TPD52: Functional Elucidation of a Novel Lipid Droplet Binding Protein in Human Hepatocytes

TPD52: functional elucidation of a novel

lipid droplet binding protein in human

hepatocytes.

Shi Bo Feng

Biochemistry, McGill University, Montreal August 2015 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master

I wish to thank Transplant Quebec with donors and their families and patients consenting to liver resection. I thank Dr. Jarred Chicoine and Dr. Robert Sladeck for the plasmid design and construction, also for making tetracycline inducible TPD52 cell lines. I would also like to thank Dr. Fariba Kalantari and Dr. Ali Fazel for providing fractionated human primary liver samples and delipidated lipid droplet fractions. I also thank Dr. Min Fu for her help with confocal microcopy, Sarita Negi for the cryosection of human liver samples, and Eun Joo Lee for training me on various techniques. Mom and Dad, thank you so much for your support all these years.

Finally, I would like to thank Dr. Robert S. Kiss and Dr. Tommy

Nilsson for accepting me into their laboratories and for supporting me in so many ways so I could complete two fulfilling years in research.

ii Abstract

TPD52: functional elucidation of a novel lipid droplet binding protein in human hepatocytes.

Shi Bo Feng McGill University, (2015) Supervisors: Robert S. Kiss & Tommy Nilsson

Lipid droplets (LDs) are intracellular vesicles involved in the storage of triglycerides (TG) and cholesteryl ester (CE). Although LDs are used for storage, they also play a dynamic role in cell metabolism. LD overaccumulation in the liver causes non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), often resulting in liver dysfunction. We identified tumor protein D52 (TPD52) as a novel LD binding protein. By immunofluorescence and western blot, TPD52 was found on LDs in both HepG2 cells and human primary liver samples. Transient transfection of TPD52-eGFP fusion protein in HepG2 and inducible stable transfection in HEK293 showed that TPD52 stably localizes to LDs induced by oleic acid and by arachidonic acid. A fluorescence recovery after photobleaching (FRAP) experiment also showed the time-dependent recruitment of TPD52 to LDs. Moreover, an in situ biotinylation identification technique (Bio-ID) identified LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) as a potential TPD52 interacting protein. LAMTOR1 is a membrane protein found on late endosomes/lysosomes known to interact with components of the Ragulator complex regulating autophagy. LAMTOR1 was shown to localize to LDs in HepG2 and human primary liver samples by immunofluorescence. Implications of a functional link between TPD52 and LAMTOR1 are discussed. TPD52 may be linked to lipophagy, which has the potential of helping understanding NAFLD. TPD52 may be the key to develop potential treatment.

iii Résumé

TPD52: élucidation fonctionnelle d'une nouvelle protéine de la gouttelette lipidique de liaison dans les hépatocytes humains.

Shi Bo Feng McGill University, (2015) Superviseurs: Robert S. Kiss & Tommy Nilsson

Les gouttelettes lipidiques (LDS) sont des vésicules intracellulaires impliquées dans le dépôt des triglycérides (TG) et l'ester de cholestéryle (CE). Bien que LDs sont utilisées pour le dépôt, ils jouent également un rôle dynamique dans le métabolisme cellulaire. LD suraccumulation dans le foie provoque la maladie non alcoolique du foie gras (SNA) et la stéatohépatite non alcoolique (NASH), ce qui entraîne souvent un dysfonctionnement hépatique. Nous avons identifié la protéine tumorale D52 (TPD52) comme une nouvelle protéine de lié à la surface de LD. Par l’immunofluorescence et Western blot, TPD52 a été trouvé sur LD dans les deux cellules HepG2 et des échantillons de foie primaires humains. La transfection transitoire de TPD52-eGFP protéine de fusion dans des cellules HepG2 et transfection stable inductible dans les cellules HEK293 a montré que TPD52 localise de manière stable à LD induite par l'acide oléique et de l'acide arachidonique. Un recouvrement de fluorescence après photoblanchiment (FRAP) a également montré le recrutement en fonction du temps de TPD52 à LD. En outre, une technique in situ d'identification de biotinylation (Bio-ID) identifié LAMTOR1 (fin endosomal / lysosomial adaptateur, MAPK et mTOR activateur 1) en tant que protéine interagissant TPD52 de potentiel. LAMTOR1 est une protéine trouvé sur les endosomes tardifs / lysosomes connus pour interagir avec des composants du complexe autophagie Ragulator régulation. LAMTOR1 a été montré à localiser à LDS HepG2 et des échantillons de foie primaires humains par immunofluorescence. Conséquences d'un lien fonctionnel entre TPD52 et LAMTOR1 sont discutées. TPD52 peut être lié à lipophagy, qui a le potentiel d'aider la compréhension de NAFLD. TPD52 peut être la clé pour développer un nouveau traitement potentiel.

iv Table of Contents

Acknowledgements ...... ii

Abstract-Résumé ...... iii

List of Illustrations ...... vii

List of Figures ...... viii

INTRODUCTION...... 1

Lipid and Lipid Droplet ...... 1 LD structure ...... 2 The PAT family proteins ...... 3 LD biogenesis ...... 4

Disease related to LD accumulation: NAFLD & NASH...... 6

TPD52 family ...... 7 Structure...... 8 TPD52 association with cancer ...... 8 TPD52 expression in different cell lines ...... 9 Potential function of TPD52 ...... 9 TPD52 and lipid metabolism ...... 11

Autophagy/Lipophagy ...... 11 Definitionof autophagy ...... 11 mTOR & Autophagy ...... 12 LAMTOR1 & Autophagy ...... 13 Autophagy, lipid metabolism and NAFLD ...... 13

Importance of the project and hypothesis ...... 14

v RESULT...... 16

Part I: TPD52, an oncogene located on the LDs under a high oleate concentration...... 16

Part II: The heterogeneity of TPD52 expression on LDs surface 22

Part III: Exogenous TPD52 is expressed under various conditions65

Part IV: TPD52 family proteins (TPD52L1 & TPD52 L2) are also found on lipid droplet ...... 33

Part V: LAMTOR1- TPD52 interacting protein that is involved in autophagy ...... 37

DISCUSSION & CONCLUSION...... 42

MATERIALS & METHODS ...... 46

REFERENCE ...... 50

vi List of Illustrations

Illustration I: LD structure ...... 3 Illustration II: Schematic representation of LD biogenesis ...... 5 Illustration III: The spectrum range of NAFLD ...... 7 Illustration IV: TPD52 gene map ...... 8

vii List of Figures

Figure 1: Endogenous TPD52 co-localizes with PLIN2 on lipid droplet in hepG2 cell line ...... 18 Figure 2: Endogenous TPD52 associates with LDs in primary human liver tissue under physiological state ...... 19 Figure 3: TPD52 expression level in human hepatic intracellular fractions shows TPD52’s localization on LDs ...... 21 Figure 4: TPD52 expression in human hepatic LD fractions is heterogeneous...... 23 Figure 5: Exogenous TPD52 is found on LDs across different time points after oleate induction ...... 27 Figure 6: Exogenous TPD52 is found on LDs generated using different lipid source ...... 29 Figure 7: FRAP analysis of TPD52 mobility on LD ...... 31 Figure 8: Both TPD52L1 and TPD52L2 are two known members of

TPD52 family that localize on LD surface ...... 34 Figure 9: TPD52L1 expression in human hepatic LD fractions is heterogeneous and differs from TPD52 expression ... 36 Figure 10: LAMTOR1 co-localizes with PLIN2 on LDs ...... 39 Figure 11: Endogenous LAMTOR1 is located on LDs of NAFLD human liver ...... 40

viii INTRODUCTION

Lipid and Lipid Droplet

Lipid droplets (LDs) are present in various eukaryotic cells as the storage organelles for excess free fatty acids and cholesterol (1). The mechanism and machinery to generate LDs is evolutionarily conserved from prokaryotic bacteria to humans (1,2). For storage purposes, free fatty acids are converted to triglycerides (TG), and cholesterol to cholesteryl ester (CE), and both are stored in LDs (1,2). However, very little is known about its formation, growth and function. For a long time, LDs were thought to serve as static “storage closets”, which collect, store, and supply lipids. Recently, more evidence has shown that LDs are complex and dynamic organelles playing an essential role in lipid homeostasis (1-3). A matured LD is an independent organelle that is contained with a limiting monolayer of phospholipid and associated LD proteins (4). LDs’ formation, fusion, fission, and intracellular motility are dynamically regulated (4). LDs show significant variation in size and composition in various cell types (3).

Even within the same cell population, LD formation is heterogeneous

(3). It is evidence of LD’s dynamic role in intracellular homeostasis since LDs may play a central role in lipolysis, cholesterol oxidation, protein synthesis, energy metabolism, and transport of membrane 1 building blocks (3). For instance, endoplasmic reticulum (ER) stress triggers LD formation and accumulation, independent of lipid arrival and consumption (5). LDs thus may play a protective role in decreasing lipotoxicity by storing toxic lipids and preventing their accumulation within the ER membrane (5). LDs might even get involved in the degradation of misfolded ER proteins since pharmacological inhibition of LD formation impaired the dislocation of other proteins (6).

LD structure:

LDs are composed of a phospholipid monolayer wrapped around a hydrophobic core of neutral lipids (Illustration I)(7). This unique monolayer structure allows hydrophobic fatty acyl tails to contact the hydrophobic neutral lipid core while the hydrophilic outer surface of

LDs (polar headgroups of phospholipids) to be exposed to the cytosol allowing the solubilization of the lipids in the aqueous environment (5).

This monolayer structure also affects the organization of LD-associated proteins. The absence of a lipid bilayer does not allow typical transmembrane proteins on LD membranes. Therefore, it is generally suggested that most LD-associated proteins are anchored by a hydrophobic stretch that is buried in the hydrophobic core (5).

2 Illustration I. LD structure. Lipid droplet contains a lipophilic neutral-lipid (TG/CE) rich core, surrounded by a phospholipid monolayer. LD associated proteins are peripheral proteins such as the PAT family of proteins.

The PAT family proteins:

The PAT family consists of five lipid droplet associated proteins identified by their binding to intracellular LDs: perilipin (PLIN1), adipose differentiation-related protein (ADRP/PLIN2), tail-interacting protein of 47 kD (TIP47/PLIN3), S3-12 (PLIN4), OXPAT (PLIN5) (8). All

PAT proteins share some sequence similarity (9). PAT proteins regulate 3 access of other proteins such as lipases to the neutral lipid in the LD core (8). They are also crucial for lipid droplet biogenesis (8). The PAT proteins are commonly used as a LD surface marker. In this project, we have used PLIN2 and PLIN3 as LD protein markers.

LD biogenesis:

LDs fulfill specific functions in different cell types. For example, in

Drosophila cells, 227 genes affecting LD formation were identified

(10). They affect LD size, morphology and distribution in the cytosol to varying degrees (10). There are many hypotheses on how LDs are formed. One postulates that LDs are formed by modification at the ER membrane. LD formation is triggered by deposition of neutral lipids

(TG and/or CE) between the cytosolic the lumenal leaflets of the ER membrane (5). Accumulation of the neutral lipids leads to the formation of an intramembrane droplet of neutral lipids between the two layers of phospholipids at the ER membrane (5). Two possible mechanisms are proposed from here. The droplet grows by structurally rearranging the monolayer ER membrane on the cytosolic side, and by segregation of proteins specific for LDs (Illustration II-A)(5). It then pinches off from the ER into the cytosol (5). Formation of LDs could also occur by forming a lens-like structure between cytosolic and lumenal leaflets of the ER membrane (Illustration II-B) (5). It then 4 detaches from the rest of the membrane resulting in the formation of a transient pore in the ER membrane (5). The released LD is thus covered by a phospholipid monolayer derived from both lumenal and cytosolic leaflets of the ER membrane (5). Alternatively, the LD could form through the budding of a transport vesicle (e.g. COPI or COPII vesicle) followed by the expansion of the hydrophobic space of the phospholipid bilayer through lipid synthesis (e.g. triglyceride formation)(2). This would lead a small patch of a lipid bilayer surrounding an intralipid droplet vesicle (2).

Illustration II. Schematic representation of LD biogenesis. Lipid accumulates between the two monolayer leaflets of the ER membrane.

5 Disease related to LD accumulation: NAFLD & NASH

Non-alcoholic fatty liver disease (NAFLD) is one of the most common diseases manifesting in accumulation of cytoplasmic LDs in human liver. NAFLD is a chronic metabolic syndrome causing major health concern in both industrialized countries and in the developing world, where between 30-50% of the adult population suffers from

NAFLD (11). It is defined by the presence of high levels of TG in liver parenchyma without inflammation in the absence of excess alcohol consumption (12). NAFLD includes a wide spectrum of disease, ranging from simple non-symptomatic steatosis (NAFL) to non- alcoholic steatohepatitis (NASH), fibrosis, and cirrhosis (Illustration.III)

(13). A more severe form of NAFLD is non-alcoholic steatohepatitis

(NASH); 20% of patients with NASH will progress to fibrosis and cirrhosis over a 15 year time period (14). In some patients, steatosis leads to cellular injury and inflammation (NASH) with a subsequent risk for progression to cirrhosis and for hepatocellular carcinoma (HCC)

(15). Furthermore, NAFLD contributes significantly to a higher rate of cardiovascular events in patients (16). Moreover, NAFLD contributes to the occurrence of type 2 diabetes (17).

6 Obesity

Steatosis

Intrahepatic triglyceride accumulation

Normal liver

Hepatocellular Fibrosis cirrhosis carcinoma

Non-alcoholic steatohepatitis (NASH)

+Inﬂammation +Hepatocellular Ballooning/Mallory-Denk bodies +Hepatocyte death

Illustration III. The spectrum range of NAFLD. (18). NAFLD is a chronic disease where intrahepatic triglyceride accumulates (steatosis)(18). It may extend to steatohepatitis where intrahepatic inflammation is observed (NASH), fibrosis cirrhosis, and even hepatocellular carcinoma (18).

TPD52 family

The tumor protein D52 family of proteins consists of small coiled- coil motif bearing proteins, which are conserved from lower organisms to human (19, 20). The TPD52 gene (found at chromosome 8q21.13) was identified through its overexpression in various human cancers around 20 years ago (19). Tumor protein D53 (TPD53, also named as

TPD52L1), and tumor protein D54 (TPD54, also named TPD52L2) can dimerize with TPD52 (19). TPD52L2 is also identified as an oncogene

7 overexpressed in various cancerous tumors (21). The TPD52 proteins are highly negatively charged molecules that are both cytosolic and peripherally membrane–bound proteins (22).

Structure:

TPD52 is 180 to 200 amino acids long hydrophilic polypeptide, and is alternatively spliced to various isoforms (Illustration.IV) (19). A

50 amino acid long coiled-coil motif helps TPD52 to homodimerize or dimerize with TPD52L1 and TPD52L2. Ser136 on TPD52 is the primary phosphorylation site for calmodulin kinase II and casein kinase II and has been shown to regulate TPD52’s localization inside a cell (19).

Illustration IV. TPD52 gene map (19) +) % .*' &%(-&# $ " %(-&#+)!+) $ ( %(-&#+ , %(-&# (*, %(-&#

TPD52 association with cancer:

TPD52 gene was first found as a gene amplified in breast tumors, where its amplification correlated with poorer outcomes in breast

8 cancer patients (19). It is thought to be a potential driver gene highly associated with regeneration (19). TPD52 overexpression is also observed in prostate tumor and matched tumor-adjacent histologically normal tissues (19). It is observed to be elevated in both high-&low- grade localized prostate cancers. Moreover, TPD52 overexpression correlates to early lethality due to prostate cancer (19). TPD52 is also found to be overexpressed in small cell lung cancer and squamous cell lung carcinoma. (19)

TPD52 expression in different cell lines:

TPD52 is endogenously expressed in many cells and used for oncology studies, such as LnCaP and C4-2 cell lines (19). Its overexpression in cancer cell lines increases cells’ tumor characteristics such as invasion, tumorgenity, proliferation, and anchorage independent growth (19). In terms of tumor treatment, TPD52 knockdown in multiple tumor cell lines significantly reduces their sensitivity to radiation treatment. (19)

Potential Function of TPD52:

TPD52 has a potential role in vesicle trafficking and exocytosis.

Ser136 phosphorylation in response to secretory stimulation of gastric parietal and pancreatic acinar cells regulates TPD52 accumulation at the plasma membrane (19). In CHO-K1 cells, TPD52 is phosphorylated 9 in a calcium dependent manner by calcium/calmodulin-dependent protein kinase (CAMK2) isoform, and the phosphorylation coincides with TPD52 rapid accumulation along the plasma membrane (23, 24).

Null and phosphomimetic mutations on Ser136 of TPD52 show that the calcium-stimulated plasma membrane accumulation of TPD52 is dependent on its phosphorylation (23). Moreover, lysosome-associated membrane protein (LAMP1) accumulation at the plasma membrane is highly regulated by TPD52 expression and its phosphorylation at

Ser136, suggesting a functional role for TPD52 phosphorylation in calcium-regulated plasma membrane trafficking (23). TPD52 also has a role in modulating lysosomal membrane protein trafficking to the plasma membrane (23). TPD52 is overexpressed in multiple cancers, but is normally abundant in exocrine cells of the pancreas, lacrimal, and submandibular glands, as well as multiple cell types throughout the gastrointestinal tract (25). Using yeast two-hybrid screening, in vitro binding assays and coimmunoprecipitations, TPD52 was shown to interact with MAL2 and annexin VI (26). MAL2 is an integral membrane protein essential for the normal transcytotic delivery of glycosylphosphatidyl inositol-anchored proteins to the apical membrane, whereas annexin VI is a calcium regulated phospholipid binding protein involved in endosome formation and trafficking (22, 10 27). TPD52L2 was reported to interact with members of the soluble N- ethylmaleimide-sensitive fusion protein attachment protein receptor

(SNARE) complex, which is necessary for membrane fusion (23).

TPD52 and lipid metabolism:

TPD52 has no obvious LD association motifs, yet recent research has shown that TPD52 interacts with known regulators of lipid storage in cancer cell lines (23). Dr. Byrne’s group recently identified PLIN2 as a direct TPD52 interacting protein in cancer cell lines, which proved

TPD52’s involvement in lipid metabolism (28). Since altered cellular metabolism is critical for cancer development, there is reason to believe TPD52 regulates lipogenesis by increasing fatty acid storage in

TG and forming greater numbers of LDs (28).

Autophagy/Lipophagy

Definition of autophagy:

There are three types of autophagy: 1) chaperone-mediated autophagy (CMA). In CMA, proteins that contain a special targeting motif, recognized by heat shock cognate protein 70 (HSC70) and co- chaperones, will be selectively delivered to lysosomes where they are internalized by lysosomal-associated membrane protein 2A (LAMP2A) 11 (29). 2) Microautophagy refers to the direct engulfment of organelles by the lysosome (29). Usually a very small portion of cytoplasm is involved in this process. 3) Autophagy, often referred to as macroautophagy, is a cellular catabolic process in response to starvation and/or other stress conditions whereby cellular components and organelles are engulfed into autophagosomes and eventually delivered to lysosomes for degradation (30). The autophagosomes, once matured and ready for degradation, are fused with endosome- lysosomes to form autolysosomes, leading to degradation of the inner membrane together with its lumenal contents (31, 32). Lysosomes function to digest intracellular debris, malfunctioning organelles and invasive microorganisms using multiple acid hydrolases (33, 34). mTOR & Autophagy:

The mammalian Target of Rapamycin (mTOR) forms a complex of proteins (mTOR complex 1 (mTORC1)) to act as essential regulators of autophagy and lipid metabolism (35). To dissect the functional pathway, three mTOR inhibitors have been commonly used:

Rapamycin, PP242 and Torin1 (36). Rapamycin is an allosteric inhibitor of mTOR but only partially inhibits mTORC1 function. PP242 and Torin

1 are catalytic inhibitors that are able to completely suppress mTORC1 via binding to ATP-binding sites (37, 38). Recent evidence suggests 12 that lysosomal function is upregulated in autophagy. This process is regulated by two critical factors: suppression of mTORC1 activity and occurrence of autophagosome-lysosome fusion (36). In other words, mTORC1 acts as a negative regulator of lysosomal function in the autophagy processes (36).

LAMTOR1 and autophagy:

As mentioned previously, mTORC1 is heavily involved in various cellular functions such as energy metabolism, autophagy, cell growth, and the trafficking/maturation of lysosomes (39). LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) is a membrane protein specifically localized to the surface of late endosomes/lysosomes that serves as an anchor for the “Ragulator” complex. The Ragulator plays crucial roles in activation of mammalian target of rapamycin complex 1 (mTORC1) on the lysosomal surface. In fact, LAMTOR1 depletion in cells leads to an aberrant lysosomal catabolism that produces excessive reactive oxygen species (ROS) causing p53-dependent apoptosis (40).

Autophagy, lipid metabolism and NAFLD:

Autophagy is essential in limiting liver injury and hepatocyte apoptosis by removing damaged intracellular organelles and accumulated LDs (41). The process of autophagy that regulates 13 intracellular lipid metabolism and digests accumulated LDs is termed lipophagy (42). Both lipophagy and lipolysis are regulated hormonally by insulin and glucagon and are increased during starvation (42). It is suggested that lipophagy may contribute to LD and TG breakdown due to the regulatory and functional similarities between lipophagy and lipolysis (42). However, another group suggests that lipophagy was necessary for the genesis of LDs rather than for the breakdown of LDs

(29, 43). Therefore, the relationship between lipophagy and NAFLD is still under debate.

Importance of the project and hypothesis

As TPD52 is found up-regulated in cancer cells and associates with LDs, it is of interest to elucidate its function and association with

NAFLD and in particular, NASH. TPD52 has never been shown to be associated with LDs until very recently in cancer cell lines (28). By elucidating its functional context, we have uncovered the potential role of TPD52 in lipid droplet metabolism. Although its effect on lipid droplet biogenesis is yet to be studied, we show in this project the expression of an oncogene bound to LDs in various cell lines including human hepatocytes and fibroblasts. We also show LAMTOR1, an interacting partner of TPD52, on LDs in hepatocytes and liver tissue.

Since LAMTOR1 is part of the Ragulator interacting with mTORC1, our 14 hypothesis is that TPD52 is involved in the lipid droplet biogenesis through mTOR pathway and lipophagy. Its interaction with LAMTOR1 may play a role in LD fusion and fission, and LD breakdown. So far, the mechanism of TPD52 in lipophagy is unknown.

15 RESULT

Part I: TPD52, an oncogene located on the LDs under a high oleate concentration

Primary human livers with varying degrees of NAFLD and NASH were subjected to subcellular fractionation, sodium didecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation, and lipid chromatography tandem mass spectrometry (LC-MS/MS) to identify novel LD associated proteins. This method was performed on over 100 liver samples. Among the novel LD associated proteins, TPD52, a gene overexpressed in various cancer tissues, was found to be highly abundant in the LD fractions of human patients’ livers. TPD52 was studied in various cancer cell lines (with no identification of subcellular localization), but a recent study has shown TPD52 localization on the

LD surface in 3T3 cell line (28), confirming our observation. Here we are the first to demonstrate that TPD52 is present on LDs in primary human liver tissue.

Using immunofluorescence, endogenous TPD52 was localized on the LDs’ surface in HepG2 cells, a human hepatocyte model (Figure 1).

Colocalization studies showed TPD52 surrounding the LD core highlighted by BODIPY fluorescence (hydrophobic fluorescent dye that fluoresces under a hydrophobic high TG/CE environment). Perilipin 2

16 (PLIN2; a LD surface marker) colocalized with TPD52 around BODIPY stained LDs (Figure 1).

It is clear that endogenous TPD52 surrounds some of the LDs inside HepG2 treated under a high oleate condition. Its colocalization with PLIN2, a common LD surface marker shows the possibility that

TPD52 is on or nearby the lipid droplet surface. However, not all LDs have TPD52. This suggests that there may be heterogeneity in TPD52 expression level. TPD52 may also be selectively recruited to LD, suggesting a potential role of TPD52 in lipogenesis or lipolysis.

17 Figure.1 Endogenous TPD52 co-localizes with PLIN2 on lipid droplet in hepG2 cell line

TPD52 expression in HepG2 hepatocytes treated with 0.4mM oleic acid

(OA) for 4 hours and fixed. Cells are stained with BODIPY 493/503 for LDs

(blue), PLIN2 (green) and TPD52 (red) and visualized by confocal microscopy.

18 TPD52’s localization to LDs was confirmed in primary human liver tissue with NAFLD (Figure 2). Human tissue sections were obtained through Transplant Quebec from liver tissue resected from patients undergoing surgery for removal of metastasis in the liver.

Figure 2. Endogenous TPD52 associates with LDs in primary human liver tissue under physiological state.

The tissue sample is frozen in isopentane chilled in liquid nitrogen to preserve its native form. The tissue is cut thinly with microtome at -20 degree Celsius and fixed using paraformaldehyde as described in

19 Materials and Methods. Immunofluorescence staining was performed to detect endogenous level of TPD52 (red) and PLIN2 (green). LDs

(blue) were detected using Bodipy 493/503 as performed in Figure 1.

Figure 2 confirms that TPD52 is located on LD surface in human liver tissue sample. It validates the results from LC-MS/MS. The fact that

TPD52 co-localizes highly (but not fully) with PLIN2 confirms the observation by Dr. Byrne’s group that reports TPD52 as a potential

PLIN2 interacting protein (28).

A western blot (WB) was done on the cell fractions of the liver tissue in order to detect the amount of TPD52 in each fraction. A

NAFLD human hepatic sample was fractionated into rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), Golgi,

Mitochondria (Mt), cytoplasmic lipid droplet (CLD). The protein concentration of each fraction was determined using the Bradford assay. Equal amounts of protein (109g/well) for each fraction were loaded for this WB. Since the samples used are intracellular fractions, we could not use GAPDH or other antibodies to verify the amount of protein loaded. It was thus essential to load equal amount of protein.

Figure 3. TPD52 expression level in human hepatic intracellular fractions shows TPD52’s localization on LDs.

Human hepatic sample was fractioned into various intracellular fractions.

The LD fraction was delipidated. Protein concentration was quantified using

Bradford protein assay. 103g protein of each fraction was loaded to ensure equal amount of protein for each fraction. The western blot was performed on liver samples from three patients and a representative sample is shown here.

A) TPD52’s size is ~23kD. From left to right: lipid droplet (LD); homogenate

(H); rough endoplasmic reticulum (SER); smooth endoplasmic reticulum

(SER); mitochondria (Mt); Golgi.

21 B) Level of TPD52 expression was determined using intensity of the band at

23kD. ImageJ gel analysis tool was used to semi-quantitatively evaluate the

intensity of the band.

We could see that TPD52 is highly expressed in the LD fraction of the NAFLD human liver tissue compare to other fractions. The homogenate fraction has some level of TPD52 expressed, which is expected. TPD52 is known as a cytoplasmic protein. Moreover, abnormally high level of LDs characterizes NAFLD liver tissue. All other cellular fractions (RER, SER, Mt, Golgi) have almost no TPD52 expressed. These results confirm what is shown in Figure 1 and Figure

2, where TPD52 is only expressed around LD. It is therefore highly possible that under high fat condition (high oleate concentration in hepatocytes and NAFLD), TPD52 is located on or near the LD surface.

Part II: the heterogeneity of TPD52 expression on LDs surface

Although TPD52 is localized to the LD surface when there is an accumulation of LDs, the expression level seems to be highly heterogeneous. TPD52 co-localizes highly, but not completely with

PLIN2 in human liver tissue. Moreover, TPD52 expression varies among different individuals as well. Figure 4 shows TPD52 expression level in six different human liver tissues. All the tissues used have a high number of LDs. However, not all are classified as NAFLD or NASH.

The 6 samples were randomly picked to show the heterogeneity of

22 TPD52 expression among individuals. The laboratory of Dr. Peter

Metrakos supplied the information on the samples using the histological scoring system for NAFLD also called Kleiner (NASH CRN) system (18). The system is used over conventional scoring systems because it covers a broader spectrum. Since only LD fractions were used for analysis, no protein loading control such as GAPDH or calnexin was used. Equal amount (103g) of LD proteins were loaded.

TPD52 expression around the LD varies from different individual. Its expression level shows no obvious correlation to the state and the histological features of liver tissue (steatosis, lobular inflammation, fibrosis). Therefore, although TPD52 is expressed around or nearby

LD, the expression levels vary in different cases. The reason for the heterogeneity of the expression is still unknown.

23 kD

LD fraction Sample Sample Sample Sample Sample Sample ONLY 1 2 3 4 5 6

23 B)

TPD52 expression level in human hepatic LD fractions

C) Lobular Steato Fibrosis % Total Human hepat Grading sis Inflammat score steatosis tissue sampl Sex Age ion Liver Femal 0 1 1A 0 LOW Sample 1 e 51 Liver LOW Sample 2 Male 77 0 0 1A 0 Liver LOW Sample 3 Male 70 0 0 1A 1 Liver 1 1 1A 15 NAFLD Sample 4 Male 51 Liver 1 2 1C 53 NAFLD Sample 5 Male 69 Liver Femal 3 2 1A 87.5 NASH Sample 6 e 55 D)

Item Score Extent

0 <5% Steatosis 1 5-33% (Amount of surface area involved by steatosis) 2 >33-66% 3 >66% 0 No foci Lobular Inflammation 1 <2 foci/200x

24 2 2-4 foci/200x 3 >4 foci/200x 0 None 1 Perisinusoidal or periportal 1A (delicate Mild, zone 3, perisinusoidal fibrosis) Moderate, zone 3, 1B (dense fibrosis) Fibrosis perisinusoidal

1C Portal/periportal Perisinusoidal and 2 portal/periportal 3 Bridging fibrosis 4 Cirrhosis Figure 4. TPD52 expression in human hepatic LD fractions is

heterogeneous.

Human hepatic LD fractions were used. LD fraction was delipidated.

Protein concentration was quantified using Bradford protein assay.

107g protein of each fraction is loaded to ensure equal amount of

protein for each fraction. The experiment was performed three

times and a representative result is shown here.

A) 6 LD fractions from different human hepatic samples were used

for WB. All LD fractions were delipidated. TPD52 antibody was used

as the primary antibody.

B) To semi-quantify level of TPD52 expression, the band at 23kDa is

used, which is the size expected and published in other TPD52

related paper. ImageJ gel analysis tool was using to semi-

quantitatively evaluate the intensity of the band.

C) The human hepatic samples used in A) were evaluated by Dr.

25 Peter Metrakos’ laboratory using the histological scoring system for

NAFLD (44). D) A table explaining the scoring system used in C) is

modified and adapted in order to explain the histological scoring

system for NAFLD (44, 45).

Part III: Exogenous TPD52 is expressed under various conditions.

From the results shown earlier, it is obvious that TPD52 is localized on LD surface. However, it is unclear whether its localization can change. LD biogenesis has different stages (formation, maturation, fusion/fission) that acquire different proteins on LDs. For instance,

ARFGAP1 was only found on LD at 4-hour incubation with oleate (46).

ARFGAP1 localized to the Golgi before 4 hours, is recruited to the LD surface and then returns to the Golgi after 8-12 hours (46). To investigate possible temporal aspects of TPD52 on LD, a plasmid expressing TPD52 with an eGFP tag was transfected into HepG2 cell line. After oleate induction, the cells were fixed at different time points to observe the localization of exogenous TPD52 after oleate induction

(Figure 5). Although partially cytosolic, TPD52 was found on LDs during the whole time course, suggesting TPD52 recruitment to LD surface might act independently of the stage of LD biogenesis.

26 Figure 5. Exogenous TPD52 is found on LDs across different time points after oleate induction.

27 HepG2 cell line is transfected with the plasmid having TPD52 gene with

eGFP tag for 24 hours. 0.5mM OA final concentration was added. Cells

were then fixed at different time points (2, 4, 6, 8, 12, 24 hours after

oleate was added to the cells) with 3% paraformaldehyde. Dotted lines

were added at picture processing step to show the membrane of each

cell.

A tetracycline inducible TPD52 expression HEK293 cell line (Flp-

In™ T-REx™ 293 Cell Line) was created to study TPD52 localization and its intracellular effect. The cell line contains the protein of interest that could be stably expressed upon tetracycline induction (generated by Dr. Jarred Chicoine and Dr. Rob Sladek). The exogenous protein expression level can be controlled by the amount of tetracycline induction.

There exists other types of lipid than oleate that could form LDs.

TG and CE may differ in their structure if the lipid source used is changed, and this may result in different LD composition. Thus, TPD52 localization may change depending on the type of lipid source available in the cell culture media. Besides oleic acid (OA), arachidonic acid (AA) was used to trigger formation of LDs (Figure 6). Exogenous TPD52 is found around LDs independent of the source of lipid available.

28 Figure 6. Exogenous TPD52 is found on LDs generated using

different lipid source.

HEK293 cell line with a tetracycline inducible eGFP tagged TPD52

(eGFP-TPD52) was used. Tetracycline was induced 24 hours to trigger

eGFP-TPD52 expression. 0.3mM OA or AA was added for 4 hours. Cells

were then fixed with 3% paraformaldehyde. LDs (red) were stained

with Nile Red.

We were also interested in testing the binding ability of TPD52 to

LD surface. It was mentioned previously that TPD52 is a cytosolic protein that is recruited to LDs for unknown reasons. Fluorescence recovery after photobleaching (FRAP) was used to quantify the

29 mobility of GFP tagged protein. This technique was performed on the tetracycline inducible eGFP-TPD52 HEK293. The fluorescence of eGFP- tagged TPD52 was bleached with a strong excitation laser in one region of the cell with LDs. The fluorescence of the region reverts when the unbleached eGFP-tagged TPD52 from outside of the region diffuses into the bleached region. (47) Pictures were taken every 6.25s using confocal microscopy. It is shown that TPD52 on LDs could recover following photobleaching (Figure 7). However, only ~70% of

TPD52 was recovered after 100 seconds. Note that after 6.25 seconds,

TPD52 associating with the LDs nearby the bleached area was reduced significantly. This suggests the recruitment of TPD52 on LDs primarily comes from other LDs nearby.

30 A)

31 B)

Figure 7. FRAP analysis of TPD52 mobility on LD.

A) HEK293 cell line with a tetracycline inducible eGFP tagged TPD52

(eGFP-TPD52) was used. Tetracycline was induced for 24 hours to trigger eGFP-TPD52 expression. 0.3mM OA was added for 24 hours.

Area in the square was the photobleached area. Unbleached reference area was marked with “R”. Area marked as “B” was used for background intensity measurement.

B) 3 fields in the bleached area were randomly chosen to measure the intensity of fluorescence using ImageJ. Averaged MOI was plotted as the percentage of intensity of the unbleached area. This experiment was repeated 3 times, and a representative sample is shown here. The live cell imaging was done at room temperature to slow down the recovery process.

32 Part IV: TPD52 family proteins (TPD52L1 & TPD52 L2) are also found on lipid droplet

Since TPD52 is found on LD surface in hepatocytes, it is expected to localize TPD52L1 and TPD52L2 to the LD surface.

Immunofluorescence staining was done on HepG2 cell line and on primary human liver tissue. HepG2 cells were also transfected with eGFP tagged plasmid to locate the exogenously expression proteins

(Figure 8). Both endogenous staining and exogenous expression reveals TPD52L1 and TPD52L2 localization on LDs. It is as expected since TPD52L1 and TPD52L2 are two proteins known to interact with

TPD52. This proves furthermore that TPD52 is located on LD surface under high oleate condition in human hepatocyte cell line and in high

LDs primary human hepatic tissue.

33 A) TPD52L1 & Human liver sample B) TPD52L2 & HepG2

C) Exogenous TPD52L1 in HepG2 D) Exogenous TPD52L2 in HepG2

Figure 8. Both TPD52L1 and TPD52L2 are two known members of TPD52 family that localize on LD surface.

A) Thin frozen sections of primary human hepatic tissue was used for immunofluorescence staining to reveal endogenous TPD52L1.

34 Bodipy493/503 (blue) was used to stain LD. PLIN2 (green) antibody

was used as a LD surface marker, and AlexaFluor596 was used as the

secondary antibody. Endogenous TPD52L1 (red) is revealed using a

rabbit polyclonal and AlexaFluor647.

B) HepG2 cells were fixed and processed for immunofluorescence 4

hours after addition of 0.5mM oleate. The cells were stained to reveal

endogenous TPD52L2 (red) using a rabbit polyclonal to TPD52L2. LDs

(blue) were stained using Bodipy493/503.

C) HepG2 cells were transfected with plasmid DNA encoding eGFP

tagged TPD52L1 for 16 hours followed by 4 hours incubation with

0.5mM oleate, and then fixed using 3% paraformaldehyde. Cells were

stained with Nile Red to reveal LDs (red).

D) HepG2 cells were transfected with plasmid DNA encoding eGFP

tagged TPD52L2 for 20 hours followed by 4 hours incubation with

0.5mM oleate and fixed. LDs (red) were stained using Nile Red.

It was previously shown that TPD52 expression level differs

between individuals (Figure 4A). In order to compare the level of

expression of the proteins of interest (TPD52 & TPD52L1), equal

amount (109g) of samples were loaded. Figure 9 shows that TPD52L1 also expresses heterogeneously in each individual. For different individuals, the level of expression varies in a significant manner.

35 Interestingly, TPD52 expression level does not correlate with TPD52L1 expression level. However, Sample 5 and Sample 6 show very low or no expression of both TPD52 and TPD52L1. This suggests these two individuals may not have any members of the TPD52 family in their liver. Both samples scored over 50% in the total percentage of steatosis and relatively high lobular inflammation comparing to the other samples

Figure 9. TPD52L1 expression in human hepatic LD fractions is heterogeneous and differs from TPD52 expression.

36 Human hepatic samples were fractionated by ultracentrifuge. LD

fraction was delipidated. Protein concentration was quantified using

Bradford protein assay. 102g protein of each fraction is loaded to ensure equal amount of protein for each fraction.

A) LD fractions from six human hepatic samples were used for WB. All samples were loaded in the same order and under the same conditions for Figure 4A) and 9A). All LD fractions were delipidated. TPD52 antibody was used as the primary antibody.

B) To semi-quantify level of TPD52 expression, the band at 22kDa is used, which is the size expected and published in other TPD52 related paper. ImageJ gel analysis tool was used to semi-quantify the amount of TPD52L1.

Part V: LAMTOR1- TPD52 interacting protein that is involved in

autophagy

In order to analyze the function of TPD52 in high oleate loading

condition, a technique called Bio-ID was employed. Bio-ID is a newly

developed tool that allows labeling of interacting proteins in vivo. It

allows the sensitive detection of both stable and transient interactions

through local diffusion of activated biotin tagged protein of interest

(48, 49). In other words, the biotin-tag of TPD52 biotinylates the

proteins in proximity under physiological conditions. The technique is

37 considered reliable since it detects proteins within 20nm of the protein of interest. Our laboratory employed this method to detect protein interactions during LDs’ biogenesis. TPD52 interacts with LAMTOR1, which is the lysosomal anchoring component of the Ragulator complex.

Since TPD52 is found on LDs in human hepatocytes, we would predict that LAMTOR1 might also localize to the LDs. HepG2 cell lines were used to detect the localization of endogenous LAMTOR1 (Figure 10).

LAMTOR1 co-localizes with PLIN2 where LDs are found. This suggests that LAMTOR1 is on LD surface. This is expected since TPD52 is found to interact with LAMTOR1 using Bio-ID.

38 Figure 10. LAMTOR1 co-localizes with PLIN2 on LDs.

HepG2 cells were fixed and processed for immunofluorescence staining

4 hours after addition of 0.4mM oleate. The staining reveals endogenous LAMTOR1 (red) using a polyclonal rabbit, PLIN2 (green) using a polyclonal guinea pig, and lipid droplet (blue) using Bodipy

493/503.

39 Figure 11. Endogenous LAMTOR1 is located on LDs of NAFLD human liver.

The tissue sample is frozen in isopentane chilled in liquid nitrogen to preserve its native form. The tissue is cut thinly with microtome and fixed using paraformaldehyde as described in Materials and Methods.

Immunofluorescence staining was performed to detect endogenous level of LAMTOR1 (red). LDs (green) are detected using Bodipy

40 493/503. The z-stack pictures were taken using confocal microscopy.

A total of 10 pictures along the z-axis were taken. 3D project was reconstructed using ImageJ.

LAMTOR1 in primary human liver tissue was also observed

(Figure 11). Thin frozen sections were generated from patients liver tissue as described in Figure 2. This is to ensure that LAMTOR1’s localization observed in Figure 10 is not due to overloading of oleate in the tissue culture model. Moreover, LAMTOR1 localization on LDs in human hepatic sample shows LAMTOR1’s potential relevance with

NAFLD an NASH. Figure 11 reveals LAMTOR1 localization on LD by immunofluorescence staining of LAMTOR1 and LDs. A 3-Dimension project was created using z-stack function of the confocal microscopy to take pictures along z-axis. It proves that LAMTOR1 is on the LD surface. LAMTOR1 makes a round ball structure around LDs. This is expected since TPD52 is found on LDs, and the Bio-ID reveals

LAMTOR1 as a potential binding partner of TPD52. However, not all

LDs have LAMTOR1 on their surfaces. We could therefore confirm

LAMTOR1 localization on LDs at steady and physiological state.

41 DISCUSSION & CONCLUSION

Proteomics on human hepatic LD fractions revealed TPD52 as a novel protein on LDs in human liver. This paper proves that TPD52 associates with LDs in cultured human hepatocytes HepG2 upon addition of oleate based on immunofluorescence. It was also found on

LDs of primary human liver revealed by both immunofluorescence staining and western blot detection. Although TPD52 shows clear localization on LDs using the above techniques, we cannot exclude the possibilities that TPD52 is localized on the nearby membrane of other organelles (ER, mitochondria, peroxisomes, and lysosomes). For this reason, electron microscopy (EM) and colocalization experiment by immunofluorescence of the above organelles and TPD52 are underway.

Not all human livers have TPD52. The expression level varies from individual for unknown reasons. By expressing exogenous TPD52 that is eGFP tagged, it was shown that TPD52 association with LDs was stable once recruited. The recruitment of TPD52 on LDs is independent of the source of lipid (oleic acid and arachidonic acid) as long as LDs are formed. Tetracycline inducible TPD52 HEK293 was used for FRAP analysis for TPD52 recovery after photobleached. The result showed that TPD52 was immediately recruited to LDs by redistribution of

TPD52 from LDs in close proximity. However, TPD52 was only 70% recovered 100 seconds after bleached compare to non-bleached LDs.

42 All of the above observations suggest TPD52’s association with LDs is relatively stable.

Other members of TPD52 family were also found on LDs after oleate induction. Exogenous TPD52L1 and TPD52L2 were both found on LDs in HepG2. Using immunofluorescence, endogenous TPD52L2 was found on LDs in HepG2. TPD52L1 associated with LDs at steady state in human liver. Just like TPD52, TDP52L1 does not express at the same level in all human livers tested. The expression level of TPD52 in human liver is different from TPD52L1. In other words, elevated TPD52 expression in human liver does not necessary correlate with high

TPD52L1 expression. It was mentioned previously that TPD52L2 could also form dimers with TPD52. Moreover, TPD52 may homodimerize.

Nevertheless, TPD52L1 and TPD52L2 localization on LDs furthermore supports TPD52’s association with LDs.

The Bio-ID technique was employed to find TPD52 interacting proteins in order to have an understanding of TPD52’s function on LDs.

This novel technique is advantageous since it identifies both stable and transient interacting proteins of TPD52 under physiological conditions.

Among many potential TPD52 binding partners, LAMTOR1 was chosen for further study. LAMTOR1 is a membrane protein found on the surface of late endosomes and lysosomes that serves as an anchoring protein for the Ragulator complex, which is related to mTORC1. The

43 latter is essential for many cellular functions such as energy metabolism and autophagy. Immunofluorescence data showed that

LAMTOR1 was also localized on LDs of HepG2 and at steady state on primary human liver tissue just like TPD52. This proves the possibility of TPD52 association with LAMTOR1 on LDs in human hepatocytes.

This suggests that TPD52 may also have a potential role on mTORC1 pathway like LAMTOR1. TPD52 is highly expressed in cancer cells. Dr.

Jenny Byrne’s group recently showed TPD52 expression on LDs in cancer cell lines, which increases TG level. (28) Since LAMTOR1 is known to bind to late endosomes and lysosomes, TPD52 may have a role in inhibiting lipophagy, and preventing energy stored in the form of TG being used for energy metabolism.

LAMTOR1 is a key regulator of mTORC1 activity. In the absence of LAMTOR1, the mTORC1 complex does not localize properly to the late endosomal/lysosomal membrane, and autophagy is aberrant, which lead to p53-dependent apoptosis (40). High levels of TPD52 have been associated with higher metastatic activity in cancer cells, suggesting that TPD52 may be providing some metabolic advantage to rapidly dividing cells (12). The novel interaction of LAMTOR1 and

TPD52 opens up an intriguing area of study. As LAMTOR1 is required for the anchoring of the Ragulator complex to the lysosome and nutrient-dependent regulation of mTORC1, and TPD52 binds the LD

44 under high lipid loading conditions, we propose that TPD52 sequesters

LAMTOR1 away from the lysosome, preventing nutrient-dependent regulation of mTORC1 by the lysosome. Alternatively, TPD52 could act as a bridge for lipophagy, by binding and recruiting LDs to the lipophagy machinery. Ser136 in TPD52 function may play a role in this regard and future experiments will test whether CAMKII stimulate or inhibit lipophagy. The relevance of TPD52 to cancer could be explained through a role in lipophagy where increased catabolism of

TGs may serve as an important energy source. Further studies are needed to elucidate TPD52’s function on LDs. For instance, it is observed that not all LDs have TPD52 on the surface. This might be due to different stages of LDs’ biogenesis, intra-LD composition or the metabolic state of the cell. LD association of TPD52 was observed in multiple human NAFLD livers. Its role in promoting cancer makes this an interesting protein to study, especially if TPD52 can be shown mechanistically to be causative of hepatocellular carcinoma.

45 MATERIALS AND METHODS

Materials

Nile Red, Triton X100, oleic acid, Tris, HCl, NacL, NA2HPO4,

KH2PO4, CaCl2, MgCl2, Tween 20, Fish skin gelatin, Saponin, mowiol,

NH4Cl, guanidine HCl, poly-L-Lysine, and HEPES were purchased form Sigma-Aldrich (Ontario, Canada). Acetone, KCl, DMSO, microscope cover glass and trypsin-EDTA were purchased from Fisher Scientific (Quebec, Canada). FuGENE HD Transfection Reagent, SDS and Tris base were purchased from Roche Ltd (Laval, Quebec, Canada). ECL detection kit was purchased from Perkin Elmer (Massachusetts, USA). PFA was from Mecalab Ltd. (Quebec, Canada). Mini-PROTEAN® TGXTM Precast gels, 2-Mercaptoethanol, Precision Plus ProteinTM WesternCTM standards, Midi Nitrocellulose Trans-Blot Turbo transfer pack, Laemmli 2X were all purchased from Bio-Rad (Ontario, Canada). MEM, Gibco DMEM, FBS, penicillin & streptomyocin, Bodipy 493/503, 0.25% trypsin-EDTA, hygromycin, and Blasticidin were purchased from Invitrogen (California, USA). The guinea pig polyclonal antibody to PLIN2 was purchased from PROGEN Biotechnik GmbH (Heidelberg,

Germany). The rabbit polyclonal antibodies to TPD52, TPD52L1, TPD52L2 were purchased from Sigma-Aldrich (Ontario, Canada). The rabbit polyclonal antibody to LAMTOR1 was from New England Biolabs, Ltd. (Ontario, Canada). AlexaFluor 488, AlexaFluor 568, AlexaFluor 594, and AlexaFluor 647 were all purchased from Dianova (Hamberg, Germany). Protein A-HRP conjugate was purchased form Bio-Rad (Ontario, Canada). TPD52-EGFP was kindly provided by Dr. Robert

46 Sladeck (McGill University and Genome Quebec Innovation Centre, M.D.).

Cell lines and culture conditions Hek293 cells were grown in DMEM with 10% FBS, 100U/mL

penicillin & 100mg/mL streptomycin, 50g/mL hygromycin, and 10g/mL blasticidin. HepG2 cell lines were grown in the DMEM with 10% FBS, 100U/mL penicillin & 100mg/mL streptomycin with 10mM HEPES. All cell lines were incubated at 37 degrees Celcius and 5%

CO2. LDs were induced by adding 0.3mM-0.5mM oleate from 100mM stock in ethanol to the media. In order to induce TPD52-eGFP in

Hek293 cell line, tetracycline was added (20ng/mL-1g/mL) directly to the DMEM media described previously without hygromycin or blasticidin for 24 hours.

Immunofluorescence and confocal microscopy For cells staining, cells were growing on glass microscope cover glass pre-coated with poly-L-Lysine. The immunofluorescence staining for cells were processed as described in (50). 1 g/mL Bodipy 493/503 and 0.2 g/mL Nile Red were added in the secondary antibodies to stain LDs. For primary human liver, tissue cryosections were cut from isopentane frozen tissue with a 6-9 micron thickness. The tissue samples were fixed using 3% PFA for 20 min. The fixed samples were merged in 6N guanidine hydrochloride for 10 min, then permeabilized using 0.2% Triton X100 for 20 min. They are blocked using 10% goat serum for 30 min. All primary antibodies were diluted in 5% goat serum and incubated on the samples for 60 min. All secondary

47 antibodies were diluted in PBS and incubated on the samples for 40 min. Coverslips were mounted in Moviol. Zeiss LSM 780 was used for image acquisition. For Alexa 488 and Bodipy 493/503, a 488 nm Argon ion laser was used. Alexa 568, Alexa 594, and Nile Red were excited using 561 nm DPSS laser.

Transfection HepG2 cells were transfected using FuGENE HD according to the

manufacturer instructions. 1 g/mL plasmid DNA was used in all HepG2 transfection. Cells were cultured in DMEM supplemented with 10% FBS only. HepG2 were transfection for 16-24 hours in all the experiments performed. EGFP were excited using the 488 nm laser.

Western blotting Samples were quantified using Bio-Rad protein Assay dye to perform Bradford assay. Wavelength of each sample was determined using spectrophotometer to measure absorbance at 595nm. Samples were prepared using 2X Laemmli and 0.7M 2-Mercaptoethanol, then boiled at 95 degrees Celcius for 15 min. All samples were run on Mini-

PROTEAN® TGXTM Precast gels at 4-10 Amp/gel. Proteins were transferred using Midi Nitrocellulose Trans-Blot Turbo kit and Trans- Blot Turbo transfer system from Bio-Rad (Ontario, Canada). Membranes were incubated overnight with primary antibody diluted in 0.1% skim milk at 4 degrees Celsius after 30 min blocking with 5% skim milk, then with Protein A-HRP conjugate diluted in 0.1 % skim milk for 2 hours at room temperature. ECL was added just before chemiluminescence detection using ImageQuant LAS 4000 from GE

48 Healthcare (Quebec, CA). The amount of protein was semi- quantitatively determined based on the intensity of the band identified using ImageJ gel analysis option.

FRAP For FRAP analysis of protein dynamic on LDs, MatTek dishes from MatTek (Massachusetts, USA) were pre-coated with poly-L-lysine. Hek293 cells were grown for at least 24 hours and imaged using LSM780 using a 63X/1.40 Oil DIC objective. After five scanning images with a 6.25 seconds interval, the selected region was bleached by 60 interactions at 100% 488 Argon ion laser followed by continuous image scanning with a 6.25 seconds interval (51). ImageJ was used for semi-quantify TPD52-eGFP. 3 areas with identical size in the bleached area was chosen and manually tracked throughout the time course. Mean gray value of each area was determined. The value was corrected with background intensity and normalized using mean gray value of unbleached area.

49 REFERENCE

1. Pol, A., Gross, S. P., & Parton, R. G. (2014). Biogenesis of the multifunctional lipid droplet: Lipids, proteins, and sites. J Cell Biol, 204(5), 635-646. doi: 10.1083/jcb.201311051 2. Kalantari, F., Bergeron, J. J., & Nilsson, T. (2010). Biogenesis of lipid droplets--how cells get fatter. Mol Membr Biol, 27(8), 462-468. doi: 10.3109/09687688.2010.538936 3. Herms, A., Bosch, M., Ariotti, N., Reddy, B. J., Fajardo, A., Fernandez-Vidal, A., . . . Pol, A. (2013). Cell-to-cell heterogeneity in lipid droplets suggests a mechanism to reduce lipotoxicity. Curr Biol, 23(15), 1489-1496. doi: 10.1016/j.cub.2013.06.032 4. Martin, S., & Parton, R. G. (2006). Lipid droplets: a unified view of a dynamic organelle. Nat Rev Mol Cell Biol, 7(5), 373-378. doi: 10.1038/nrm1912 5. Hapala, I., Marza, E., & Ferreira, T. (2011). Is fat so bad? Modulation of endoplasmic reticulum stress by lipid droplet formation. Biol Cell, 103(6), 271-285. doi: 10.1042/BC20100144 6. Arrese, E. L., Saudale, F. Z., & Soulages, J. L. (2014). Lipid Droplets as Signaling Platforms Linking Metabolic and Cellular Functions. Lipid Insights, 7, 7-16. 7. Leber, R., Zinser, E., Zellnig, G., Paltauf, F., & Daum, G. (1994). Characterization of lipid particles of the yeast, Saccharomyces cerevisiae. Yeast, 10(11), 1421-1428. doi: 10.1002/yea.320101105 8. Bickel, P. E., Tansey, J. T., & Welte, M. A. (2009). PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores. Biochim Biophys Acta, 1791(6), 419-440. doi: 10.1016/j.bbalip.2009.04.002 9. Hickenbottom, S. J., Kimmel, A. R., Londos, C., & Hurley, J. H. (2004). Structure of a lipid droplet protein; the PAT family member TIP47.

50 Structure, 12(7), 1199-1207. doi: 10.1016/j.str.2004.04.021 10. Guo, Y., Walther, T. C., Rao, M., Stuurman, N., Goshima, G., Terayama, K., . . . Farese, R. V. (2008). Functional genomic screen reveals genes involved in lipid-droplet formation and utilization. Nature, 453(7195), 657-661. doi: 10.1038/nature06928 11. Liu, C. J. (2012). Prevalence and risk factors for non-alcoholic fatty liver disease in Asian people who are not obese. J Gastroenterol Hepatol, 27(10), 1555-1560. doi: 10.1111/j.1440- 1746.2012.07222.x 12. Dowman, J. K., Tomlinson, J. W., & Newsome, P. N. (2010). Pathogenesis of non-alcoholic fatty liver disease. QJM, 103(2), 71- 83. doi: 10.1093/qjmed/hcp158 13. Khedmat, H., & Taheri, S. (2011). Non-alcoholic steatohepatitis: An update in pathophysiology, diagnosis and therapy. Hepat Mon, 11(2), 74-85. 14. Angulo, P. (2010). Long-term mortality in nonalcoholic fatty liver disease: is liver histology of any prognostic significance? Hepatology, 51(2), 373-375. doi: 10.1002/hep.23521 15. Torres, D. M., Williams, C. D., & Harrison, S. A. (2012). Features, diagnosis, and treatment of nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol, 10(8), 837-858. doi: 10.1016/j.cgh.2012.03.011 16. Stepanova, M., & Younossi, Z. M. (2012). Independent association between nonalcoholic fatty liver disease and cardiovascular disease in the US population. Clin Gastroenterol Hepatol, 10(6), 646-650. doi: 10.1016/j.cgh.2011.12.039 17. Targher, G., Bertolini, L., Padovani, R., Poli, F., Scala, L., Tessari, R., . . . Falezza, G. (2006). Increased prevalence of cardiovascular disease in Type 2 diabetic patients with non-alcoholic fatty liver disease. Diabet Med, 23(4), 403-409. doi: 10.1111/j.1464-5491.2006.01817.x

51 18. Lavallard, V. J., & Gual, P. (2014). Autophagy and Non-Alcoholic Fatty Liver Disease. Biomed Res Int, 2014, 120179. doi: 10.1155/2014/120179 19. Byrne, J. A., Frost, S., Chen, Y., & Bright, R. K. (2014). Tumor protein D52 (TPD52) and cancer-oncogene understudy or understudied oncogene? Tumour Biol, 35(8), 7369-7382. doi: 10.1007/s13277-014-2006-x 20. Cao, Q., Chen, J., Zhu, L., Liu, Y., Zhou, Z., Sha, J., . . . Li, J. (2006). A testis-specific and testis developmentally regulated tumor protein D52 (TPD52)-like protein TPD52L3/hD55 interacts with TPD52 family proteins. Biochem Biophys Res Commun, 344(3), 798-806. doi: 10.1016/j.bbrc.2006.03.208 21. Yang, M., Wang, X., Jia, J., Gao, H., Chen, P., Sha, X., & Wu, S. (2015). Tumor protein D52-like 2 contributes to proliferation of breast cancer cells. Cancer Biother Radiopharm, 30(1), 1-7. doi: 10.1089/cbr.2014.1723 22. Thomas, D. D., Kaspar, K. M., Taft, W. B., Weng, N., Rodenkirch, L. A., & Groblewski, G. E. (2002). Identification of annexin VI as a Ca2+- sensitive CRHSP-28-binding protein in pancreatic acinar cells. J Biol Chem, 277(38), 35496-35502. doi: 10.1074/jbc.M110917200 23. Thomas, D. D., Martin, C. L., Weng, N., Byrne, J. A., & Groblewski, G. E. (2010). Tumor protein D52 expression and Ca2+- dependent phosphorylation modulates lysosomal membrane protein trafficking to the plasma membrane. Am J Physiol Cell Physiol, 298(3), C725-739. doi: 10.1152/ajpcell.00455.2009 24. Chew, C. S., Chen, X., Zhang, H., Berg, E. A., & Zhang, H. (2008). Calcium/calmodulin-dependent phosphorylation of tumor protein D52 on serine residue 136 may be mediated by

52 CAMK2delta6. Am J Physiol Gastrointest Liver Physiol, 295(6), G1159-1172. doi: 10.1152/ajpgi.90345.2008 25. Groblewski, G. E., Yoshida, M., Yao, H., Williams, J. A., & Ernst, S. A. (1999). Immunolocalization of CRHSP28 in exocrine digestive glands and gastrointestinal tissues of the rat. Am J Physiol, 276(1 Pt 1), G219-226. 26. Wilson, S. H., Bailey, A. M., Nourse, C. R., Mattei, M. G., & Byrne, J. A. (2001). Identification of MAL2, a novel member of the mal proteolipid family, though interactions with TPD52-like proteins in the yeast two- hybrid system. Genomics, 76(1-3), 81-88. doi: 10.1006/geno.2001.6610 27. De Marco, M. C., Martin-Belmonte, F., Kremer, L., Albar, J. P., Correas, I., Vaerman, J. P., . . . Alonso, M. A. (2002). MAL2, a novel raft protein of the MAL family, is an essential component of the machinery for transcytosis in hepatoma HepG2 cells. J Cell Biol, 159(1), 37-44. doi: 10.1083/jcb.200206033 28. Kamili, A., Roslan, N., Frost, S., Cantrill, L. C., Wang, D., Della-Franca, A., . . . Byrne, J. A. (2015). TPD52 expression increases neutral lipid storage within cultured cells. J Cell Sci. doi: 10.1242/jcs.167692 29. Shibata, M., Yoshimura, K., Furuya, N., Koike, M., Ueno, T., Komatsu, M., . . . Uchiyama, Y. (2009). The MAP1-LC3 conjugation system is involved in lipid droplet formation. Biochem Biophys Res Commun, 382(2), 419-423. doi: 10.1016/j.bbrc.2009.03.039 30. Mehrpour, M., Esclatine, A., Beau, I., & Codogno, P. (2010). Autophagy in health and disease. 1. Regulation and significance of autophagy: an overview. Am J Physiol Cell Physiol, 298(4), C776-785. doi: 10.1152/ajpcell.00507.2009 31. Yang, Z., & Klionsky, D. J. (2010). Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol, 22(2), 124-131. doi: 10.1016/j.ceb.2009.11.014 32. Singh, R., & Cuervo, A. M. (2011). Autophagy in the cellular energetic

53 balance. Cell Metab, 13(5), 495-504. doi: 10.1016/j.cmet.2011.04.004 33. Lubke, T., Lobel, P., & Sleat, D. E. (2009). Proteomics of the lysosome. Biochim Biophys Acta, 1793(4), 625-635. doi: 10.1016/j.bbamcr.2008.09.018 34. Mindell, J. A. (2012). Lysosomal acidification mechanisms. Annu Rev Physiol, 74, 69-86. doi: 10.1146/annurev-physiol-012110-142317 35. Soliman, G. A. (2011). The integral role of mTOR in lipid metabolism. Cell Cycle, 10(6), 861-862. 36. Zhou, J., Tan, S. H., Nicolas, V., Bauvy, C., Yang, N. D., Zhang, J., . . . Shen, H. M. (2013). Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome- lysosome fusion. Cell Res, 23(4), 508-523. doi: 10.1038/cr.2013.11 37. Thoreen, C. C., Kang, S. A., Chang, J. W., Liu, Q., Zhang, J., Gao, Y., . . . Gray, N. S. (2009). An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem, 284(12), 8023-8032. doi: 10.1074/jbc.M900301200 38. Feldman, M. E., Apsel, B., Uotila, A., Loewith, R., Knight, Z. A., Ruggero, D., & Shokat, K. M. (2009). Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol, 7(2), e38. doi: 10.1371/journal.pbio.1000038 39. Nada, S., Mori, S., Takahashi, Y., & Okada, M. (2014). p18/LAMTOR1: a late endosome/lysosome-specific anchor protein for the mTORC1/MAPK signaling pathway. Methods Enzymol, 535, 249-263. doi: 10.1016/b978- 0-12-397925-4.00015-8 40. Malek, M., Guillaumot, P., Huber, A. L., Lebeau, J., Petrilli, V., Kfoury, A., . . . Manie, S. N. (2012). LAMTOR1 depletion induces p53-dependent apoptosis via aberrant lysosomal activation. Cell Death Dis, 3, e300. doi: 10.1038/cddis.2012.39

54 41. Ding, W. X., Li, M., & Yin, X. M. (2011). Selective taste of ethanol-induced autophagy for mitochondria and lipid droplets. Autophagy, 7(2), 248-249. 42. Singh, R., Kaushik, S., Wang, Y., Xiang, Y., Novak, I., Komatsu, M., . . . Czaja, M. J. (2009). Autophagy regulates lipid metabolism. Nature, 458(7242), 1131-1135. doi: 10.1038/nature07976 43. Kwanten, W. J., Martinet, W., Michielsen, P. P., & Francque, S. M. (2014). Role of autophagy in the pathophysiology of nonalcoholic fatty liver disease: a controversial issue. World J Gastroenterol, 20(23), 7325-7338. doi: 10.3748/wjg.v20.i23.7325 44. Kleiner, D. E., Brunt, E. M., Van Natta, M., Behling, C., Contos, M. J., Cummings, O. W., . . . Sanyal, A. J. (2005). Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology, 41(6), 1313-1321. doi: 10.1002/hep.20701 45. Pittsburgh University UPMC (2015). Transplant Pathology Internet Services. from http://tpis.upmc.com/changebody.cfm?url=/tpis/schema/NAFLD2006.jsp 46. Gannon, J., Fernandez-Rodriguez, J., Alamri, H., Feng, S. B., Kalantari, F., Negi, S., . . . Nilsson, T. (2014). ARFGAP1 Is Dynamically Associated with Lipid Droplets in Hepatocytes. PLoS One, 9(11), e111309. doi: 10.1371/journal.pone.0111309 47. Zheng, C.-Y., Petralia, R. S., Wang, Y.-X., & Kachar, B. (2011). Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons. Journal of Visualized Experiments: JoVE, (50), 2568. doi:10.3791/2568 48. Lambert, J. P., Tucholska, M., Go, C., Knight, J. D., & Gingras, A. C. (2015). Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin- associated protein complexes. J Proteomics, 118, 81-94. doi:

55 10.1016/j.jprot.2014.09.011 49. Dingar, D., Kalkat, M., Chan, P. K., Srikumar, T., Bailey, S. D., Tu, W. B., . . . Raught, B. (2015). BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors. J Proteomics, 118, 95- 111. doi: 10.1016/j.jprot.2014.09.029 50. Fullekrug, J., Suganuma, T., Tang, B. L., Hong, W., Storrie, B., & Nilsson, T. (1999). Localization and recycling of gp27 (hp24gamma3): complex formation with other p24 family members. Mol Biol Cell, 10(6), 1939-1955. 51. Gong, J., Sun, Z., Wu, L., Xu, W., Schieber, N., Xu, D., . . . Li, P. (2011). Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites. J Cell Biol, 195(6), 953-963. doi: 10.1083/jcb.201104142