Sculpting blood cells with actomyosin forces: The central role of myosin-II in biophysical regulation of megakaryopoiesis and early hematopoiesis

The human body generates 3 x 1010 nucleated blood cells, 2 x 1013 anucleated red blood cells and 1 x 1011 platelets (plts) every day. A number of soluble factors that control different aspects of hematopoiesis have been discovered, but the recapitulation of the ex vivo environment to program hematopoietic stem cells (HSCs) has been less successful. Bone marrow (BM) niches offer diverse mechanical cues to influence their resident cells, but their roles have been rarely investigated in the context of the hematopoietic system compared to large adherent cells, likely due to the impression that most blood cells are ‘non-adherent’ in culture and soft. However, most hematopoietic cells nevertheless express cytoskeletal and adhesion components. Given that cells sense mechanical cues predominantly via cellular contractility, this dissertation work investigates roles of non-muscle myosin-II (NMM-II), the major ATPase-dependent motor protein that converts biochemical to mechanical signals, in regulating two different ends of the adult hematopoiesis—megakaryocyte (MK) differentiation that leads to plt generation, and early HSC differentiation. Selective inhibitors against NMM-II, including blebbistatin, were used to first establish the importance of NMM-II in regulating both MK maturation with plt generation, and early HSC maintenance and differentiation. Then, a series of experiments were done to characterize the effects of NMM-II-mediated biophysical factors on hematopoiesis, including cytokinesis, cell membrane elasticity and matrix elasticity sensing. NMM-II can be regulated physiologically by the phosphorylation of the heavy chain. The site-specific signaling of the NMM-II phosphorylation is a common post-translational mechanism that can deactivate NMM-II in both mature MKs and early HSCs. The dissertation work introduces novel quantitative analyses of both global protein and mRNA expression profiles to define functional potentials of MKs in generating plts and engraftment potentials of HSCs in molecular terms. The functionality of MKs and HSCs modulated by NMM-II inhibition was tested by direct transplantation of human cells into xenograft mouse models for potential biomedical applications. In all, this work highlights NMM-II as a critical node for the biophysical regulation of adult hematopoiesis and identifies salient features of BM niches that can be used to enrich for functional MKs and HSCs.