Cell motility influences many biological processes. During development, cells move in an organized manner to give rise to specific organs, and disruption of this process can lead to serious abnormalities. Directed cell movements are also associated with angiogenesis, neuronal re-arrangements, wound healing, and the movement of macrophages to sites of infection. One of the most lethal manifestations of uncontrolled cell motility is metastasis. In muscle, myosin is the protein which converts the chemical energy of ATP into the mechanical energy of movement. Myosin is also present in non-muscle cells and is believed to be responsible for most types of cell locomotion. In order to understand how cell motility is regulated, therefore, it is essential to study regulation of the myosin molecule. Dictyostelium is a good system for investigating this problem because the amoebae can be grown easily, are amenable to genetic analysis, and exhibit chemotaxis. The heavy chain of the amoebae myosin is phosphorylated in vivo, and this modification inhibits self-assembly and ATPase activity. In contrast, phosphorylation of the light chain enhances ATPase activity. The goals of this research are to understand how the cell regulates the level of phosphorylation, and how this modification exerts its effect on myosin function. To qualtitate changes in phosphorylation during chemotaxis, cells are grown in radioactive phosphate and myosin is rapidly purified by immunoprecipitation. Because the level of phosphorylation is ultimately controlled by specific kinases and phosphorylation is ultimately controlled by specific kinases and phosphatases it is important to purify these enzymes. Studies on the regulation of these enzymes should help elucidate the chain of events which links an extracellular signal to changes in myosin phosphorylation. These purified enzymes also provide the tools for altering levels of phosphorylation in vitro. Current in vitro studies concern the effects of phosphorylation on ATPase activity and assembly. In addition studies are being carried out to determine if the supramolecular organization of molecules within the thick filament can control ATPase activity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM031907-04
Application #
3280322
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1983-04-01
Project End
1989-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
4
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Northwestern University at Chicago
Department
Type
School of Medicine & Dentistry
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
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Aguado-Velasco, C; Kuczmarski, E R (1993) Contraction of reconstituted Dictyostelium cytoskeletons: an apparent role for higher order associations among myosin filaments. Cell Motil Cytoskeleton 26:103-14
Kuczmarski, E R; Palivos, L; Aguado, C et al. (1991) Stopped-flow measurement of cytoskeletal contraction: Dictyostelium myosin II is specifically required for contraction of amoeba cytoskeletons. J Cell Biol 114:1191-9
Kuczmarski, E R; Routsolias, L; Parysek, L M (1988) Proteolytic fragmentation of Dictyostelium myosin and localization of the in vivo heavy chain phosphorylation site. Cell Motil Cytoskeleton 10:471-81
Watanabe, T K; Kuczmarski, E R; Reddy, J K (1987) Myosin from pancreatic acinar carcinoma cells. Isolation, characterization and demonstration of heavy- and light-chain phosphorylation. Biochem J 247:513-8
Kuczmarski, C A; Orlina, A R; Delahanty, L K et al. (1987) Instability of red cell shape associated with the absence of membrane glycophorin C. Vox Sang 52:36-42
Kuczmarski, E R; Tafuri, S R; Parysek, L M (1987) Effect of heavy chain phosphorylation on the polymerization and structure of Dictyostelium myosin filaments. J Cell Biol 105:2989-97
Kuczmarski, E R; Pagone, J (1986) Myosin specific phosphatases isolated from Dictyostelium discoideum. J Muscle Res Cell Motil 7:510-6
Kuczmarski, E R (1986) Partial purification of two myosin heavy chain kinases from Dictyostelium discoideum. J Muscle Res Cell Motil 7:501-9