Microtubules are critical nanoscale structural features of eukaryotic cells. These cytoskeletal elements confer shape and motility to the cells. They serve as organizers of subcellular structure, as tracks along which nanoscale molecular motors move subcellular organelles (including chromosomes and vesicles) in actively directed fashion from one place in the cell to another, and as key components of motile cellular appendages (cilia and flagella). Microtubules are made up of a unique class of proteins called tubulins. Tubulins come in alpha- and beta- isoforms. Vertebrates produce 6-7 distinct beta-tubulin gene products that are well conserved across species and are differentially expressed among various tissues. The existence of such distinct beta-tubulin gene products has led to the hypothesis that different tubulins subserve different functions. However, most of the data to date suggest that the different isotypes are functionally interchangeable for microtubule assembly. In contrast to these earlier studies, however, recent work has demonstrated that class V beta-tubulin, which is normally a low abundance but widely distributed protein, has a potent ability to disrupt microtubules and inhibit cell proliferation when overexpressed in cells. The goal of this research project is to determine the natural function of normal (low) cellular levels of beta-5-tubulin. The experiments are directed toward asking whether a low abundance of beta-5-tubulin is necessary for cell proliferation and toward elucidation of the mechanism by which beta-5-tubulin affects microtubule assembly. Inhibitory RNAs will be used to reduce or eliminate beta-5-tubulin expression, and the effects on cell growth and cell cycle progression will be examined. Because microtubule assembly is dramatically disrupted in cells that overexpress beta-5-tubulin, it is hypothesized that beta-5-tubulin is used by cells to keep the microtubules in a dynamic state. This idea will be tested by using a fluorescently-marked microtubule-binding protein, GFP-MAP4, to measure the dynamic aspects (growth and shrinkage) of microtubules in living cells that over- or under-express beta-5-tubulin. Finally, site-directed mutagenesis of beta-5-tubulin will be used to identify structural elements that are critical for the protein's effect on microtubule dynamics. Mapping the critical residues to the published crystal structure of the most abundant beta- tubulin isoform will provide important clues about the subunit interactions that are likely to be impacted by incorporation of the beta-5-tubulin into microtubules.
Intellectual Merit: These studies will provide important new information about an understudied and potentially critical beta-5-tubulin subunit for microtubule assembly. Understanding the putative involvement of beta-5-tubulin in modulating the assembly and disassembly of microtubules may be the first step in uncovering a previously unappreciated mechanism that cells use to regulate the dynamic function of microtubules by altering the composition of their constituent tubulin proteins.
Broader Impacts: The project will serve as a vehicle for training postdoctoral and graduate students. In addition, new fundamental knowledge and understanding of how cells regulate microtubule assembly may lead to as-yet unanticipated benefits to society through a variety of disparate means, e.g., biomimetic engineering of self-assembling nanostructures or genetic engineering of crop plants (since microtubules are the primary target of certain types of agricultural herbicides).
