Characterization of an NBEAL2-Interacting Partner

Characterization of an NBEAL2-interacting partner

Kevin To

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Biochemistry University of Toronto

Characterization of an NBEAL2-interacting partner

Kevin To Master of Science Department of Biochemistry University of Toronto

2018

Abstract

Platelets are small, abundant blood cells involved in many processes. Loss of function of

Neurobeachin-like 2 (NBEAL2) is causative for Gray Platelet Syndrome (GPS), where patient platelets lack the crucial secretory α-granules. Thus, NBEAL2 is required for α-granule formation in platelet precursor megakaryocytes, but the functions and interaction partners of this protein are largely unknown. Here, I verified VAMP-associated protein A (VAPA) as an

NBEAL2-binding protein. Co-immunoprecipitation (Co-IP) experiments confirmed the

NBEAL2-VAPA interaction, which was mapped to at least five atypical FFAT motifs located within the PH-BEACH domain of NBEAL2. Structural modeling and docking simulations implicated one motif, termed ncFFAT*, however, Co-IP experiments with individual acidic residues in ncFFAT* (E2218K and D2224K) suggested that these residues are not uniquely involved. Immunofluorescence microscopy of human megakaryocytes and megakaryocytic cells suggested a partial interaction of NBEAL2 and VAPA. These studies suggest that NBEAL2-

VAPA interactions may facilitate platelet α-granule formation.

Acknowledgements

I would like to express my deepest appreciation to my supervisor, Dr. Walter Kahr, for his guidance, inspiration, and unending patience during my graduate experience. I am grateful to my supervisory committee Dr. Angus McQuibban and Dr. Aleixo Muise for their insight, guidance, and constructive criticisms. The work presented here would not have been possible without the contributions of my colleagues, former and current, from the Kahr and Trimble Labs. I am especially grateful to Dr. Ling Li for her boundless enthusiasm and positivity that allowed me to persevere through challenging times. I owe much of my technical proficiency to her wonderful teaching. I am also grateful to Dr. Fred Pluthero for insightful discussions, microscopy expertise, and boundless knowledge in random scientific facts. Thank you for unending support and enthusiasm. I thank other colleagues Stephanie Nguyen, Carrie Chen, Marko Drobac, and Richard Liu for their helpful advice and support, especially Richard Lo for providing the initial results for my project; Dr. William Trimble for insightful discussions; and to all members of the Trimble Lab for helpful advice and sharing of reagents. Last, but not least, I would like to thank my parents Hoa and Tieu for their faith and support throughout my graduate experience.

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Table of Contents

Acknowledgements...... iii Table of Contents ...... iv List of Tables ...... vii List of Figures ...... viii List of Appendices ...... x List of Abbreviations ...... xi Chapter 1: Introduction ...... 1 1.1 Platelets: effectors of haemostasis and other processes ...... 1 1.2 Platelet secretory granules ...... 3 1.3 Platelet production by megakaryocytes...... 5 1.4 The endosome is a crucial structure in α-granule production ...... 7 1.4.1 Endosome maturation facilitates α-granule formation and release ...... 7 1.4.2 α-Granules are sorted at the endosome and trafficked into proplatelet shafts ...... 8 1.5 NBEAL2 is implicated in α-granule formation as its absence causes Gray Platelet Syndrome ...... 10 1.6 NBEAL2 is predicted to have roles in vesicular and protein-scaffolding events ..... 13 1.7 Rationale and hypothesis ...... 14 Chapter 2: Materials and methods ...... 15 2.1 Lab reagents, antibodies, and plasmids ...... 15 2.2 Cell maintenance and growth ...... 15 2.3 Plasmid cloning ...... 17 2.3.1 Yeast plasmids ...... 17 2.3.2 Cloning of mammalian overexpression constructs ...... 17 2.4 Yeast-2-Hybrid screen...... 19 2.5 Mammalian transfections and preparing cell lysates ...... 22 2.6 Co-Immunoprecipitation preparation and procedures...... 23 2.6.1 Co-IP requiring pre-conjugated α-FLAG and control IgG beads ...... 23 2.6.2 Co-IP requiring self-conjugated α-HA, α-MYC, and control IgG beads ..... 24 2.7 Sodium dodecyl sulfate-polyacrylamide gel eletrophoresis and western blotting .... 24 2.8 Culturing and differentiation of human megakaryocytes ...... 25 2.9 Immunofluorescent microscopy ...... 25 2.10 Structural prediction and protein-protein docking simulations ...... 26 Chapter 3: Results ...... 27 3.1 Yeast-two-hybrid screening ...... 27 3.1.1 The majority of NBEAL2 truncations cloned into the Yeast-2-Hybrid pGBKT7 vector were unstable in DH5α E. coli ...... 27 3.1.2 NBEAL2 Fragment C was expressed in the yeast system and did not exhibit reporter autoactivation ...... 29 3.1.3 A small variety of binding partners were screened in Y2H with NBEAL2

Fragment C as bait ...... 31 3.2 Verifying candidate binding partners to NBEAL2 ...... 33 3.2.1 Cross-referencing binding partners from Y2H and affinity purification-mass spectrometry screens showed no overlap ...... 33 3.2.2 VAPA, among a list of AP-MS candidates, could be an NBEAL2-interacting protein ...... 33 3.3 Verifying the NBEAL2-VAPA interaction ...... 35 3.3.1 Full length MYC-VAPA can co-immunoprecipitate with HA-VAPB ...... 35 3.3.2 Full length MYC-VAPA can co-immunoprecipitate with GFP-NBEAL2- FLAG in HEK293 and NBEAL2-stable Dami cells ...... 35 3.3.3 Endogenous VAPA can Co-IP with GFP-NBEAL2-FLAG in NBEAL2- stable Dami cells ...... 39 3.4 Characterizing the interaction between NBEAL2 and VAPA ...... 40 3.4.1 HA-VAPB does not Co-IP with GFP-NBEAL2-FLAG in HEK293 cells ... 40 3.4.2 The PH-BEACH domains of NBEAL2 may be responsible for the VAPA interaction ...... 40 3.4.3 HA-C5 is predicted to harbour several atypical FFAT motifs for VAPA- binding ...... 42 3.5 Searching for relevant ncFFAT motifs in the NBEAL2-VAPA interaction ...... 45 3.5.1 The structure of the PH-BEACH regions in NBEAL2 is predicted to be homologous to that of NBEA ...... 45 3.5.2 The PH-BEACH regions of NBEAL2 and NBEA share similar ncFFAT motifs ...... 45 3.5.3 Predicted ncFFAT motifs could be surface-exposed on NBEAL2 ...... 48 3.5.4 Protein docking simulations implicate the ncFFAT* motif of NBEAL2 in VAPA-binding ...... 49 3.6 Identifying the important residues in the ncFFAT* motif of NBEAL2 for VAPA- binding ...... 52 3.6.1 Glu2218 and Asp2224 may be involved in VAPA-binding ...... 52 3.6.2 Glu2218 and Asp2224 do not individually mediate binding to VAPA ...... 53 3.7 Determining the in vivo localization pattern of VAPA in megakaryocytes ...... 55 3.7.1 Immunofluorescence microscopy suggested that VAPA partially-localized with GFP-NBEAL2-FLAG and RAB7 in the megakaryocytic NBEAL2- stable Dami cells ...... 55 3.7.2 Immunofluorescence microscopy suggested that VAPA partially localizes with secretory granules, the late endosomes, and NBEAL2 in human bone marrow-derived megakaryocytes ...... 55 Chapter 4: Discussion and future directions ...... 60 4.1 Understanding NBEAL2 function requires the identification of binding partners ... 60 4.2 Use of Yeast-2-hybrid screen to detect potential NBEAL2-binding partners ...... 60 4.3 The Y2H and AP-MS interaction lists are mutually exclusive ...... 63 4.4 NBEAL2 likely interacts with VAPA through an atypical FFAT motif...... 64 v

4.5 A transient NBEAL2-VAPA interaction...... 68 4.6 The potential binding specificity for VAPA as opposed to VAPB ...... 69 4.7 VAPA could be involved in granule formation in human megakaryocytes ...... 69 4.8 Possible roles of NBEAL2 in regulated transports through VAPA ...... 72 Chapter 5: Concluding remarks ...... 74 References ...... 76 Appendices ...... 83

List of Tables Table 1. List of antibodies used in Co-IP and western blotting ...... 15 Table 2. List of antibodies used in immunofluorescence studies ...... 16 Table 3. List of cells used ...... 17 Table 4. List of common lab reagents and equipment used ...... 18 Table 5. List of constructs, plasmid backbones, inserts and cloning sites used ...... 20 Table 6. List of PCR Primers and target plasmids used ...... 21 Table 7. Summary of interaction results obtained from 3 individual mating events with pGBKT7-C as bait against the human bone marrow library ...... 32 Table 8. Summary of interaction results obtained from 3 individual mating events with empty pGBKT7 against the human bone marrow library...... 32 Table 9. The PH-BEACH domains of NBEAL2 are predicted to contain at least five non-canonical FFAT motifs ...... 44 Table 10. NBEAL2 is predicted to also contain non-canonical FFAT motifs outside of the PH-BEACH region ...... 44

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List of Figures

Figure 1. Normal platelets on a blood film ...... 1 Figure 2. Platelet activation and hemostasis ...... 2 Figure 3. Platelet secretory granules ...... 3 Figure 4. Platelet ultrastructure ...... 4 Figure 5. An overview of thrombopoiesis ...... 6 Figure 6. Molecular organization of the mammalian endosome ...... 9 Figure 7. GPS patient platelets are devoid of α-granules ...... 11 Figure 8. Model of α-granule formation ...... 12 Figure 9. Schematic of NBEAL2 functional domains ...... 13 Figure 10. Domain architecture of BEACH domain containing proteins ...... 14 Figure 11. Schematic of the NBEAL2 cloning strategy for Y2H ...... 27 Figure 12. The verification of NBEAL2 construct instability through DNA restriction enzyme digestion ...... 28 Figure 13. pGBKT7-C construct is expressed in AH109 yeast cells ...... 30 Figure 14. pGBKT7-C tested negative for reporter autoactivation ...... 31 Figure 15. MYC-VAPAΔ co-immunoprecipitated with GFP-NBEAL2-FLAG in HEK293 cells ...... 34 Figure 16. MYC-VAPA co-immunoprecipitated with VAPB in HEK293 cells ...... 36 Figure 17. MYC-VAPA co-immunoprecipitated with GFP-NBEAL2-FLAG in HEK293 cells ...... 37 Figure 18. Full length MYC-VAPA co-immunoprecipitated with GFP-NBEAL2-FLAG in Dami cells ...... 38 Figure 19. Endogenous VAPA co-immunoprecipitated with GFP-NBEAL2-FLAG in the NBEAL2-stable Dami cells ...... 39 Figure 20. HA-VAPB did not co-immunoprecipitate with GFP-NBEAL2-FLAG in HEK293 cells ...... 41 Figure 21. MYC-VAPAΔ co-immunoprecipitated with specific domain containing fragments of GFP-NBEAL2-FLAG in HEK293 cells ...... 43 Figure 22. The PH-BEACH regions of NBEAL2 contains a prominent binding pocket ...... 46

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Figure 23. Sequence alignment of NBEAL2 and NBEA show that some predicted non-canonical FFAT motifs were conserved within the PH-BEACH regions ...... 47 Figure 24. All predicted non-canonical FFAT motifs were found on the surface of the PH-BEACH regions of NBEAL2 ...... 48 Figure 25. Summary of protein-protein docking simulations with the PH-BEACH regions of NBEAL2 and the MSP domain of VAPA ...... 49 Figure 26. The VAPA MSP domain is predicted to bind at the ncFFAT* motif of NBEAL2 ...... 51 Figure 27. Two acidic ncFFAT* motif residues are predicted to be on the surface of the PH-BEACH region of NBEAL2...... 52 Figure 28. Co-IP experiments using positive and negative controls resolved as expected ...... 53 Figure 29. Single amino acid mutations E2218K or D224K did not prevent MYC- VAPAΔ binding to HA-C5 ...... 54 Figure 30. VAPA partially co-localized with GFP-NBEAL2-FLAG and RAB7 in NBEAL2-stable Dami cells ...... 56 Figure 31. VAPA partially localized with secretory vesicles in human megakaryocytes ...... 58 Figure 32. VAPA partially colocalized with NBEAL2 and RAB7 in human megakaryocytes ...... 59 Figure 33. Overview of lipid composition in membranes of compartments associated with cell-endocytic pathway ...... 73 Figure 34. A hypothetical model of NBEAL2 function through interacting with VAPA...... 75

List of Appendices Figure A1. The PH-BEACH region of NBEA a contains a prominent binding pocket...... 83 Figure A2. Predicted non-canonical FFAT motifs were found on the surface of the PH-BEACH regions of NBEA ...... 83 Figure A3. Summary of protein-protein docking simulations with the PH-BEACH regions of NBEA and the MSP domain of VAPA ...... 84 Figure A4. The VAPA MSP domain is predicted to bind at a similar ncFFAT* motif of NBEA...... 85 Figure A5. Two acidic residues in the similar ncFFAT* motif are also predicted to be on the surface of the PH-BEACH regions of NBEA ...... 86 Figure A6. Mouse α-VAPA antibody colocalized well with rabbit α-VAPA antibody in HEK293 cells ...... 87 Table A1. The PH-BEACH regions of NBEA is predicted to contain at least five non- canonical FFAT motifs like NBEAL2 ...... 88 Table A2. NBEA was predicted to contain non-canonical FFAT motifs in other domains of the protein ...... 88 Table A3 Protein-protein docking simulations suggested the involvement of electrostatic and hydrophobic interactions ...... 89

List of Abbreviations

AD transcriptional Activation Domain Ade Adenine ADP Adenosine Diphosphate AP Autophagosome AP-MS Affinity Purification-Mass Spectrometry ARC Arthrogryposis, Renal tubular dysfunction, and Cholestasis ARM Armadillo domain BDCP BEACH-domain containing family of proteins BEACH Beige and Chediak-Higashi domain BLAST Basic Local Alignment Search Tool BSA Bovine Serum Albumin CCV Clathrin-Coated Vesicles CD61 Integrin β3 CD62P P-Selectin CD63 Lysosomal Associated Protein 3 CHS Chediak-Higashi Syndrome CIC Clathrin-Independent Carrier CIP Calf Intestinal Phosphatase Co-IP Co-Immunoprecipitation CON-A Concanavalin-A-like lectin domain COPB2 Coatomer Protein Complex Subunit Beta 2 DBD DNA Binding Domain DMEM Dulbecco’s Modified Eagle’s Media DNA Deoxyribunucleic Acid DTS Dense Tubular System ECL Enhanced Chemiluminescence ECM Extracellular Membrane ECV Endosomal Carrier Vesicle EDTA Ethylenediaminetetraacetic acid EE Early Endosome EEA1 Early Endosome-Associated 1 EM Electron Microscopy ER Endoplasmic Reticulum ESCRT Endosome Sorting Complex Required for Transport FBS Fetal Bovine Serum FFAT Phenylalanine-Phenylalanine Acidic Tract GFP Green Fluorescent Protein GPS Gray Platelet Syndrome HEK293 Human Embryonic Kidney 293 cells His Histidine HRP Horse Radish Peroxidise

HSC Hematopoietic Stem Cell IF Immunofluorescence microscopy ILV Intralumental Vesicles IMDM Iscove’s Modified Dulbecco’s Media IMS Invaginated Membrane System KO Knock-Out LAMP1 Lysosome-Associated Protein 1 LB Lysogeny Broth LB-amp LB supplemented with ampicillin antibiotic LB-kan LB supplemented with kanamycin antibiotic LE Late Endosome LRBA Lipopolysaccharide-responsive, beige-like anchor LYS Lysosome LYST Lysosomal Trafficking regulator MCS Membrane Contact Site MK Megakaryocyte MSP Major Sperm Protein domain MT Microtubule mVAPA Mouse monoclonal α-VAPA antibody MVB Multivesicular Body NBEA Neurobeachin NBEAL2 Neurobeachin-like 2 ncFFAT Non-Canonical FFAT OCS Open Canalicular System ORP1L Oxysterol-binding protein-Related Protein-1L OSBP Oxysterol-Binding Protein P4H Prolyl-4-Hydroxylase P4HB Prolyl-4-Hydroxylase Beta peptide PBS Phosphate-Buffered Saline PCC Pearson’s Correlation Coefficient PCR Polymerase Chain Reaction PF4 Platelet Factor 4 PFA Paraformaldehyde PH Pleckstrin-Homology domain PM Plasma Membrane PMA Phorbol 12-myristate 13-acetate PHYRE 2 Protein Homology/analogy Recognition Engine V2.0 QDO Quadruple amino acid Drop-Out ROCK1 Rho-associated protein kinase 1 RPM Revolutions Per Minute rVAPA Rabbit α-VAPA antibody SCAMP3 Secretory Carrier Associated Membrane Protein 3 SD Synthetic Dropout

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SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Eletrophoresis SERCA3 Sarco/Endoplasmic Reticulum Calcium ATPase 3 SNX Sorting Nexins STARD3 Steroidogenic Acute Regulatory protein related lipid transfer Domain-3 STARD3NL STARD3 N-terminal-Like TBS-T Tris-buffered saline-Tween TGN Trans-Golgi Network TMD Trans-Membrane Domain TPO Thrombopoietin Trp Tryptophan TSP-1 Thrombospondin-1 VAP VAMP-Associated Protein VAPA VAMP-Associated Protein A VAPB VAMP-Associated Protein B VWF Von Willebrand Factor WD40 Tryptophan-aspartate dipeptide repeats Y2H Yeast-2-Hybrid YPDA Yeast, Peptone, Dextrose, Adenine media

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Chapter 1: Introduction

1.1 Platelets: effectors of haemostasis and other processes

Platelets are small, abundant blood cells that lack nuclei but are nevertheless metabolically active and capable of influencing a wide range of cells and physiological processes (Figure 1) (1,2). The platelet plasma membrane (PM) contains a variety of lipids and receptors that allow these cells to detect and respond to key changes in their environment. For example, when a blood vessel is damaged, platelets detect the exposed area and undergo a profound transformation that involves three key aspects: adhesion, secretion, and aggregation (Figure 2). Circulating platelets adhere to the vascular sub-endothelium via interactions with collagen and released proteins (e.g. Von Willebrand Factor; VWF). Adherent cells then transition from their resting discoid shape to a more irregular globular form, which can extend spiky filopodia (3,4). These activated platelets promote the localized generation of thrombin, which catalyzes the transformation of plasma fibrinogen into fibrin strands. Platelets bind both fibrinogen and fibrin and become activated in a complex interplay of protein phosphorylation, lipid conversion

Figure 1. Normal platelets on a blood film. Blood from a healthy neonatal patient shows platelets (black arrow heads) stained dark blue with Romanowsky (Wright-Giemsa) stain when visualized via bright field microscopy. Image was adapted from Urban, Li et al 2012 (5). Reprinted with permission.

1 and Ca+2 mobilization events within platelets and triggers the discharge of secretory vesicle contents ranging from small molecules such as adenosine diphosphate (ADP) and serotonin to large proteins like VWF and Factor V (6-8). Platelet activation and secretion contributes to the establishment of positive feedbacks where autocrine and paracrine stimuli locally amplify platelet aggregation and stimulation until a hemostatic clot has formed to close the vessel wound. In addition to their well-known role as regulators of hemostasis, platelets are also recognized as key contributors to other processes. This includes host immune defenses as well as the progression of cardiovascular disease (9-11). Thus, platelets play key roles in both health and disease, and much of their influence comes from their ability to secrete a wide range of bioactive molecules.

Figure 2. Platelet activation and hemostasis. Platelet response to vascular damage can be depicted as having three stages. In the first stage (left), damage to the vascular endothelium exposes collagen and extracellular membrane (ECM) proteins that are detected by platelet surface receptors that signal cells to adhere to the site of injury. The second stage (centre) involves the secretion of platelet proteins at the site of injury that recruits other cells and initiates the coagulation cascade. In the last stage (right), coagulation generates a localized fibrin mesh that combines with platelets to form a hemostatic plug to stop bleeding. Platelet secretion also stimulates wound healing and immune responses. Image was obtained from Semple et al 2011 (12). Reprinted with permission.

1.2 Platelet secretory granules

When examined via electron microscopy, a distinctive feature of platelets is the presence of electron-dense vesicles, or granules. Platelets contain three recognizable classes of granules that vary in abundance and cargo: α-granules, δ-granules, and lysosomes (Figure 3). Appearing at a frequency of 50-80 per platelet, α-granules are the most abundant and they primarily contain proteins, including factors involved in hemostasis such as VWF and Thrombospondin-1 (TSP-1) (13-15). Appearing at roughly 8 per platelet, δ-granules contain small molecules such as serotonin, calcium and ADP, which can activate platelets and other cells (16). The least numerous platelet granules are the lysosomes, which carry proteases involved in the remodelling of blood clots (17). In addition to their transport and secretory roles, α-granules also act as an internal membrane reservoir, along with the platelet open canalicular system (OCS, Figure 4). The OCS

Figure 3. Platelet secretory granules. Platelets contain three distinct classes of secretory granules, each with a distinctive set of cargo. Alpha-granules (α) are the most abundant and contain proteins involved in processes including adhesion, hemostasis and immunity. Dense granules (δ) contain nucleotides and bioactive molecules including ADP and Ca+2 that activate other cells. Lysosomes (λ) carry proteases and glycosidases that remodel blood blots. Image was obtained from Yeaman 2014 (18). Reprinted with permission.

3 is a network of membrane tubules, generated from invaginations of the plasma membrane, that functions both in platelet spreading and in secretory granule release (19). Vesicular fusion with the OCS combines granule membranes with the surface of activated platelets, allowing them to dramatically increase their surface area, over which come granule contents are spread (20).

Figure 4. Platelet ultrastructure. Transmission electron microscopy (EM) shows a cross-section of a resting human platelet, where the discoid shape is maintained by a peripheral microtubule coil (MT). Platelets contain extensive internal membrane networks including the open canalicular system (OCS) and the dense tubular system (DTS). Platelets also contain numerous secretory vesicles including α-granules (G), δ-granules (DB) and lysosomes, as well as mitochondria (M). Occasionally, platelets can be seen with masses of glycogen particles (Gly) that make up the electron-dense nucleoid and coated vesicles (CV). Image was obtained from White 1999 (20). Reprinted with permission.

1.3 Platelet production by megakaryocytes

Platelets originate from large precursor cells called megakaryocytes (MKs). The MK is one of the largest and rarest cell to arise from hematopoietic stem cells (HSCs) in the bone marrow but MKs can also be found in the in the yolk sac, liver, and spleen of the early fetus (21- 23). During maturation, MKs undergo the long and complex process of thrombopoiesis that is regulated primarily by the cytokine thrombopoietin (TPO) (24). The goal of thrombopoiesis is to ensure that platelets are packaged with the correct constituents for their functions within the body. Thus, abnormal maturation of MKs often leads to abnormal platelets.

Within the initial stages of thrombopoiesis, before the MK has the capacity to release platelets, MKs undergo repeated cycles of endomitotic replication to increase cytoplasmic mass as well as DNA content (Figure 5A-B). Unique to MKs, endomitosis is both similar and different from regular mitosis of somatic cells. On one hand, endomitosis is followed by prophase, metaphase, and anaphase, however, endomitosis differs in that it bypasses the telophase and cytokinesis stages (25,26). As a result, multiple iterations of DNA replication with the inability to divide the cell leads to the development of a polyploid nucleus, containing upwards to 128N compliment of DNA (27,28). A single endomitotic event spans roughly 9.3 hours from start to finish and the repeated cycling of endomitosis generates a significant amount of platelet proteins which are packaged into the maturing platelets (27). In this time frame, MKs also develop an internal membrane network that is distinct from the Endoplasmic Reticulum (ER) and the Golgi Apparatus known as the invaginated membrane system (IMS) (29,30). The IMS initially forms from the invagination of the plasma membrane as one continuous structure near the early polyploid nucleus and accumulate membranes as the MK matures, possibly through Golgi vesicles and ER contacts (30). The plasma membrane together with the IMS provides the necessary deposit of membrane for the formation of thousands of platelets (31).

Mature polyploid MKs produce pseudopodial extensions, 1-4 μm in diameter, called proplatelets, which extend away from the cell body (Figure 5C-D) (9). This process commences from a single site on the MK surface where one or more broad pseudopodia form from microtubules (9,32). These microtubules are responsible for the elongation of proplatelets and extend into the free end where it is seen to form a loop and reconvene back into the proplatelet shaft (9). The result is a platelet-sized swelling at the bulbous proplatelet tips measuring 3-5µm

Figure 5. An overview of thrombopoiesis. Megakaryocyte maturation begins with multiple cycles of endomitosis to generate large quantities of organelles, proteins and cytoplasmic mass (A-B). Microtubules are then arranged at the cell periphery and assemble into extensions called pseudopods that project away from the cell body (C). Pseudopods transition into longer extensions called proplatelets where branches serve to amplify platelet production. At this stage, secretory granules are trafficked into proplatelet ends and migrate toward blood vessels (D). Within the later stages, megakaryocytes convert its cytoplasm into masses of proplatelets and extrude the nucleus where platelets are released into circulation (E). Adapted from Thon and Italiano 2012 (33). Reprinted with permission.

6 in diameter from which platelets emerge (9,34). Microtubule loops are also found studded along proplatelet shafts that serve to elongate into new proplatelet branches (9). This branching process acts to amplify platelet production. As proplatelet extensions grow more numerous and lengthy, the megakaryocyte cytoplasm is converted into a complex and interconnected network of proplatelets (Figure 5E). The multi-lobed nucleus however, gets compressed and is extruded with very little cytoplasm ultimately leaving numerous platelet-sized swellings linked by thin strands of cytoplasm (9). At this stage, MKs physically resemble a string of beads. These proplatelet extensions project through bone marrow sinuses to contact blood vessels where the shear forces of the blood stream will fragment the extensions into nascent platelets (9,35). The whole process, from the start to the complete conversion of MK cytoplasm, takes approximately 12-16 days in humans (36).

1.4 The endosome is a crucial structure in α-granule production

The development of α-granules, as well as δ-granules, begins within MKs and continues in circulating platelets. In maturing MKs, vesicles from the trans-Golgi network and those from the plasma membrane contribute cargo to the maturing α-granules (37). Many α-granule cargo proteins and chemokines are synthesized by MKs, including VWF, P-selectin, Platelet Factor 4 (PF4), RANTES and NAP-2 (38). Other α-granule cargo proteins are taken up by receptor- mediated endocytosis, for example fibrinogen is endocytosed via MK integrin αIIbβ3 cell surface receptors (37,39,40). All granule cargo passes to late endosome/multi-vesicular bodies (LE/MVB), transient endosomal structures associated with the sorting of endocytosed and synthesized proteins in many cell types (41). In MKs, MVBs are equally important in secretory granule production (42).

1.4.1 Endosome maturation facilitates α-granule formation and release

Vesicular constituents, including membrane-bound and soluble proteins, destined to be stored within α-granules initially transition to and fuse with a persistent internal structure known as the early endosome (EE, Figure 6A). Characterized by networks of tubulo-cisternae elements rich in phosphatidylinositol-3-phosphates, the EE is the first of two trafficking hubs that organize cargoes originating from the trans-Golgi network (TGN) and the PM (43). The small GTPase RAB5 along with its effector VPS34/p150 are key components that help maintain this

7 organelle and its function (43). Open communication with the PM and TGN are facilitated through the bi-directional exchange of vesicles, converging upon the multimeric retromer complex, in the recycling and retrograde transport routes (44). The unidirectional sorting of proteins into LEs at the anterograde route requires a slightly different array of factors including the Endosome Sorting Complex Required for Transport (ESCRT) complexes, Secretory Carrier Associated Membrane Protein 3 (SCAMP3), and Sorting Nexins (SNXs) to list a few (43).

It is understood that ESCRT-mediated invaginations on the EE membrane act to partition proteins within intraluminal vesicles (ILVs) that will later detach as clusters to form free- floating MVBs. Free MVBs at this stage, containing numerous internal vesicles, are referred to as the early MVB-I and travel along microtubule tracks aided by dynein/kinesin motors (Figure 6B) (42,45). Exactly how the LEs are formed from MVBs is not clear and remain a highly debated area but evidence suggests that it is partly regulated by the oscillatory movement of the dynein/kinesin motor proteins and the switching from RAB5 to RAB7 GTPases (Figure 6C) (46,47). Recently, the ER has been proposed to facilitate this process through the formation of membrane contact sites (MCS) and promote non-vesicular signalling and lipid transport events. At the crux of MCSs are the family of ER-resident VAMP- associated protein (VAP) proteins, VAPA and VAPB, that tether to other organelle-specific partners including the LE-membrane- anchored Steroidogenic Acute Regulatory protein related lipid transfer Domain-3 (STARD3), STARD3 N-terminal-Like (STARD3NL), and the Oxysterol-binding protein-Related Protein-1L (ORP1L) proteins (48,49). These ER-LE tethers may provide a mechanism of lipid-based regulation since low cholesterol levels appear to impede LE/MVB mobility and maturation (49,50). None-the-less, the internal structure of the MVB-I compartment will mature with time and develop electron-dense matrixes in addition to internal vesicles. This mature structure is called the MVB-II and represents the second trafficking hub within MKs of which to further sort cargoes (42). Mature secretory vesicles, either α-granules, δ-granules, or lysosomes are then dispatched from the LE/MVB-II to be trafficked into the maturing platelets (42).

1.4.2 α-Granules are sorted at the endosome and trafficked into proplatelet shafts

The exact stage where α-granule constituents are sorted within the endosomal structures, leading up to granule release, is not well understood. The endocytosis of fibrinogen into the recycling endosome/EE and its presence within mature α-granules predict that specificity occurs

8 early in endosome maturation (39). This is also reasonable for endogenously synthesized constituents that originate from the TGN. The observation that MVB-II structures contain concentrated patches of electron-dense material reminiscent of granules suggests that the MVB- I functions in collecting an initially dispersed set of granule-specific constituents. The gradual maturation of the endosome into the MVB-II then concentrates these constituents into a pre-α- granule structure and later, the release of mature granules.

Figure 6. Molecular organization of the mammalian endosome. The endocytic pathway can be divided into three stages of which proteins traverse through. Vesicles carrying synthesized and endocytosed proteins/constituents converge in the early endosome where they are sorted into intraluminal vesicles (A). Vesicles detach from endosomal tubules as structures containing similar internal vesicles as the early endosome called the early multivesicular body-I which then fuses with the late endosome (B). The late endosome/multivesicular body-II contains electron- dense material and continues to sort constituents before they are released throughout the cell (C). Numbers represent specific Rab GTPases associated with organelles/trafficking events. Known trafficking routes are indicated through black and red arrows. Acronyms include clathrin-coated vesicles (CCV), clathrin-independent carriers (CIC), early endosome (EE), endosomal carrier vesicle/multivesicular body (ECV/MVB), late endosome (LE), lysosomes (LYS), autophagosomes (AP), trans-golgi network (TGN), and endoplasmic reticulum (ER). Diagram was obtained from Scott et al 2014 (43). Reprinted with permission.

MKs utilize a diverse set of mechanisms to package an equally diverse set of α-granule- specific cargoes. Membrane-anchored proteins, such as P-selectin, contain a sorting sequence within its cytoplasmic tail region that allows proper trafficking into storage vesicles (51,52). Some soluble proteins and cytokines including PF4 and RANTES also contain a short signal sequence for this very purpose (53). Other small and soluble cargoes may require a mediator, a glycosaminoglycan called serglycin, to sequester cationic regions as a method of retaining cargo within the α-granules (54). Larger soluble proteins including VWF are proposed to incorporate into α-granules through homo-aggregation where it is only found in discrete tubular structures within the granules (55,56). Although the sorting of a select number of cargo proteins are known, the trafficking of other known α-granule constituents remains an active field of research.

An essential part of platelet production is the distribution of α-granules and other organelles into the maturing platelets. Mature granules and organelles travel in a bi-directional manner, along the microtubule tracks of proplatelet extensions likely powered by the microtubule motor protein kinesin (34). These constituents exhibit pausing and frequent changes in the direction of movement but, are eventually captured by microtubule loops at the terminal ends (34). Trafficking within proplatelet shafts are also mediated, although indirectly, through a microtubule-sliding mechanism and add a second modality to granule distribution (57,58). Organelles are propelled through proplatelet shafts by being situated on microtubule segments as microtubules extend the proplatelet protrusions. Together, these two mechanisms facilitate platelet maturation and their release into circulation.

1.5 NBEAL2 is implicated in α-granule formation as its absence causes Gray Platelet Syndrome

Inherited platelet disorders have been vital in developing our current understanding of the mechanisms of platelet granule biogenesis. Gray Platelet Syndrome (GPS) is one such bleeding disorder that is clinically characterized by macrothrombocytopenia and pale-appearing platelets on peripheral blood smears due to the deficiency of α-granules (Figure 7) (59,60). Patients afflicted with GPS present a heterogeneous set of symptoms where nearly half of all patients present with severe bleeding (61,62). However, the development of myelofibrosis is almost definite in GPS patients and the severity of which is strongly correlated with age (61). The onset of myelofibrosis is predicted to be caused by the non-specific release of platelet growth factors from platelet α-granules into the bone marrow environment (61). In addition to 10 the requirement of α-granule proteins in the formation of a robust platelet plug, of which GPS patients lack, the cause of GPS is likely due to the inability of platelets or MKs to retain α- granule cargoes (61).

The genetics of GPS has been well characterized and can appear as autosomal dominant, autosomal recessive, or X-linked disorders (63-65). The most prevalent form, however, is an autosomal recessive mode of inheritance caused by the loss of function in a protein called Neurobeachin-like 2 (NBEAL2) (63,66,67). Mutations of NBEAL2 that lead to GPS can present as non-sense, missense, splice site, and frameshift mutations (63,66,67). Mouse models attempting to recapitulate GPS through the knock-out of NBEAL2 have been quite successful, showing that the lack of NBEAL2 led to low platelet counts and enlarged, agranular platelets like their human counterparts (68-70). The absence of VWF and TSP-1 cargo proteins in the NBEAL2 KO platelets led to the hypothesis that NBEAL2 mediates the sorting of cargoes into maturing α-granules since the granule membrane appeared intact (68,70). Another disorder known as Arthrogryposis, Renal tubular dysfunction, and Cholestasis (ARC) syndrome present

Figure 7. GPS patient platelets are devoid of α-granules. Under transmission electron microscopy, normal and healthy patient platelets are classically characterized as small and granular containing an abundance of α-granules shown through the white arrow heads (A). However, GPS patient platelets are enlarged, agranular, and vacoulated (B). Image was adapted from Kahr et al 2011 (63). Reprinted with permission.

11 platelets that lack both α-granule-specific soluble cargo proteins and the granule membrane due to the loss of function of VPS33B-VPS16B (5). Since the loss of NBEAL2 function only causes the loss of soluble cargoes, it is possible that NBEAL2 acts downstream of VPS33B-VPS16B (5,68). As such, the current working model is that the VPS33B-VPS16B complex is required for the formation of the precursor α-granule membrane, a step that must be completed before cargoes can be sequestered inside, while the role of NBEAL2 may be in sequestration (71) (Figure 8).

Figure 8. Model of α-granule formation. Endogenously-synthesized and endocytosed granule cargo, originating from the trans-Golgi network (TGN) and early endosomes respectively, converge in multivesicular bodies (MVBI, II). These organelles give rise to granule precursors and NBEAL2 is required for loading/maturation of α-granules. Image was obtained from Chen et al 2017 (71). Reprinted with permission.

1.6 NBEAL2 is predicted to have roles in vesicular and protein-scaffolding events

NBEAL2 is a 302 kDa protein containing 2754 amino acids and several domains that are consistent with the predicted role of cargo sorting and organization (Figure 9). Most noticeably, NBEAL2 contains a Beige and Chediak-Higashi (BEACH) domain that classifies it as a member of the BEACH-domain containing family of proteins (BDCPs) (72). Similar to other members within this family, the BEACH domain of NBEAL2 is preceded by a Pleckstrin- Homology (PH) domain that is known to associate with phosphoinositides of the membrane (73). The BEACH domain of NBEAL2 is also followed by WD40 repeats consisting of multiple short motifs, each ending with a tryptophan-aspartate dipeptide. WD40 repeats are present in regulatory proteins of the cell cycle, signal transduction, and vesicular trafficking pathways where this domain serves as a scaffold for protein interactions (74). Upstream of these aforementioned domains is a Concanavalin-A-like lectin (CON-A) domain proposed to be involved in oligosaccharide binding within the secretory pathway (75). Lastly, the CON-A domain is flanked on both sides by two Armadillo (ARM) domains that are predicted to be also involved in protein scaffolding (76).

The functions of BDCPs are poorly understood. The nine family members identified in vertebrates share similar domain architectures, and BDCPs have also been identified in lower eukaryotes including yeast, Dictyostelium, Caenorhabditis elegans and Drosophila (72). The presence of a single BDCP in yeast with a vacuolar protein sorting function indicates an ancestral function in vesicular trafficking, and several vertebrate BDCPs are predicted to have such roles (77). Loss of function of the Lysosomal Trafficking regulator (LYST) is causative for

Figure 9. Schematic of NBEAL2 functional domains. NBEAL2 is a 302kDa protein that contains 2754 amino acids in total. Two ARM domains, in blue, are found between residues 310-525 and 937-1102. A single Con-A domain, in yellow, is found between residues 572-853. Several WD repeats, in orange, are found between 1515-1555, 2457-2499, 2500-2539, 2551- 2592, and 2685-2724. A PH domain, in gray, is located between residues 1918-2003. The characteristic BEACH domain, in green, is found between residues 2053-2345.

Chediak-Higashi Syndrome (CHS), characterized by enlarged lysosomes in granulocytes and a deficiency of platelet δ-granules, likely arising from defective vesicle fusion/fission and membrane signalling (78-80). Mutations in Neurobeachin (NBEA), a close homologue to NBEAL2 that is largely expressed in nervous tissues, have been implicated in autism, where the loss of NBEA function causes aberrant neurotransmitter release (81,82). Alterations of lipopolysaccharide-responsive, beige-like anchor (LRBA) function affect autophagy, required for recycling of cellular components (83). LYST, NBEA, LRBA, and NBEAL2 share other protein domains in addition to the BEACH domain, including a PH domain and several WD repeats (Figure 10). Other proteins within this family have proposed functions in protein scaffolding,

1.7 Rationale and hypothesis

Studies of human GPS and mouse models implicate NBEAL2 in α-granule formation, and this protein and its BDCP family members contain functional domains predicted to be involved in vesicle-related processes. Thus, I predict that NBEAL2 is involved in the maturation of α-granules via interactions with granule precursors and other proteins associated with cargo loading, tethering and/or other processes. Identifying interaction partners will yield key insights into NBEAL2 function. Here, I investigated the interaction of NBEAL2 with a potential interacting partner, VAPA.

Figure 10. Domain architecture of BEACH domain containing proteins. This schematic compares LYST, NBEA, LRBA and NBEAL2 proteins. The scale bar represents 250 amino acids. This image was adapted from Cullinane et al 2013 (72). Reprinted with permission.

Chapter 2: Materials and methods

2.1 Lab reagents, antibodies, and plasmids

All antibodies used in western blotting and Co-immunoprecipitation (Co-IP) experiments are listed in Table 1 along with the product identifiers and dilutions. All antibodies used in immunofluorescence (IF) studies are listed in Table 2 with the product identifiers and dilutions. All bacteria and mammalian cells are listed in Table 3 with the appropriate identifiers. All commonly used lab reagents are listed in Table 4. All basic reagents used in relation to yeast growth and selection were obtained from Bishop and Clontech respectively. All DNA restriction enzymes and polymerase chain reaction (PCR) reagents were purchased from NEB and used as directed. All PCR primers were ordered from IDT Integrated DNA Technologies. All plasmids constructs and their cloning sites are listed in Table 5.

2.2 Cell maintenance and growth

DH5α competent cells were grown in Lysogeny Broth (LB) media or on agar at 30 °C or 37 °C supplemented with 10 μg/mL kanamycin or 100 μg/mL ampicillin antibiotic overnight. If

Table 1. List of antibodies used in Co-IP and western blotting.

Primary antibodies

Antibody Source Clonal Clone/catalogue Company Dilution

α-CD61 Mouse Monoclonal Y2/51 Dako Cytomation 1:1000 α-FLAG Mouse Monoclonal M2 Sigma Aldrich 1:1000 α-HA Rabbit Monoclonal A190-208A Bethyl 1:1000 α-MYC Mouse Monoclonal 9e10 Covance 1:1000 α-VAPA Rabbit Polyclonal HPA009174 Sigma Aldrich 1:1000

Secondary Antibodies

Antibody Source Conjugate Company Dilution

α-Mouse Donkey HRP Jackson ImmunoResearch 1:5000 α-Rabbit Donkey HRP Jackson ImmunoResearch 1:5000

15 bacterial cells were not harvested from LB-agar, plates were wrapped in parafilm and stored at 4 °C until needed. Yeast AH109, Y183 donors, and diploid mates were all grown at 30 °C with or without shaking at 200 revolutions-per-minute (rpm) in the specified media outlined by the manufacturer. Human Embryonic Kidney 293 (HEK293) cells were cultured in Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 5% fetal bovine serum (FBS) in T-75 flasks. Dami cells that stably express GFP-NBEAL2-FLAG were cultured in Iscove’s modified DMEM supplemented with 8% Horse Serum and 1mg/mL of puromycin antibiotic. Both

HEK293 and Dami cells were grown at 37 °C and with 5% CO2.

Table 2. List of antibodies used in immunofluorescence studies.

Primary antibodies

Antibody Source Clonal Clone/catalogue Company Dilution

α-CD61 Mouse Monoclonal Y2/51 Dako Cytomation 1:200 α-CD62P Goat Polyclonal SC-6941 Santa Cruz 1:100 α-CD63 Mouse Monoclonal H5C6 Hybridoma Bank 1:100 α-EEA1 Rabbit Monoclonal C45B10 Cell Signalling 1:200 α-GFP Mouse Monoclonal A11120 Invitrogen 1:100 α-LAMP1 Mouse Monoclonal H4A3 Hybridoma Bank 1:100 α-NBEAL2 Rabbit Monoclonal EPR14501(B) Abcam 1:100 α-RAB7 Rabbit Polyclonal #2094 Cell Signalling 1:100 α-SERCA3 Mouse Monoclonal SC-81759 Santa Cruz 1:100 α-VAPA Rabbit Polyclonal HPA009174 Sigma Aldrich 1:50 α-VAPA Mouse Polyclonal Ab172371 Abcam 1:50 α-VWF Sheep Polyclonal AHP062 Bio-Rad 1:500

Secondary Antibodies

Antibody Source Wavelength Company Dilution

α-Mouse Donkey 488 Invitrogen 1:5000 α-Rabbit Donkey 488 Invitrogen 1:5000 α-Rabbit Donkey 568 Invitrogen 1:5000

2.3 Plasmid cloning

2.3.1 Yeast plasmids

The cloning of yeast plasmids was performed using the same protocol, with varying buffers and restriction enzymes depending on the reactions. The human NBEAL2 gene (Entrez Gene 23218) was PCR amplified with PFU Ultra II polymerase as per manufacturer’s recommendations using 3 sets of DNA primers listed in Table 6. PCR inserts and pGBKT7 plasmids were double-digested with NdeI and BamHI restriction enzymes and gel-purified from 1% agarose gels using the Mackery-Nagel PCR clean-up Gel Extraction kit. The backbone pGBKT7 vector was then dephosphorylated with calf intestinal phosphatase (CIP) for 1 hour at 37 °C and gel-purified again before ligating together with purified inserts using T4 DNA Ligase overnight at 16 °C. Ligation mixtures were transformed into competent DH5α bacterial cells using heat-shock and plated on LB-agar supplemented with kanamycin (LB-kan) overnight. Screened colonies were isolated and inoculated in fresh LB-kan liquid media overnight before harvesting through the Plasmid Miniprep as outlined by Geneaid. Plasmid constructs were then sequence-verified.

2.3.2 Cloning of mammalian overexpression constructs

The cloning of mammalian plasmids followed similarly to the yeast plasmids mentioned previously. The cloning of the NBEAL2 gene into the PLJM1 vector, with an N-terminal green

Table 3. List of cells used.

Cells Origin Company

AH109 Yeast Clontech Dami Mammalian ATCC DH5α Bacterial Invitrogen Human Embryonic Kidney 293 (HEK-293) Mammalian ATCC STBL3 Bacterial Invitrogen

17 fluorescent protein (GFP) and a C-terminal 3xFLAG epitope, was done by another graduate student (Richard Lo) in the lab using AgeI and SacI restriction cut sites. The cloning of NBEAL2 fragments C1-C6 was also done by Richard Lo, using the XhoI and NotI cut sites, into the pCMV-HA vector. To clone MYC-VAPA and MYC-VAPAΔ, primers were used to PCR amplify the VAPA gene from a pre-purchased VAPA-MYC-DDK construct and inserted into the pCMV-MYC vector using EcoRI and Not I cut sites. To clone HA-VAPB, primers were used to

Table 4. List of common lab reagents and equipment used.

Reagent Company

Agarose Amresco Ampicillin OmniPur Dako Fluorescent mounting medium Dako Cytomation Dulbecco’s Modified Eagle’s Media (DMEM) Wisent Bioproducts Fetal Bovine Serum (FBS) Wisent Bioproducts Horse Serum (HS) Wisent Bioproducts Iscove’s Modification of DMEM Wisent Bioproducts JetPRIME Transfection reagents Polyplus Transfection Kanamycin-sulfate OmniPur Lysogeny Broth (LB) Wisent Bioproducts PCR clean-up Gel Extraction kit Machery-Nagel Phorbol 12-myristate 13-acetate (PMA) Invitrogen Presto Mini Plasmid kit Geneaid Protease inhibitor cocktail Roche PFU Ultra II polymerase Agilent Technologies Protein G sepharose beads GE Healthcare Salmon Sperm DNA Sigma Aldrich Thrombopoietin (TPO) Kirin Brewery Company Triton-X-100 Bioshop Trypsin/EDTA Wisent Bioproducts Tween20 Sigma Aldrich

18 amplify the VAPB gene from a pre-purchased VAPB-MYC-DDK construct to insert it into the pCMV-HA vector using EcoRI and NotI cut sites. All ligation mixtures were transformed into competent DH5α bacterial cells using heat-shock and plated on LB-agar supplemented with ampicillin (LB-amp) overnight. Screened colonies were isolated and inoculated in fresh LB-amp liquid media overnight before harvesting through the Plasmid Miniprep as outlined by Geneaid. All plasmid constructs were then sequence-verified. To generate the HA-C5 (D2224K) and HA-C5 (E2218K) mutants, site-directed mutagenesis was performed with near-complimentary primers. Respective primers were designed such that a single amino acid mutation is situated in the center of the primer and PCR was carried out as mentioned above using HA-C5 as the template vector. PCR reaction mixtures were then digested with DpnI restriction enzyme to remove methylated HA-C5 template and subsequently transformed into DH5α bacteria for overnight selection on LB-amp agar plates. Bacteria were picked the following day and inoculated in LB-amp liquid media to be grown overnight. Overnight cultures were harvested using the Presto Mini Plasmid as per manufacturer recommendations and plasmids were sequence verified.

2.4 Yeast-2-Hybrid screen

The following protocols can be found in the AH109 Yeast Handbook by Clontech. Briefly, to transform AH109 yeast cells with each of the three NBEAL2 bait constructs described previously, AH109 cells were grown from glycerol stock onto plates containing rich media Yeast extract, peptone, dextrose, adenine (YPDA; 20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose, 0.1 g/L NaOH, 20 g/L agar, 0.003% adenine hemisulfate in distilled water) over several days at 30 °C. Single colonies were picked and grown in 5 mL YPDA overnight and used to inoculate 300 mL YPDA the next day such that the starting OD600 was 0.1. The cultures were then incubated with shaking until OD600 reached 0.5-0.7 before the yeast cells were pelleted at 1,500 x g for 5 minutes. The cells were then washed with distilled water and pelleted again. The cells were then re-suspended in 1.5 mL 1x TE/LiOAc buffer (0.01 M Tris, pH 7.5, 0.001 M Ethylenediaminetetraacetic acid (EDTA), 0.1 M lithium acetate, in distilled water) and 100 μL aliquots were used for each transformation reaction. A mixture of 50 μg of salmon sperm DNA, denatured by boiling for 5 minutes at 95 °C, and 100 ng of the bait DNA were added to each of the 100 μL aliquots of AH109 cells. A volume of 300 μL of 1x

Table 5. List of constructs, plasmid backbones, inserts and cloning sites used.

Name Plasmid Insert Cloning sites Company GFP-NBEAL2- Pljm1 NBEAL2 (1-2754aa, AgeI, PacI FLAG Entrez 23218) HA-C1 pCMV-HA NBEAL2 (1-550aa) XhoI, NotI HA-C2 pCMV-HA NBEAL2 (530-903aa) XhoI, NotI HA-C3 pCMV-HA NBEAL2 (877-1403aa) XhoI, NotI HA-C4 pCMV-HA NBEAL2 (1350-1903aa) XhoI, NotI HA-C5 pCMV-HA NBEAL2 (1877-2400aa) XhoI, NotI HA-C5 (D2224K) pCMV-HA NBEAL2 (1877-2400aa) XhoI, NotI HA-C5 (E2218K) pCMV-HA NBEAL2 (1877-2400aa) XhoI, NotI HA-C6 pCMV-HA NBEAL2 (2384-2754aa) XhoI, NotI pCMV-HA-Empty pCMV-HA BD Bioscience (PT3283-5) pCMV-MYC-Empty pCMV- BD MYC Bioscience (PT3282-5) pGBKT7-Empty pGBKT7 Clontech (630443) pGBKT7-A pGBKT7 NBEAL2 (1-861aa) BamHI, NdeI pGBKT7-B pGBKT7 NBEAL2 (860-1666aa) BamHI, NdeI pGBKT7-C pGBKT7 NBEAL2 (1600-2754aa) BamHI, NdeI MYC-VAPA pCMV- VAPA (1-249aa) XhoI, NotI MYC MYC-VAPAΔ pCMV- VAPA (1-218aa) EcoRI, NotI MYC MYC-VAPB pCMV- VAPB (1-243aa) EcoRI, NotI MYC VAPA-MYC-DDK pCMV6- VAPA (1-249aa, SgfI, MluI Origene Entry NM_194434) (RC214308) VAPB-MYC-DDK pCMV6- VAPB (1-243aa, SgfI, MluI Origene Entry NM_004738) (RC20051)

TE/LiOAc/PEG buffer (0.01 M Tris, pH 7.5, 0.001 M EDTA, 0.1 M lithium acetate, in 50% PEG-3350) was then added, mixed by inversion, and incubated on ice for 30 minutes. Subsequently, 70 μL dimethyl sulfoxide (DMSO) was added to each transformation reaction and was subjected to a heat shock at 42 °C for 15 minutes. A volume of 200 μL was removed from the mixture and plated onto selective growth media SD/-Trp (6.7 g/L yeast nitrogen base with ammonium sulphate, without amino acids, 0.6 g/L –Trp dropout mix, 20 g/L glucose, 20 g/L agar in distilled water). The plates were incubated for 2-3 days until colonies were observed. Colonies were picked, and bait toxicity was assessed visually by the relative size of colonies as specified by Clontech. Auto-activation controls were conducted through the Urea/SDS lysis method followed by western blots of the lysates. To perform mating, a concentrated overnight culture (OD600 > 0.8) of the bait strain (AH109 cells transformed with NBEAL2 bait constructs) was prepared through inoculation in a 50mL aliquot of SD/-Trp media overnight,

Table 6. List of PCR Primers and target plasmids used.

Name Sense Sequence Plasmid

NBEAL2 Forward AAAAAACATATGATGGCCGCCTCGGAGCGG pGBKT7 A (1- Reverse AAAAAAGGATCCTCACAGGTCCAGGCAGATGTTGTT 861aa) NBEAL2 Forward AAAAAACATATGGACCTGTCCCCCAGTCATGGGCTT B (860- Reverse AAAAAAGGATCCCTAGTAGGCCTGGGCATGCAGCTG 1666aa) NBEAL2 Forward AAAAAACATATGGTGAGATTGCACATGCTGCTACAG C (1600- Reverse AAAAAAGGATCCCTATCAGCGCGCCTCAGTAGGGTT 2754aa) NBEAL2 Forward CCGGAATTCTTCTACTTTCCTAAATTCCTGGAGAACCAGAAC pCMV- C5 HA Reverse GTTCTGGTTCTCCAGGAATTTAGGAAAGTAGAAGAATTCCGG (D2224K) NBEAL2 Forward GTGAAGGAGCTCATCCCGAAATTCTTCTACTTTCCTGAC C5 Reverse GTCAGGAAAGTAGAAGAATTTCGGGATGAGCTCCTTCAC (E2218K) VAPA Forward TTTAACTCGAGGAATGGCGTCCGCCTCAG pCMV- MYC Reverse TATAATGCGGCCGCTCACAAGATGAATTTCCCT VAPAΔ Forward GGGGGAATTCAAATGGCGTCCGCCTCAGGG Reverse TGCGGCCGCTCATGCAGTTGAGGTTGATCC VAPB Forward AAAAAAGAATTCCGATGGCGAAGGTGGAGCAGGTCCTG pCMV- HA Reverse AAAAAAGCGGCCGCCTACAATGCAATCTTCCCAATAATTAC

21 shaking at 250rpm. Cells were collected at 1,500 x g for 5 minutes and re-suspended in 5 mL of residual liquid by gently pipetting. One frozen aliquot of the pre-transformed Human Bone Marrow Library (Clontech) in the Y187 yeast recipient strain was thawed at room temperature and added to 50 mL of 2x YPDA. Both the AH109 and the Y187 cells were mixed together and then incubated overnight with gentle swirling (30-50 rpm) to allow for mating. The mating culture was collected the next day at 1,000 x g for 10 minutes and re-suspended in 10 ml of 0.5x YPDA-Kanamycin. A small aliquot of mates was spread in 1/10, 1/100, 1/1000, and 1/10,000 dilutions for each SD/-Trp, SD/-Leu, and SD/-Leu/-Trp plates and incubated for 3-5 days to calculate mating efficiencies. The rest of the mates were plated onto high stringency Quadruple amino acid Drop-Out (QDO) plates (SD/-Ade/-His/-Leu/-Trp, with X-α-gal) at 200 μL per plate and incubated at 30 °C until colonies appeared (1 to 2 weeks). Positive mate colonies were re-streaked on QDO plates a second time to ensure the correct phenotype and grown for another 3-5 days. Plasmids from these colonies were isolated first by obtaining 5 mL of overnight liquid cultures and pelleting the cells at 1,500 x g for 5 minutes. The pellets of cells were re-suspended in 150 μL of plasmid isolation buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, 1 mM EDTA). To lyse the yeast cells, 0.3g of glass beads and 400 μL of phenol:chloroform:isoamyl solution were added, mixed by vortexing for 2 minutes, and centrifuged at maximum speed for 5 minutes. The aqueous upper phase was collected, an additional 150 μL of the plasmid isolation buffer was added to the remaining solution containing beads, mixed by inversion, and centrifuged again for 5 minutes. The combined aqueous phases were applied onto mini-prep columns and eluted. Yeast plasmid DNAs were amplified through retransformation into competent DH5α bacterial cells and sequenced at the ACGT Corporation sequencing facility. Results were identified through a Basic Local Alignment Search Tool (BLAST) search (84).

2.5 Mammalian transfections and preparing cell lysates

To prepare for single and co-transfection, HEK293 cells were grown as indicated above and seeded onto 10 cm petri dishes at approximately 70-80% confluence the day prior to transfection. Cells were transfected with constructs the following day using JetPrime® as instructed by the manufacturer’s guidelines and grown for a total of 48 hours for the transfection of one construct. In co-transfection, both DNA plasmids of interest were transfected at the same time and at a mass ratio of 1:1 for most constructs. However, the co-transfection of HA-C5 and

MYC-VAPAΔ was done sequentially at a mass ratio of 50:1; HA-C5 was transfected on the first day where cells were given 24 hours to recover, followed by the transfection of MYC-VAPAΔ the next day, and another 24 hours before harvest. For transfections in Dami cells, cells were stimulated immediately after transfection with 50 ng/mL of TPO and 50 ng/mL of Phorbol 12- myristate 13-acetate (PMA).

To prepare lysates from adherent HEK293 cells, cells were first washed twice with ice- cold Phosphate-Buffered Saline (PBS) and gently scraped off in 1 ml of lysis buffer (25 mM Tris HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, supplemented with 1x Protease inhibitor cocktail) using a cell scraper. Scraped cells were collected in an Eppendorf tube and incubated on ice for 30 minutes. The lysates were then cleared at 16,000 x g for 10 minutes at 4 °C to pellet cell debris. The supernatant was kept for experiments and the pellet was discarded.

2.6 Co-Immunoprecipitation preparation and procedures

2.6.1 Co-IP requiring pre-conjugated α-FLAG and control IgG beads

When α-FLAG (Sigma A2220) and/or mouse IgG (Sigma A0919) beads were required, washing and blocking of the beads were performed prior to lysate preparations. Approximately 20 µL of beads per Co-IP condition (which includes 10 µL of beads and 10 µL of preservative solution) were aliquoted into fresh eppendorf tubes and cleared at 3000 x g for 30 seconds. The supernatant was discarded, and the beads were washed 3 times with 1x ice-cold PBS by gentle inversion, allowing the beads to sit on ice for 3 minutes prior and after the clearing steps. Washed beads were then blocked in ice-cold PBS containing 1% Bovine Serum Albumin (BSA) with end-over-end rotation at 4 °C for 1 hour. Blocked beads were then cleared once at 1000 x g for 5 seconds and the supernatant was removed such that a small amount of liquid remained. Prior to adding washed and blocked beads to the prepared lysates, BSA was added to the lysates such that the final concentration was 1% (w/v). A small percentage of the prepared lysate (>2%) was saved as “Input” and the rest were divided equally as needed (>300uL per Co-IP condition). Washed beads were then added to the lysates and incubated with end-over-end rotation at 4 °C for 2 hours. Following the 2-hour incubation, beads were cleared for 5 seconds at 1000 x g in a chilled centrifuge and allowed to settle on ice for 5 minutes. The resulting supernatant was discard such that 100 μL of bead solution remained in the eppendorf tube. 1mL of ice-cold PBS was gently aliquoted into the bead solution and where the beads were gently 23 inverted to resuspend the beads (generally takes ~ 6-8 inversions). Beads were then allowed to sit on ice for another 5 minutes before they were cleared again at 1000 x g for 5 seconds. Washing of the beads was performed a total of 3 times. After the last wash step, most of the supernatant was discard so that approximately 20 μL of bead solution remained. Proteins were eluted off the beads by adding 20 μL of 2x sample buffer (20% v/v glycerol, 2% w/v SDS, 65 mM Tris-HCl, pH 6.8, 0.001% w/v Bromophenol Blue), supplemented with 1% β- mercaptoethanol, and denatured for 10 minutes at 95 °C.

2.6.2 Co-IP requiring self-conjugated α-HA, α-MYC, and control IgG beads

After preparation of cleared lysates, the lysates were split evenly (>300 μL per Co-IP condition) into chilled eppendorf tubes and 1 µg of the respective antibodies were added into the lysates. BSA solution was also added to the lysate to a working concentration of 1% (w/v). The lysate-antibody solution was incubated with end-over-end rotation at 4 °C for 2 hours. In the meantime, 20 μL of unconjugated protein-G sepharose bead slurry (per Co-IP condition) was aliquoted and washed in ice-cold PBS exactly as mentioned above for pre-conjugated beads. Washed beads were then blocked in ice-cold PBS with 1% (w/v) BSA in PBS with end-over- end rotation at 4 °C for 1 hour. Blocked beads were then washed once as mentioned above. When the initial 2-hour incubation is finished, washed and blocked, unconjugated beads were aliquoted into each Co-IP condition containing lysate, antibody, and BSA. The beads were incubated for another hour at 4 °C. The following washing and clearing steps followed exactly as mentioned above for pre-conjugated beads.

2.7 Sodium dodecyl sulfate-polyacrylamide gel eletrophoresis and western blotting

All sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) and western blotting was performed using the same protocol with various antibodies depending on the experiment. Samples were treated with SDS sample buffer (20% v/v glycerol, 1% v/v β- mercaptoethanol, 2% w/v SDS, 65 mM Tris-HCl, pH 6.8, 0.001% w/v Bromophenol Blue) and boiled for 10 min at 95 °C. Samples were then loaded onto polyacrylamide gels (4% stacking, 10% resolving), and run at 200V for approximately 45 minutes or until the dye front reached the end of the gel. The gel was subsequently transferred onto nitrocellulose membranes at 100V for 1.5 hours or 30V overnight. Membranes were blocked in Tris-buffered saline-Tween (TBS-T;

20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% v/v Tween 20) containing 5% w/v skim milk for 40 min, then incubated with primary antibody in blocking solution for 1 hour. Membranes were then washed with TBS-T for 5 minutes for a total of 3 washes followed by incubation with horse radish peroxidise (HRP)-conjugated secondary antibody in blocking solution for 40 min. The membranes were washed again 3 times for 10 minutes each in TBS-T, and treated with enhanced chemiluminescence (ECL; Biorad) reagent before being exposed in the LICOR Odessy® Fc Imaging System.

2.8 Culturing and differentiation of human megakaryocytes

Culturing and differentiation of megakaryocytes were performed by another graduate student (Richard Lo) described previously (85). Briefly, G-CSF-mobilized blood progenitor cells from bone marrow transplant donors were purified for CD34+ cells using the CD34 MicroBeads kit (Miltenyi Biotec). The resulting cells were cultured in Iscove’s Modified Dulbecco’s Media (IMDM, Gibco) supplemented with 10% BIT (Stemcell Technologies), 2 mM L-glutamine (Wisent), 1% Penicillin-Streptomycin (Wisent), and 50 ng/mL Thrombopoietin (a gift from the Kirin Brewery Company). On day 8 of culture, cells were seeded onto coverslips coated with 1:6 dilution of Matrigel (Corning) in DMEM in 12-well plates. Cells were harvested, fixed, and stained on day 12 of culture.

2.9 Immunofluorescent microscopy

NBEAL2-stable Demi cells were seeded onto glass coverslips and stimulated with 50 ng/mL PMA and 50 ng/mL TPO for 2 days prior to staining. For CD34+ human megakaryocytes, cells were harvested at day 12 of culture mentioned above. All coverslips were washed gently twice with PBS and fixed in 4% paraformaldehyde (PFA) at room temperature for 20 minutes. Cells were then gently washed 3 times with PBS before they were permeabilized with 0.1% Triton X-100 in PBS for 30 minutes at room temperature. Cells were subsequently washed 3 times again with PBS for 5 minutes each and incubated with blocking solution (2% horse serum and 2% w/v BSA in PBS) for 1 hour at room temperature. Coverslips were then incubated with the appropriate primary antibody in blocking solution overnight at 4 °C followed by 3 washes in PBS for 5 minutes each the following day. Subsequently, the coverslips were incubated with the appropriate fluorescent secondary antibody for 1 hour at room temperature in

25 the dark. Cells were then treated with DAPI for 1 minute and washed 3 times in PBS for 10 minutes each. Where appropriate, cells were also treated with fluorescently-labelled primary antibodies, either through Zenon Alexa 555 (Thermofisher, Cat# 25005) or APEX Alexa 488 (Cat#10468), after DAPI for 1 hr and followed with 3 washes in PBS 10 minutes each. If conjugated primary antibodies were added, a post-fixation step was performed that was also equivalent to the initial fixation mentioned earlier. The fixed samples were mounted on microscope slides using DAKO or Prolong Diamond Antifade (Thermofisher, Cat# 36965) mountant solution and allowed to set at room temperature over night and in the dark. Images were taken using the Quorum Spinning Disk Confocal microscope with the 63x oil immersion objective. All images were visualized in the Volocity software and deconvolved before the Pearson’s Correlation Coefficients (PCC) were calculated for each cell.

2.10 Structural prediction and protein-protein docking simulations

The crystal structure of the PH-BEACH regions of NBEAL2 (1877-2400aa) was modelled using the Protein Homology/analogy Recognition Engine V2.0 (PHYRE 2) server and downloaded as a pdb file for docking simulations (86). Protein-protein docking simulations were analyzed through the ZDock server using the PHYRE 2 structure of NBEAL2 or the crystal structure of NBEA (1MI1) combined with the crystal structure of the VAPA MSP domain (2RR3) (87). All structures were prepared and visualized in PyMol.

Chapter 3: Results

3.1 Yeast-two-hybrid screening

3.1.1 The majority of NBEAL2 truncations cloned into the Yeast-2-Hybrid pGBKT7 vector were unstable in DH5α E. coli

The mechanism by which NBEAL2 mediates α-granule formation is not currently known but NBEAL2 harbors several domains that have been implicated in the formation of multi-protein complexes (74,76). To identify binding partners of NBEAL2, a graduate student (Richard Lo) in the lab had previously performed a preliminary yeast-2-hybrid (Y2H) screen using the C- and N-terminal halves of NBEAL2 as bait against a human bone marrow library. The Y2H screen yielded only a few candidate partners likely due to unreliable construct expression in the yeast system. From these preliminary results, I predicted that NBEAL2 constructs expressing even smaller fragments may be more productive and yield a larger list of possible binding partners (88).

Using PCR, I generated three smaller fragments encompassing clusters of domains, annotated as Fragments A, B, and C (Figure 11). Each fragment of NBEAL2 was cloned into the pGBKT7 vector using NdeI and BamHI cloning sites however, only cloning of Fragment C (pGBKT7-C) was successful. To troubleshoot, restriction enzyme digestions of the pGBKT7-A construct, expected to contain the NBEAL2 Fragment A (~2.5 kilobase-pairs (kbps)) were performed. A single ligation mixture of pGBKT7 and NBEAL2 Fragment A was transformed

Figure 11. Schematic of the NBEAL2 cloning strategy for Y2H. The NBEAL2 protein was divided into three fragments: Fragment A spanned from amino acid residue 0-861, Fragment B spanned residues 860-1666, and Fragment C spanned residues 1600-2754. All fragments were cloned into the N-terminally MYC-tagged pGBKT7 bait vector using the NdeI and BamHI restriction cut sites. Each domain is represented with a color: blue for the ARM domains (310- 525aa and 937-1102aa), yellow for the Con-A domain (572-853aa), orange for the WD40 repeats (1515-1555aa, 2457-2499aa, 2500-2539aa, 2551-2592aa, and 2685-2724aa), gray for the PH domain (1918-2003aa), and green for the BEACH domain (2053-2345). This schematic is not drawn to scale. 27 into DH5α E. coli and positive clones were selected with kanamycin. Surviving colonies were then grown in liquid culture for 16 hours, and cells harvested for DNA digestion the following day. Intact pGBKT7 lacking NBEAL2 (pGBKT7-empty, ~7.3 kbps) served as a control. As expected, DNA digestion reactions resolved in agarose gels showed single and distinct bands upwards of 7 kbps for the pGBKT7-empty vector when digested with NdeI, BamHI, or a combination of both enzymes (double-digestion, Figure 12). NBEAL2 Fragment A (Insert) was expected to be indigestible with NdeI and BamHI due to the lack of a recognition sequence and resolved as a single band at a predicted size of 2.5 kbps. Similar double-digestion reactions performed for pGBKT7-A plasmids harvested from Colony A showed one faint and two distinct DNA bands that did not correspond in size with pGBKT7-empty at 7.3 kbps or with Fragment A

Figure 12. Verification of NBEAL2 construct instability through DNA restriction enzyme digestion. A singe ligation mixture of sticky-end pGBKT7 and NBEAL2 Fragment A was ligated together overnight using T4 DNA Ligase enzyme. The overnight ligation mixture was transformed into DH5α E. coli bacteria and plated on LB-Kan agar plates to select for positive clones overnight. Four colonies were picked and grown in liquid culture for 16 hours before the plasmids were harvested and digested. Plasmids were digested for 2 hours at 37 °C with either NdeI, BamHI, or both enzymes. Intact pGBKT7 (pGBKT7-empty) served as a positive digestion control and Nbeal2 Fragment A (Insert) served as a negative digestion control. Digestion reactions were resolved on a 1% w/v agarose gel and visualized with ethidium bromide.

28 at 2.5 kbps. This digestion pattern appeared to be inconsistent between different batches of cells, Colony B-D, transformed with the same pGBKT7-A ligation mixtures. The ligation mixture used prior to transformation and without any restriction enzymes was then resolved on 1% w/v agarose and showed multiple bands that likely indicated the natural, higher-ordered structures of plasmids and suggested that ligation was successful (89). The same results were also observed when restriction enzyme digestions were performed with pGBKT7-B ligation mixtures. Bacterial cloning has been studied extensively and it is not uncommon to have unstably-cloned fragments due to the features of the cloning vectors, the unintentional production of toxic proteins, repetitious nucleotides, or strong secondary structures (90-92). To allow for the propagation of unstable plasmids, bacteria were also grown at a lower temperature of 30 °C, as opposed to 37 °C, and were unsuccessful. Modifications to the cloning strategy including altered primers and altered truncations yielded the same result. Unlike fragments A and B, fragment C is likely stable and was subsequently used as bait in my Y2H screen while fragments A and B were omitted.

3.1.2 NBEAL2 fragment C was expressed in the yeast system and did not exhibit reporter autoactivation

To determine whether NBEAL2 Fragment C was suitable for Y2H, the corresponding pGBKT7-C vector was transformed into the AH109 yeast strain (AH109-C) and controlled experiments were performed. To check for expression of the bait-fusion protein, I first tried the freeze-thaw lysis method used by alumnus from our lab but, was unable to see any protein bands on western blot when probed with the α-MYC antibody. Due to my low lysis efficiency, I then tried mechanical disruption and bulk protein precipitation through the TCA lysis method however, the results remained the same. Subsequent lysis with Urea-SDS worked the best and showed that pGBKT7-C was expressed in the yeast system, albeit weakly (Figure 13). NBEAL2 Fragment C and Gal4 DNA binding domains were expected to have a molecular weight of 128 kDa and 18 kDa respectively. As such, the combined fusion protein is predicted to resolve at 146 kDa. The largest and most distinct band migrated relatively close to the 135 kDa protein ladder band, with a multiplicity of bands appearing underneath. No protein bands were observed for untransformed AH109 cells as expected, confirming that the NBEAL2 Fragment C-bait fusion protein was expressed in the yeast system.

AH109 yeast cells were engineered to lack the enzymes to synthesize a select number of amino acid nutrients. If the pGBKT7-C bait-fusion protein non-specifically interacted with either of the Adenine (Ade) or Histidine (His) amino acid reporter genes, then AH109-C is expected to survive in amino acid-deficient conditions. In addition, Tryptophan (Trp) was used as a nutrient transformation marker, which is supplied by the pGBKT7 vector to the yeast cells. Untransformed AH109 yeast cells would then served as a negative control. As expected, two different AH109-C yeast colonies (annotated as 2-2 and 3-2) placed on synthetic dropout (SD) agar lacking only the Trp nutrient (SD/-Trp) showed abundant growth (Figure 14). These colonies appeared large, translucent, and beige in color with cells concentrated near the center of the cluster. AH109-C cells placed under Trp, Ade, and His deficiency (SD/-Trp/-Ade/-His) showed no growth and confirmed that NBEAL2 Fragment C did not exhibit non-specific activation of reporter genes. Untransformed AH109 cells were not able to grow under any of the

Figure 13. pGBKT7-C construct is expressed in AH109 yeast cells. AH109 cells were transformed with the pGBKT7-C plasmid and grown to an OD600 of 0.577 before being lysed using the SDS/Urea method. Untransformed AH109 cells were lysed under the same condition and used as a negative control. 20% of the total lysate was loaded into a 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The blot was probed for the GAL4-MYC- NBEAL2-Fragment-C fusion protein (146 kDa) using the α-MYC antibody (9e10, 1:1000 dilution). The expected molecular weights of proteins are indicated with the black arrow heads.

30 conditions mentioned above as expected. Together, results verified that NBEAL2 Fragment C was suitable for Y2H.

3.1.3 A small variety of binding partners were screened in Y2H with NBEAL2 Fragment C as bait

AH109-C yeasts were mated with the Y187 donor cells that were purchased pre- transformed with cDNA of a human bone marrow library. Respective yeast mates were screened on stringent Quadruple amino acid Drop-Out (-His/-Leu/-Trp/-Ade; QDO) plates. A total of 29 positive interaction colonies through 3 experimental trials were detected using the X-α-Gal assay. All positive mates were identified by plasmid sequencing and nucleotide BLAST search where the results are tabulated in Table 7. The results from the negative control mating of AH109 cells transformed with intact pGBKT7, without an NBEAL2 insert, with Y187 cells are tabulated in Table 8. The average mating efficiencies were calculated to be 2.9% and 2.8% respectively, which was at the lower spectrum of the predicted efficiency of 2-5% by the manufacturer Clontech. Candidates obtained from negative control mating largely consisted of Hemoglobin and Ferritin. Mating with NBEAL2 Fragment C screened one consistent protein

Figure 14. pGBKT7-C tested negative for reporter autoactivation. To assess whether NBEAL2 Fragment C exhibited non-specific activation of yeast reporter genes, AH109 cells were transformed with pGBKT7-C and plated on SD/-Trp and SD/-Trp/-Ade/-Trp nutrient deficient agar plates and incubated for 3 days. Untransformed AH109 cells were used as a negative control. Blue boxes enclose the area where yeast cells were placed. Two individual colonies of AH109-C yeast cells were streaked in each condition, annotated with the numbers 2-2 and 3-2. Back arrow heads point towards a single cluster of yeast cells that were seen.

31 known as beta subunit of the Prolyl-4-Hydroxylase beta peptide (P4HB). Less frequent but unique partners include CDC20, CD63, CD74, and Myosin 1F. Sequencing and nucleotide BLAST search of some positive mates showed proteins unrelated to humans for example, the Kunitz-type Protease Inhibitor B from the Solanum berthaultii plant. Since these partners were likely irrelevant in the human bone marrow, they were grouped together into the “Others” category. The ‘Others’ category also included ones that tested positive in the X-α-Gal assay but were not able to be sequenced due to the failure of the T7 sequencing primer to recognize the pGBKT7 promoter. Thus, I reasoned that these mates were likely false-positives of the screen.

Table 7. Summary of interaction results obtained from 3 individual mating events with pGBKT7-C as bait against the human bone marrow library. The full names of proteins are shown where space allows but protein acronyms are also used. “Other” denoted mates that were unsuccessfully sequenced or appeared as bacterial proteins through BLAST search. The average mating efficiency was 2.9% across 3 events. Mating #1 Mating #2 Mating #3 Protein Name Freq Protein Name Freq Protein Name Freq Hemoglobin 3 Hemoglobin 3 HMBS 3 CDC20 1 CD63 2 Hemoglobin 3 P4HB 2 Ferritin Light Chain 1 Ferritin Light Chain 2 CD74 1 P4HB 1 MYO1F 1 Ferritin Light Chain 1 Other 1 P4HB 1 Other 1 Other 2

Table 8. Summary of interaction results obtained from 3 individual mating events with empty pGBKT7 against the human bone marrow library. The full names of proteins are shown where space allows but protein acronyms are also used. “Other” denoted mates that were unsuccessfully sequenced or appeared as bacterial proteins through BLAST search. The average mating efficiency was 2.8% across 3 events. Mating #1 Mating #2 Mating #3 Protein Name Freq Protein Name Freq Protein Name Freq Hemoglobin 2 Hemoglobin 2 Ferritin Light Chain 2 Other 3 Ferritin Light Chain 1 Hemoglobin 1 Other 1

3.2 Verifying candidate binding partners to NBEAL2

3.2.1 Cross-referencing binding partners from Y2H and affinity purification-mass spectrometry screens showed no overlap

To supplement our list of binding partners, a graduate student in the lab (Richard Lo) performed affinity purification-mass spectrometry (AP-MS) of the NBEAL2 protein within the megakaryocytic Dami cells in parallel to my Y2H screen. Affinity purification was performed using α-FLAG beads towards stably expressing GFP-NBEAL2-FLAG in the megakaryocytic Dami cells (NBEAL2-stable Dami) where affinity purified eluates were trypsinized and analyzed through mass spectrometry. This screen was repeated a total of three times to obtain a large list of binding partners. I used this list to identify potential binding partners to NBEAL2. However, comparisons of protein binding partners obtained through Y2H screen with the list of partners that were obtained from AP-MS did not reveal any overlapping proteins.

3.2.2 VAPA, among a list of AP-MS candidates, could be an NBEAL2-interacting protein

Many binding partners identified through AP-MS can be categorized into several broad groupings based on function: intracellular signalling, actin/microtubule structural dynamics, vesicular trafficking, and others. From Y2H however, the list was less diverse. Several proteins were selected from each grouping to be verified using Co-IP based on a combination of three factors: reproducibility, the uniqueness of the hit defined as a hit that does not appear in the negative control pull-down, and their corresponding protein functions. Most of the proteins selected for verification including SCAMP3, Rho-associated protein kinase 1 (ROCK1), and Coatomer Protein Complex Subunit Beta 2 (COPB2) were unable to Co-IP with NBEAL2. However, VAPA was able to Co-IP with NBEAL2 (Figure 15). The family of VAP (VAMP- associated protein) proteins are ubiquitously expressed with known roles in lipid transport, vesicular trafficking, and neurotransmitter release (48,93). The ability for VAPA to tether the ER to the LE and influence LE maturation through lipid transfer makes it an interesting NBEAL2-binding protein to study in terms of MK maturation and α-granule formation (50).

Using PCR, I cloned a truncated, N-terminal MYC-tagged VAPA construct that lacked the transmembrane domain (TMD; MYC-VAPAΔ) to optimize for Co-IP (Figure 15A). The MYC-VAPAΔ and GFP-NBEAL2-FLAG vectors were co-transfected into HEK293 cells and Co-IP was performed with α-FLAG and α-MYC beads. The distinct protein band corresponding

33 to GFP-NBEAL2-FLAG in the input lane suggested that this construct was expressed well in HEK293 cells but not as well as MYC-VAPAΔ (Figure 15B). The appearance of GFP- NBEAL2-FLAG in the α-FLAG-IP and MYC-VAPAΔ in the α-MYC-IP lanes confirmed that precipitations using the corresponding beads were successful. The presence of MYC-VAPAΔ in the α-FLAG-IP lane, which was more intense than that seen in the negative control IgG immunoprecipitation, indicated that MYC-VAPAΔ co-immunoprecipitated with GFP- NBEAL2- FLAG. In the reverse Co-IP, a very faint protein band corresponding to GFP- NBEAL2-FLAG was present in the α-MYC-IP lane, that was absent in the negative control

Figure 15. MYC-VAPAΔ co-immunoprecipitated with GFP-NBEAL2-FLAG in HEK293 cells. To verify the VAPA-NBEAL2 interaction seen from AP-MS results, a VAPA construct was generated lacking the TMD at residues 221-242 (MYC-VAPA∆). VAPA, The Major Sperm Protein domain between residues 14-118 and the coiled-coil domain between residues 162-198, of VAPA remained intact (A). HEK293 cells were co-transfected with MYC-VAPAΔ and GFP- NBEAL2-FLAG vectors and Co-IP was repeated using pre-conjugated α-FLAG and self- conjugated α-MYC beads. Pre-conjugated IgG beads served as a negative control. Eluates were resolved on 10% SDS-PAGE and membranes were probed for MYC-VAPAΔ with α-MYC (9e10, 1:1000 dilution) and for GFP-NBEAL2-FLAG with α-FLAG (M2, 1:1000 dilution) antibodies simultaneously (B). All conditions were performed in parallel. The expected molecular weights of proteins are indicated with the black arrow heads.

MYC-IgG pull-down, indicated that GPF-NBEAL2-VAPA co-immunoprecipitated with MYC- VAPAΔ. It was noted that the co-immunoprecipitation of MYC-VAPAΔ with GFP-NBEAL2- FLAG was more consistent than the co-immunoprecipitation of GFP-NBEAL2-FLAG with MYC-VAPAΔ. Nevertheless, these results suggested that MYC-VAPAΔ interacted with GFP- NBEAL2-FLAG in HEK293 cells.

3.3 Verifying the NBEAL2-VAPA interaction

3.3.1 Full length MYC-VAPA can co-immunoprecipitate with HA-VAPB

To determine whether the N-terminal MYC-tag perturbs known VAPA-interacting partners, I decided to evaluate binding to the well-known partner VAPB (VAMP-associated protein B) (48). Through PCR, I generated an N-terminally MYC-tagged, full length VAPA construct (MYC-VAPA) using XhoI and NotI cloning sites. I also cloned full length VAPB with an N-terminal HA-tagged (HA-VAPB). HEK293 cells were co-transfected with both MYC- VAPA and HA-VAPB constructs and Co-IP was performed where α-MYC beads were used to immunoprecipitate MYC-VAPA. Although both proteins were under the influence of equivalent promoters, MYC-VAPA was expressed slightly more abundantly than HA-VAPB as shown in the Input lane (Figure 16). There was a visible VAPA band in the IgG pull-down, but the large amount of MYC-VAPA in the α-MYC-IP lane suggested that immunoprecipitation of MYC- VAPA with the beads was successful. The presence of HA-VAPB in the α-MYC-IP lane, not seen in the negative control IgG pull-down, suggested that HA-VAPB was able to co- immunoprecipitate with MYC-VAPA. Interestingly, only a small amount HA-VAPB co- immunoprecipitated with MYC-VAPA as can be seen with the drastically different bands intensities in α-MYC-IP lane. This Co-IP result suggested that the MYC-epitope tag did not interfere with the interaction between VAPA and VAPB.

3.3.2 Full length MYC-VAPA can co-immunoprecipitate with GFP-NBEAL2-FLAG in HEK293 and NBEAL2-stable Dami cells

Previous results suggested that MYC-VAPAΔ, a truncated VAPA construct, was able to co-immunoprecipitate with GFP-NBEAL2-FLAG and that N-terminal MYC-tag did not disrupt the known interaction between VAPA and VAPB. Thus, I predicted that the interaction between GFP-NBEAL2-FLAG and full length VAPA will also remain intact. To confirm this, HEK293

35 cells were co-transfected with MYC-VAPA and GFP-NBEAL2-FLAG constructs and Co-IP was repeated with α-FLAG and α-MYC beads (Figure 17). As expected, the precipitation of GFP-NBEAL2-FLAG using beads was successful since a distinct and intense protein band above the 245 kDa ladder marker, like in the Input, was seen in the α-FLAG-IP lane. The presence of MYC-VAPA in the α-MYC-IP also suggested that precipitation with α-MYC beads was successful. The presence of MYC-VAPA in the α-FLAG-IP lane, noticeably more intense than that seen in the negative control IgG pull-down, suggested that MYC-VAPA was able to Co-IP with GFP-NBEAL2-FLAG. In the reverse Co-IP condition however, GFP-NBEAL2- FLAG protein signal was not present in the α-MYC-IP lane. Multiple repetitions of this Co-IP showed that GFP-NBEAL2-FLAG inconsistently co-immunoprecipitated with MYC-VAPA,

Figure 16. MYC-VAPA co-immunoprecipitated with VAPB in HEK293 cells. VAPA and VAPB constructs were generated with an N-terminal MYC and HA tag respectively and co- transfected into the HEK293 system. Self-conjugated α-MYC beads were used to immunoprecipitate MYC-VAPA where self-conjugated IgG beads served as a negative control. Eluates were resolved on a 10% SDS-PAGE gel where the membrane was blotted for HA- VAPB using α-HA antibodies (A190-208A, 1:1000 dilution) and for MYC-VAPA using α- MYC antibodies (9e10, 1:1000 dilution) simultaneously. All conditions were performed in parallel. The expected molecular weights of proteins are indicated with the black arrow heads.

36 similar to previous results shown here with MYC-VAPAΔ.

Next, I transfected NBEAL2-stable Dami cells with MYC-VAPA to utilize a closer-to- endogenous expression of the NBEAL2 protein in a relevant cell. Co-IP experiments were repeated with α-FLAG and α-MYC beads as before. The decreased expression of GFP- NBEAL2-FLAG was expected in the Input lane and the presence of a protein band corresponding to MYC-VAPA indicated that expression was reasonable (Figure 18). The presence of GFP-NBEAL2-FLAG in the α-FLAG-IP and MYC-VAPA in the α-MYC-IP lanes suggested that precipitations with the corresponding beads were successful. Consistent with Co- IP in the HEK293 system, the appearance of an intense protein band corresponding to MYC- VAPA, not as defined in the negative control IgG pull-down, confirmed that MYC-VAPA co- immunoprecipitated with GFP-NBEAL2-FLAG in NBEAL2-stable Dami cells. In the reverse pull-down, GFP-NBEAL2-FLAG was not seen in the α-MYC-IP lane nor in the corresponding

Figure 17. MYC-VAPA co-immunoprecipitated with GFP-NBEAL2-FLAG in HEK293 cells. To verify that the full length VAPA protein can exhibit NBEAL2-binding, HEK293 cells were co-transfected with Gfp-Nbeal2-Flag and Myc-Vapa constructs. Co-IP experiments were repeated with pre-conjugated α-FLAG and self-conjugated α-MYC beads. Pre-conjugated IgG beads served as a negative control. Eluates were resolved on a 10% SDS-PAGE and the membrane was probed for MYC-VAPA using α-MYC (9e10, 1:1000 dilution) antibodies and for GFP-NBEAL2-FLAG using α-FLAG (M2, 1:1000 dilution) antibodies simultaneously. All conditions were performed in parallel. The expected molecular weights of proteins are indicated with the black arrow heads.

37 corresponding to MYC-VAPA indicated that expression was reasonable (Figure 18). The presence of GFP-NBEAL2-FLAG in the α-FLAG-IP and MYC-VAPA in the α-MYC-IP lanes suggested that precipitations with the corresponding beads were successful. Consistent with Co- IP in the HEK293 system, the appearance of an intense protein band corresponding to MYC- VAPA, not as defined in the negative control IgG pull-down, confirmed that MYC-VAPA co- immunoprecipitated with GFP-NBEAL2-FLAG in NBEAL2-stable Dami cells. In the reverse pull-down, GFP-NBEAL2-FLAG was not seen in the α-MYC-IP lane nor in the corresponding negative IgG pull-down. These results confirmed that full length MYC-VAPA could Co-IP with GFP-NBEAL2-FLAG in the relevant cell type.

Figure 18. Full length MYC-VAPA co-immunoprecipitated with GFP-NBEAL2-FLAG in Dami cells. To verify that full length MYC-VAPA can interact with GFP-NBEAL2-FLAG in a relevant cell type, NBEAL2-stable Dami cells were transfected with MYC-VAPA constructs. Co-IP experiments were repeated with pre-conjugated α-FLAG and self-conjugated α-MYC beads. Pre-conjugated IgG beads served as a negative control. Eluates were resolved on a 10% SDS-PAGE and the membrane was probed for MYC-VAPA using α-MYC (9e10, 1:1000 dilution) and for GFP-NBEAL2-FLAG using α-FLAG (M2, 1:1000 dilution) antibodies sequentially in that order. Both blots depicted above are cropped from the same larger blot. All conditions were performed in parallel. The expected molecular weights of proteins are indicated with the black arrow heads.

3.3.3 Endogenous VAPA can Co-IP with GFP-NBEAL2-FLAG in NBEAL2-stable Dami cells To confirm that VAPA interacts with NBEAL2, NBEAL2-stable Dami cell lysates were used in Co-IP experiments where GFP-NBEAL2-FLAG was immunoprecipitated with α-FLAG beads and probed for endogenous VAPA. The presence of protein bands corresponding to GFP- NBEAL2-FLAG and endogenous VAPA indicated that these two proteins were expressed well in the NBEAL2-stable Dami cells (Figure 19). The presence of GFP-NBEAL2-FLAG in the α- FLAG-IP lane also suggested that immunoprecipitation was successful. The presence of endogenous VAPA in the α-FLAG-IP, not seen in the negative control IgG pull-down, suggested

Figure 19. Endogenous VAPA co-immunoprecipitated with GFP-NBEAL2-FLAG in the NBEAL2-stable Dami cells. Co-IP experiments were conducted to reconfirm a list of hits following AP-MS where GFP-NBEAL2-FLAG from NBEAL2-stable Dami cells was immunoprecipitated with pre-conjugated α-FLAG beads. Pre-conjugated IgG beads (FLAG- IgG) served as a negative control. Eluates were resolved on a 10% SDS-PAGE gel and the membrane was probed for endogenous VAPA (HPA009174, 1:1000 dilution) and α-FLAG (M2, 1:1000 dilution) simultaneously. VAPA is predicted to be 28 kDa and GFP-NBEAL2-FLAG is 329 kDa. All conditions were performed in parallel. The expected molecular weights of proteins are annotated on the right slide of images following black arrow heads.

39 that endogenous VAPA co-immunoprecipitated with GFP-NBEAL2-FLAG in the NBEAL2- stable Dami cells. The respective protein band intensities however, were only a fraction of those seen in the input lane and suggested that Co-IP conditions were suboptimal. Results shown here recapitulated the AP-MS results obtained by the previously mentioned graduate student (Richard Lo).

3.4 Characterizing the interaction between NBEAL2 and VAPA

3.4.1 HA-VAPB does not Co-IP with GFP-NBEAL2-FLAG in HEK293 cells

The family of VAP proteins, VAPA and VAPB, have been known to form homo- and heterodimers at the ER membrane (48). Both VAPA and VAPB harbor the same domains, similar structure, and share 63% sequence similarity (94). It is possible that NBEAL2 may also interact with VAPB, however, the AP-MS screen did not show VAPB as a potential binding partner. To determine whether NBEAL2 had specificity for VAPB, HEK293 cells were co- transfected with GFP-NBEAL2-FLAG and HA-VAPB constructs and Co-IP experiments were conducted with α-FLAG and α-HA beads in HEK293 cells. As could be seen in the Input lane, HEK293 cells were able to express HA-VAPB very well however, no visible protein band was seen that corresponded to GFP-NBEAL2-FLAG (Figure 20). This suggested that the expression of GFP-NBEAL2-FLAG construct was very weak. However, the appearance of a single distinct protein band above the 245 kDa ladder marker in the α-FLAG-IP lane suggested that immunoprecipitation was a success regardless. In addition, a single distinct protein band corresponding to HA-VAPB in the α-HA-IP lane also suggested that immunoprecipitation was successful. The absence of the HA-VAPB protein band from the α-FLAG-IP and the absence of GFP-NBEAL2-FLAG from the α-HA-IP lanes suggested that HA-VAPB could not Co-IP with GFP-NBEAL2-FLAG.

3.4.2 The PH-BEACH domains of NBEAL2 may be responsible for the VAPA interaction

To further characterize the interaction between NBEAL2 and VAPA, NBEAL2 truncations were generated each harboring individual or sets of domains. Cloning of NBEAL2 into six fragments labelled C1 through to C6, each with an N-terminal HA-tag, was done by another graduate student (Richard Lo) in the lab (Figure 21A). HEK293 cells were co- transfected with a combination of one NBEAL2 truncation with MYC-VAPAΔ. The presence of

HA-C1, HA-C2, HA-C3, HA-C4, HA-C5, and HA-C6 protein bands within their respective α- HA-IP lanes confirmed that these constructs expressed and immunoprecipitated successfully using α-HA beads (Figure 21B-G). The absence of MYC-VAPAΔ protein bands in the α-HA- IP lanes of the HA-C1, HA-C2, and HA-C6 constructs suggested that these portions of NBEAL2 were not individually responsible for MYC-VAPAΔ-binding (Figure 21B-C, G). Although MYC-VAPAΔ appeared present in the α-HA-IP lane of the HA-C3 and HA-C4 conditions, these protein bands were indistinguishable and less intense when compared to the negative control IgG pull-down, which suggested that MYC-VAPAΔ did not interact with these constructs (Figure

Figure 20. HA-VAPB did not co-immunoprecipitate with GFP-NBEAL2-FLAG in HEK293 cells. To determine whether NBEAL2 had specificity towards only VAPA, its homologue VAPB was used in Co-IP experiments. HEK293 cells were co-transfected with HA-VAPB and GFP-NBEAL2-FLAG constructs. Pre-conjugated α-FLAG and self-conjugated α-HA beads were used in precipitation where pre-conjugated IgG beads served as negative control. Eluates were resolved on a 10% SDS-PAGE gel and membranes were probed for HA-VAPB using α- HA (A190-208A, 1:1000 dilution) and for GFP-NBEAL2-FLAG using α-FLAG (M2, 1:1000 dilution) antibodies sequentially in that order. Both large blots depicted above are from the same larger blot. Cell lysates were resolved on a separate SDS-PAGE gel and was included as a cropped image. The expected molecular weights of proteins are indicated with the black arrow heads.

21D-E). However, the appearance of MYC-VAPAΔ in the α-HA-IP experiment with the HA-C5 construct was more intense than the IgG control suggested that this region bound to MYC- VAPAΔ (Figure 21F). Interestingly, the HA-C5 construct was expressed as two protein bands, roughly 9 kDa apart from each other, in the HEK293 cells when the actual protein was expected to resolve at 58 kDa. Taken together, these domain mapping experiments suggested that HA-C5 is required for MYC-VAPAΔ-binding.

3.4.3 HA-C5 is predicted to harbour several atypical FFAT motifs for VAPA-binding

It is well known that VAPA interact with proteins that harbor a two-phenylalanine acidic tract (FFAT) motif through its cytosolic Major Sperm Protein (MSP) domain (48). The canonical FFAT motif contains a canonical core sequence of “E-F-F-D-A-x-E” where ‘x’ represented any amino acid, all flanked by regions rich in acidic residues upstream and downstream however, residues within this sequence can be flexible (48). To determine whether NBEAL2 contained a FFAT motif within the HA-C5 construct, I followed two approaches: a manual search for a FFAT-like sequence and a subsequent algorithm-based search. In a manual search for the exact canonical sequence, I found one non-canonical/atypical sequence (ncFFAT) sharing the first three residues “E-F-F” of the canonical sequence however, the other 4 residues “Y-F-P-D” were only similar and not identical to the canonical sequence. I referred to this sequence as the ncFFAT* motif. I later implemented Murphy and Levine’s searching algorithm, which identified four other ncFFAT motifs (first 4 rows of Table 9) (48). All the predicted motifs identified through the algorithm showed weak binding scores between 4 and 5 and were spread out across the PH-BEACH region including 2 in the BEACH domain, one in the PH domain, and one situated between these two domains. However, the ncFFAT* motif did not appear among the 4 predicted motifs. It was noted by the authors that the FFAT motif searching algorithm returns the top 4 predictions based on binding scores (48). Therefore, it was possible that the absence of the ncFFAT* motif from the predictions was due to a lower predicted binding score. To determine whether the ncFFAT* motif that I discovered initially could also be identified as a ncFFAT motif through the algorithm, a smaller portion of the PH-BEACH region from residues 2152-2400 was analyzed. Interestingly, the ncFFAT* motif appeared as a predicted atypical FFAT motif with a weaker binding score (last row of Table 9). To determine whether ncFFAT motifs were also predicted outside of the PH-BEACH region of the NBEAL2 protein, full length NBEAL2

Figure 21. MYC-VAPAΔ co-immunoprecipitated with specific domain containing fragments of GFP-NBEAL2-FLAG in HEK293 cells. To elucidate the domain(s) required in the NBEAL2-VAPA interaction, domain mapping experiments were performed. Nbeal2 truncations were generated by another graduate student (Richard Lo) with N-terminal HA-tags and are annotated here as C1-C6 (A). HEK293 cells were co-transfected with a combination of one NBEAL2 truncation (either HA-C1 (B), HA-C2 (C), HA-C3 (D), HA- C4 (E), HA-C5 (F), or HA-C6 (G)) and MYC-VAPA∆ for Co-IP experiments. Precipitations were performed using self-conjugated α-HA and IgG beads. Eluates were resolved on 10% SDS-PAGE gels and membranes were probed for MYC-VAPAΔ with α-MYC (9e10, 1:1000 dilution) and for fragments of NBEAL2 with α-HA (A190-208A, 1:1000 dilution) antibodies in that order. Every set of conditions within a single co-transfection were run in parallel. The cropped blots within each set of co-transfections above originated from the same larger membrane. The expected molecular weights of proteins are indicated with the arrow heads.

43 was analyzed using the searching algorithm. Of the 4 predicted motifs, the algorithm returned three ncFFAT motifs within the Con-A domain where the remaining motif was situated in the BEACH domain (Table 10). Like the motifs predicted in the PH-BEACH region, these ncFFAT motifs in the CON-A domain all appeared to have weak binding scores to VAPA however, they tended to score slightly better than most motifs in the PH-BEACH region.

Table 9. The PH-BEACH domains of NBEAL2 are predicted to contain at least five non- canonical FFAT motifs. Using the FFAT motif searching algorithm published by Murphy and Levine (48), several non-canonical FFAT motifs were identified within the PH-BEACH region (residues 1877-2400) of NBEAL2. The FFAT motifs are given as a total of 15 amino acids consisting of 6 residues upstream of a 7-residue core followed by 2 residues downstream. Binding scores range from 0-2 for strong binding, 2.5-4 for OK binding, 4-7 for weak binding, and 7+ for poor binding.

Amino acid Domain FFAT motif sequence (6 N-term + 7 Core + 2 C-term) Binding score position 2264 BEACH RQALESEYVSAHLHE 4 1941 PH VTTQNVYFYDGSTER 4.5 2042 --- LRPPSQGYLSSRSPQ 5 2294 BEACH EEALNVFYYCTYEGA 5 2212 BEACH VKELIPEFFYFPDFL 5.5

Table 10. NBEAL2 is predicted to also contain non-canonical FFAT motifs outside of the PH- BEACH regions. Using the FFAT motif searching algorithm published by Murphy and Levine (48), several non-canonical FFAT motifs were identified outside of the PH-BEACH region when the full NBEAL2 (residues 1-2754) were submitted. The FFAT motifs are given as a total of 15 amino acids consisting of 6 residues upstream of a 7-residue core followed by 2 residues downstream. Binding scores range from 0-2 for strong binding, 2.5-4 for OK binding, 4-7 for weak binding, and 7+ for poor binding.

Amino acid Domain FFAT motif sequence (6 N-term + 7 Core + 2 C-term) Binding score position 638 CONA SGSGFEAFFTAAGTL 3.5 627 CONA QRKQLYSFFTSSGSG 4 2264 BEACH RQALESEYVSAHLHE 4 802 CONA ELGAVAIFHEALQAT 4.5

3.5 Searching for relevant ncFFAT motifs in the NBEAL2-VAPA interaction

3.5.1 The structure of the PH-BEACH regions in NBEAL2 is predicted to be homologous to that of NBEA

It is possible that some of the predicted ncFFAT motifs are required for binding to VAPA while others are sequestered from the surface of the NBEAL2 protein because of folding. The identification of surface-exposed motifs would require a resolved crystal structure; however, no such structure exists for NBEAL2. The only PH-BEACH crystal structures currently available were generated for two other BDCPs: LRBA and NBEA. Protein BLAST alignment determined that the PH-BEACH region of NBEAL2 shared 35% identity and 52% amino sequence similarity to NBEA. Residues 1877-2400 of NBEAL2 were analyzed for protein structural homology predictions through PHYRE 2 and results suggested that this structure was highly homologous to the PH-BEACH regions of NBEA, with 100% confidence and covering 78% of the submitted residues. When the structures were visualized using computer software modeling (PyMol), the predicted structure of the PH-BEACH region in NBEAL2 appeared to contain a large binding pocket, circled in red, at the center of one face of the protein that was not seen elsewhere (Figure 22). The PH-BEACH structure of NBEA also contained a similar binding pocket (Figure A1).

3.5.2 The PH-BEACH regions of NBEAL2 and NBEA share similar ncFFAT motifs

The structural similarity and the presence of similar domains between NBEAL2 and NBEA suggested that NBEA may also interact with VAPA. The protein sequences for the PH- BEACH regions of NBEA (residues 2147-2563) was analyzed in the FFAT-searching algorithm and several ncFFAT motifs were discovered, with binding scores between 3.5 and 6. Of the 5 predicted ncFFAT motifs for NBEA, 3 were predicted to reside within the BEACH domain and 1 in the PH domain (first 4 rows of Table A1). Like NBEAL2, when the full protein sequence of NBEA was analyzed, the algorithm predicted several ncFFAT motifs outside of the PH- BEACH region including some within the CON-A and ARM domains (Table A2). The binding scores for the full-length protein screen was also classified as weak, between 3 and 6, similar to what was seen for NBEAL2.

To determine whether NBEAL2 and NBEA shared similar ncFFAT motifs, protein sequence alignment was performed using whole protein sequences. Within the PH-BEACH

45 regions of NBEAL2 and NBEA, similar ncFFAT motifs were present in both proteins (Figure 23). These motifs appeared to be positioned within the same regions compared to each other, including the motifs colored in red and green. However, the opposite was also true – two motifs predicted within NBEAL2 were not predicted to be within NBEA including the ones colored in purple and cyan. As well, two motifs that were predicted within NBEA were not predicted to be within NBEAL2 and included the ones colored in blue and yellow. To determine whether NBEA contained a similar motif to the ncFFAT* motif (colored in magenta) that was discovered earlier, a smaller region of NBEA from residues 2398-2476 was analyzed instead of the full-length protein. Interestingly, NBEA was predicted to harbor a ncFFAT motif that was not previously detected (last row of Table A1). As well, this novel ncFFAT motif appeared within the same vicinity as the ncFFAT* motif of NBEAL2. This novel ncFFAT motif of NBEA scored higher in VAPA binding compared to the other motifs like its NBEAL2 counterpart.

Figure 22. The PH-BEACH regions of NBEAL2 contains a prominent binding pocket. Residues 1877-2400 of NBEAL2, analyzed in PHYRE 2 for structural homology prediction, generated a structure using NBEA as a template due to high homology. The above images are 3D surface models, visualized in PyMol, depicting the possible binding pocket in the center panel. Images were rotated either to the clockwise (+90°) or counterclockwise (-90°) based on the center panel. The red circle depicts a potential binding pocket on the protein.

NBEAL2 1918 LVLSAECQLVTVVAVVPGLLEVTTQNVY 1947 NBEA 2417 VVLSTPAQLIAPVVVAKGTLSITTTEIY 2179

NBEAL2 1948 FYDG------STERVETEEGIGYDFRRPLAQLREVHLRRFNLRRSALELFFIDQANYFLNFPCKVGTTPVSSPSQTP 2018 NBEA 2180 FEVDEDDSAFKKIDTKVLAYTEGL--HGKWMFSEIRAVFSRRYLLQNTALEVFMANRTSVMFNFPDQATVKKVVY--SLP 2255

NBEAL2 2019 RPQPGPIPPHTQVRNQVYSWLLRLRPPSQGYLSSRSPQEMLRASGLTQKWVQREISNFEYLMQLNTIAGRTYNDLSQYPV 2098 NBEA 2180 RVGVGTSYGLPQARR------ISLATPRQLYKSSNMTQRWQRREISNFEYLMFLNTIAGRTYNDLNQYPV 2319

NBEAL2 2099 FPWVLQDYVSPTLDLSNPAVFRDLSKPIGVVNPKHAQLVREKYESFEDPAgtIDKFHYGTHYSNAAGVMHYLIRVEPFTS 2178 NBEA 2320 FPWVLTNYESEELDLTLPGNFRDLSKPIGALNPKRAVFYAERYETWEDDQ--SPPYHYNTHYSTATSTLSWLVRIEPFTT 2397

NBEAL2 2179 LHVQLQSGRFDCSDRQFHSVAAAWQARLESPADVKELIPEFFYFPDFLENQNGFDLGCLQLtNEKVGDVVLPPWASSPED 2258 NBEA 2398 FFLNANDGKFDHPDRTFSSVARSWRTSQRDTSDVKELIPEFYYLPEMFVNSNGYNLGVRED-EVVVNDVDLPPWAKKPED 2476

NBEAL2 2259 FIQQHRQALESEYVSAHLHEWIDLIFGYKQRGPAAEEALNVFYYCTYEGAVDLDHVTDERERKALEGIISNFGQTPCQLL 2338 NBEA 2477 FVRINRMALESEFVSCQLHQWIDLIFGYKQRGPEAVRALNVFHYLTYEGSVNLDSITDPVLREAMEAQIQNFGQTPSQLL 2556

NBEAL2 2339 KEPHPT 2345 NBEA 2557 IEPHPP 2563

Figure 23. Sequence alignment of NBEAL2 and NBEA show that some predicted non-canonical FFAT motifs were conserved within the PH-BEACH regions. The PH-BEACH regions of NBEAL2 (1918-2345aa) and NBEA (2147-2563aa) are shown from the full alignment performed using COBALT. Dashed lines represented gaps predicted during sequence alignment. Unique non-canonical FFAT motifs were highlighted in different colors including red, purple, blue, yellow, magenta, green, and cyan. Predicted FFAT motifs and their sequences refer to tables 3 and 5. Numbers present amino acid positions within the whole protein.

3.5.3 Predicted ncFFAT motifs could be surface-exposed on NBEAL2

To look for relevant ncFFAT motifs in VAPA binding, all the ncFFAT motifs were mapped onto the predicted structure of the PH-BEACH regions within NBEAL2. Interestingly, all five of the predicted ncFFAT motifs appeared on the surface of NBEAL2, although on different parts of the structure (Figure 24). As well, two motifs depicted in magenta and green were situated within proximity to the binding pocket mentioned earlier. Mapping onto NBEA with the corresponding motifs showed a similar result (Figure A2). In addition to the presence of the magenta and green motifs, a third motif in yellow was also located near this potential binding pocket in NBEA. Like those seen for NBEAL2, other NBEA ncFFAT motifs appeared spread within the protein.

Figure 24. All predicted non-canonical FFAT motifs were found on the surface of the PH- BEACH regions of NBEAL2. Predicted non-canonical FFAT motifs were mapped onto the crystal structure of the PH-BEACH regions in NBEAL2, predicted by PHYRE 2, to resolve for relevant motifs in VAPA binding. The presence of all predicted motifs on the surface suggested that any one or more motifs may be involved. The above images are 3D surface models, visualized in PyMol. Images were rotated either to the clockwise (+90°) or counterclockwise (- 90°) based on the center panel. All colors are coordinated with the ncFFAT motif sequences shown in Figure 23. The presence of the same color across both proteins indicated similar motifs. Non FFAT protein surfaces are shown in gray.

3.5.4 Protein docking simulations implicate the ncFFAT* motif of NBEAL2 in VAPA-binding

It was possible that one or more of the predicted ncFFAT motifs within the PH-BEACH regions of NBEAL2 are implicated in binding to VAPA. To screen for likely motifs in binding, the crystal structure of the VAPA MSP domain (2RR3) and the PHYRE 2-prediction of the PH- BEACH structure of NBEAL2 were analyzed through the ZDock protein-protein docking server. Of the 10 best binding simulations generated, 9 stimulations implicated at least one ncFFAT motif in VAPA-binding (Figure 25). These ncFFAT motifs included the ones represented in magenta, green, and red while the cyan and purple motifs were not predicted to be involved. The remaining simulation, categorised as “Other”, suggested that VAPA interacted with NBEAL2 at a novel site. Interestingly, these docking simulations also suggested that the NBEAL2-VAPA

Figure 25. Summary of protein-protein docking simulations with the PH-BEACH regions of NBEAL2 and the MSP domain of VAPA. The crystal structure of the VAPA MSP domain (2RR3) and the PHYRE 2-prediction, of the PH-BEACH region within NBEAL2, were analyzed for potential binding sites through the ZDock server. The 10 best binding predictions were tabulated and presented in a Venn diagram as the frequency of simulations that implicated one or more ncFFAT motif. All colors are coordinated with the ncFFAT motif sequences shown in Figure 23. Gray represents the collective “Others” group. The “Others” group represented simulations that did not bind to the predicted ncFFAT motifs.

49 interaction may be a combined effort of two motifs involving the magenta (ncFFAT*) and green motifs. The magenta (ncFFAT*) motif was the only motif implicated within the top 3 simulations while the red motif was implicated in the fourth prediction (Figure 26, Table A3). Overall, the magenta (ncFFAT*) motif was predicted to be implicated in 7 of 10 predictions and suggested that the ncFFAT* motif was likely involved in binding. Since the canonical FFAT motifs interacted with residues Lys45, Thr47, Lys87, Met89, and Lys118 on the VAPA MSP domain, these docking simulations were analyzed further to determine if these residues were also implicated (95,96). Some predictions suggested the role of none, one or several of the residues mentioned above due to its presence in the binding interface of NBEAL2 and VAPA, all of which generally did not involve Lys188 (Table A3). However, some binding predictions suggested the involvement of other residues but, are at least suggestive that the interaction between VAPA and NBEAL2 could require both electrostatic and hydrophobic forces like canonical motifs (97).

Docking simulations through the ZDock server were also performed for the PH-BEACH regions of NBEA as a comparison to NBEAL2. Like predictions for NBEAL2, the majority of predictions implicated one or more ncFFAT motifs in VAPA binding however, a larger number of simulations, 4 of 10 simulations, implicated a novel binding site (Figure A3). As well, the binding simulations also suggested that the interaction between the VAPA MSP domain and the PH-BEACH regions of NBEA could be mediated by various combinations of two ncFFAT motifs but not a combination of 3 or 4 motifs together. Of the top 3 binding simulations, only 2 predictions implicated the combined effort of the magenta and green motif while the top prediction suggested a novel binding site (Figure A4). Although the PH-BEACH regions of NBEAL2 is predicted to be highly homologous with the PH-BEACH regions of NBEA, these results suggested that it may have different VAPA-binding properties.

Figure 26. The VAPA MSP domain is predicted to bind at the ncFFAT* motif of NBEAL2. The crystal structure of the VAPA MSP domain (2RR3) and the PHYRE2-prediction, of the PH-BEACH regions within NBEAL2, were analyzed for potential binding sites through the ZDock server. The top five binding predictions are shown here in order from ranked #1 (A), ranked #2 (B), ranked #3 (C), and ranked #4 (D). The above images are 3D surface models, visualized in PyMol, depicting the ncFFTA* motif in the center panel of each row. Images were rotated either to the clockwise (+90°) or counterclockwise (-90°) based on the center panel. All colors co-ordinated exactly with the ncFFAT motif sequences shown in Figure 23 except for orange which represents the VAPA MSP domain and non-FFAT protein surfaces are shown in gray.

3.6 Identifying the important residues in the ncFFAT* motif of NBEAL2 for VAPA-binding

3.6.1 Glu2218 and Asp2224 may be involved in VAPA-binding

The interaction between the VAPA MSP domain and the canonical FFAT motifs requires an initial non-specific electrostatic attraction that is then followed by hydrophobic interactions (97). To determine whether the ncFFAT* motif in the PH-BEACH portion of NBEAL2 exposed acidic residues onto the protein surface, the PHYRE 2-structural prediction was re-analyzed with the ncFFAT* motif residues “V-K-E-L-I-P-E-F-F-Y-F-P-D-F-L” at positions 2212-2226. Interestingly, only four residues of the ncFFAT* motif were exposed on the surface of NBEAL2: Glu2218, Tyr2221, Phe2222, and Asp2224 (Figure 27). These four resides were situated in the ncFFAT core-sequence and not in the flanking regions. Half of these exposed residues were characterized as hydrophobic while the remaining were acidic amino acids. The rest of the ncFFAT* motif appeared to be sequestered inside the protein by protein folding. Like in NBEAL2, the ncFFAT*-like motif in NBEA exposed 4 core residues while the other residues were sequestered under the surface of the protein (Figure A5). As well, 2 of the 4 residues were acidic and the remaining 2 residues were hydrophobic. Interestingly, these exposed acidic residues on NBEAL2 and NBEA lined up perfectly with each other. (Figure 22).

Figure 27. Two acidic ncFFAT* motif residues are predicted to be on the surface of the PH- BEACH region of NBEAL2. PHYRE 2 structural prediction, generated from the analysis of residues 1877-2400, of the PH-BEACH region of NBEAL2, suggested that many of the residues were located inside the protein except for Glu2218, Tyr2221, Phe2222, and Asp2224. The above images are 3D surface models, visualized in PyMol, in an orientation that centers on the ncFFAT* motif colored in magenta as in Figure 23. The inset is a zoomed-in picture depicting the surface-exposed residues of interest. 52

3.6.2 Glu2218 and Asp2224 do not individually mediate binding to VAPA

To determine if the exposed acidic residues Glu2218 and Asp2224 are responsible for the NBEAL2-VAPA interaction, site-directed mutagenesis experiments were performed. Single amino acid mutations were generated in the HA-C5 construct, changing the glutamic acid at position 2218 into a lysine (HA-C5(E2218K)) or changing the aspartic acid at position 2224 into lysine (HA-C5(D2224K)). HEK293 cells were co-transfected with MYC-VAPAΔ and either of the mutagenesis constructs. Un-transfected HEK293 cell lysates served as a double-negative transfection control and transfections with either non-mutated HA-C5 construct or MYC- VAPAΔ served as single-transfection controls. Co-IP experiments were repeated with α-HA and IgG beads where all conditions were divided onto two separate SDS-PAGE gels. As expected, the double-negative transfection control Co-IP experiment did not show non-specific protein bands when probed with α-MYC or α-HA antibodies (lanes 1-3 in Figure 28 and Figure 29).

Figure 28. Co-IP experiments using positive and negative controls resolved as expected. HEK293 cells were transfected with either non-mutated HA-C5, MYC-VAPAΔ alone, both MYC-VAPAΔ, or neither constructs as control experiments. Co-IP was performed with self- conjugated α-HA and negative IgG beads. Eluates for double-negative transfection and non- mutated HA-C5 positive co-transfection were divided between this blot and the blot in Figure 29. All eluates were resolved on 10% SDS-PAGE and membranes were probed for MYC- VAPAΔ with α-MYC (9e10, 1:1000 dilution) and for HA-C5 with α-HA (A190-208A, 1:1000 dilution) antibodies sequentially in that order. All blots derived from the same large blot and all conditions were performed in parallel. The expected molecular weights of proteins are indicated with the black arrow heads.

The Co-IP of MYC-VAPAΔ in the co-transfection condition, that was visibly more intense that that of the IgG pull-down, indicated that MYC-VAPAΔ interacted with the C5 portion of NBEAL2 (lanes 4-6 in Figure 28 and Figure 29). The absence of a MYC-VAPAΔ in the single transfection of HA-C5 was expected (lanes 6-9 in Figure 28). The absence of HA- C5 in the single transfection of MYC-VAPAΔ was also expected (lanes 10-12 in Figure 28). When MYC-VAPAΔ was probed with the α-MYC antibody, small amounts of MYC-VAPAΔ were seen, that was visibly more intense and distinct than that the IgG control, for both the mutation conditions (lanes 7-12 in Figure 29). However, the VAPA signal in the HA-C5(E2218K) immunoprecipitation appeared greater than that the negative IgG Co-IP control. As well, the VAPA signal in the HA-C5(D2224K) appeared weaker than the positive non-mutated C5 condition (lane 4-6 in Figure 29). These results suggest that Glu2218 and Asp2224 are not uniquely required in the interaction between HA-C5 and MYC-VAPAΔ.

Figure 29. Single amino acid mutations E2218K or D224K did not prevent MYC-VAPAΔ binding to HA-C5. HEK293 cells were co-transfected with a combination of MYC-VAPAΔ and either HA-C5(E2218K) or HA-C5(D2224K). Co-IP was performed with self-conjugated α-HA and negative IgG beads. Eluates for double-negative transfection and unmutated HA-C5 positive co-transfection were divided between this blot and the blot in Figure 28. Eluates were resolved on 10% SDS-PAGE and membranes were probed for MYC-VAPAΔ with α-MYC (9e10, 1:1000 dilution) and for HA-C5 with α-HA (A190-208A, 1:1000 dilution) antibodies sequentially in that order. All blots derived from the same large blot and all conditions were performed in parallel. The expected molecular weights of proteins are indicated with the black arrow heads.

3.7 Determining the in vivo localization pattern of VAPA in megakaryocytes

3.7.1 Immunofluorescence microscopy suggested that VAPA partially-localized with GFP- NBEAL2-FLAG and RAB7 in the megakaryocytic NBEAL2-stable Dami cells

To further investigate whether VAPA is a potential NBEAL2-binding partner, megakaryocytic Dami cells analyzed using immunofluorescence (IF) microscopy. NBEAL2- stable Dami cells were stimulated with TPO and PMA for 2 days before fixation, permeabilization, and incubation with the appropriate primary antibodies. Across all conditions, VAPA appeared in a reticular pattern throughout all cells while GFP-NBEAL2-FLAG exhibited a largely cytoplasmic and punctated pattern with some exhibiting membrane-like patterns (Figure 30A). The average Pearson’s Correlation Coefficient (PCC) of VAPA against GFP- NBEAL2-FLAG was 0.463 where the lower and upper 95% confidence interval was 0.248 to 0.654 respectively. Lysosome-Associated Protein 1 (LAMP1) was used to stain lysosomes and showed a defined, organelle-like pattern (Figure 30B). The average PCC of VAPA against LAMP1 was 0.210 where the lower and upper 95% confidence interval was 0.065 to 0.361. RAB7 was used to stain late endosomes (LE) and showed an abundant, vesicular pattern sometimes clustered together in parts of the cell (Figure 30C). The average PCC of VAPA against Zenon-555 tagged RAB7 was 0.468 where the lower and upper 95% confidence interval was 0.299 to 0.636. Lysosomal Associated Protein 3 (CD63) was used to stain the δ-granules and showed distinct, vesicular structures (Figure 30D). The average PCC of VAPA against CD63 was 0.201 where the lower and upper 95% confidence interval was 0.014 to 0.389. Sarco/endoplasmic Reticulum Calcium ATPase 3 (SERCA3) was used to stain the ER and showed a vast reticular pattern (Figure 30E). The average PCC of VAPA against SERCA3 was 0.775 where the lower and upper 95% confidence interval was 0.632 to 0.930. Overall, the IF studies showed that VAPA colocalized with GFP-NBEAL2-FLAG and RAB7 more than with CD63 and LAMP1 positive cellular compartments (Figure 30F).

3.7.2 Immunofluorescence microscopy suggest that VAPA partially localizes with secretory granules, the late endosomes, and NBEAL2 in human bone marrow-derived megakaryocytes

Since Dami cells are a megakaryocytic cell line that lack α-granule-like structures, human bone marrow-derived megakaryocytes were stained to investigate the localization of VAPA to α-granules. Peripheral mobilized CD34+ cells from bone marrow transplant donors were collected and CD34+ primary cells were immunomagnetically purified and differentiated 55 into megakaryocytes for 12 days, and fixed for IF staining. VAPA staining again showed reticular pattern throughout all cells. Similarly, SERCA3 also showed a reticular staining pattern with substantial co-localization with VAPA (Figure 31A). The average PCC of SERCA3 against

Figure 30. VAPA partially co-localized with GFP-NBEAL2-FLAG and RAB7 in NBEAL2- stable Dami cells. Megakaryocytic Dami cells stably expressing GFP-NBEAL2-FLAG were stimulated for 2 days prior to fixation and staining with α-GFP (A11120, 1:100 dilution, green, A), α-LAMP1 (H4A3, 1:100 dilution, green, B), α-CD63 (H5C6, 1:100 dilution, green, C), α- RAB 7 non-covalently conjugated to Zenon-555 (#2094, 1:100 dilution, green, D), and α- SERCA3 (SC-8097, 1:100 dilution, green, E), co-stained with α-VAPA (HPA009174, 1:50 dilution, red) primary antibodies. Pearson’s Correlation Coefficients (PCC) were calculated against the co-stained markers and visualized with the mean and 95% confidence intervals (F). The small panels show individual red, green and colocalization signals. The larger panel shows the merged image. Images are mid Z-slices of cells. 56

VAPA was 0.799 where the lower and upper 95% confidence interval was 0.736 to 0.862. LAMP1 stained large organelle-like structures clustered between the nuclear lobes of the cell (Figure 31B). The average PCC of LAMP1 against VAPA was 0.410 where the lower and upper 95% confidence interval was 0.345 to 0.476. VWF was used as an α-granule cargo stain and showed a punctated and clustered staining pattern (Figure 31C) The average PCC of VWF against VAPA was 0.527 where the lower and upper 95% confidence interval was 0.364 to 0.691. Integrin β3 (CD61) served as a megakaryocyte membrane stain which showed a distinct peripheral staining pattern (Figure 31D). The average PCC of CD61 against VAPA was 0.221 where the lower and upper 95% confidence interval was 0.005 to 0.438. P-selectin (CD62P) served as an α-granule-specific membrane marker and showed a membranous staining pattern (Figure 31E). The average PCC of CD62P against VAPA was 0.434 where the lower and upper 95% confidence interval was 0.222 to 0.647. CD63 stained localized vesicles around the cell. The average PCC of CD63 against VAPA was 0.380 where the lower and upper 95% confidence interval was 0.265 to 0.495. Overall, VAPA IF signal appeared to partially localize with the secretory granule markers of human megakaryocytes (Figure 31F).

To determine if VAPA colocalized with NBEAL2 and RAB7 in human megakaryocytes (as noted in Dami cells) IF microscopy was performed in these cells. Since the α-VAPA antibody, used with Dami cells above, and the α-NBEAL2 antibody are both rabbit antibodies, a mouse α-VAPA (mVAPA) antibody was purchased from Abcam and verified in HEK293 cells. Unlike the rabbit VAPA (rVAPA) antibody, which showed extensive reticular staining that is reminiscent of the ER, mVAPA antibody staining showed a more punctated pattern with some membrane-like structures (Figure A6A). The average PCC of both VAPA stains was 0.752 where the lower and upper 95% confidence interval was 0.712 to 0.792 (Figure A6B). Human megakaryocytes were stained for NBEAL2, EEA1 (marker for early endosomes), or RAB7 and co-stained for VAPA using the mVAPA antibody mentioned above. VAPA staining with the mVAPA antibody again showed a punctated pattern throughout all cells imaged. NBEAL2 also showed a punctated pattern like that observed in NBEAL2-stable Dami cells (Figure 32A). The average PCC of NBEAL2 against VAPA was 0.306 where the lower and upper 95% confidence interval was 0271 to 0.341. Staining for EEA, showed a dispersed and punctated pattern (Figure 32B). The average PCC of EEA1 against VAPA was 0.173 where the lower and upper 95% confidence

Figure 31. VAPA partially localized with secretory vesicles in human megakaryocytes. Human megakaryocytes were harvested from human bone marrow donors, differentiated for 12 days, and harvested for IF staining. Cells were stained with SERCA3 (SC-8097, 1:100 dilution, green, A), LAMP1 (H4A3, 1:100 dilution, green, B), α-VWF (AHP062, 1:500 dilution, green, C), α-CD61 (Y2/51, 1:100 dilution, green, D), α-CD62P (SC-6941, 1:100 dilution, green, E), and α-CD63 (H5C6, 1:100 dilution, green, F), while co- stained with α-VAPA (HPA009174, 1:50 dilution, red) primary antibodies. Pearson’s Correlation Coefficients (PCC) were calculated against the co-stained markers and visualized with the mean and 95% confidence intervals (G). Each fluorescent panel consists of smaller panels showing green staining, red staining, and a panel for colocalization. The larger panel in each set shows the superposition of all color channels. Each image is a mid Z-slice of cells. 58 interval is 0.137 to 0.209. Staining of RAB7 was abundant and showed a vesicular pattern that mainly clustered at the center of the cell (Figure 32C). The average PCC of RAB7 against VAPA was 0.347 where the lower and upper 95% confidence interval was 0.306 to 0.389. Overall, VAPA colocalized more with NBEAL2 and the LE RAB7 marker than with the early endosome marker EEA1 (Figure 32D).

Figure 32. VAPA partially colocalized with NBEAL2 and RAB7 in human megakaryocytes. Megakaryocytes were derived from CD34+ primary cells harvested from bone marrow donors, differentiated with TPO for 12 days then fixed for IF staining. Cells were stained with α- NBEAL2 (EPR14501(B), 1:100 dilution, green, A), α-EEA1 (C45B10, 1:200 dilution, green, B), and α-RAB 7 (#2094, 1:100 dilution, green, C) while co-stained with α-VAPA (ab172371, 1:50 dilution, red) primary antibodies. Pearson’s Correlation Coefficients (PCC) were calculated against the co-stained markers and visualized with the mean and 95% confidence intervals (D). Each fluorescent panel consists of smaller panels showing green staining, red staining, and a panel for colocalization. The larger panel in each set shows the superposition of all color channels. Each image is a mid Z-slice of cells.

Chapter 4: Discussion and future directions

4.1 Understanding NBEAL2 function requires the identification of binding partners

GPS was first characterized in 1971. Patients exhibit macrothrombocytopenia, bone marrow fibrosis and easy bruising, and show pale-appearing platelets on blood films (59,61). The pale appearance of GPS platelets is due to a deficiency of α-granules. In 2011, our lab and two others simultaneously reported the identification of GPS-causative mutations in NBEAL2 (63,66,67). Subsequent work in our lab has focused on how the protein NBEAL2, contributes to megakaryocyte maturation and α-granule formation for the production of platelets.

NBEAL2 is a member of the BDCP family, which share the BEACH domain but have diverse cellular roles where it is difficult to demonstrate common functions among these proteins (72). NBEAL2 is a large protein with multiple functional domains including two protein-protein scaffolding platforms: the ARM domains and the WD40 β-propeller (72). Studies of a murine NBEAL2 knockout models of GPS have demonstrated the importance of this protein in α-granule cargo trafficking/retention, maintenance of internal membrane structures (e.g. the IMS), and megakaryocyte maturation (68). These wide-ranging effects indicate that NBEAL2 may interact with a wide range of cellular structures (e.g. transport vesicles) and proteins. Determining potential NBEAL2 binding partners should advance our understanding of how NBEAL2 may function together with the binding proteins, during MK development and α-granule biogenesis.

4.2 Use of Yeast-2-hybrid screen to detect potential NBEAL2-binding partners

The Y2H system is a well-established screening technique available for identifying potential binding proteins. It was originally based on the modularity of the Gal4 yeast transcriptional activator protein (98). The protein of interest, the bait, is covalently linked to the DNA Binding Domain (DBD) of Gal4, while the prey protein is attached to the transcriptional Activation Domain (AD) (98). The reconstitution of the Gal4 DBD and AD through the interaction of bait and prey proteins drives reporter gene expression to allow growth on specific media and/or a colorimetric reaction (98). The bait and prey protein fusions are required to interact with each other in the nucleus of the yeast cell, a non-native environment that may inhibit some proteins (98). The assumption that the fusion proteins will not interfere with the

60 activity of one another is also something to consider when using Y2H. Since its inception, the Y2H system has undergone many modifications, including the use of auxotrophic markers and other reporters, that have enhanced its stringency and usability (98).

For my research, I utilized the Y2H system to screen for NBEAL2-interacting proteins. Following the work from one graduate student (Richard Lo) who tried Y2H with two halves of NBEAL2 and obtained very few candidate partners, I reasoned that improved bait protein expression may yield a more comprehensive list and attempted to generate three smaller truncations of NBEAL2 to be used in Y2H. However, I was only able to clone one of the three NBEAL2 truncations, which harbored the PH-BEACH-WD40 domains (Fragment C, Figure 11). The likely reason is that these fragments (truncations A and B) were unstable in bacteria during the cloning stages, which was reinforced through restriction enzyme DNA digestion experiments (Figure 12). I predict that altered nucleotide sequences and/or deletions of a part of the constructs led to the appearance of varied digestion products although every cell was transformed from the same aliquot of ligation mixture. The use of another bacterial propagation strains could have resolved this issue however, this problem is unlikely because the DH5α strain has been widely used. Alternatively, Fragment A and B may unintentionally generate toxic products and the cloning of even smaller truncations of the NBEAL2 gene, possibly 5 fragments, could serve to minimize this likelihood (90-92). However, an appropriate cloning strategy should also consider that clusters of domains, such as the WD40 repeats, need to included into one fragment to preserve its native function as a protein-protein interaction scaffold (99). It would therefore be difficult to design small truncations that preserve the conformation of domains while maintaining specificity to the NBEAL2 protein.

Y2H was performed with NBEAL2 Fragment C as it was the only truncation that was cloned successfully. The NBEAL2 Fragment C-bait fusion protein was expected to have a molecular weight of 146 kDa however, my western blot showed a distinct band appearing slightly lower at 135 kDa (Figure 13). Nevertheless, it appeared that the fusion protein was expressed and subsequent reporter autoactivation tests suggested that the activity of the bait protein was not altered through its fusion to NBEAL2 (Figure 14). Y2H screening generated a small list of potential candidates, the majority of which included hemoglobin and ferritin light chain. The prevalence of these proteins in the negative mating condition implied that these proteins were contaminants of the system (Table 7-8). As mentioned, the separation of the WD40 repeats may have disrupted a crucial protein-protein binding platform. Considering that 61

WD40 repeats tend to be within the vicinity of one another, my cloning design of Fragment C excluded the lonesome WD40 repeat at residues 1515-1555 on the assumption that it was too far removed from the others (74). However, the necessity of this domain cannot be ruled out until the crystal structure of NBEAL2 has been generated and studied. It is possible that all 5 WD40 domains are needed to form a 5-bladed β-propeller fold, where the PH-BEACH domain is looped out. It is also conceivable that omitting Fragments A and B from the Y2H screen meant looking at a small population of binding partners. Finally the truncations may have caused improper folding causing aggregation and toxicity in yeast (98).

The C-terminal portion of NBEAL2, appeared to interact with a small set of proteins of diverse function. Potentially interesting binding partners included P4HB, CD63, and Myosin 1F. P4HB is a multifunctional tetramer, consisting of two alpha and two beta subunits, well known in catalyzing the hydroxylation of proline residues in collagen synthesis and in hypoxia-induced wound healing (100,101). Its abundance in the blood plasma and its presence in platelets suggest that P4HB may be an α-granule cargo protein, consistent with the role that NBEAL2 mediates α-granule maturation, however this needs to be verified (102-104). The interaction between NBEAL2 and the δ-granule-specific membrane protein CD63 was unexpected based on the current model (Figure 8). Although the formation of α- and δ-granules likely requires distinct machinery, for example VPS33A-VPS16A in δ-granule formation as opposed to VPS33B- VPS16B in α-granule formation, there may be components common to both pathways (71). NBEAL2 may be one such component, which follows CD63 early in MK development and later interacts specificity with α-granules through other interacting proteins. Lastly, our mouse model of GPS predicted that NBEAL2 could function as a negative regulator of cargo protein release, because VWF was observed to be secreted to the outside of NBEAL2 KO MKs (68). Myosin 1F is an atypical myosin motor protein abundantly expressed in immune cells, where its absence caused the non-specific release of neutrophil granular proteins (105). Recently, myosin 1F was also discovered to be involved in micropinocytosis (106). The possibility that NBEAL2 acts as a negative regulator of granule release through myosin 1F is an idea not far off from the function of NBEA (82).

4.3 The Y2H and AP-MS interaction lists are mutually exclusive

Co-current with my Y2H screens, a graduate student in the lab (Richard Lo) performed an AP-MS using megakaryocytic Dami cells that stably expressed GFP-NBEAL2-FLAG. AP- MS is a screening technique with the same end goal as Y2H however, with important differences. It involves the immunoprecipitation of the protein of interest with antibody- conjugated beads from a total cell lysate, where subsequent binding partners are identified using mass spectrometry. While Y2H identifies binary and possibly tertiary interactions, AP-MS is advantageous in that it can identify whole protein complexes in a relevant cell type and in the native cytosolic environment (98). However, AP-MS is only robust in identifying strong binding partners and is not optimal for weak partners whereas Y2H can be modified to detect either. As a result, both the Y2H and AP-MS screening techniques have distinct advantages that can be complementary in the search for NBEAL2-interacting proteins (98).

I predicted that the high stringency of my Y2H screen, using 4 auxotrophic and 1 colorimetric reporter, would lead to the detection of relatively strong binding partners to NBEAL2 (Table 7). Due to likely problems inherent in the yeast system and my cloning strategy mentioned above, only a small number of partners were identified and with very few repeated hits. To select for the most likely interactions, my Y2H screen was compared with the AP-MS screen and I was not able to identify binding partners that was observed using both techniques. An important difference between the two techniques was the use of the full length NBEAL2 protein in AP-MS compared to only Fragment C in the Y2H screen. In addition to the differences in the environment where interactions occur, my design of Fragment C also excluded the upstream WD40 repeat. As mentioned, it is possible that this design altered the β- propeller structure, however the PH and BEACH domains should have remained intact (Figure 11). Crystal structures of the PH-BEACH region in the LRBA and NBEA BDCPs proposed that although the PH and BEACH domains are separate linear entities, the intricate association of the PH domain with the BEACH domain in the globular protein prevents its classical function in recognizing membranes (15). Thus, disruption of the WD40 structure may disrupt the whole native structure of the area.

4.4 NBEAL2 likely interacts with VAPA through an atypical FFAT motif

To home in on relevant NBEAL2-binding partners, I screened possible binding partners from AP-MS based on 3 criteria: uniqueness, reproducibility, and protein function. These criteria led me to interrogate VAPA as a potential NBEAL2-binding partner where a combination of Co-IP and IF experiments substantiated their interaction. Co-IP experiments were first conducted in HEK293 cells using a truncated VAPA construct that lacked the C- terminal TMD. This truncated VAPA construct was initially developed since various detergents have varied effects on folding and solubilization of membrane proteins (107). Thus, a construct lacking the TMD should optimize Co-IP conditions and aid in the detection of NBEAL2-VAPA. In addition, it is reasonable to assume that the interaction between VAPA and NBEAL2 was independent of the TMD because NBEAL2 was a cytosolic protein. The results shown here suggested that truncated MYC-VAPAΔ protein interacts with NBEAL2 when overexpressed in HEK293 cells however, the experiment did not recapitulate the conditions from which the AP- MS screening was performed (Figure 15). I needed to demonstrate that endogenous VAPA can co-immunoprecipitate with NBEAL2 in the NBEAL2-stable Dami cells to confirm that this is likely a true interaction. To confirm that NBEAL2 interacts with VAPA, I conducted three subsequent Co-IP experiments. 1) Co-IP experiments with full length MYC-VAPA confirmed that full length VAPA interacted with NBEAL2 in the HEK293 cells (Figure 17). 2) Co-IP of full length MYC-VAPA with NBEAL2 in NBEAL2-stable Dami cells confirmed this interaction (Figure 18). 3) Co-IP of VAPA with NBEAL2 in cultured NBEAL2-stable Dami cells in the absence of transient transfection confirmed that endogenous, full length VAPA protein could bind to NBEAL2 expressed at a near-endogenous level (Figure 19). This last Co- IP experiment with endogenous VAPA thus completely reproduced the AP-MS result and confirmed that VAPA is a NBEAL2-interacting protein.

It was interesting, however, that GFP-NBEAL2-FLAG and endogenous VAPA immunoprecipitated in near equimolar ratios (Figure 19). Whether the interaction between VAPA and NBEAL2 in a native environment of a megakaryocyte required one of each protein remains to be studied further however, results from the docking simulations of the VAPA MSP domain to PH-BEACH regions of NBEAL2 suggested that this may not be the case (Figure 25). None-the-less, the Dami cell is the best system currently available to study protein interactions in the context of a megakaryocyte and the verification that binding occurred within such a

64 system adds authenticity to the nature of their interaction. In addition, IF staining of GFP- NBEAL2-FLAG, using antibodies against GFP, and endogenous VAPA showed that these two proteins were partially localized together within the NBEAL2-stable Dami cells with an average PCC of 0.463 (Figure 30A). Incomplete co-localization was expected in part due to the different locations of the two proteins inside the cell – VAPA being anchored to the ER and NBEAL2 being widely distributed as a cytosolic protein. NBEAL2 and VAPA likely interact with a whole host of other proteins in the megakaryocyte to explain the low PCC value obtained. Nevertheless, this result does suggest that VAPA can potentially interact with NBEAL2 inside a megakaryocytic cell. Along with the Co-IP experiments, these results suggested that one of the possible functions associated with NBEAL2 may be facilitated through its interaction with VAPA.

Since MYC-VAPAΔ constructs were frequently used in domain mapping experiments, one can argue that the N-terminal MYC-tag on VAPA could affect the structure of the MSP domain and thus artificially influencing future experiments. To this end, I verified that the N- terminal MYC-tag did not affect the interaction between VAPA and its well-known binding partner VAPB however, this epitope tag may only affect the MSP domain and leave the TMD and CC domains, which are required for VAPA-VAPB dimerization, intact (48). It may have been better to confirm the unaltered structure of the MSP domain through Co-IP of a known interactor to this domain and one example could have been the oxysterol-binding protein (OSBP) (108). However, my ability to Co-IP HA-VAPB with MYC-VAPA suggested that, at the very least, the N-terminal MYC-tag did not drastically alter the entire protein structure (Figure 16). Another control experiment that is not presented here is a Co-IP experiment utilizing a C-terminally tagged VAPA construct. This would be especially relevant for experiments conducted with the VAPA TMD truncation since epitopes situated in place of the TMD would not be expected to cause drastic structural changes. If the small MYC peptide were to prevent the binding of VAPA with NBEAL2, then my Co-IP experiments would not have been successful. In addition, the ability to Co-IP endogenous, full length VAPA with NBEAL2 further confirmed that the N-terminal MYC-tag did not interfere with this interaction (Figure 19). The possibility exists that the MYC-tag may artificially enhance the co- immunoprecipitation of my two proteins by non-specific binding to the beads, possibly explaining the consistent appearance of MYC-tagged VAPA protein bands in my IgG Co-IP controls in NBEAL2-stable Dami cells. Some immunoblots however showed variable VAPA

65 band intensities in the IgG lanes on the same blot under similar experimental conditions (Figure 17-18) and suggested that these residual VAPA bands were likely due to insufficient washing steps and that the N-terminal MYC tag unlikely perturbed the overall VAPA protein structure.

VAPA-binding was observed to be to the C5 PH-BEACH portion of NBEAL2 following domain mapping experiments. It was interesting that the PH-BEACH regions of NBEAL2 could be important in binding to VAPA because up until very recently, no other protein has been identified to bind to the BEACH domain of this BDCP family (109). Subsequent motif predictions suggested the presence of several motifs within this area of the protein and the possibility that one or more of the predicted ncFFAT motifs could be responsible for the interaction of NBEAL2 with VAPA (Figure 25). Contradictory to my domain mapping results however, the FFAT motif prediction also discovered 3 ncFFAT motifs located within the Con-A domain (Table 10). These motifs were also predicted to interact with VAPA with slightly stronger affinities than those within the C5 BEACH domain containing portion of NBEAL2. Co-IP experiments of HA-C2 containing the CON-A domain, with VAPA did not show MYC- VAPAΔ with HA-C2 (Figure 21C). However, these Co-IP experiments need to be repeated due to suboptimal Co-IP conditions to confirm or disprove my results. Considering that the FFAT motif predictions were made through the primary protein structure as opposed to 3D conformations, not every ncFFAT motif would be expected to bind to VAPA and that it is possible that VAPA binds solely to the PH-BEACH regions of NBEAL2 (48).

Surprisingly, surface mapping of the motifs onto the PHYRE 2 structural prediction of the PH-BEACH regions did not show a motif that was completely sequestered. Through protein docking simulations, I filtered all possibilities down to one likely motif in the interaction, the ncFFAT* motif. Interestingly, the ncFFAT* motif was not initially identified in the FFAT motif searching algorithm but rather, was discovered through manually looking for double- phenylalanine residues within the NBEAL2 sequence. However, this did not imply that the ncFFAT* motif is not a FFAT-like motif and the analysis of a smaller portion of the C5 sequence with the same algorithm verified that this sequence indeed resembled the canonical FFAT motif. Its absence from the list of initial predictions can be explained by the high predicted binding score (Table 9). It was noted by Murphy and Levine that their algorithm only presents the 4 best motifs, where a low binding score predicted strong interactions, in binding to VAP proteins (48). The ncFFAT* motif, not observed by the initial algorithm search, presented

66 another important caveat of the prediction algorithm in that quite possibly, the PH-BEACH portion of NBEAL2 may contain more than 5 ncFFAT motifs. These unidentified motifs may not be true FFAT motifs since they deviate drastically from the canonical sequence to generate such a high binding score, correlating to very weak binding. Needless-to-say, future experiments aiming to implicate ncFFAT motifs not initially identified through Murphy and Levine’s algorithm in NBEAL2 or beyond need to be verified by mutagenesis and Co-IP experiments. Thus, it appeared that I was fortunate to have identified the ncFFAT* motif because most of the protein docking simulations implicated this motif in some way (Figure 25).

Unlike the other ncFFAT motifs predicted, most of the ncFFAT* motif sequence appeared to be buried within the protein, with only a fraction of the sequence exposed to the surface (Figure 27). Not surprisingly, this was also seen for the PH-BEACH regions of NBEA (Figure A5). This model proposed that the acidic flanking regions of the motif are not involved directly in the interaction, which is contrary to canonical FFAT motifs but is also true to its classification as non-canonical/atypical. My site-directed mutagenesis experiments were unable to definitively implicate the ncFFAT* motif in VAPA binding however, it is certainly a possibility. The basis of the VAPA-FFAT motif interaction centers around electrostatic interactions, and although the NBEAL2 ncFFAT* motif is atypical, I predict that its interaction with VAPA could also be mediated in a similar manner (97). A closer analysis of the docking simulations suggested that most of the binding predictions involved the electro-negative surface of NBEAL2 and the electro-positive surface of VAPA, as well as hydrophobic interactions (Table A3). Glu2218Lys and Asp2224Lys single amino acid mutants were generated in hopes to ablate the electrostatic interaction between C5 and VAPA, however, Co-IP experiments suggested that neither of the two amino acid changes abrogated the interaction. An explanation for this could be that, more than one amino acid change is required to completely abolish the interaction. Here VAPA may be interacting with both Glu2218 and Asp2224 residues simultaneously, similar to other binding partners (96). To prevent the initial electrostatic interaction, future experiments could be designed to generate mutant constructs that replaces both Glu2218 and Asp2224 with alternative amino acids. The mutants that I have generated were conservative in nature, the substitution to non-polar, uncharged alanine or basic residues is also a possibility to completely abolish the interaction. It is also possible that the interaction between VAPA and NBEAL2 may be mediated by other sites in addition to the predicted ncFFAT* motif (Figure 26, Table A3). Accordingly, ablation of one site alone may not

67 completely prevent the interaction between VAPA and NBEAL2. The significance of these other binding sites will have to be confirmed by additional experiments.

4.5 A transient NBEAL2-VAPA interaction

It was noted that the Co-IP experiments conducted throughout my thesis generated inconsistent results, especially when performing the reverse Co-IP that immunoprecipitated the smaller VAPA protein and attempted to co-immunoprecipitate the larger NBEAL2 protein. Frequently, these reverse Co-IP experiments were unable to co-immunoprecipitate NBEAL2 (Figure 17-18) but on occasions, a small amount did immunoprecipitate (Figure 15). However, the forward Co-IP involving the precipitation of the larger NBEAL2 and co-precipitating the smaller VAPA usually generated more consistent Co-IP results. For this reason, these conditions were chosen for most of the experiments. The inconsistent reverse Co-IP experiments suggested that under certain cellular conditions, a small amount of VAPA from the total VAPA protein pool will bind with NBEAL2, among a host of other proteins. This kind of interaction may be transient to allow switching of various VAPA-binding partners, possibly explaining the weak binding scores associated with predicted ncFFAT motifs in NBEAL2 (Table 9-10). The more consistent forward Co-IP experiments suggests that NBEAL2 bound to VAPA much more frequently. Taken together, this would suggest that the NBEAL2-VAPA interaction could be more important to the NBEAL2 function than to the VAPA function in cells.

FFAT motif predictions of NBEAL2 presented several atypical motifs spread throughout the PH-BEACH region and even across the entire protein (Table 9-10). A commonality between all predicted ncFFAT motifs is that they likely bind weakly to VAPA, which may also explain the inconsistent Co-IP experiments mentioned above. Weak/transient protein-protein interactions define many signalling and regulatory pathways of the cell that require quick and dynamic control (110). The transient nature of the NBEAL2-VAPA interaction may follow suite in a yet-to-be recognized pathway that is related to α-granule development. It could also be possible that the weak interaction with VAPA may have exhibited selection pressure to evolve multiple ncFFAT motifs within the PH-BEACH region of NBEAL2 (48). Reinforcing this idea was the fact that protein-protein docking simulations implicated more than one ncFFAT motif in VAPA binding (Figure 25).

4.6 The potential binding specificity for VAPA as opposed to VAPB

VAPA and VAPB are homologous ER-resident membrane-anchor proteins that share many aspects. Like VAPA, VAPB also shares the same domain architecture and functions in establishing membrane contact sites (48,94). It is therefore likely that a VAPA-interacting protein may also interact with VAPB through the FFAT motif, however, my Co-IP experiment of NBEAL2 and VAPB suggested that this may not be the case (Figure 20). Although VAPA was able to Co-IP with NBEAL2, the inability for VAPB to Co-IP implied that the NBEAL2- VAPA interaction was specific – selecting one homologue over the other. This result was also confirmed by the absence of VAPB in the initial AP-MS screen. It is possible that the fusion of epitope tags onto either NBEAL2 or VAPB could alter the native conformation of the respective proteins. Indeed, my experiment alone cannot rule out those possibilities and further experiments involving Co-IP with a C-terminally HA-tagged VAPB construct should be conducted in parallel to verify the apparent binding specificity of NBEAL2.

The functional significance for this selectivity is unclear however, VAPA and VAPB may mediate slightly different pathways. Mutations in VAPB have been linked to type 8 amyotrophic lateral sclerosis while VAPA has yet to be implicated in a disease phenotype (111). One possibility is that VAPB function cannot replace the loss of VAPA function, resulting in embryonic lethality, and thus VAPA mutations have never been observed. The VAP proteins appear to be distinct and is indicated by the fact that VAPA cannot completely rescue the loss of VAPB function in cells resulting in type 8 amyotrophic lateral sclerosis. Cell survival rates associated with each protein was demonstrated in rat hippocampal neuronal cells. Knockdown of VAPA resulted in the death of ~80% of cultured neurons while there was no effect on survival following VAPB knockdown (112). The relative importance of either VAP proteins in specific cells and tissues, and the relevance of interactions with specific proteins such as NBEAL2 remains to be studied further.

4.7 VAPA could be involved in granule formation in human megakaryocytes

Immunostaining of NBEAL2-stable Dami cells was suggested the possibility that the potential interaction between VAPA and NBEAL2 exist within megakaryocytes. The studies also suggested that VAPA may mediate function at the LE due to its partial colocalization to the

69 vicinity of RAB7 (Figure 30C). All associations observed through IF however, should not be interpreted as a direct interaction between two proteins – rather that the proteins may be in close proximity within cells. The direct interaction between NBEAL2 and VAPA was confirmed earlier through Co-IP experiments but the same cannot be said for VAPA and RAB7. Alternatively, VAPA has been reported to interact directly with the endosomal membrane proteins STARD3 and STARD3NL (49). As well, the ER-membrane cholesterol-binding ORP protein ORP1L is a RAB7-binding protein and the indirect association between VAPA and RAB7 may be mediated by a platform that bring these components together (49,113). None-the- less, this association appeared consistent with previous reports that VAPA tethers the ER to the endosome and my results are the first to extend this possibility in the context of megakaryocytic cells (49). Thus, it is enticing to postulate that VAPA may also influence endosome maturation in MKs (50). Since the MK endosomes give rise to the 3 classes of secretory vesicles, α- granules, δ-granules and lysosomes, NBEAL2-stable Dami cells were co-stained for VAPA and these respective compartments. Immunostaining of α-granules, however, was not performed since Dami cells lack true α-granule-like structures and several α-granule specific proteins (unpublished lab results). CD63 and LAMP1 immunostaining showed little colocalization of the δ-granules and lysosomes respectively with VAPA and suggested that VAPA is less likely associated with these structures (Figure 30B, D).

In the search to further confirm the possible involvement of VAPA in α-granule formation, human MKs cultures derived from healthy human donors (isolated and cultured by another graduate student Richard Lo) were co-staining with intracellular marker proteins. Preliminary results here indicated a partial association of VAPA with all 3 of the endosome- related organelles, contrary to the staining results seen in the Dami cells. This included LAMP1 (Figure 31B), CD62P which marked the α-granule membrane (Figure 31E), and CD63 (Figure 31F). In comparison, it appeared that VAPA localized slightly more to CD62P compared to LAMP1 and CD63 (Figure 31G). Staining for the α-granule cargo protein VWF also suggested that VAPA tended to associate slightly more prominently with α-granule-related compartments (Figure 31C).

Differences in the associations observed between VAPA and LAMP1/CD63 in human MKs as opposed to Dami cells may result from different stages of maturation at which MKs were fixed or the fact that Dami cells are not true megakaryocytes. VAPA associates closely

70 with the endosomes throughout maturation and possibly during the packaging of secretory granules, explaining similar localization patterns with all three granule markers seen for human MK cells (114). However, the associations may fade once megakaryocytes are at a more mature stage of secretory granule production. Dami cells likely represent a terminally differentiated mature megakaryocytic-like cell with δ-granules and lysosomes but not distinct α-granules. It is apparent that further IF microscopy studies are required to look at stage specific maturation and association of NBEAL2 with VAPA to confirm my preliminary observations. Taken together it is conceivable that NBEAL2 is an important α-granule-specific protein that targets VAPA to α- granule-like structures and facilitate α-granule maturation. Immunostaining of VAPA and NBEAL2, showed a stronger association in the NBEAL2-stable Dami cells compared to human MKs (Figure 32D). This difference is likely attributed to the different antibodies used between these IF experiments. Although the mouse α-VAPA antibody co-localized very well with the rabbit α-VAPA antibody, previously used to co-stain Dami and some MK cells, the mouse α- VAPA antibody lacked a reticular ER-staining pattern (Figure A6). The reticular staining pattern seen with the rabbit α-VAPA antibody is likely because of the detection of a variety of VAPA epitopes as opposed to the mouse α-VAPA, which may detect a different set of epitopes. Confirmation that the reticular pattern seen using the rabbit α-VAPA represented the ER was demonstrated by co-staining with SERCA3, a well-known ER resident membrane protein (Figure 30E, Figure 31A). Unfortunately, there is no reliable mouse α-NBEAL2 antibody that is currently available, but one possibility is to covalently conjugate fluorophores onto rabbit α- NBEAL2 or rabbit α-VAPA. Thus, the current IF microscopy of NBEAL2 and VAPA in human MKs is largely constrained by the availability of good IF antibodies.

Nevertheless, my staining results suggest that VAPA is likely associated with the early development of megakaryocytes where granule constituents have not yet matured, to ultimately associate with NBEAL2 for α-granule maturation. These experiments need to be confirmed with the following goals. Firstly, using human MKs for immunofluorescence microscopy, we need to evaluate the largest and most mature cells in the population. This may be achieved by utilizing gradients to isolate the largest cells. Secondly, future studies should explore and optimize the conjugation of fluorophores onto either VAPA and NBEAL2 antibodies.

4.8 Possible roles of NBEAL2 in regulated transports through VAPA

The family of VAP proteins have been studied extensively in the context of lipid transfer events since many of the known VAP-interactors are lipid binding/transfer proteins. The current model proposes that the ER maintains a bulk reservoir of lipids that functions as a distributor to other compartments through the VAP-mediated formation of membrane contact sites (114). The close apposition (10-30nm) of two adjacent membranes then brings together the necessary components for lipid exchange (48). Such non-vesicular transfer events are crucial in establishing lipid gradients across the cell and in the normal functioning of organelles including the Golgi apparatus, mitochondria, peroxisomes, and endosome (114). Since Co-IP and IF results here suggested that VAPA is likely a true NBEAL2-binding partner, it is possible that the NBEAL2-VAPA interaction is indirectly related to lipid exchange.

Despite the high trafficking rate of constituents through the endosomes, which receive membrane-bound vesicles of various compositions from multiple different sources, the endosomes can maintain a non-random distribution of membrane lipids (115). Unique combinations of phosphatidylserines, raft lipids, and phosphoinositides define the identity of different endosomal compartments and the ability for membrane compartments to maintain this gradient is important in cargo transport (Figure 33). Thus, future experiments should look towards evaluating the effects of inhibiting the NBEAL2-VAPA interaction on endosomal lipid content. A good starting point should be in evaluating cholesterol content at the endosomes following inhibition of the NBEAL2-VAPA interaction. Results from HeLa cells indicated that LE motility and thus maturation is dependent on cholesterol transport where the accumulation of cholesterol at the LE inhibited the trafficking of LE (50). The formation of the ER-LE contact site through the ER membrane protein Protrudin and the LE RAB7 results in the delivery of the microtubule motor Kinesin-1 to the LE (116). The involvement of VAP proteins in this contact site is unknown but suggests that the formation of ER-LE contact sites may be involved in the transfer of motor proteins in addition to lipids. The generation of a mutant NBEAL2 protein, that lacks only the ability to interact with VAPA, will be pivotal for these experiments.

The formation of ER contact sites is not restricted to intracellular compartments of the cell but, have also been found to connect to the PM. Muscles cells contain a specialized ER, the sarcoplasmic reticulum, where the ER-PM contact sites are widely accepted to coordinate Ca+2 influx from the PM for contraction events (117). In other non-specialized cells, ER-PM contact

72 sites have been studied extensively in Ca+2 homeostasis as well as non-vesicular lipid transfer (118). Since the MK demarcation membrane system (DMS) initially forms from invaginations of the PM in as early as the first endomitotic cycle and maintains this surface connection throughout maturation, it is possible that ER-PM contact sites may also facilitate DMS development in the megakaryocyte (30). Interestingly, NBEAL2 KO mice MKs presented a marked reduction in platelet territories, areas within the cell defined by the DMS, but how NBEAL2 contributed to this process is unclear (68). The DMS is believed to curate membranes from anterograde vesicles from Golgi apparatus and the close apposition of the DMS to the ER at multiple sites suggested non-vesicular communication between the two compartments (30). Thus, it is also possible that NBEAL2 may also regulate α-granule biogenesis at the DMS through interactions with VAPA at the ER membrane.

Figure 33. Overview of lipid composition in membranes of compartments associated with cell- endocytic pathway. Unique combinations of lipids maintain the identities of the plasma membrane, recycling endosomes (RE), static early endosomes (SEE), dynamic early endosomes (DEE), late endosomes (LE), and lysosome-related organelles (Lys). This lipid-specific identity is regulated by the family of RAB GTPases (various shades of gray). Multiple pathways bring various lipids towards the endosomal compartment ranging from clatherin-coated pits (CCP) that generate clatherin-coated vesicles (CCV), clatherin-independent carriers (CLiC), or biosynthetic pathway (not shown). Image was obtained from Arumugam and Kaur 2017 (115). Reprinted with permission.

Chapter 5: Concluding remarks

Due to technical cloning difficulties, Y2H screening was only performed using the C- terminal portion of NBEAL2 against the human bone marrow library. This screen generated a small list of interacting proteins that did not overlap with the laboratory’s AP-MS screen performed concurrently. After a focused assessment to determine probable NBEAL2-binding proteins, I selected VAPA as a likely candidate. I verified the AP-MS pull-down results in NBEAL2-stable Dami cells – demonstrating the ability to Co-IP VAPA with NBEAL2. These results were further investigated using IF microscopy demonstrating partial colocalization between the two proteins in cells. To study this interaction further, I confirmed that the N- terminal MYC-tag did not appear to alter the VAPA proteins ability to bind with VAPB via Co- IP experiments suggesting that the overall conformation of VAPA was largely unperturbed. Domain mapping experiments using truncated the NBEAL2 protein fragments suggested that VAPA interacted at the PH-BEACH region of NBEAL2. This region of NBEAL2 was predicted to contain at least 5 different atypical FFAT motifs, one or all of which may be implicated in binding VAPA. Due to the lack of a NBEAL2 crystal structure, PHYRE 2 predictions of the PH-BEACH region in NBEAL2 were generated to create a model and suggested a striking homology to NBEA. Attempts to filter out surface-sequestered FFAT motifs using the PHYRE 2 predictions were unsuccessful. However, docking simulations predicted that the ncFFAT* motif, among others, may be important in the interaction of NBEAL2 with VAPA. Site directed mutagenesis experiments with isolated single amino acid changes Glu22218Lys and Asp2224Lys within the ncFFAT* motif suggested that these residues are not exclusively required for NBEAL2 binding to VAPA. Future experiments generating multiple amino acid changes at this and other FFAT motives will provide insights into the exact location of the VAPA binding site within NBEAL2.

The functional significance of the NBEAL2-VAPA interaction remains to be determined. Preliminary immunofluorescence microscopy studies of human megakaryocytes confirmed that VAPA broadly associates with endosome-related compartments including α- granules, δ-granules, and lysosomes. Since VAPA has been implicated in several lipid trafficking events, it is possible that NBEAL2 confers α-granule specificity by binding VAPA in

74 the vicinity of α-granules. This interaction is predicted to facilitate the proper maturation of the endosomes, and thus of α-granules, in a yet to be defined pathway in MKs (Figure 34).

Figure 34. A hypothetical model of NBEAL2 function through interacting with VAPA. The Endoplasmic Reticulum extends throughout the cell and comes in close apposition to a number of different sites including the plasma membrane and the Late Endosomes. Endogenously synthesized α-granule constituents transit through the Endoplasmic Reticulum, Golgi Apparatus, Early Endosomes, and the Late Endosomes/MVB before being packaged into mature α-granules in the megakaryocyte. The interaction between NBEAL2 and VAPA may target the Endoplasmic Reticulum to α-granules, possibly contributing to α-granule maturation in a yet to be defined pathway.

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Appendices

Figure A1. The PH-BEACH region of NBEA a contains a prominent binding pocket. The crystal structure of the PH-BEACH region of NBEA (1MI1, B) is shown above. The PHYRE 2 prediction of NBEAL2 structure resembles that of NBEA, including the binding pocket. 3D surface models visualized in PyMol show the possible binding pocket (red circle), other images were rotated either clockwise (+90 °) or counter-clockwise (-90 °).

Figure A2. Predicted non-canonical FFAT motifs were found on the surface of the PH-BEACH regions of NBEA. Predicted non-canonical FFAT motifs were mapped onto the crystal structure of the PH-BEACH regions in NBEA (1MI1, B). Like NBEAL2, all predicted non-canonical FFAT motifs were surface-exposed, suggesting potential involvement in VAPA binding. 3D surface models visualized in PyMol; rotated +90 ° or -90 ° from center panel. Colors co-ordinate with ncFFAT motifs shown in Figure 23. The presence of the same color across both proteins indicates similar motifs. Irrelevant protein surfaces are shown in gray.

Figure A3. Summary of protein-protein docking simulations with the PH-BEACH regions of NBEA and the MSP domain of VAPA. The crystal structure of the VAPA MSP domain (2RR3) and the PH-BEACH regions within NBEA (1MI1) were submitted for binding simulations through the zDOCK server. The 10 binding predictions were tabulated and presented in a Venn diagram as the frequency of simulations that implicated one or more ncFFAT motif. All colors co-ordinated exactly with the ncFFAT motif sequences shown in Figure 23. except for gray to represent the collective “Others” group. The “Others” group represented simulations that did not bind to the predicted ncFFAT motifs.

Figure A4. The VAPA MSP domain is predicted to bind at a similar ncFFAT* motif of NBEA. The crystal structure of the VAPA MSP domain (2RR3) and the PH-BEACH regions within NBEA (1MI1) were submitted for binding simulations through the zDOCK server. The top three binding predictions are shown here in order from ranked #1 (A), ranked #2 (B), and ranked #3 (C). Of the top three simulations shown, only one suggested a novel VAPA binding site (A) while the others suggested a similar ncFFAT* motif that was for in NBEAL2 docking simulations. The above images are 3D surface models, visualized in PyMol, depicting the possible binding pocket in the center panel of each row. Images were rotated either to the clockwise (+90 °) or counterclockwise (-90 °) based on the center panel. All colors co-ordinated exactly with the ncFFAT motif sequences shown in Figure 23 except for orange which represents the VAPA MSP domain and irrelevant protein surfaces are shown in gray.

Figure A5. Two acidic residues in the similar ncFFAT* motif are also predicted to be on the surface of the PH-BEACH regions of NBEA. The crystal structure of NBEA (1MI1) suggested that many of the residues were located inside the protein except for Glu2426, Try2430, Leu2431, and Glu2433. The above images are 3D surface models, visualized in PyMol, in an orientation that centers on a similar ncFFAT* motif, as that found in NBEAL2, colored in magenta like in Figure 23. The inset is a zoomed-in picture depicting the surface-exposed residues of interest.

Figure A6. Mouse α-VAPA antibody colocalized well with rabbit α-VAPA antibody in HEK293 cells. Mouse α-VAPA antibody was purchased to stain human megakaryocytes along with rabbit NBEAL2. HEK293 cells were co-stained with the new mouse VAPA (mVAPA, M01, 1:50 dilution) antibody and the previous rabbit (rVAPA, HPA009174, 1:50 dilution) antibody to verify that they stained the same proteins (A). Pearson’s Correlation Coefficients were calculated against the co-stained markers and visualized with the mean and 95% confidence intervals (D). Each fluorescent panel consists of smaller panels showing green staining, red staining, and a panel for colocalization. The larger panel in each set shows the superposition of all color channels. Each image is a mid Z-slice of cells

Table A1. The PH-BEACH region of NBEA is predicted to contain at least five non- canonical FFAT motifs like NBEAL2. Using the FFAT motif searching algorithm published by Murphy and Levine (48), at least five non-canonical FFAT motifs were identified in the PH-BEACH regions of NBEA (2147-2563aa). The FFAT motifs are given as a total of 15 amino acids consisting of 6 residues upstream of a 7-residue core followed by 2 residues downstream. Binding scores range from 0-2 for strong binding, 2.5-4 for OK binding, 4-7 for weak binding, and 7+ for poor binding.

Amino acid Domain FFAT motif sequence (6 N-term + 7 Core + 2 C-term) Binding score position 2178 PH SITTTEIYFEVDEDD 3.5 2488 BEACH RMALESEFVSCQLHQ 4.0 2372 BEACH PYHYNTHYSTATSTL 4.5 2392 BEACH IEPFTTFFLNANDGK 5.0 2421 BEACH VKELIPEFYYLPEMF 6.0

Table A2. NBEA was predicted to contain non-canonical FFAT motifs in other domains of the protein. Using the FFAT motif searching algorithm published by Murphy and Levine (48), several non-canonical FFAT motifs were identified NBEA when searched with the full protein sequence. The FFAT motifs are given as a total of 15 amino acids consisting of 6 residues upstream of a 7-residue core followed by 2 residues downstream. Binding scores range from 0-2 for strong binding, 2.5-4 for OK binding, 4-7 for weak binding, and 7+ for poor binding.

Amino acid Domain FFAT motif sequence (6 N-term + 7 Core + 2 C-term) Binding score position 2178 PH SITTTEIYFEVDEDD 3.5 1017 ARM SPESETDYPVSTDTR 4.0 2488 BEACH RMALESEFVSCQLHQ 4.0 392 CONA QLGAVYVFSEALNPA 4.5

Table A3. Protein-protein docking simulations suggested the involvement of electrostatic and hydrophobic interactions. To clarify the residues responsible for the protein docking simulations obtained through ZDock, each of the 10 binding prediction were analyzed for surface-exposed residues at the binding interface. The binding interface was defined here as to the interacting surfaces of the VAPA MSP domain (2RR3) and the PHYRE 2 structure of the PH-BEACH region of NBEAL2. Residues on VAPA that are known to bind FFAT motifs, including Lys45, Thr47, Lys87, and Met89 are shaded in grey. Residues on NBEAL2 were shaded in different colors to coordinate with the predicted non-canonical FFAT motifs shown in Figure 23. Docking Residues on VAPA Residues on NBEAL2 Rank 1 K17, K45 T47, D79, N81, E82, K83, E1960, G1961, I1962, Y1964, R1968, S84, K85, K87, M89, R120, V122, E124, A1996, Y1998, V2173, E2174, E2218, M125, P126, N127, E128 Y2221, F2222, D2224, E2227, Q2229, N2230, G2231, F2232, D2233, L2234, N2241 2 T23, T47, A48, R50, R51, D77, D79, L2226, E2227, N2228, Q2229, F2232, K85, K87 D2246, S2255, E2257 3 D24, R50, C53, N57, Q74, N81 E1960, I1962, V2173, E2174, F2222, D2225, Q2229, N2230, G2231, F2232, D2233 4 D24, V25, P49, R51, C53, R55, Q74, Y1949, D1950, E1960, G1961, I1962, E127 G1963, Y1964, D1965, D1994, Y1998, Q2229 5 D24, K45, T47, R50, R51, C53, R55, E1960, I1962, G1963, D1965, R1967, M72, K87, E101, K107, K110, P111, D1994, Q1995, A1996, Y1998, R2041, F2076, E2077, Y2221, F2222, D2224 6 T23, R50, D77, D79 E2227, Q2229, G2231, F2232, D2233, E2257 7 T27, P35, S36, K39, P56, D62, S65, Q1995, F2076, E2077, E2218, Y2221, V67, T68, P97, N98, S100, M102, F2222, P2223, D2224, E2227, Q2229, F2232, D2246, P2256, Q2261, R2264, E2268 8 T47, R51, C53, R55, N57, S58, G59, I60, E1960, D1994, Q1995, F2076, E2077, M72, Q74, P75, D77, K85, H86, S100, S2178, Y2221, F2222, D2224, Q2229, M102, N2230, L2234 9 R50, D77, D79, H86 E2227, Q2229, G2231, F2232, D2233, K2243, E2257 10 K43, P49, C53, R55, M72, E103, S1923, E1925, L1938, E1940, Y1947, D1950, S1952, Y1964