Microtubules exhibit equilibrium and treadmilling reactions which may be integral to many of their cytoplasmic functions. Understanding the physiological regulation of the microtubule steady state is therefore arguably of paramount importance to our understanding of the molecular mechanisms of microtubule behavior. We propose to further study one regulatory system which profoundly affects microtubule stability and equilibrium behavior. In the presence of substoichiometric amounts of a regulatory factor, microtubules become extremely stable to millimolar calcium, to cold temperature and to assembly inhibiting drugs. These microtubules are referred to as """"""""cold stable."""""""" The cold stable microtubule regulatory material in turn is apparently subject to control mechanisms, losing its activity when exposed to Ca++ calmodulin or upon phosphorylation. We have isolated a discrete polypeptide complex which contains the stabilizing activity, and we propose to use biochemical and cell biological approaches to characterize the mechanisms by which it regulates microtubule equilibria. Our major goals are presently to determine the parameters that regulate the interaction of this complex with microtubules, to determine if it contains active subcomponents, and to determine if this complex represents a microtubule regulatory mechanism common to many cell types. Our overall goal is to determine how such a regulatory mechanism can serve to integrate microtubule behavior into the overall physiological response of cells to calmodulin and kinase activating stimuli.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028189-06
Application #
3275463
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1980-07-01
Project End
1988-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
6
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
075524595
City
Seattle
State
WA
Country
United States
Zip Code
98109
Margolis, R L; Rauch, C T; Pirollet, F et al. (1990) Specific association of STOP protein with microtubules in vitro and with stable microtubules in mitotic spindles of cultured cells. EMBO J 9:4095-102
Pirollet, F; Rauch, C T; Job, D et al. (1989) Monoclonal antibody to microtubule-associated STOP protein: affinity purification of neuronal STOP activity and comparison of antigen with activity in neuronal and nonneuronal cell extracts. Biochemistry 28:835-42
Vogel, D G; Margolis, R L; Mottet, N K (1989) Analysis of methyl mercury binding sites on tubulin subunits and microtubules. Pharmacol Toxicol 64:196-201
Job, D; Rauch, C T; Margolis, R L (1987) High concentrations of STOP protein induce a microtubule super-stable state. Biochem Biophys Res Commun 148:429-34
Pirollet, F; Job, D; Margolis, R L et al. (1987) An oscillatory mode for microtubule assembly. EMBO J 6:3247-52
Pirollet, F; Job, D; Margolis, R L (1987) Biochemical assay of microtubule mean length. Anal Biochem 167:352-7
Garel, J R; Job, D; Margolis, R L (1987) Model of anaphase chromosome movement based on polymer-guided diffusion. Proc Natl Acad Sci U S A 84:3599-603
Margolis, R L; Job, D; Pabion, M et al. (1986) Sliding of STOP proteins on microtubules: a model system for diffusion-dependent microtubule motility. Ann N Y Acad Sci 466:306-21
Margolis, R L; Rauch, C T; Job, D (1986) Purification and assay of cold-stable microtubules and STOP protein. Methods Enzymol 134:160-70
Margolis, R L; Rauch, C T; Job, D (1986) Purification and assay of a 145-kDa protein (STOP145) with microtubule-stabilizing and motility behavior. Proc Natl Acad Sci U S A 83:639-43

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