The long term goal of this work is to understand the molecular basis for the regulation of Arp2/3 complex. Arp2/3 complex is an essential component of the actin assembly machinery because of its ability to nucleate branched actin filaments in response to cellular signals. Arp2/3 complex is involved in a number of processes that occur in healthy cells, such as endocytosis and motility of growth cones, but also plays roles in host cell infection by bacterial pathogens and the metastasis of tumor cells. The activity of Arp2/3 complex is tightly regulated in vivo, and numerous activators and inhibitors of the complex have been discovered, including WASp/Scar family proteins and coronin family proteins. Despite the central importance of the Arp2/3 complex, the molecular mechanism of its activation is still unknown due to the lack of biochemical and high resolution structural information of partially or fully activated states of the complex. Here we propose to use structural, biochemical and cell biological approaches to determine a precise molecular mechanism by which Arp2/3 complex and its regulatory proteins create branched actin networks. We will pursue this goal through the following three specific aims: 1.) Determine the key structural elements that allow Arp2/3 complex to bind to mother and daughter filaments of actin;2.) Determine how WASp/Scar family proteins activate Arp2/3 complex;and 3.) Determine how Coronin proteins inhibit Arp2/3 complex. Our approach will be to use mutational analysis and biochemical assays to precisely map the interactions of S. pombe Arp2/3 complex with mother and daughter filaments of actin. We will use mass spectrometry, x-ray crystallography and mutational analysis to determine where WASp/Scar family proteins bind to Arp2/3 complex and how they recruit the first actin monomer for the daughter filament. Finally, we will use biochemical and structural methods to determine how yeast coronin proteins inhibit Arp2/3 complex and influence its interactions with actin filaments.

Public Health Relevance

In this work, we are studying at the molecular level cellular machinery that controls actin polymerization. Bacteria and viruses use this machinery to infect human cells and cancerous cells depend on it to spread. Therefore, improving our understanding of the molecules that constitute this machinery will contribute to our understanding of diseased states in humans and how to treat them.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM092917-03
Application #
8291013
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Deatherage, James F
Project Start
2010-07-01
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
3
Fiscal Year
2012
Total Cost
$263,025
Indirect Cost
$74,925
Name
University of Oregon
Department
Biochemistry
Type
Organized Research Units
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
Helgeson, Luke A; Prendergast, Julianna G; Wagner, Andrew R et al. (2014) Interactions with actin monomers, actin filaments, and Arp2/3 complex define the roles of WASP family proteins and cortactin in coordinately regulating branched actin networks. J Biol Chem 289:28856-69
Luan, Qing; Nolen, Brad J (2013) Structural basis for regulation of Arp2/3 complex by GMF. Nat Struct Mol Biol 20:1062-8
Liu, Su-Ling; May, Jordan R; Helgeson, Luke A et al. (2013) Insertions within the actin core of actin-related protein 3 (Arp3) modulate branching nucleation by Arp2/3 complex. J Biol Chem 288:487-97
Hetrick, Byron; Han, Min Suk; Helgeson, Luke A et al. (2013) Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change. Chem Biol 20:701-12
Wagner, Andrew R; Luan, Qing; Liu, Su-Ling et al. (2013) Dip1 defines a class of Arp2/3 complex activators that function without preformed actin filaments. Curr Biol 23:1990-8
Liu, Su-Ling; Needham, Karen M; May, Jordan R et al. (2011) Mechanism of a concentration-dependent switch between activation and inhibition of Arp2/3 complex by coronin. J Biol Chem 286:17039-46