Endomitotic Megakaryocytes form a Midzone in Anaphase but Have a Deficiency in Cleavage Furrow Formation

Abstract

Megakaryocyte differentiation is marked by development of progressive polyploidy and accumulation of large nuclear mass and cytoplasmic volume. During differentiation, megakaryocytes undergo repeated incomplete cell cycles in which mitosis is aborted in late anaphase with failure of cytokinesis, termed endomitosis. Recent studies have postulated that failure of Aurora-B kinase to localize to the spindle midzone is responsible for endomitosis in megakaryocytes. In diploid cells, the translocation of Aurora-B kinase is critical for positioning of the cleavage furrow, in part through its phosphorylation of the Rho family GTPase activating protein MgcRacGAP which in turn alters activity of RhoA. However, we have previously demonstrated that Aurora-B kinase localizes to centromeres and is functional in endomitotic megakaryocytes. Here, we show that endomitotic megakaryocytes form midzone structures that recruit Aurora-B kinase and its substrate MgcRacGAP. Although many cells with polyploid anaphases showed cortical localization of Aurora-B kinase, we did not observe accumulation of RhoA in furrows or formation of an actin ring. When mitotic exit was induced by inhibition of cdk1, diploid control cells formed furrows exhibiting cortical RhoA but megakaryocytes exited endomitosis without evidence of furrowing. Therefore, localization of Aurora-B kinase to the midzone is normal in endomitotic megakaryocytes but furrowing is abnormal. These data suggest that endomitotic MKs fail to complete cytokinesis due to aberrant regulation of furrowing at a step subsequent to the localization of Aurora-B kinase, possibly involving the activation or localization of RhoA. This work explores the mechanism of a normally occurring furrowing defect in a non-malignant primary cell.