The research objective is to elucidate the molecular mechanisms by which Ca2+- and polyphosphoinositide (PPI)-dependent changes in actin filament length regulate the defense functions of lung macrophages. Particular emphasis will be focused on gelsolin, a 80 kDa PPI- and Ca binding protein first identified in rabbit lung macrophages. Gelsolin fragments actin filaments in the presence of uM Ca2+, and its activity is inhibited by PPI. Through regulation of actin filament length and polymerization, cytoplasmic gelsolin can cause reorganization of the actin cytoskeleton. Since both Ca2+ and PPI levels change transiently in cells following agonist stimulation, gelsolin may be the key control point in many cytological events. First, we will continue to characterize the functional domains of gelsolin. We have identified by limited proteolysis three actin binding sites and distinct Ca2+ and PPI- regulatory sites as well. We will focus on a gelsolin domain (designated CT28N) which binds to the side of actin filaments causing distortions which may contribute to PPI-inhibitable severing. We will study the effect of CT28N on actin filament morphology, and identify its PPI- and actin binding sites by NTCB cleavage. We will identify interactive sites between actin and gelsolin's 3 actin binding domains by chemical crosslinking, and prepare gelsolin crystals for analysis by X-ray crystallography. Second, we will use recombinant DNA technology to obtain further information about the boundaries as well as the interactions between gelsolin domains. Initially, mutated gelsolins with end deletions will be expressed in eukaryotic cells by gene transfection. We will capitalize on the fact that a gelsolin variant is secreted, so that it can be analyzed in the culture medium directly. Subsequently, specific alterations will be made by site-directed mutagenesis. Third, we will over/underexpress cytoplasmic gelsolin to assess its role in vivo. Gelsolin sense and antisense DNA will be transfected into fibroblasts and macrophage-like cells, and changes in cell shape and motility determined. We will look for compensatory changes in actin and other cytoskeletal proteins, to determine if there are mechanisms for maintaining a proper balance between them. Fourth, we will determine if the expression of cytoplasmic gelsolin changes under physiological and pathological conditions to gain further insight about its role in the cytoplasm. Our data indicate that cytoplasmic gelsolin expression decreases during the S phase of cell cycle, and after A23187 or cycloheximide treatment. The molecular basis for these changes will be determined.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL029113-10
Application #
3340296
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1989-09-01
Project End
1993-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
10
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Onoda, K; Yin, H L (1993) gCap39 is phosphorylated. Stimulation by okadaic acid and preferential association with nuclei. J Biol Chem 268:4106-12
Yu, F X; Lin, S C; Morrison-Bogorad, M et al. (1993) Thymosin beta 10 and thymosin beta 4 are both actin monomer sequestering proteins. J Biol Chem 268:502-9
Onoda, K; Yu, F X; Yin, H L (1993) gCap39 is a nuclear and cytoplasmic protein. Cell Motil Cytoskeleton 26:227-38
Yu, F X; Sun, H Q; Janmey, P A et al. (1992) Identification of a polyphosphoinositide-binding sequence in an actin monomer-binding domain of gelsolin. J Biol Chem 267:14616-21
Yu, F X; Zhou, D M; Yin, H L (1991) Chimeric and truncated gCap39 elucidate the requirements for actin filament severing and end capping by the gelsolin family of proteins. J Biol Chem 266:19269-75
Yu, F X; Johnston, P A; Sudhof, T C et al. (1990) gCap39, a calcium ion- and polyphosphoinositide-regulated actin capping protein. Science 250:1413-5
Johnston, P A; Yu, F X; Reynolds, G A et al. (1990) Purification and expression of gCap39. An intracellular and secreted Ca2(+)-dependent actin-binding protein enriched in mononuclear phagocytes. J Biol Chem 265:17946-52
Yin, H L; Janmey, P A; Schleicher, M (1990) Severin is a gelsolin prototype. FEBS Lett 264:78-80
Kwiatkowski, D J; Janmey, P A; Yin, H L (1989) Identification of critical functional and regulatory domains in gelsolin. J Cell Biol 108:1717-26
Sutoh, K; Yin, H L (1989) End-label fingerprintings show that the N- and C-termini of actin are in the contact site with gelsolin. Biochemistry 28:5269-75

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