Studies of Vesicle Trafficking Following Functional Depletion of Clathrin: Use of Clathrin Light Chain-FKBP-Binding Domain Chimera Open Access

Grossniklaus, Emily Jane (2010)

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Eukaryotic cells use vesicle carriers to target and transfer membrane proteins between selected intracellular compartments in a process that is generally referred to as vesicle trafficking. The clathrin triskelion molecule plays a critical role during vesicle trafficking, as it coats vesicles early in formation and recruits molecular machinery to help pinch the coated pits off from the plasma membrane, creating a fully formed and mature vesicle. A host of adaptor proteins work together with clathrin, interacting with membrane-bound proteins to create sites of high protein concentration that attract clathrin and initiate vesicle formation. Due to the precise and specific nature of adaptor protein interactions with membrane-bound receptors, adaptor proteins are largely responsible for determining the final protein composition of each vesicle.

An outstanding question is how the single protein clathrin regulates specific vesicle formation and composition in concert with these adaptor proteins. It is generally accepted that the AP-1 and AP-2 adaptor proteins require interaction with the clathrin triskelion for successful vesicle formation, but less is known about the role of AP-3 in vesicle biogenesis pathways. In my project, I have tested the hypothesis that AP-3 participates in alternative trafficking pathways that do not require functional clathrin. Clathrin function was perturbed using a recombinant clathrin light chain containing a modified FKBP domain that is susceptible to oligomerization following treatment by the drug AP20187. This recombinant clathrin molecule was expressed in mammalian cell lines in culture and rendered non-functional by AP20187 prior to subcellular fractionation and immunofluorescent microscopy experiments. I observed that the affects of clathrin disruption on AP-3 concentration in clathrin coated vesicle fractions was unique from the affects of clathrin on AP-1 and AP-2. This finding contrasts with the current notion that all clathrin-adaptor interactions are mechanistically similar. Rather, this data supports an alternative hypothesis that the kinetics of AP-3 interactions of clathrin are slower than those of clathrin with AP-1 and AP-2, or that AP-3 participates in clathrin-independent trafficking pathways.

Table of Contents

Table of Contents

  1. Introduction

a. The Discovery of Clathrin and its Proposed Role in Coat Formation...1

b. Stepwise Mechanism of Vesicle Formation and Generalized Trafficking Pathway...1

c. The Clathrin Triskelion...2

d. Understanding the Coat: Layers, Function and Known Interactions...2

e. Adaptor Structure...3

f. Adaptor Function...4

g. The Debate over AP-3...5

h. Significance of Clathrin Coated Vesicles to Cellular Transport and the Question Addressed by this Thesis...6

  1. Experimental Aims

a. Specific Objectives...9

b. Hypotheses...9

3. Experimental Methods

a. PC12 Cell Line: Formation of Construct and Transfection...11

b. Cell Culture...12

c. AP20187 Drug Treatment...13

d. Clathrin Coated Vesicle Isolation...13

e. Protein Concentration Analysis...14

f. Coomassie Staining...15

g. Immunoblot Analysis...16

h. Immunofluorescence Microscopy...18

4. Results

a. Effect of AP20187 on Protein Concentration in Clathrin Coated Vesicle Fractions...21

b. Effect of AP20187 on Protein Content in Clathrin Coated Vesicle Fractions: Cargoes, Adaptor Proteins and Clathrin Cages...22

c. Effect of AP20187 on Clathrin Heavy Chain and mCherry Colocalization by Immunofluorescent Microscopy...23

d. Effect of AP20187 on CLC and AP-3 Colocalization in Immunoflourescent Microscopy...24

5. Discussion...26

6. Acknowledgments...29

7. References...40

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