One of the unanswered questions in cell biology is--how do mammalian cells organize radial microtubule arrays in the interphase, and during the cell division cycle, how do they transform interphase microtubule (MT) array into the mitotic apparatus? This proposal focuses on the determination of mechanisms responsible for the organization of MTs by microtubule organizing centers (MTOC's) in mammalian cells. The specific aims are driven by the general hypothesis that a conserved component of MTOC's, gamma-tubulin, organizes cellular MT arrays by initiating MT assembly and/or attaching MTS to the centrosomes. We will first determine if in vitro synthesized gamma-tubulin interacts with the MT ends using biochemical and immunocytochemical approaches. Using this biochemical assay and the in vitro synthesis of systematically altered alanine scanning mutant gamma-tubulin molecules, we will determine which surface residues within gamma-tubulin are important for MT interactions. This information will be necessary to understand the precise relationship of MT attachment to the centrosome, an organelle that contains 10 4 molecules of gamma- tubulin as its integral component. Next, using a gene replacement strategy in the fission yeast, S. pombe, we will test the ability of gamma-tubulin mutations to functionally replace the yeast gamma-tubulin. Our next aim is based upon the observation that gamma-tubulin over -expression in mammalian cells produces MTS throughout eh cytoplasm and it uncouples the two normally coordinated phases of the cell cycle, the M-phase and the S-phase. By producing extracentrosomal microtubules in vivo, we are in a unique position to test the hypothesis that the mitotic check point "reads" the kinetochore-MT interactions. Finally, if funded for five years, we will exploit any conditional alleles of gamma-tubulin generated in the alanine- scanning mutagenesis by initiating an extragenic suppressor analysis to identify cellular proteins that interact with gamma-tubulin. The basis for these experiments is founded in promising preliminary studies including isolation of mammalian gamma-tubulin cDNA, development of specific antibodies that seem to block gamma-tubulin function, and the successful use of the in vitro transcription/translation for the synthesis of this rare protein. We are now in an excellent position to rigorously examine the hypothesis that gamma-tubulin organizes cellular MT arrays by initiating MT assembly and attaching MTs to the centrosomes. Since gamma- tubulin seems to be a ubiquitous component of MTOC's from as diverse systems as the ciliated epithelia, mouse embryo, and plant cells, the results of this study will be of interest to a diverse group of investigators including oncologists, reproductive and developmental biologists.