Little is known about how erythroblasts extrude their nuclei to become reticulocytes at the conclusion of erythroid differentiation. Landmark events of this process include chromatin condensation, movement of the nucleus to the plasma membrane and partitioning of proteins to the reticulocyte. We propose to study erythroblasts during terminal differentiation using murine bone marrow and splenic erythroblasts from Friend virus-infected mice to: 1) Determine mechanisms of nuclear reorganization by analyzing the localization and integrity of protein components of important nuclear subdomains, including nuclear pores, nuclear matrix and RNA splicing centers; testing nuclear function, such as protein import; and characterizing chromatin protein and DNA from early and late erythroblasts. 2) Determine whether centrosomes undergo specific changes in protein composition and maintain their capacity to nucleate microtubule growth by assessing centrosomal sublocalization and morphology microscopically; characterizing centrosomal proteins in purified centrosomes; performing microtubule nucleation assays; and analyzing the morphology of mitotic spindles and the fidelity of anaphasic daughter chromosome separation in mitotic erythroblasts. 3) Determine mechanisms of protein sorting that regulate the protein content of plasma membranes of reticulocytes and extruded nuclei by examining protein sorting in normal enucleating erythroblasts, in erythroblasts after perturbing actin polymerization and in erythroblasts from mice with red cell cytoskeletal protein abnormalities. For our experiments we will use conventional, confocal, and electron microscopy, flow cytometry, GFP live cell imaging, Western blot analysis and micropipette microdeformation techniques. Successful accomplishment of these research objectives will provide fundamental insights into biochemical mechanisms which prepare nuclei for enucleation and which regulate protein sorting to reticulocytes. Moreover, they will provide the basis for future studies examining whether perturbations in these processes contribute to the pathobiology of hematologic diseases including hereditary spherocytosis, hereditary elliptocytosis and sickle cell disease.
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