Regulation of the actin cytoskeleton by cell signaling pathways is important for cellular movement and function. The activation of one Rho GTPase, Cdc42, leads to the rapid and transient polymerization of a highly-branched actin network nucleated by Arp2/3 complex. The event creates a myosin-independent force that pushes forward the membrane at the leading edge of motile cells. While much is known about the activation and deactivation of this process, important questions remain. How is formation of branched networks localized to the leading edge of the cell and not from actin filaments deeper in the cortex and in stress fibers? What protects selected populations of actin filaments from severing and pointed-end depolymerization by ADF/cofilin? Is there a hierarchy of filament stability? The goal of the research is to understand the cascade of events leading to the formation and maintenance of stable actin filaments in the cell. The research will explore the roles of ADF/cofilin, Arp2/3 complex, tropomyosin and tropomodulin, together with the actin nucleotide, in defining the pathway from nascent, branched actin filaments to stable filaments. There are four specific aims. 1) How are branched filaments converted to stable unbranched filaments and how do long, unbranched filaments remain unbranched?2) How are the pointed ends of stable actin filaments protected from depolymerization?3) How are long actin filaments in the deep cell cortex and stress fibers protected from severing and pointed end depolymerization by ADF/cofilin while those in the branched actin network are not?4) To determine the localization of tropomyosin isoforms in relation to Arp2/3 complex, cofilin, and regions of dynamic remodeling of the actin cytoskeleton. The methods for Aims 1-3 include measurement of actin polymerization and depolymerization using actin labeled with a fluorescent probe as well as microscopic analysis of fluorescently-labeled actin filaments.
Aim 4 will localize the proteins as well as free filament ends in cells using indirect immunofluorescence. The results will help understand how the cytoskeleton is remodeled during complex functions such as leucocyte chemotaxis and growth factor-stimulated cellular outgrowth, processes important in both normal and pathological states.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063257-15
Application #
6621897
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Deatherage, James F
Project Start
1985-09-30
Project End
2006-02-28
Budget Start
2003-03-01
Budget End
2004-02-29
Support Year
15
Fiscal Year
2003
Total Cost
$304,090
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Neurosciences
Type
Schools of Medicine
DUNS #
617022384
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
Kostyukova, Alla S; Hitchcock-Degregori, Sarah E; Greenfield, Norma J (2007) Molecular basis of tropomyosin binding to tropomodulin, an actin-capping protein. J Mol Biol 372:608-18
Wawro, Barbara; Greenfield, Norma J; Wear, Martin A et al. (2007) Tropomyosin regulates elongation by formin at the fast-growing end of the actin filament. Biochemistry 46:8146-55
Hitchcock-DeGregori, Sarah E; Greenfield, Norma J; Singh, Abhishek (2007) Tropomyosin: regulator of actin filaments. Adv Exp Med Biol 592:87-97
Kostyukova, Alla S; Choy, Andy; Rapp, Brian A (2006) Tropomodulin binds two tropomyosins: a novel model for actin filament capping. Biochemistry 45:12068-75
Gupton, Stephanie L; Anderson, Karen L; Kole, Thomas P et al. (2005) Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin. J Cell Biol 168:619-31
Greenfield, Norma J; Kostyukova, Alla S; Hitchcock-DeGregori, Sarah E (2005) Structure and tropomyosin binding properties of the N-terminal capping domain of tropomodulin 1. Biophys J 88:372-83
Kostyukova, Alla S; Rapp, Brian A; Choy, Andy et al. (2005) Structural requirements of tropomodulin for tropomyosin binding and actin filament capping. Biochemistry 44:4905-10
Kostyukova, Alla S; Hitchcock-DeGregori, Sarah E (2004) Effect of the structure of the N terminus of tropomyosin on tropomodulin function. J Biol Chem 279:5066-71
Bharadwaj, Shantaram; Hitchcock-DeGregori, Sarah; Thorburn, Andrew et al. (2004) N terminus is essential for tropomyosin functions: N-terminal modification disrupts stress fiber organization and abolishes anti-oncogenic effects of tropomyosin-1. J Biol Chem 279:14039-48
Akkari, P Anthony; Song, Yuhua; Hitchcock-DeGregori, Sarah et al. (2002) Expression and biological activity of Baculovirus generated wild-type human slow alpha tropomyosin and the Met9Arg mutant responsible for a dominant form of nemaline myopathy. Biochem Biophys Res Commun 296:300-4

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