In the proposed research, nucleated erythrocytes serve as a model experimental system for studying biogenesis of the cytoskeleton and its relationship to cellular morphogenesis. Formation of the marginal band of microtubules (MTs) and its function in effecting the change of cell shape from spheroidal erythroblast to flattened ellipsoid are the focus of the work. One major objective is to analyze the mechanism of MT bundling that underlies marginal band formation, using our recently developed in vitro MT assembly of MT protein extracted from erythrocyte cytoskeleton at low temperature, or from isolated marginal bands. The analysis includes identification and structural localization of MT- associated proteins, particularly tau, that may be involved in inter-MT cross-bridging. Antibodies, high salt, and proteases are utilized as potential bundling inhibitors or """"""""unbundling"""""""" agents, and gel electrophoresis, immunoblotting, and immunofluorescence for protein identification and localization. Another major objective is to test our working hypothesis for the natural sequence of stages in marginal band biogenesis, and their functional correlation with changes in erythroblast morphology during the transition from spheroid to flattened ellipsoid. This work exploits the anemic Xenopus system, in which large numbers of erythroblasts undergo marginal band biogenesis and cellular morphogenesis in the circulation during recovery from experimentally induced anemia. Of particular interest are singly- and doubly-pointed erythroid cells containing correspondingly pointed incomplete marginal bands, that we think may be morphogenetic intermediates. Experiments include labeling of erythroblast nuclei and/or membranes to determine the morphogenetic sequence in vivo, testing of predicted MT polarity in pointed cells, and studying the causal relationship between marginal band biogenesis and cell shape changes. The work seeks a better understanding of mechanisms involved in the biogenesis and function of the cytoskeleton during cellular morphogenesis. It is anticipated that the information obtained will help clarify current controversy regarding MT bundle formation and function, and will improve understanding of MT dynamics and cytoskeletal function in eucaryotic cells in general.
