The final objective of the research proposed in this application is to determine whether the different beta-tubulin isotypes expressed in brain are functionally equivalent and interchangeable of if they possess distinct functional properties. The research focuses primarily on Class III beta-tubulin (betaIII). The amino acid sequence of betaIII differs from the other four beta-tubulin isotypes (gene products) expressed in brain by ~ 10%. Recent work from the applicant's laboratory has established that betaIII expression is neuron-specific. The onset of expression occurs immediately after proliferating neuroblasts complete their final mitotic cycle and initiate neurite outgrowth. Although betaIII is expressed in both the brain and testes, only betaIII in brain is posttranslationally modified by phosphorylation and glutamylation within its C-terminal isotype-defining domain. Moreover, of the five beta-tubulin isotypes expressed in brain, only betaIII is phosphorylated. Therefore, beta-tubulin in neurons is phosphorylated during development, but beta-tubulin in glia is not similarly modified. Both modifications are developmentally regulated. These facts strongly suggest that betaIII has unique functional properties. The goals of this proposal are: 1) To sequence by tandem mass spectrometry the neonatal and adult forms of betaIII to determine: a) the temporal sequence in which betaIII is modified by phosphorylation and glutamylation, and b) the relative abundance of the two modified forms of betaIII at different times during development. 2) To sequence the C-terminal domains of the other four brain beta-tubulin isotypes to determine: a) at what sites, b) when and c) to what extent these isotypes are modified during development. 3) To obtain antibodies which recognize either: a) phosphorylated betaIII, or b) glutamylated betaIII. 4) To begin to isolate the enzyme which glutamylates betaIII. 5) To perform in vitro polymerization assays to determine: a) how betaIII-depletion and b) how betaIII enrichment alters the kinetics of microtubule assembly. 6) To study in cultured cells betaIII expression over time to determine which charge variants are associated with the soluble, cytoskeletal and particulate fractions.
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