The long term goal of this project is to understand the regulation of cytoskeletal and contractile functions by studying the control of the assembly and disassembly of actin filaments (F-actin). The approach we have been taking is to use cytochalasins (fungal metabolites with anti-motility activity) as tools to identify cellular components that affect actin polymerization. We previously found that these compounds bind with high affinity to the fast growing end of F-actin, thereby strongly affecting the rates of filament assembly and disassembly as well as filament length distribution in vitro. Moreover, we were able to isolate from skeletal muscle a protein (designated as capactin) that affects actin assembly and disassembly in a manner that closely resembles that of the cytochalasins. In the proposed research, we plan to study the structure and function of capactins in muscle and nonmuscle cells in the following ways. (A) We will determine the biochemical structure of the protein by analyzing its primary and subunit structures, active site, isoelectric isoforms, etc. cDNA encoding capactin will be isolated and sequenced. Monoclonal antibodies against different epitopes on the protein will be prepared. We will compare the properties of capactins from muscle and nonmuscle cells, and between this class of proteins and other actin modulating proteins such as gelsolin. (B) We will study the localization of capactins in adult and developing muscle and in nonmuscle tissues by immunofluorescence and by incorporation of labelled capactin into cellular and cell-free systems. The appearance of the protein and its messengers will also be studied under a variety of physiological conditions with the use of Western and Northern blots. (C) The interaction of capactin with actin and with other cellular components will be studied with biophysical and pharmacological techniques.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM022289-16
Application #
3271062
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1978-05-01
Project End
1992-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
16
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Arts and Sciences
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
McCleverty, Clare J; Lin, Diane C; Liddington, Robert C (2007) Structure of the PTB domain of tensin1 and a model for its recruitment to fibrillar adhesions. Protein Sci 16:1223-9
Hiura, K; Lim, S S; Little, S P et al. (1995) Differentiation dependent expression of tensin and cortactin in chicken osteoclasts. Cell Motil Cytoskeleton 30:272-84
Chuang, J Z; Lin, D C; Lin, S (1995) Molecular cloning, expression, and mapping of the high affinity actin-capping domain of chicken cardiac tensin. J Cell Biol 128:1095-109
Davis, S; Lu, M L; Lo, S H et al. (1991) Presence of an SH2 domain in the actin-binding protein tensin. Science 252:712-5
Ottlinger, M E; Lin, S (1988) Clostridium difficile toxin B induces reorganization of actin, vinculin, and talin in cultured cells. Exp Cell Res 174:215-29
Wachsstock, D H; Wilkins, J A; Lin, S (1987) Specific interaction of vinculin with alpha-actinin. Biochem Biophys Res Commun 146:554-60
Mitchell, M J; Laughon, B E; Lin, S (1987) Biochemical studies on the effect of Clostridium difficile toxin B on actin in vivo and in vitro. Infect Immun 55:1610-5
Magargal, W W; Lin, S (1986) Transformation-dependent increases in endogenous cytochalasin-like activity in chicken embryo fibroblasts infected by Rous sarcoma virus. Proc Natl Acad Sci U S A 83:8201-5
Zachary, J M; Cleveland, G; Kwock, L et al. (1986) Actin filament organization of the Dunning R3327 rat prostatic adenocarcinoma system: correlation with metastatic potential. Cancer Res 46:926-32
Casella, J F; Maack, D J; Lin, S (1986) Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments. J Biol Chem 261:10915-21