CONTROL OF MICROTUBULE ASSEMBLY IN VITRO AND IN VIVO

  • Luduena, Richard F (PI)

Project: Research project

Project Details

Description

Tubulin, the subunit protein of microtubules, consists of an alpha and beta
subunit, each of which exists as various isotypes, differing from each
other in their amino acid sequences and their post-translational
modifications. The fundamental aim of this proposal is to test the
hypothesis that these differences are functionally significant. We shall
focus on the various beta isotypes and on two of the post-translational
modifications undergone by the tubulin molecule. The function of one of
these, the phosphorylation of betaIII-tubulin, is not known. The other
modification, the covalent addition of glutamate residues to the gamma-
carboxyl groups of glutamates in alpha and betaIII, is not understood, and
is unique among known proteins. We have used monoclonal antibodies to
purify tubulin isotypes and found that they exhibit dramatic differences
among themselves in their assembly and drug-binding properties, and their
cellular distributions. We have also made the first accurate measurements
of phosphate content of individual tubulin isotypes and found that only
betaIII, and the alpha and binding to betaIII, are phosphorylated. In this proposal, we intend to carry out the following specific aims: 1.
To determine the role of tubulin phosphorylation by locating the phosphate
groups in the primary structure of tubulin, removing the phosphates,
preparing antibodies capable of distinguishing between phosphorylated and
non-phosphorylated tubulin, and separating these two forms of tubulin to
determine their properties in vitro. 2. To examine the interaction of
tubulin isotypes with proteins involved in microtubule assembly and
function. For each form of tubulin, we shall measure its binding to, and
its effects on the activities of, proteins mediating microtubule function,
such as kinesin and cytoplasmic dynein. We shall distinguish the effects
of the various tubulin isotypes and of phosphorylated and non-
phosphorylated tubulin. 3. To determine the cellular and subcellular
distributions of the beta isotypes and of phosphorylated and non-
phosphorylated tubulin. We shall use monoclonal antibodies to determine
the distributions of the various forms of tubulin in cells. We shall see
how the apparent function of the microtubule in vivo, whose isotype
composition and state of phosphorylation we will determine, is correlated
with the known functions of those isotypes in vitro. 4. To identify and
purify the enzymes involved in the glutamylation of tubulin. The
purification of the enzymes that carry out this unique modification will be
a major step toward understanding the role of glutamylation.
StatusFinished
Effective start/end date4/1/793/31/96

Funding

  • National Institutes of Health: $113,731.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $138,157.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $127,842.00

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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