Clathrin coated vesicles are the most intensively studied carriers of membrane traffic in eukaryotic cells and the most prominent form of traffic from the plasma membrane to endosomes (endocytosis). Clathrin-coated pits and vesicles were the first membrane-traffic system recognized and analyzed in detail, and they have become a paradigm for efforts to understand molecular mechanisms of other modes of vesicular transport. We have recently combined high spatial-resolution snapshots from cryoEM with data from high temporal-resolution light microscopy (with single-molecule sensitivity) to answer one long-standing question about the way the clathrin pathway operates: how does the uncoating ATPase, Hsc70, together with its co-chaperone, auxilin, catalyze coat disassembly? Each of the Aims of this proposal seeks to exploit the single-molecule sensitivity of in vitro, single-particle methods as used in that work, to analyze mechanisms of general significance for intracellular membrane traffic.
In Aim 1, we will determine the molecular mechanism by which auxilin detects membrane scission.
In Aim 2, we will use in vitro reconstitution experiments to dissect the steps of coated pit initiation and growth.
In Aim 3, we will study the molecular organization of Eps15 and its partners (epsin, intersection, FCH01/2) -- a complex, network-like assembly, with multiple cross-interacting components.

Public Health Relevance

Clathrin-mediated endocytosis is the route by which cells take up transferring, immunoglobulins, LDL, hormones, and signaling receptors. It is a pathway frequently usurped for cell entry by viruses and bacterial toxins, as well as a one that might be exploited in designing next-generation vectors for vaccines and gene therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM036548-30S1
Application #
8898937
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
1986-04-01
Project End
2015-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
30
Fiscal Year
2014
Total Cost
$131,133
Indirect Cost
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Böcking, Till; Aguet, François; Rapoport, Iris et al. (2014) Key interactions for clathrin coat stability. Structure 22:819-29
Ivanovic, Tijana; Boulant, Steeve; Ehrlich, Marcelo et al. (2011) Recruitment of cellular clathrin to viral factories and disruption of clathrin-dependent trafficking. Traffic 12:1179-95
Böcking, Till; Aguet, François; Harrison, Stephen C et al. (2011) Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating. Nat Struct Mol Biol 18:295-301
Yu, Anan; Xing, Yi; Harrison, Stephen C et al. (2010) Structural analysis of the interaction between Dishevelled2 and clathrin AP-2 adaptor, a critical step in noncanonical Wnt signaling. Structure 18:1311-20
Xing, Yi; Bocking, Till; Wolf, Matthias et al. (2010) Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly. EMBO J 29:655-65
Guan, Rong; Dai, Han; Han, Dai et al. (2010) Structure of the PTEN-like region of auxilin, a detector of clathrin-coated vesicle budding. Structure 18:1191-8
Rapoport, Iris; Boll, Werner; Yu, Anan et al. (2008) A motif in the clathrin heavy chain required for the Hsc70/auxilin uncoating reaction. Mol Biol Cell 19:405-13
Yu, Anan; Rual, Jean-Francois; Tamai, Keiko et al. (2007) Association of Dishevelled with the clathrin AP-2 adaptor is required for Frizzled endocytosis and planar cell polarity signaling. Dev Cell 12:129-41
Ma, Yu May; Boucrot, Emmanuel; Villen, Judit et al. (2007) Targeting of AMSH to endosomes is required for epidermal growth factor receptor degradation. J Biol Chem 282:9805-12
Cheng, Yifan; Boll, Werner; Kirchhausen, Tomas et al. (2007) Cryo-electron tomography of clathrin-coated vesicles: structural implications for coat assembly. J Mol Biol 365:892-9

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