Enucleation of human erythroblasts involves non-muscle myosin IIB

Abstract

Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various celldivision inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34 ؉ cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts. IntroductionDuring erythropoiesis, stem cells undergo lineage specific commitment and generate erythroid progenitor cells through cellular division events including nuclear (mitosis) and cytoplasmic (cytokinesis) division. These progenitor cells consist of immature and mature erythroid progenitors, the burst-forming unit-erythroid (BFU-E) and the colony-forming unit-erythroid (CFU-E), respectively. The BFU-E can be considered as a progenitor of the CFU-E. Indeed, after 6 to 7 days in culture, cells generated from human BFU-E have all the functional characteristics of CFU-E 1 . After an additional 6 to 7 days in culture, human CFU-E proliferate and differentiate into mature erythroblasts. 1 Terminally differentiated erythroblasts in mammals expel their nuclei via a process termed enucleation, becoming reticulocytes and subsequently mature erythrocytes. The nucleus separates from the remainder of the cell and is phagocytosed by reticular cells such as macrophages (for a review, see Chasis et al 2 ).Enucleation of erythroblasts is thought to occur through a process similar to cytokinesis. Several general principles apply to cytokinesis. Firstly, the microtubule cytoskeleton plays an important role in both the choice and positioning of the division site. Once this site is chosen, the local assembly of the actomyosin contractile ring remodels the plasma membrane. Finally, membrane trafficking to, and membrane fusion at the division site result in the physical separation of the daughter cells, a process termed abscission (for reviews, see Barr et al 3 and Glotzer et al 4 ). Although modulation of the actomyosin cytoskeleton is crucial for proper cytokinesis, there is a paucity of information regarding how non-muscle myosin II contributes to enucleation.Several investigations have studied the mol...

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

“…Therefore, it is likely that non-muscle myosin IIA and B have two distinct functions, both in human and murine megakaryopoiesis. These two myosin II isoforms have previously been shown to have distinct roles in cadherin junction of the epithelial cells 31 or in the erythroblast enucleation 32 . However, MYH10 silencing does not explain the entire polyploidization process.…”

Isolation and functional characterization of human erythroblasts at distinct stages: implications for understanding of normal and disordered erythropoiesis in vivo

“…To date, many studies on enucleation have used mixed populations of murine or human erythroblasts. [31][32][33][34] In those studies, it is difficult to dissect out the effect of genetic or chemical manipulation on proliferation, differentiation, or enucleation. Because purified orthochromatic erythroblasts lack proliferation capability, they provide a powerful resource for studying the enucleation process per se.…”

Extrinsic and intrinsic control by EKLF (KLF1) within a specialized erythroid niche

“…Although an abnormal level of circulating nucleated definitive cells are observed in embryos deficient for the DNASE2A, MAF or palladin macrophage proteins (Kawane et al, 2001;Kusakabe et al, 2011;Liu et al, 2007), ablating the majority of macrophage cells does not automatically yield an abundance of nucleated red cells in circulation (Chow et al, 2013;Ramos et al, 2013). In addition, there are a number of structural, enzymatic and chromatin-associated erythroid cell factors that play an intrinsic role in morphologically preparing the cell for proper nuclear extrusion (Ji et al, 2011;Keerthivasan et al, 2011;Konstantinidis et al, 2012;Ney, 2011;Ubukawa et al, 2012;von Lindern, 2006). In the case of EKLF, the abundance of nucleated red cells in circulation seen in its absence or in the CDA KLF1/E325K patients may not only result from defective function in the island macrophage, but also as part of the global erythroid-specific panoply of EKLF targets that include ones important for these final maturation steps.…”