Actin is involved in generation of contractile force, cell shape determination, cytokinesis, and determination of cell polarity. Proper actin fuction, essential for cell viability, depends on a certain degree of filament stability and conformational flexibility. The dynamic nature of the actin monomer, both free and within the actin filament, is required for allosteric regulation of actin, both internally, and imposed upon it by an array of actin regulatory proteins. A manifestation of this regulation is the difference in functional behavior of yeast vs. muscle actin. They are 87% identical and have virtually superimposable structures. Yet they differ in parameters such as speed of polymerization, propensity of filaments to fragment, rate of nucleotide exchange, rate of phosphate release following bound nucleotide hydrolysis (a determinant of filament dynamics), ability to activate myosin, and response to actin binding proteins such as cofilin and Arp2/3 complex. To understand at the molecular level, the ways in which actin filament dynamics are regulated, one must first understand the way in which conformational changes are transmitted within the protein and how differences in these communication networks result in the behavioral differences of actin isoforms. We will use a series of yeast/muscle hybrid actins, manufactured by site-directed mutagenesis, to better understand the roles of actin's two domains in controlling the behavior of the actin monomer in the G-form and in the filament. With yeast as a model system, we will extend our initial work with such hybrid proteins using a combination of cell biological and protein chemical approaches such as polymerization kinetics, covalent crosslinking, and hydrogen/deuterium exchange to address the following aims. We will delineate the role of a subdomain 1 column of amino acids in regulation of domain/domain interactions. We will assess the effect of filament fragmentation on cytoskeletal function and how the balance between inherent filament stability and cofilin severing affects cytoskeletal behavior. We will examine the importance of two proposed intermonomer ionic interactions on filament stability, and we will assess the role of two critical subdomain 4 residues on the behavior of subdomains 1 and 2 in their interaction with actin binding proteins. Health Significance: This work will provide new insight into the molecular basis of actin regulation which, when abnormal, leads to pathological states such as cancer, muscle disease, and hearing loss.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033689-26
Application #
7786244
Study Section
Special Emphasis Panel (ZRG1-CB-G (02))
Program Officer
Gindhart, Joseph G
Project Start
1984-07-01
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2012-03-31
Support Year
26
Fiscal Year
2010
Total Cost
$361,945
Indirect Cost
Name
University of Iowa
Department
Biochemistry
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Lee, Cho-Yin; Lou, Jizhong; Wen, Kuo-Kuang et al. (2016) Regulation of actin catch-slip bonds with a RhoA-formin module. Sci Rep 6:35058
Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S et al. (2011) Functional adaptation between yeast actin and its cognate myosin motors. J Biol Chem 286:30384-92
Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema et al. (2011) Mutant profilin suppresses mutant actin-dependent mitochondrial phenotype in Saccharomyces cerevisiae. J Biol Chem 286:41745-57
Kudryashov, Dmitri S; Grintsevich, Elena E; Rubenstein, Peter A et al. (2010) A nucleotide state-sensing region on actin. J Biol Chem 285:25591-601
Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema et al. (2010) A potential yeast actin allosteric conduit dependent on hydrophobic core residues val-76 and trp-79. J Biol Chem 285:21185-94
Scoville, Damon; Stamm, John D; Altenbach, Christian et al. (2009) Effects of binding factors on structural elements in F-actin. Biochemistry 48:370-8
Stokasimov, Ema; Rubenstein, Peter A (2009) Actin isoform-specific conformational differences observed with hydrogen/deuterium exchange and mass spectrometry. J Biol Chem 284:25421-30
Wen, Kuo-Kuang; Rubenstein, Peter A; DeMali, Kris A (2009) Vinculin nucleates actin polymerization and modifies actin filament structure. J Biol Chem 284:30463-73
Stokasimov, Ema; McKane, Melissa; Rubenstein, Peter A (2008) Role of intermonomer ionic bridges in the stabilization of the actin filament. J Biol Chem 283:34844-54
Wen, Kuo-Kuang; McKane, Melissa; Houtman, Jon C D et al. (2008) Control of the ability of profilin to bind and facilitate nucleotide exchange from G-actin. J Biol Chem 283:9444-53

Showing the most recent 10 out of 58 publications