light on its interaction with RNA and if it establishes a similar

RNautophagy process in the fly system as it does in the mammals (Aizawa et al., 2016; Fujiwara,

Furuta, et al., 2013; Fujiwara, Kikuchi, et al., 2013; Hase et al., 2015). Pull-down experiments on cell lysates can be designed to identify the interacting proteins through mass spectrometry analysis and using genetic screens to develop relationship between Lamp-1 and these molecular partners to solve for its biochemical significance in fruit-fly and where in the autophagic pathway it becomes indispensable for maintaining cellular homeostasis of the organism. The cell-free system suggested earlier for studying the interaction of lysosomes with autophagosomes can also be used to study the uptake of RNA by the lysosomes derived from D. melanogaster in the presence and absence of

Lamp-1 in an ATP-dependent manner as observed in mammalian systems. To this end, the expression of certain ribonucleoproteins (RNPs) and RNA-binding proteins (RBPs) involved in RNA metabolism can be compared in the presence and absence of Lamp-1 to study the effects on RNA homeostasis in the LOF mutant flies and establish correlation with lysosomes as well.

Furthermore, the clinicopathological features of Danon disease in humans become more pronounced with age (Sugie et al., 2002). Therefore, it will be interesting to characterize the larval crawling ability with increasing age to see if larva deteriorate in their ability to crawl. Moreover, in this study, only the fat body cells were analyzed for cellular phenotype. Since the mice models of

LAMP-1/LAMP-2 double knockouts had accumulation of autophagic vacuoles in their cardiac, brain 72 and skeletal muscles, we can continue to observe the representative cardiac, brain and skeletal muscle tissues in the LOF mutant larvae and flies to observe if the cellular phenotype stays consistent. We can also observe the cardiac health of these mutant flies through established molecular protocols

(Ocorr, Vogler, & Bodmer, 2014).

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APPENDIX. LIST OF PRIMERS AND DATABASES USED IN THE STUDY

Table A1: List of primers used. Name Type 5’ – SEQUENCE – 3’

RpL32con Forward 5’-AAGATCGTGAAGAAGCGCAC-3’

Reverse 5’-CGTAACCGATGTTGGGCATC-3’

Lamp-1 Forward 5’-TTCGGCATAGCAACAGAAGGA-3’

Reverse 5’-CCATACCTCACCAATGCCGT-3’

Table A2: Databases used for phylogenetic analysis Name Accession # Species Common name

Lamp-1 P11279 Homo sapiens human

Lamp-2C NP_001116078 Homo sapiens human

Lamp-1 P11438 Mus musculus mouse

Lamp-2 P17047 Mus musculus mouse

Lamp-1 Q9V9S0 Drosophila melanogaster fruit fly

LMP-1 Q11117 Caenorhabditis elegans nematode

LMP-2 Q9GYK0 Caenorhabditis elegans nematode

Lamp A0A0K0J3R7 Brugia malayi roundworm

Lamp Q9U5N4 Lumbricus rubellus earthworm

Lamp-1 XP_005107131.1 Aplysia californica California sea hare

Lamp XP_014790028.1 Octopus bimaculoides California two-spot octopus

Lamp-1 XP_015759380.1 Acropora digitifera Staghorn coral

Lamp-1 XP_002158873.2 Hydra vulgaris fresh-water polyp 82

Table A2: (continued)

Name Accession # Species Common name

Amphimedon Lamp-1 XP_011407957.1 sponge queenslandica

Lamp-2 XP_013087471.1 Biomphalaria glabrata freshwater snail