Signaling and cytoskeletal requirements in erythroblast enucleation

Abstract

To understand the role of cytoskeleton and membrane signaling molecules in erythroblast enucleation, we developed a novel analysis protocol of multiparameter high-speed cell imaging in flow. This protocol enabled us to observe F-actin and phosphorylated myosin regulatory light chain (pMRLC) assembled into a contractile actomyosin ring (CAR) between nascent reticulocyte and nucleus, in a population of enucleating erythroblasts. CAR formation and subsequent enucleation were not affected in murine erythroblasts with genetic deletion of Rac1 and Rac2 GTPases because of compensation by Rac3. Pharmacologic inhibition or genetic deletion of all Rac GTPases altered the distribution of Factin and pMRLC and inhibited enucleation. Erythroblasts treated with NSC23766, cytochalasin-D, colchicine, ML7, or filipin that inhibited Rac activity, actin or tubulin polymerization, MRLC phosphorylation, or lipid raft assembly, respectively, exhibited decreased enucleation efficiency, as quantified by flow cytometry. As assessed by high-speed flow-imaging analysis, colchicine inhibited erythroblast polarization, implicating microtubules during the preparatory stage of enucleation, whereas NSC23766 led to absence of lipid raft assembly in the reticulocyte-pyrenocyte border. In conclusion, enucleation is a multistep process that resembles cytokinesis, requiring establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocyte and pyrenocyte. (Blood. 2012;119(25): 6118-6127) IntroductionErythropoiesis in mammals concludes with the dynamic process of enucleation, by which the orthochromatic erythroblast generates a reticulocyte that will mature to become a red blood cell, and a pyrenocyte, a membrane-encased nucleus surrounded by a thin rim of cytoplasm. [1][2][3] After enucleation, the reticulocytes are released into the bloodstream, whereas the pyrenocytes expose apoptotic signals on their surface, resulting in engulfment and degradation by the central macrophage of the erythroblastic island. 3,4 The mechanism of enucleation has been a long-standing matter of investigation and remains controversial.Early electron microscopy studies suggested that enucleation may be analogous to cytokinesis, pointing to the resemblance of the cytoplasmic constriction between incipient reticulocyte and pyrenocyte to the cleavage furrow at the equatorial region of a mitotic cell. 5 Koury et al demonstrated with electron and immunofluorescent microscopy, using mouse splenocytes infected with the anemia-inducing strain of Friend virus, that F-actin bundles concentrate at the furrow behind the extruding nucleus, and that cytochalasin-D, a potent inhibitor of actin polymerization, inhibits enucleation. 6 In parallel, a quantitative study by Chasis et al showed that inhibition of microtubule polymerization by colchicine stalls enucleation in vivo and in vitro in rat bone marrow. 7 A recent study by Keerthivasan et al using primary mouse and human erythroblasts...

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.…”

Survivin: a new player during erythroblast maturation

“…Konstantinidis et al suggested that the fusion of lipid rafts upon docking of autophagosomes with the cytoplasmic membrane is an important process during the terminal phase of enucleation. 7 This cartoon is based on Figure 6C in the study of Keerthivasan et al support of a direct role of this latter complex in enucleation, the authors found that knockdown of the genes reduces the efficiency of enucleation of primary human erythroblasts. Based on their own findings, they confirm that overexpression of survivin in cells dramatically increases the ratio of enucleated cells.…”

“…[1][2][3][4][5][6] It has been recently reported that, in the very last stage of enucleation, not only proteins but also specialized regions of the lipid bilayer, known as lipid rafts, as well as organelles participate in the extrusion of the nucleus. 7 Approximately 120 million reticulocytes are generated each minute in our body. Macrophages phagocyte and recycle a similar number of extruded nuclei and other organelles, such as mitochondria.…”

Translational control mediated by hnRNP K links NMHC IIA to erythroid enucleation

“…NMHC IIA, but not IIB or IIC could be detected in megakaryocytes and platelets (Marigo et al, 2004;Maupin et al, 1994) where NMHC IIA is crucial for organelle distribution (Pertuy et al, 2014). Enucleation of erythroid cells is promoted by actinomyosin ring contraction between the incipient reticulocyte and the nucleus that is induced by Rac-GTPase-dependent myosin regulatory light chain phosphorylation (Ji et al, 2008;Konstantinidis et al, 2012;Koury et al, 1989). Because we noticed increased enucleation of hnRNP-K-depleted K562 cells (Fig.…”