9603730 Olmsted The long range goal of the proposed research is to understand how microtubule- associated proteins that are not motors participate in the function and organization of microtubules and the structuring of the cytoplasm. These studies focus on MAP 4, a microtubule-associated protein that is expressed during early development and in specific adult tissues in mice. The working hypothesis is that the functions of this ubiquitous protein are context-specific; MAP 4 structure and functions in cycling cells may differ from those in differentiated tissues. The basic approach will be to dissect how variations which naturally arise either by alternative splicing or by post-translational modification affect MAP 4 behavior within cells. Chimeras will be generated in which MAP 4 constructs are modified at sites corresponding to characterized post-translational modifications or which reflect primary sequence variations observed in specific tissues. Living cells transfected with MAP 4 constructs tagged with green fluorescent protein or its spectral variants will be analyzed to deduce how such changes in MAP structure affect microtubule dynamics in interphase and mitotic cells. Other aspects of cytoplasmic organization, including the distribution of cytoskeletal elements or major membranous organelles, will also be analyzed to assess whether specific domains of MAP 4 may interact with non-microtubular structures. These studies should provide fundamental information on whether this ubiquitous protein has multiple roles depending on its place of expression. Microtubules comprise one of the three major cytoskeletal systems in eukaryotic cells, and one of two such systems known to be involved in intracellular motility phenomena by serving as specific "tracks" along which ATP-powered molecular motors (dyneins and kinesins) move. In addition to the motor proteins, other specific proteins ("Microtubule- Associated Proteins, or MAPs) are known bind to microtubules, although their functions are far from established. Dr. Olmsted will carry out functional studies of one of these MAPs, termed MAP-4. The structure of the MAP-4 protein is known to vary among different tissues, as a result of alternative splicing (different ways in which the cell "reads" the gene) and/or posttranslational modifications; however, the functional significance of these variations is unknown. These functional studies will employ state-of-the-art approaches such as site-directed mutagenesis and "green fluorescent protein" (a marker that allows one to literally see, in a microscope, exactly where the MAP-4 protein is located within the cell). Evidence increasingly suggests that these various cytoskeletal systems are not independent, but rather are dependent upon one another in terms of their function within cells. The results of these studies are expected to help us understand MAP-4 function and its possible role in coordinating microtubule function with various cellular structures, including other cytoskeletal systems. ***
