This is a proposal to study the significance of tubulin variants in cell development and to determine, through the use of tubulin variants, the mechanisms of microtubule function in differentiation. Chicken erythrocytes contain a unique microtubule organelle, the marginal band, that is composed of a distinctive tubulin variant whose expression and assembly are closely associated with red cell differentiation. Erythrocyte tubulin is the only tubulin variant distinct from brain tubulin that has been isolated and shown to exhibit different biochemical characteristics and different assembly properties in vitro. Nucleated erythrocytes, like other cells, contain multiple tubulin isoforms or variants, but it is not known if the variants perform different functions or if the variants are """"""""sorted"""""""" in common cytoplasm. The role and function of microtubule-associated proteins (MAPs) in cell development are also largely unknown. These questions will be examined in developing chicken erythroblasts which contain two principal tubulin variants and which use microtubules for at least two distinct purposes -- mitosis and marginal band formation. There are three project goals: (1) The expression of tubulin variants in development. We will examine the relationship between erythrocyte tubulin synthesis and cell development, determine if there is differential expression of beta tubulin variants, and study the dynamics of alpha and beta tubulin subunit usage in erythroblasts. (2) Mechanisms of microtubule dynamics. We will use brain and erythrocyte tubulin subunit sorting in vitro and evaluate the significance of these activities in living cells. These observations will tell us if tubulin variants are sorted, have different assembly properties, and serve different purposes. (3) Erythrocyte MAPs as microtubule bundling factors. A functional assay based on microtubule bundling will be used to identify and define the activity of microtubule bundling factors in erythrocytes. These studies will indicate if erythrocytes contain specific MAPs and tell us whether tubulin variants differ in their interaction with MAPs, thus providing new insights on the role of MAPs and the erythrocyte tubulin variant in marginal band formation.
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