The long-term objective of this proposal is to elucidate the function of microtubules, which are involved in cell division, cell motility, morphogenesis, and ciliary/flagellar function. Microtubules and intermediate filaments (IFs) are affected in disease and by toxins and chetnotherapeutic drugs; also, antibodies against IFs provide tools for tumor diagnosis. Cilia and flagella are present in cells of the respiratory and reproductive tracts, and in sensory cells of the auditory, olfactory and visual systems. This proposal rests on the discovery of a novel set of proteins, tektins, that form filaments in flagellar microtubules from marine invertebrates. These microtubules contain a specialized 3- protofilament (pf) ribbon, composed principally of tubulin, three tektins and two 77/83kD polypeptides. Tektins are strikingly similar in their properties to IFs. There are four specific aims to determine how these proteins are assembled into the pf-ribbons, and ultimately what significance tektins may have for microtubule function: 1. To map the arrangement of proteins forming the tektin-tubulin protofilament ribbon. In one approach chemical cross-linking will be used to map nearest neighbor proteins within the pf-ribbon. Cross-linked proteins will be identified by sodium- dodecyl-sulfate polyacrylamide-gel-electrophoresis and immunoblotting. In second approach, pf-ribbons will be stained with gold-conjugated monoclonal antibodies specific for tubulin, tektins or the 77/83kD pair and examined by electron microscopy (EM) 2. To study the reassoeiation of tektins and tektin-tubulin complexes. In vitro experiments will be conducted to reassemble the tektins, tubulin and the 77/83kD proteins into filaments or ribbons. Other approaches will also be taken to promote formation of ordered structures and to examine potential interaction of tektins and tubulin. 3. To study the molecular structure of tektin polypeptides and tektin-tubulin filament assemblies. Low- angle metal shadowing will be used to determine the shape and dimensions of tektin subunits and oligomers. Fourier-EM and immuno-EM techniques will be used to analyze the structure of tektin filaments, tektin-tubulin protofilament ribbons and reassociated structures. 4. To sequence the tektins and to study their expression and function using cillogenesis. The sequence will be completed for an existing cDNA clone for one of the tektins and for clones of the other two tektins, and these sequences will be compared to those of IF proteins. Sequences of the putative tektins will be identified as such b) comparison with direct amino acid sequence data. Finally, using the cDNA probes, the expression and role of tektins in sea urchin embryonic ciliogenesis will be investigated.
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