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Regulation of clathrin-coated vesicle nucleation

Thieman, James Robert (2011) Regulation of clathrin-coated vesicle nucleation. Doctoral Dissertation, University of Pittsburgh.

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    Abstract

    Clathrin-mediated endocytosis is a selective pathway for the entry of transmembrane proteins into the cell through the generation of a short-lived vesicular intermediate. Cells and tissues depend on this process for obtaining nutrients, modulation of signaling and cell migration. The clathrin-coated structure intermediate is assembled on the plasma membrane from a cohort of 20-30 distinct proteins that aid in cargo selection, scaffolding, membrane bending and scission of the vesicle. Exactly how these complex assemblies are nucleated at the plasma membrane remains unclear although the lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) plays an important role by anchoring many of the endocytic components. The work in this thesis helps to clarify the nucleation phase by describing the molecular details of the interaction between a PtdIns(4,5)P2-generating lipid kinase PIPKIgamma and the heterotetrameric clathrin adaptor AP-2. By engaging a subdomain on the AP-2 beta2 subunit appendage, the kinase is strategically positioned at assembly sites to generate PtdIns(4,5)P2 and drive coat assembly forward. Clathrin binds to the same subdomain on the beta2 appendage but with a higher apparent affinity. I therefore invoke a model in which PtdIns(4,5)P2 production for nucleation is negatively regulated by PIPKIgamma displacement from AP-2 by clathrin at later stages of assembly. I also demonstrate that a cargo-sorting alternate adaptor that binds to the other subsite on the AP-2 beta2 appendage is not subject to displacement by clathrin during clathrin-coated vesicle budding, ensuring non-competitive cargo incorporation into the vesicle. Finally, the PtdIns(4,5)P2-binding EFC domain proteins FCHO1 and FCHO2 have been proposed to act as dedicated nucleators of clathrin-coated structures on the plasma membrane. I demonstrate in multiple cell lines that these proteins are not invariantly required for placement of clathrin-coated assemblies on the plasma membrane despite being early arriving components themselves. FCHO1/2 are involved in the regulation of the size and number of these assemblies in some cellular contexts. My data support the model of PtdIns(4,5)P2 regulated, not protein regulated, nucleation of clathrin-coated structures; however multiple parallel pathways may contribute to initiation of endocytic buds.


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    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmail
    Committee ChairAridor, Meiraridor@pitt.edu
    Committee MemberBisello, Alessandroalb138@pitt.edu
    Committee MemberApodaca, Gerard Lgla6@pitt.edu
    Committee MemberTraub, Lintontraub@pitt.edu
    Committee MemberWatkins, Simon Cswatkins@pitt.edu
    Title: Regulation of clathrin-coated vesicle nucleation
    Status: Unpublished
    Abstract: Clathrin-mediated endocytosis is a selective pathway for the entry of transmembrane proteins into the cell through the generation of a short-lived vesicular intermediate. Cells and tissues depend on this process for obtaining nutrients, modulation of signaling and cell migration. The clathrin-coated structure intermediate is assembled on the plasma membrane from a cohort of 20-30 distinct proteins that aid in cargo selection, scaffolding, membrane bending and scission of the vesicle. Exactly how these complex assemblies are nucleated at the plasma membrane remains unclear although the lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) plays an important role by anchoring many of the endocytic components. The work in this thesis helps to clarify the nucleation phase by describing the molecular details of the interaction between a PtdIns(4,5)P2-generating lipid kinase PIPKIgamma and the heterotetrameric clathrin adaptor AP-2. By engaging a subdomain on the AP-2 beta2 subunit appendage, the kinase is strategically positioned at assembly sites to generate PtdIns(4,5)P2 and drive coat assembly forward. Clathrin binds to the same subdomain on the beta2 appendage but with a higher apparent affinity. I therefore invoke a model in which PtdIns(4,5)P2 production for nucleation is negatively regulated by PIPKIgamma displacement from AP-2 by clathrin at later stages of assembly. I also demonstrate that a cargo-sorting alternate adaptor that binds to the other subsite on the AP-2 beta2 appendage is not subject to displacement by clathrin during clathrin-coated vesicle budding, ensuring non-competitive cargo incorporation into the vesicle. Finally, the PtdIns(4,5)P2-binding EFC domain proteins FCHO1 and FCHO2 have been proposed to act as dedicated nucleators of clathrin-coated structures on the plasma membrane. I demonstrate in multiple cell lines that these proteins are not invariantly required for placement of clathrin-coated assemblies on the plasma membrane despite being early arriving components themselves. FCHO1/2 are involved in the regulation of the size and number of these assemblies in some cellular contexts. My data support the model of PtdIns(4,5)P2 regulated, not protein regulated, nucleation of clathrin-coated structures; however multiple parallel pathways may contribute to initiation of endocytic buds.
    Date: 20 July 2011
    Date Type: Completion
    Defense Date: 09 June 2011
    Approval Date: 20 July 2011
    Submission Date: 18 July 2011
    Access Restriction: No restriction; The work is available for access worldwide immediately.
    Patent pending: No
    Institution: University of Pittsburgh
    Thesis Type: Doctoral Dissertation
    Refereed: Yes
    Degree: PhD - Doctor of Philosophy
    URN: etd-07182011-221052
    Uncontrolled Keywords: clathrin lattice; initiation; plasma membrane; siRNA
    Schools and Programs: School of Medicine > Cell Biology and Molecular Physiology
    Date Deposited: 10 Nov 2011 14:52
    Last Modified: 19 Jun 2012 11:59
    Other ID: http://etd.library.pitt.edu/ETD/available/etd-07182011-221052/, etd-07182011-221052

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