Using genetics approaches to understand the structural basis for actin filament generation by the Arp2/3 complex ; The Arp2/3 complex is an actin nucleation factor conserved across evolution. In vitro, the Arp2/3 complex is activated by Wiskott-Aldrich syndrome family proteins and initiates the polymerization of a new filament at a distinct angle from an existing filament, resulting in a unique actin branch structure. These structures have also been observed in vivo and are thought to be critical for protrusive force generation during cell motility. The overall goal of this program project is to understand the detailed structural and biochemical mechanisms by which the Arp2/3 complex carries out its function. This project contributes genetic approaches to structure/function analysis of the Arp2/3 complex. Most of the aims involve using yeast genetic techniques to generate mutations or tags in various subunit of the Arp2/3 complex. Biochemical analysis and structural characterization (by NMR and electron cryo-microscopy, etc.) of the mutant or tagged complexes will allow testing specific models for Arp2/3 complex activation and actin nucleation, as well as defining the structural changes that occur during activation. In the last aim, we will use mouse embryonic stem cell genetics to generate cell lines bearing mutations in an Arp2/3 complex subunit that could affect actin branch formation, morphology or stability. Characterization of the dynamics and structure of branch junctions in fibroblasts differentiated from these cell lines wilt provide key insights into the role of the Arp2/3 complex in the generation and organization of actin filaments during cell motility.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM066311-04
Application #
7551239
Study Section
Special Emphasis Panel (ZRG1)
Project Start
Project End
Budget Start
2006-06-01
Budget End
2007-05-31
Support Year
4
Fiscal Year
2006
Total Cost
$339,000
Indirect Cost
Name
Yale University
Department
Type
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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Wang, Pei-Shan; Chou, Fu-Sheng; Ramachandran, Sreekumar et al. (2016) Crucial roles of the Arp2/3 complex during mammalian corticogenesis. Development 143:2741-52
Pollard, Thomas D (2016) Actin and Actin-Binding Proteins. Cold Spring Harb Perspect Biol 8:
Page, Christopher; Hanein, Dorit; Volkmann, Niels (2015) Accurate membrane tracing in three-dimensional reconstructions from electron cryotomography data. Ultramicroscopy 155:20-26
Jurgenson, Christopher T; Pollard, Thomas D (2015) Crystals of the Arp2/3 complex in two new space groups with structural information about actin-related protein 2 and potential WASP binding sites. Acta Crystallogr F Struct Biol Commun 71:1161-8
Li, Yongchao; Wang, Pei-Shan; Lucas, George et al. (2015) ARP2/3 complex is required for directional migration of neural stem cell-derived oligodendrocyte precursors in electric fields. Stem Cell Res Ther 6:41
Tee, Yee Han; Shemesh, Tom; Thiagarajan, Visalatchi et al. (2015) Cellular chirality arising from the self-organization of the actin cytoskeleton. Nat Cell Biol 17:445-57
Suraneni, Praveen; Fogelson, Ben; Rubinstein, Boris et al. (2015) A mechanism of leading-edge protrusion in the absence of Arp2/3 complex. Mol Biol Cell 26:901-12
Volkmann, Niels; Page, Christopher; Li, Rong et al. (2014) Three-dimensional reconstructions of actin filaments capped by Arp2/3 complex. Eur J Cell Biol 93:179-83
Volkmann, Niels (2014) The joys and perils of flexible fitting. Adv Exp Med Biol 805:137-55

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