Studies on Cytoskeletal Function in the Generation and Maintenance of Cell Shape

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In the case of mature mammalian erythrocytes (red blood cells), the cells have lost their nuclei during differentiation, and their candy-like discoid shape is maintained by a complex cytoskeletal network on the cytoplasmic face of the plasma membrane. In other animals, including vertebrates such as birds, fish and amphibians, and also certain mollusks that possess hemoglobin-bearing blood cells (Vesicomyid clams, or "blood clams"), the mature erythrocytes maintain their nuclei, and the shapes of these cells (usually flattened ellipsoids) are maintained by a submembranous band of microtubules termed the marginal band. This work examines how cells maintain or change their shape, using nucleated erythrocytes of Xenopus, axolotls, and "blood clams" as a model system because of their constancy of shape and relative simplicity of cytoskeletal organization. Major questions addressed are: (1) how is the characteristic flattened ellipsoidal shape of these cells generated? and (2) how is this morphology maintained? With respect to the first, Dr. Cohen's laboratory has previously found that immature singly- and doubly-pointed erythroid cells containing correspondingly pointed, incomplete marginal bands of microtubules occur in many species. Experiments are designed to test the hypothesis that these represent true cytoskeletal intermediates and to establish the morphogenetic sequence, using bromodeoxyuridine labeling of differentiating erythroblasts (immature red cells) in the larval amphibian spleen (axolotl), in circulating blood of anemic adults (Xenopus), and in erythroid cell cultures from both sources. With respect to the second question, in recent work, Dr. Cohen's laboratory has discovered that the hemoglobin-bearing erythrocytes of "blood clams" undergo a drastic transformation of cell shape from flattened ellipsoids to wrinkled spheroids that is induced by a natural factor in the "blood" (hemolymph) and is remarkably reversible. These cells are particularly interesting because they not only contain marginal bands, but also have marginal band-associated centrosomes (microtubule organizing centers; centrioles). A variety of experiments will examine cytoskeletal function and its control in this natural reversible change of cell shape, and additional data on cytoskeletal organization will be obtained by fluorescence localization of selected cytoskeletal proteins, both in this system and in the two amphibian systems. Of particular interest are proteins characteristic of microtubule organizing centers, centrin and gamma tubulin, the localization of which will shed light on centrosome function in cell shape maintenance. The work is expected to provide basic new information on the function of ubiquitous cytoskeletal elements. This information will be of importance not only in understanding how nucleated red blood cells maintain their shape and volume, but also for its potential to contribute important new insights to the knowledge base of how microtubules influence cell shape in eukaryotic cells in general. Dr. Cohen routinely involves both undergraduate and graduate students in his research, both in his laboratory at Hunter College and during the summer months at the Marine Biological Laboratory in Woods Hole, Massachusetts. Thus, this project is expected to contribute significantly to the integration of research and education.