The mechanism of microtubule-related motility is being approached through a detailed study of the chemistry of tubulin and its interactions with itself and other proteins. We ultimately hope to be able to define the dynamics of tubulin synthesis, storage, mobilization, organelle-specific utilization, and recycling in order to better understand the role of microtubules in both cell division and cell differentiation. Four major projects will be emphasized in this continuing study. We will extend our differential detergent binding methodology for tubulin variant separation through: a) high-resolution acid-urea-Triton electrophoretic separation; b) isoelectric focusing in the presence of certain anionic, cationic, and zwitterionic detergents; c) charge-shift electrophoresis of dimeric tubulin; and d) chromatographic resolution of tubulins in accord with relative hydrophobicity. How many tubulin variants occur in different microtubules, are some shared, and what is their fate in development? We will continue primary structural studies of organelle-specific tubulines through the separation and analysis of unique peptide sequences obtained by enzymatic or chemical cleavage of tubulin subunits. What kinds of sequence variation are involved in organelle-specificity and how do these correlate with microtubule properties? We will resume our investigation of basal body and basal rootlet biochemistry through: a) characterization of tubulin solubilized from the isolated molluscan basal apparatus, comparing it with ciliary axonemal tubulins; b) characterization of rootlet protein with respect to self-assembly, interaction with basal microtubules, and the effect of calcium ions on rootlet length and periodicity; and c) reconstitution of outer doublet microtubules onto basal body templates. What is the role of the basal apparatus in ciliary assembly and function? We will explore the disposition of membrane tubulin in natural and reconstituted ciliary membranes through: a) vectorial labeling and specific cleavage to map polypeptide topology; b) incorporation of tubulin fragments into lipid bilayers; c) determination of specific lipids associated with membrane tubulin; and d) interaction of tubulin-containing membranes with cytoplasmic proteins. How is tubulin associated with the membrane, topologically, and how does it interact with cellular constituents?
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