The interactions of molecular tracks and motors result in movements in cells. Fundamental to our understanding of the way in which motility is accomplished in cells is a detailed description not only of the atomic structures of the complexes involved, but also the way in which the activity of these complexes is regulated in response to the needs of the cell. The long term goals of the work described in this application are to understand in structural terms, the way in which myosin motors are regulated. Three types of myosin-based regulation will be investigated: (I) Regulation mediated by light chain phosphorylation will be investigated by examining a number of smooth muscle myosin constructs. (ii) Regulation by light chain dissociation will be examined in brush border myosin l and myosin V. (iii) Regulation by heavy chain phosphorylation will be investigated in Acanthamoeba and Dictyostelium myosin ls. The methods to be used include cryoelectron microscopy, helical image analysis and modeling using the x-ray structures of the myosin head and regulatory domain. The experiments have been designed to visualize actomyosin conformations which are the basis of the regulatory mechanism. Atomic models of the regulated and activated states will be built from the EM and X-ray data. The results should provide detailed insights into the structural basis for regulation in myosin involved in uterine contractions, blood pressure maintenance and intestinal peristalsis (smooth muscle myosin), and in intracellular trafficking and endocytosis (myosin ls and Vs). Some exploratory experiments on dynein are planned.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR039155-11
Application #
2472268
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1988-02-01
Project End
2003-01-31
Budget Start
1998-02-01
Budget End
1999-01-31
Support Year
11
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Littlefield, Kimberly P; Ward, Andrew B; Chappie, Joshua S et al. (2008) Similarities and differences between frozen-hydrated, rigor acto-S1 complexes of insect flight and chicken skeletal muscles. J Mol Biol 381:519-28
Dang, Thanh X; Milligan, Ronald A; Tweten, Rodney K et al. (2005) Helical crystallization on nickel-lipid nanotubes: perfringolysin O as a model protein. J Struct Biol 152:129-39
Manuell, Andrea L; Yamaguchi, Kenichi; Haynes, Paul A et al. (2005) Composition and structure of the 80S ribosome from the green alga Chlamydomonas reinhardtii: 80S ribosomes are conserved in plants and animals. J Mol Biol 351:266-79
Roger, Benoit; Al-Bassam, Jawdat; Dehmelt, Leif et al. (2004) MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain. Curr Biol 14:363-71
Rouiller, I; Pulokas, J; Butel, V M et al. (2001) Automated image acquisition for single-particle reconstruction using p97 as the biological sample. J Struct Biol 133:102-7
Rouiller, I; Butel, V M; Latterich, M et al. (2000) A major conformational change in p97 AAA ATPase upon ATP binding. Mol Cell 6:1485-90
Wilson-Kubalek, E M (2000) Preparation of functionalized lipid tubules for electron crystallography of macromolecules. Methods Enzymol 312:515-9
Wilson-Kubalek, E M; Brown, R E; Celia, H et al. (1998) Lipid nanotubes as substrates for helical crystallization of macromolecules. Proc Natl Acad Sci U S A 95:8040-5
Jontes, J D; Ostap, E M; Pollard, T D et al. (1998) Three-dimensional structure of Acanthamoeba castellanii myosin-IB (MIB) determined by cryoelectron microscopy of decorated actin filaments. J Cell Biol 141:155-62
Bernstein, S I; Milligan, R A (1997) Fine tuning a molecular motor: the location of alternative domains in the Drosophila myosin head. J Mol Biol 271:1-6

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