The overall purpose of this study is to understand how cells use actin- sequestering proteins to regulate levels of G- and F-actin and the organization of the cytoskeleton.
The specific aims are, first, to investigate the structural basis for actin sequestration by thymosin Beta4 (Tbeta4) by identifying sites of contact through chemical crosslinking studies, by determining the packing of molecules in macrocrystals of the actin-TBeta4 complex, and by growing crystals suitable for structure determination by x-ray diffraction.
The second aim i s to determine how anchorage dependent cells such as fibroblast regulate the concentration of actin-sequestering proteins and G and F- actin levels in response to changes in substrate attachment and cell density; the third, related aim is to identify the actin-sequestering protein in fibroblasts.
The fourth aim i s to investigate the relationship between actin sequestering proteins and the changes in G- and F-actin levels in cells that undergo cytoskeletal reorganization in response to growth factors. This will be studied in PC12 cells which extend neurites when stimulated by nerve growth factor; some clones increase the transcription of Tbeta4 mRNA before and during neurite extension while others do not. We will use transfection with plasmids containing Tbeta4 cDNA (sense and antisense) to manipulate Tbeta4 levels in the presence and absence of growth factor stimulation and determine the effects on G-actin levels and on neurite extension. The fifth aim is to determine whether homologs of Tbeta4 are responsible for maintaining the large pools of sequestered actin in specific invertebrate cells of interest, using echinoderm eggs, an insect cell line, and the protozoan Dictyostelium discoideum. The regulation of G-actin pools and of cytoskeletal structure is intimately related to the regulation of cell attachment, growth, locomotion and cytokinesis. These processes are fundamental aspects of medically significant problems such as hemostasis, inflammation, the immune response and of normal and metastatic growth.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR040840-07
Application #
2390513
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1991-04-01
Project End
1999-03-31
Budget Start
1997-04-01
Budget End
1999-03-31
Support Year
7
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Nagamalleswari, K; Safer, D (2000) Sequestered actin in chick embryo fibroblasts. Mol Cell Biochem 209:63-7
Niu, M; Nachmias, V T (2000) Increased resistance to apoptosis in cells overexpressing thymosin beta four: A role for focal adhesion kinase pp125FAK. Cell Adhes Commun 7:311-20
De La Cruz, E M; Ostap, E M; Brundage, R A et al. (2000) Thymosin-beta(4) changes the conformation and dynamics of actin monomers. Biophys J 78:2516-27
Devineni, N; Minamide, L S; Niu, M et al. (1999) A quantitative analysis of G-actin binding proteins and the G-actin pool in developing chick brain. Brain Res 823:129-40
Safer, D (1999) Undecagold cluster labeling of proteins at reactive cysteine residues. J Struct Biol 127:101-5
Golla, R; Philp, N; Safer, D et al. (1997) Co-ordinate regulation of the cytoskeleton in 3T3 cells overexpressing thymosin-beta4. Cell Motil Cytoskeleton 38:187-200
Niu, M Y; Mills, J C; Nachmias, V T (1997) Development of polarity in human erythroleukemia cells: roles of membrane ruffling and the centrosome. Cell Motil Cytoskeleton 36:203-15
Safer, D; Sosnick, T R; Elzinga, M (1997) Thymosin beta 4 binds actin in an extended conformation and contacts both the barbed and pointed ends. Biochemistry 36:5806-16
Safer, D; Chowrashi, P K (1997) Beta-thymosins from marine invertebrates: primary structure and interaction with actin. Cell Motil Cytoskeleton 38:163-71
Sanger, J M; Golla, R; Safer, D et al. (1995) Increasing intracellular concentrations of thymosin beta 4 in PtK2 cells: effects on stress fibers, cytokinesis, and cell spreading. Cell Motil Cytoskeleton 31:307-22

Showing the most recent 10 out of 19 publications