Regulation of actin polymerization by tropomodulin-3 controls megakaryocyte actin organization and platelet biogenesis

Abstract

AbstractThe actin cytoskeleton is important for platelet biogenesis. Tropomodulin-3 (Tmod3), the only Tmod isoform detected in platelets and megakaryocytes (MKs), caps actin filament (F-actin) pointed ends and binds tropomyosins (TMs), regulating actin polymerization and stability. To determine the function of Tmod3 in platelet biogenesis, we studied Tmod3−/− embryos, which are embryonic lethal by E18.5. Tmod3−/− embryos often show hemorrhaging at E14.5 with fewer and larger platelets, indicating impaired platelet biogenesis. MK numbers are moderately increased in Tmod3−/− fetal livers, with only a slight increase in the 8N population, suggesting that MK differentiation is not significantly affected. However, Tmod3−/− MKs fail to develop a normal demarcation membrane system (DMS), and cytoplasmic organelle distribution is abnormal. Moreover, cultured Tmod3−/− MKs exhibit impaired proplatelet formation with a wide range of proplatelet bud sizes, including abnormally large proplatelet buds containing incorrect numbers of von Willebrand factor-positive granules. Tmod3−/− MKs exhibit F-actin disturbances, and Tmod3−/− MKs spreading on collagen fail to polymerize F-actin into actomyosin contractile bundles. Tmod3 associates with TM4 and the F-actin cytoskeleton in wild-type MKs, and confocal microscopy reveals that Tmod3, TM4, and F-actin partially colocalize near the membrane of proplatelet buds. In contrast, the abnormally large proplatelets from Tmod3−/− MKs show increased F-actin and redistribution of F-actin and TM4 from the cortex to the cytoplasm, but normal microtubule coil organization. We conclude that F-actin capping by Tmod3 regulates F-actin organization in mouse fetal liver-derived MKs, thereby controlling MK cytoplasmic morphogenesis, including DMS formation and organelle distribution, as well as proplatelet formation and sizing.