Membrane forces and key protein determinants of hematopoietic cell function: Lamins and myosin-II in hematopoiesis and CD47 in immunotherapy of cancer.

摘要

Hematopoiesis in human bone marrow generates every second about 10 5--106 anucleated platelets and red blood cells as well as nucleated white blood cells that are capable of infiltrating distant tissues. The thesis begins in the marrow with a description of (1) nuclear membrane 'lamina' physicochemical properties that influence marrow-to-circulation trafficking, and proceeds to detail (2) the physicochemical roles of membrane cortex 'myosin' in key marrow processes of motility and division as well as platelet biogenesis and disease. The thesis finishes with (3) studies of macrophages in peripheral tissues far from the marrow and aspects of how such cells distinguish 'foreign' cells from 'self' cells.;Collaborative studies show the lamin-A:B ratio controls nuclear viscoelasticity and in turn cell trafficking relevant to marrow escape. Additionally, differential lamin expression can direct erythroid and megakaryocyte differentiation. Secondly, xenografts show that MIIB is required for blood cell generation, while MIIA is required for long-term HSC/P engraftment. MII inhibition by blebbistatin prior to xenotransplantation enriches for long-term hematopoietic multilineage reconstituting cells, and also multi-nucleated megakaryocytes. Cone and plate rheometry demonstrates an optimal shear stress for platelet-like-particle generation from MKs that is enhanced by MII inhibition, and that MIIA heavy chain phosphorylation at S1943 is shear sensitive. Micropipette aspiration of MKs with mutations of the MIIA gene, MYH9, recapitulates MYH9-RD macrothrombocytopenia. Comparing MYH9-RD patient and normal donor platelets shows a similarity in pre/pro-platelets when normals are treated with blebbistatin. These findings provide evidence that regulation of MIIA activity through S1943 phosphorylation is critical to proper MK fragmentation and proplatelet fission to generate platelets of normal size and number.;The dissertation concludes with an investigation of an immunotherapy approach to treat peripheral solid tumors by controlling tumor cell CD47 expression and administering an anti-human polyclonal IgG antibody. These in vivo xenograft models provide a mechanism of selective tumor clearance driven by FcR stimulation of macrophages. In all, this work highlights biophysical factors of cortical and nuclear membranes that govern hematopoietic differentiation and trafficking, normal and pathological thrombopoiesis, and a mechanism of phagocytic clearance of cancer cells through CD47 attenuation and species specific but epitope non-specific antibody infusion.