The role of microtubules in development and the mechanism of microtubule-based motility will be approached through a detailed study of the chemistry of tubulin and its interactions with itself and with other proteins. The classes and amounts of tubulin in cells during development, the state of tubulin pools before, during, and after microtubule assembly into spindles or cilia, tubulin association with accessory proteins to form specific organelles such as basal bodies or ciliary axonemes, the association of tubulin with membranes, and the interaction of tubulin with dynein are basic questions that this project has been exploring. There will be four major aims during this project period. First, studies of reverse-phase HPLC separation and analysis of tubulin subunits will be extended by systematically varying stationary-phase and solvent conditions, performing separations with new detergent-modified systems, exploring co- chromatography to detect protein-protein interaction, and separating large organelle-specific peptides in an attempt to define distinguishing regions of the tubulin molecules. Second, further studies on tektins, a new class of tubulin-associated proteins, will involve fractionation of ciliary remnants, nearly devoid of tubulin, to ascertain the location of tektin filaments, a systematic study of the reassembly properties of tektin subunits and the reassembly of tubulin on tektin filaments in an attempt to produce doublet microtubules and, finally, an investigation of possible spindle tektin synthesis at prophase. Third, studies on ciliary membrane tubulin will be continued in order to determine what chemical differences distinguish membrane from axonemal tubulin. We will study protein topology with membrane probes, determine tubulin interaction with specific lipids, and study the possible interaction of tubulin-containing membranes with cytoplasmic proteins. Finally, studies of a neuronally-controlled dynein from mussel gill lateral cilia will involve a thorough characterization of both the inner and outer arm dynein molecules and a 10S phosporylated entity that co-extracts with dynein, an analysis of the specificity of phosphorylation of 3 presumptive light chains, and a determination of possible dynein-associated kinases and phosphatases.
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