Myosin-ll inhibition and soft 2D matrix maximize multinucleation and cellular projections typical of platelet-producing megakaryocytes

摘要

Cell division, membrane rigidity, and strong adhesion to a rigid matrix are all promoted by myosin-ll, and so multinucleated cells with distended membranes—typical of megakaryocytes (MKs)- seem predictable for low myosin activity in cells on soft matrices. Paradoxically, myosin mutations lead to defects in MKs and plate lets. Here, reversible inhibition of myosin-ll is sustained over several cell cycles to produce 3- to 10-fold increases in polyploid MK and a number of other cell types. Even brief inhibition generates highly distensible, proplatelet-like projections that fragment readily un der shear, as seen in platelet generation from MKs in vivo. The effects are maximized with collagenous matrices that are soft and 2D, like the perivascular niches in marrow rather than 3D or rigid, like bone. Although multinucleation of other primary hematopoi etic lineages helps to generalize a failure-to-fission mechanism, lineage-specific signaling with increased polyploidy proves possible and novel with phospho-regulation of myosin-ll heavy chain. Label free mass spectrometry quantitation of the MK proteome uses a unique proportional peak fingerprint (ProPF) analysis to also show upregulation of the cytoskeletal and adhesion machinery critical to platelet function. Myosin-inhibited MKs generate more platelets in vitro and also in vivo from the marrows of xenografted mice, while agonist stimulation activates platelet spreading and integrin αllbβ3. Myosin-ll thus seems a central, matrix-regulated node for MK-poiesis and platelet generation.