In mammalian erythroblastic islands (EI's), erythroid cells proliferate, differentiate, and enucleate while contacting and surrounding a central macrophage (CM). Dynamic adhesive interactions involving erythroblasts (Eb's) and CM's are essential for normal erythropoiesis, but the molecular mechanisms regulating these interactions are poorly understood. This proposal uses state-of-the-art biophysical methods to investigate the best characterized of these adhesive interactions:
Aim 1 studies the interaction between Eb 1421 integrin and CM VCAM-1;
Aim 2 studies interactions between Eb ICAM-4 and CM 1v and 1L22 integrins;
Aim 3 studies interactions between Eb 1421 and 1521 integrins and fibronectin and between Eb 1421 and Eb ICAM-4. """"""""Zhu- Golan"""""""" two-dimensional affinity (2D Kd) analysis, fluorescence photobleaching recovery, and supported phospholipid bilayers reconstituted with receptor constructs are used to measure the 2D Kd, average lateral mobility, and cell surface expression of counterreceptor molecules on cells adherent to the bilayers. Single particle tracking is used to measure the lateral motion of individual receptor molecules in Eb and CM plasma membranes, and thereby to estimate the fractions of molecules that are tightly confined (e.g., cytoskeletally attached), loosely confined (e.g., localized to lipid raft domains), and freely diffusing in the membrane. Laser optical tweezers are used to measure the fraction of receptors that are cytoskeletally attached and the magnitudes of molecular forces retarding lateral diffusion of these molecules. Confocal fluorescence microscopy and single-cell fluorescence resonance energy transfer are used to measure protein-protein associations involving receptors, CD47, and cytoskeletal regulators.
Each Aim characterizes the physical properties of integrin-mediated adhesive interactions at the level of single molecules in human and mouse EI's, investigates how changes in these physical properties modulate cell-cell and cell-matrix interactions during Eb differentiation, studies the influence of physiologic and pathologic growth factors and cytokines on these adhesive interactions, and elucidates the molecular mechanisms that regulate integrin-mediated adhesion. This research will help to explain derangements in erythrocyte underproduction (anemia) and overproduction (erythrocytosis), and may lead to therapies for disorders of erythropoiesis such as anemia of chronic inflammation, myelodysplastic syndrome, and polycythemia vera.
Red blood cells are made in the bone marrow within structures called erythroblastic islands. The research proposed in this application aims to characterize the properties of molecules on cells in erythroblastic islands that are critically important for normal red blood cell production. Results from this research may lead to safer and more effective treatments for anemia, including improvements in the clinical use of erythropoietin, new and improved treatments for bone marrow disorders such as myelodysplastic syndrome and polycythemia vera, and the development of culture systems for artificial blood production.
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