Actin Cytoskeleton Regulators Interact with the Hermansky-Pudlak Syndrome Complex BLOC-1 and its Cargo Phosphatidylinositol-4-kinase Type II Alpha Open Access

Abstract

Vesicle biogenesis machinery components such as coat proteins can interact with the actin cytoskeleton for cargo sorting into multiple pathways. However, whether or not these interactions are a general requirement for the diverse endosome traffic routes is unknown. In this dissertation, I identified actin cytoskeleton regulators as previously unrecognized interactors of complexes associated with the Hermansky-Pudlak syndrome. Two complexes mutated in the Hermansky-Pudlak Syndrome, AP-3 and BLOC-1, interact with and are regulated by the lipid kinase PI4KIIα. I therefore hypothesized that PI4KIIα interacts with novel regulators of these complexes. To test this hypothesis, I immunoaffinity purified PI4KIIα from isotope-labeled cell lysates (SILAC) to quantitatively identify interactors. Strikingly, PI4KIIα isolation preferentially co-enriched proteins that regulate the actin cytoskeleton, including guanine exchange factors for Rho family GTPases such as RhoGEF1 and several subunits of the WASH complex. I biochemically confirmed several of these PI4KIIα interactions. Importantly, BLOC-1 complex, WASH complex, RhoGEF1, or PI4KIIα depletions altered the content and/or subcellular distribution of the BLOC-1-sensitive cargoes PI4KIIα, ATP7A, and VAMP7. I conclude that the Hermansky-Pudlak syndrome complex BLOC-1 and its cargo PI4KIIα interact with regulators of the actin cytoskeleton. Exploring these interactions will provide insight into the regulation of actin polymerization at endosomes and the pathogenic mechanisms of human diseases such as pigmentation disorders, neurocutaneous syndromes, and neurodegeneration.

Table of Contents

Table of Contents

Chapter I. General Introduction. 1

Overview and significance 2 Fundamental Principles of Membrane Trafficking 4 Endocytic and secretory organelles exchange components by membrane trafficking 4 Adaptor protein complexes link coat complexes with cargo at specific subcellular domains 5 Figure 1. Adaptor-mediated vesicular transport creates and maintains unique organelle composition. 8 AP-2-dependent vesicle budding at the plasma membrane 8 Figure 2. The canonical vesicle budding process: AP-2-dependent clathrin-mediated endocytosis. 10 The role of actin polymerization in AP-2-dependent vesicle budding 11 The adaptor protein complex-3 (AP-3) 13 Figure 3. Composition of the adaptor protein complex-3 (AP-3). 14 AP-3 deficiency causes the Hermansky-Pudlak syndrome 15 The biogenesis of lysosome-related organelles complex-1 (BLOC-1) 18 Figure 4. Subunit composition of the biogenesis of lysosome-related

organelles complex-1 (BLOC-1). 19

Possible functions of the BLOC-1 complex 19

Possible molecular mechanisms of BLOC-1 function 20 The phosphatidylinositol 4-kinase type IIα is a cargo and a regulator of AP-3 and BLOC-1 22 Initial characterization of phosphatidylinositol kinases 23 The type II phosphatidylinositol 4-kinases 24 Figure 5. Domain architecture of phosphatidylinositol4-kinase type IIa

(PI4KIIa). 25

PI4KIIa is a component of the canonical Wnt signaling pathway 25

Figure 6. Model for regulation of PI4KIIa activity by the Wnt signaling pathway. 26

PI4KIIa is a cargo and a regulator of the BLOC-1 and AP-3 complexes 27

Figure 7. Model for regulation of the AP-3 and BLOC-1 complexes by PI4KIIa. 28

The actin cytoskeleton and its regulators 29 Actin monomers polymerize into structural units critical for cellular function 29 Regulation of actin polymerization in vivo: small GTPases and guanine exchange factors 31 Figure 8. The RhoA GTPase cycle and regulation by Rho guanine exchange

factor-1 (RhoGEF1). 35

The Arp 2/3 complex nucleates and organizes actin filaments 35

Nucleation-promoting factors (NPFs) 36

Figure 9. Nucleation of actin polymerization by the Arp2/3 complex and

nucleation-promoting factors. 38

The Wiskott-Aldrich Syndrome Proteins 38

The WASP and Verprolin Homolog (WAVE) Proteins 39 WASP and SCAR Homolog (WASH) 40

Localization 41

Function 42

Mechanism of action 43

Figure 10. Proposed mechanism of action for WASH-dependent membrane scission. 44 Figure 11. Potential roles for the actin cytoskeleton in endosomal sorting. 45

Regulation 46

Figure 12. The WAVE and WASH actin nucleation-promoting factors are incorporated into structurally analogous complexes. 47 Contributions of this dissertation research 49

Chapter II.The WASH Complex, an Endosomal Arp2/3 Activator, Interacts with the Hermansky-Pudlak Syndrome Complex BLOC-1 and its Cargo Phosphatidylinositol-4-kinase Type II Alpha 51

Abstract 52 Introduction 53 Results 57

The PI4KIIα interactome enriches actin regulatory proteins 57

Biochemical and genetic confirmation of PI4KIIα-interacting proteins 61 The WASH complex and filamentous actin reside on PI4KIIa- positive early endosomes 65

The WASH complex modulates the targeting of BLOC-1

cargoes 67

Discussion 70

Materials and Methods 77

Antibodies and cell culture 77

DNA Expression Constructs 78

Immunoprecipitation and immunoaffinity chromatography 78

Transient protein knockdown and immunoblot analysis 80

Immunofluorescence microscopy 81

Cell fractionation 84

Surface labeling and streptavidin pulldowns 84

Computational and statistical analysis 86

Acknowledgements 87

Figures 88

Figure 1. The PI4KIIα interactome enriches actin regulatory proteins. 88

Figure 2. Biochemical confirmation of PI4KIIα-interacting proteins. 89

Figure 3. Genetic confirmation of PI4KIIα-interacting proteins. 90

Figure 4. PI4KIIα, the WASH complex subunit strumpellin, and actin

cytoskeleton components co-reside at early endosomes. 91

Figure 5. PI4KIIα co-localizes with the WASH complex. 94

Figure 6 . Depletion of pallidin and strumpellin alter endosomal

morphology. 95

Figure 7. WASH complex depletion alters the subcellular distribution

of BLOC-1 cargoes. 96

Supplementary Figure 1. 97

Supplementary Table 1 . SILAC Mass spectrometry Data for PI4KIIα Interactors 99 Supplementary Table 2 . Functional Ontology Terms Enriched in the PI4KIIα Interactome 104

Supplementary Table 3. Antibodies used in this Study. 114

Chapter III. Discussion 116

Summary of findings and contributions to the field 118

Figure 1 . Model of AP-3- and BLOC-1-dependent vesicle biogenesis prior

to this dissertation research. 120

Figure 2. Model for AP-3- and BLOC-1 vesicle biogenesis based on this dissertation research. 122 Novel hypothesis #1: PI4KIIα and BLOC-1 concurrently recruit the WASH complex to AP-3-BLOC-1 vesicle biogenesis pathway 123

Figure 3. Proposed interactions between PI4KIIa, PI4P, the WASH complex,

and BLOC-1. 126 Novel hypothesis #2: The Arp2/3- and WASH complex-dependent polymerization of actin regulates the cargo sorting into and/or membrane scission of tubular vesicle intermediates 126

Table 1 . Subunits of the Arp2/3 and WASH complexes co-isoslate with

PI4KIIα. 130 Novel hypothesis #3: The BLOC-1 complex acts as a tubule-stabilizing factor 131 Novel hypothesis #4: PI4KIIα and BLOC-1 recruit guanine exchange factors as activators of actin polymerization 135

Figure 4. Model for RhoGEF1 activation of the WASH complex. 138

Table 2. Upstream and downstream elements of Rho family GTPase

signaling co-isolate with PI4KIIα. 139

Chapter IV. References 144

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Cell
Keyword	Vesicular Trafficking Actin Cytoskeleton Hermansky-Pudlak Syndrome
Committee Chair / Thesis Advisor	Faundez, Victor, Emory University
Committee Members	Li, Lian, Emory University Sale, Winfield S, Emory University Eaton, Douglas C, Emory University Kowalczyk, Andrew, Emory University

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