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• W2214952942 abstract "•Dynein was expressed during terminal differentiation of human erythroblasts.•Inhibition of dynein blocked the cell proliferation of colony-forming units–erythroid and enucleation.•Dynein was distributed in aggregates on the periphery of the nucleus in erythroblasts.•Cell polarization was blocked in the dynein inhibitor-treated erythroblasts.•Enucleation of human erythroblasts requires cell polarization by dynein. Mammalian erythroblasts undergo enucleation through a process thought to be similar to cytokinesis. Microtubule-organizing centers (MTOCs) mediate organization of the mitotic spindle apparatus that separates the chromosomes during mitosis and are known to be crucial for proper cytokinesis. However, the role of MTOCs in erythroblast enucleation remains unknown. We therefore investigated the effect of various MTOC inhibitors on cytokinesis and enucleation using human colony-forming units–erythroid (CFU-Es) and mature erythroblasts generated from purified CD34+ cells. We found that erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA), a dynein inhibitor, and monastrol, a kinesin Eg5 inhibitor, as well as various inhibitors of MTOC regulators, including ON-01910 (Plk-1), MLN8237 (aurora A), hesperadin (aurora B), and LY294002 (PI3K), all inhibited CFU-E cytokinesis. Among these inhibitors, however, only EHNA blocked enucleation. Moreover, terminally differentiated erythroblasts expressed only dynein; little or none of the other tested proteins was detected. Over the course of the terminal differentiation of human erythroblasts, the fraction of cells with nuclei at the cell center declined, whereas the fraction of polarized cells, with nuclei shifted to a position near the plasma membrane, increased. Dynein inhibition impaired nuclear polarization, thereby blocking enucleation. These data indicate that dynein plays an essential role not only in cytokinesis but also in enucleation. We therefore conclude that human erythroblast enucleation is a process largely independent of MTOCs, but dependent on dynein. Mammalian erythroblasts undergo enucleation through a process thought to be similar to cytokinesis. Microtubule-organizing centers (MTOCs) mediate organization of the mitotic spindle apparatus that separates the chromosomes during mitosis and are known to be crucial for proper cytokinesis. However, the role of MTOCs in erythroblast enucleation remains unknown. We therefore investigated the effect of various MTOC inhibitors on cytokinesis and enucleation using human colony-forming units–erythroid (CFU-Es) and mature erythroblasts generated from purified CD34+ cells. We found that erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA), a dynein inhibitor, and monastrol, a kinesin Eg5 inhibitor, as well as various inhibitors of MTOC regulators, including ON-01910 (Plk-1), MLN8237 (aurora A), hesperadin (aurora B), and LY294002 (PI3K), all inhibited CFU-E cytokinesis. Among these inhibitors, however, only EHNA blocked enucleation. Moreover, terminally differentiated erythroblasts expressed only dynein; little or none of the other tested proteins was detected. Over the course of the terminal differentiation of human erythroblasts, the fraction of cells with nuclei at the cell center declined, whereas the fraction of polarized cells, with nuclei shifted to a position near the plasma membrane, increased. Dynein inhibition impaired nuclear polarization, thereby blocking enucleation. These data indicate that dynein plays an essential role not only in cytokinesis but also in enucleation. We therefore conclude that human erythroblast enucleation is a process largely independent of MTOCs, but dependent on dynein. Mammalian erythropoiesis culminates in enucleation, an incompletely understood process entailing the expulsion of the nucleus from the cytoplasm of erythroblasts. During erythropoiesis, stem cells undergo lineage-specific commitment and generate erythroid progenitor cells through cellular division events that include both nuclear (mitosis) and cytoplasmic (cytokinesis) division. These progenitor cells consist of burst-forming units–erythroid (BFU-Es) and their progeny, colony-forming units–erythroid (CFU-Es) [1Hebiguchi M. Hirokawa M. Guo Y.M. et al.Dynamics of human erythroblast enucleation.Int J Hematol. 2008; 88: 498-507Crossref PubMed Scopus (32) Google Scholar, 2Oda A. Sawada K. Druker B.J. et al.Erythropoietin induces tyrosine phosphorylation of Jak2, STAT5A, and STAT5B in primary cultured human erythroid precursors.Blood. 1998; 92: 443-451Crossref PubMed Google Scholar, 3Sawada K. Krantz S.B. Kans J.S. et al.Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin.J Clin Invest. 1987; 80: 357-366Crossref PubMed Scopus (168) Google Scholar, 4Sawada K. Krantz S.B. Dai C.H. et al.Transitional change of colony stimulating factor requirements for erythroid progenitors.J Cell Physiol. 1991; 149: 1-8Crossref PubMed Scopus (32) Google Scholar]. Then over an additional 6 to 7 days, the CFU-Es proliferate and differentiate into mature erythroblasts [1Hebiguchi M. Hirokawa M. Guo Y.M. et al.Dynamics of human erythroblast enucleation.Int J Hematol. 2008; 88: 498-507Crossref PubMed Scopus (32) Google Scholar, 5Ubukawa K. Guo Y.M. Takahashi M. et al.Enucleation of human erythroblasts involves non-muscle myosin IIB.Blood. 2012; 119: 1036-1044Crossref PubMed Scopus (62) Google Scholar]. During terminal differentiation, mammalian erythroblasts undergo enucleation, becoming reticulocytes and, subsequently, mature erythrocytes. The expelled nuclei are phagocytosed by reticular cells such as macrophages (for a review, see [6Chasis J.A. Mohandas N. Erythroblastic islands: Niches for erythropoiesis.Blood. 2008; 112: 470-478Crossref PubMed Scopus (357) Google Scholar]). The process of enucleation is thought to be similar to cytokinesis, and many of the general principles of cytokinesis apply to enucleation. In both cytokinesis and enucleation, the cytoskeleton is key in the choice and positioning of the division site. Once this site is chosen, there is local assembly of a contractile actomyosin ring, during which nonmuscle myosin IIB remodels the plasma membrane [5Ubukawa K. Guo Y.M. Takahashi M. et al.Enucleation of human erythroblasts involves non-muscle myosin IIB.Blood. 2012; 119: 1036-1044Crossref PubMed Scopus (62) Google Scholar]. Trafficking of the necessary components to the division site and membrane fusion lead to the physical separation of the daughter cells (for a review, see [7Glotzer M. The molecular requirements for cytokinesis.Science. 2005; 307: 1735-1739Crossref PubMed Scopus (568) Google Scholar, 8Barr F.A. Gruneberg U. Cytokinesis: placing and making the final cut.Cell. 2007; 131: 847-860Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar]). Wang et al. reported that nuclear polarization regulated by phosphoinositide 3-kinase (PI3K) is important for the enucleation [9Wang J. Ramirez T. Ji P. et al.Mammalian erythroblast enucleation requires PI3K-dependent cell polarization.J Cell Sci. 2012; 125: 340-349Crossref PubMed Scopus (41) Google Scholar]. During polarization, the nucleus becomes displaced to one side of the cell, near the plasma membrane, while actin becomes restricted to the other side, where dynamic cytoplasmic contractions generate pressure that pushes the viscoelastic nucleus through a narrow constriction in the cell surface, forming a bud. These findings implicate PI3K and its products in the polarization and enucleation of erythroblasts [9Wang J. Ramirez T. Ji P. et al.Mammalian erythroblast enucleation requires PI3K-dependent cell polarization.J Cell Sci. 2012; 125: 340-349Crossref PubMed Scopus (41) Google Scholar]. Microtubule-organizing centers (MTOCs) function as sites where microtubule formation begins, as well as locations where free ends of microtubules are attracted to proteins anchored in the cytoplasmic membrane [10Brinkley B.R. Microtubule organizing centers.Annu Rev Cell Biol. 1985; 1: 145-172Crossref PubMed Scopus (301) Google Scholar]. The most notable MTOCs are the centrosomes formed during interphase and the mitotic spindle poles. Thus, MTOCs are essential for proper cytokinesis. The degree to which MTOCs contribute to the enucleation of terminally differentiated erythroblasts remains unknown, however. γ-Tubulin is a member of the tubulin family and is important for nucleation and polar orientation of microtubules [10Brinkley B.R. Microtubule organizing centers.Annu Rev Cell Biol. 1985; 1: 145-172Crossref PubMed Scopus (301) Google Scholar, 11Mardin B.R. Schiebel E. Breaking the ties that bind: New advances in centrosome biology.J Cell Biol. 2012; 197: 11-18Crossref PubMed Scopus (99) Google Scholar]. γ-Tubulin is an abundant component of centrosomes and spindle pole bodies, where it is found within γ-tubulin ring complexes, which chemically mimic the plus end of microtubules, thus enabling microtubules to bind [12Rodionov V. Nadezhdina E. Borisy G. Centrosomal control of microtubule dynamics.Proc Natl Acad Sci U S A. 1999; 96: 115-120Crossref PubMed Scopus (131) Google Scholar, 13Tanenbaum M.E. Medema R.H. Mechanisms of centrosome separation and bipolar spindle assembly.Dev Cell. 2010; 19: 797-806Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar]. Another molecule, the motor protein dynein, mediates unidirectional movement toward MTOCs (minus end of microtubules), whereas kinesins “walk” along microtubule filaments toward the plus end, often enabling transport of cargo from the cell center toward the periphery [14Splinter D. Tanenbaum M.E. Lindqvist A. et al.Bicaudal D2, dynein, and kinesin-1 associate with nuclear pore complexes and regulate centrosome and nuclear positioning during mitotic entry.PLoS Biol. 2010; 8: e1000350Crossref PubMed Scopus (211) Google Scholar, 15Gomes E.R. Jani S. Gundersen G.G. Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells.Cell. 2005; 121: 451-463Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 16Laan L. Rothb S. Dogteromb M. End-on microtubule–dynein interactions and pulling- based positioning of microtubule organizing centers.Cell Cycle. 2015; 11: 3750-3757Crossref Scopus (35) Google Scholar]. The motor functions of both dynein and kinesins are known to support cytokinesis, but their roles in erythroblast enucleation remain unclear [17Wordeman L. How kinesin motor proteins drive mitotic spindle function: Lessons from molecular assays.Semin Cell Dev Biol. 2010; 21: 260-268Crossref PubMed Scopus (115) Google Scholar, 18Hirokawa N. Tanaka Y. Kinesin superfamily proteins (KIFs): Various functions and their relevance for important phenomena in life and diseases.Exp Cell Res. 2015; 334: 16-25Crossref PubMed Scopus (153) Google Scholar, 19Vicente J.J. Wordeman L. Mitosis, microtubule dynamics and the evolution of kinesins.Exp Cell Res. 2015; 334: 61-69Crossref PubMed Scopus (56) Google Scholar]. In the present study, therefore, we investigated the contributions made by dynein, kinesin, Eg5, and several MTOC regulatory proteins to the process of human erythroblast enucleation. Here we illustrate that although the motor proteins dynein and Eg5 and the various MTOC regulatory proteins tested were all needed for CFU-E cytokinesis (proliferation), only dynein was necessary for the cell division and nuclear polarization during terminal erythroblast differentiation and enucleation. Enucleation is thus independent of most MTOC-related proteins, but dependent on dynein. Granulocyte colony-stimulating factor (G-CSF)-mobilized human peripheral blood CD34+ cells were purified from healthy volunteers as described previously and stored in liquid nitrogen until required. Informed consent was obtained from all subjects prior to their entry into this study, and the study was pre-approved by the Akita University Graduate School of Medicine Committee for the Protection of Human Subjects [2Oda A. Sawada K. Druker B.J. et al.Erythropoietin induces tyrosine phosphorylation of Jak2, STAT5A, and STAT5B in primary cultured human erythroid precursors.Blood. 1998; 92: 443-451Crossref PubMed Google Scholar]. To generate erythroid progenitor cells, CD34+ cells were thawed and prepared for culture as previously described [2Oda A. Sawada K. Druker B.J. et al.Erythropoietin induces tyrosine phosphorylation of Jak2, STAT5A, and STAT5B in primary cultured human erythroid precursors.Blood. 1998; 92: 443-451Crossref PubMed Google Scholar]. Cells were cultured in Iscove's modified Eagle's medium (IMDM), erythroid medium containing 20% fetal calf serum (FCS), 10% heat-inactivated pooled human AB serum, 1% bovine serum albumin (BSA), 10 μg/mL insulin, 0.5 μg/mL vitamin B12, 15 μg/mL folic acid, 50 nmol/L β-mercaptoethanol (β-ME), 50 U/mL penicillin, and 50 μg/mL streptomycin in the presence of 50 ng/mL interleukin (IL)-3, 50 ng/mL stem cell factor (SCF), and 2 IU/mL erythropoietin (EPO). Cells were maintained at 37°C in a 5% CO2 incubator. On day 7 of culture (D7), the cells were harvested and washed three times with IMDM containing 0.1% BSA and stored at 4°C until required. The maturation level of the D7 cells was similar to that of CFU-Es, as reported elsewhere [2Oda A. Sawada K. Druker B.J. et al.Erythropoietin induces tyrosine phosphorylation of Jak2, STAT5A, and STAT5B in primary cultured human erythroid precursors.Blood. 1998; 92: 443-451Crossref PubMed Google Scholar, 3Sawada K. Krantz S.B. Kans J.S. et al.Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin.J Clin Invest. 1987; 80: 357-366Crossref PubMed Scopus (168) Google Scholar]. Hereafter, D7 cells will be referred to as CFU-Es. Because the cells were not completely synchronized, the effects of inhibitors on cell proliferation and the enucleation ratio (describe in the Supplementary Methods, online only, available at www.exphem.org) were decided after incubation for 24 h. Aliquots of CFU-Es were then cultured in erythroid medium with EPO alone, without β-mercaptoethanol, SCF, or IL-3 to induce differentiation with or without various the inhibitors of cell division. The volume of inhibitor and control solutions (H2O or dimethyl sulfoxide [DMSO]) was fixed at 5% of the erythroid medium. The final concentration of DMSO in the erythroid medium was 0.1% (v/v), as higher concentrations are toxic to human erythroid progenitors. The cells were harvested at various times, washed three times with IMDM containing 0.1% BSA, resuspended in IMDM containing 0.1% BSA, and stored at 4°C until required. The cells were classified based on the whether the nucleus was localized at the center of the cells (centered); the cells were spherical containing a condensed nucleus located to one side, close to the plasma membrane (polarized); or the cells were enucleated (reticulocytes). Other cell types, such as multinucleate cells and cells with condensed apoptotic nuclei, were classified as “other.” Images of cells classified as described are provided in Supplementary Figure E1 (online only, available at www.exphem.org). The fractions of each cell type were calculated as described in the previous section. Results are presented as the means ± SD of three independent experiments. Immunochemical distributions of γ-tubulin and dynein were determined using confocal microscopy as described above. D10 cells were cultured in the presence or absence of 400 μmol/L erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA). Additional methods and details of are described in the Supplementary Methods. We first examined the effects of inhibitors of cell division on primary human CFU-Es generated from purified CD34+ cells. Given that cellular division consists of both mitotic and cytokinetic events and inhibition of either step should block cell proliferation, effective inhibitors were defined as those that blocked CFU-E proliferation. Figure 1A and B illustrates total cell numbers and morphology when human CFU-Es were cultured without inhibitors. EHNA, a specific inhibitor of dynein ATPase activity, completely blocked cell proliferation (Fig. 1C). Among the cells incubated with a motor protein inhibitor, the fraction of cells in G1/G0/S phase (46.0 ± 0.6% for EHNA, as illustrated in Fig. 1D) was little changed from control, though there was a significant reduction in the mitotic fraction (4n<). The morphology of the EHNA-treated cells was similar to that of control cells at 0 hours (Fig. 1E). No multinucleate cells were observed among the cells incubated with 400 μmol/L EHNA. To avoid the possibility of a nonspecific effect of EHNA on the proliferation of CFU-Es, dynein and α-tubulin expression was analyzed using Western blotting (Supplementary Figure E2, all supplementary figures are online only, available at www.exphem.org), whereas expression of CD71 and glycophorin A (GPA) was analyzed using fluorescence-activated cell sorting (FACS) (Supplementary Figure E3). There was no nonspecific effect on the expression of dynein or α-tubulin. The levels of CD71 and GPA expression indicated that EHNA inhibited cell proliferation. We also examined the effect of EHNA on gene expression of GATA1 and GLUT1 (Supplementary Figure E4), but found no significant changes in cells cultured with or without EHNA for 24 h. Mitotic kinesin Eg5, serving as a control motor protein moving in the opposite direction of dynein, was blocked by monastrol, which was previously reported to affect cell proliferation [5Ubukawa K. Guo Y.M. Takahashi M. et al.Enucleation of human erythroblasts involves non-muscle myosin IIB.Blood. 2012; 119: 1036-1044Crossref PubMed Scopus (62) Google Scholar, 20Penningroth S.M. Cheung A. Bouchard P. et al.Dynein ATPase is inhibited selectively in vitro by erythro-9-[3-2-(hydroxynonyl)]adenine.Biochem Biophys Res Commun. 1982; 104: 234-240Crossref PubMed Scopus (48) Google Scholar, 21Sarli V. Giannis A. Targeting the kinesin spindle protein: basic principles and clinical implications.Clin Cancer Res. 2008; 14: 7583-7587Crossref PubMed Scopus (124) Google Scholar]. Our present findings confirm those earlier results (Supplementary Figure E5A–C). Or results also indicate that EHNA selectively inhibits dynein with no nonspecific effects. No multinucleate cells were observed after treatment with high concentrations of EHNA, which indicates that EHNA does not affect actin polymerization. Thus, EHNA specifically blocks human CFU-E proliferation as effectively as monastrol [5Ubukawa K. Guo Y.M. Takahashi M. et al.Enucleation of human erythroblasts involves non-muscle myosin IIB.Blood. 2012; 119: 1036-1044Crossref PubMed Scopus (62) Google Scholar]. ON-01910, an inhibitor of Plk1, which is an early trigger for G2/M transition; MLN8237, an inhibitor of aurora A, which is associated with centrosome maturation and separation and, thus, regulates spindle assembly and stability; and hesperadin, an inhibitor of aurora B, which functions in the attachment of the mitotic spindle to the centromere all completely blocked CFU-E proliferation (Fig. 2A). Cell cycle analysis revealed that ON-01910 reduced the cell fraction in G1/G0 phase, leading to accumulation of cells in S (13.7 ± 1.9%) and G2/M (24.7 ± 1.9%) phases, and dramatically increased the dead cell fraction (32.8 ± 3.2%) (Fig. 2B). Many CFU-Es treated with ON-01910 for 48 hours exhibited nuclear fragmentation characteristic of early apoptosis (Fig. 2C). Treatment with MLN8237 also significantly reduced the cell fraction in G1/G0 phase (35.4 ± 1.3%), resulting in the accumulation of cells in G2/M phase and an increase in dead cells (Fig. 2B). Hesperadin treatment led to an even greater reduction in G1/G0 cells (27.2 ± 4.5%) and a corresponding increase in G2/M cells (20.0 ± 2.9%). Abnormally shaped nuclei and multinuclear cells also appeared among hesperadin-treated CFU-Es (Fig. 2C). Consistent with earlier reports, we found that the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 blocks CFU-E proliferation with an increase in the dead cell fraction (10.8 ± 1.9%) as compared with control (5.9 ± 1.2%) (Supplementary Figure E5D and E) [9Wang J. Ramirez T. Ji P. et al.Mammalian erythroblast enucleation requires PI3K-dependent cell polarization.J Cell Sci. 2012; 125: 340-349Crossref PubMed Scopus (41) Google Scholar, 22Haseyama Y. Ki Sawada Oda A. et al.Phosphatidylinositol 3-kinase is involved in the protection of primary cultured human erythroid precursor cells from apoptosis.Blood. 1999; 94: 1568-1577Crossref PubMed Google Scholar]. LY294002 also induced significant morphologic changes, as illustrated in Supplementary Figure E5F. These results indicate that blocking MTOCs completely inhibits the proliferative capacity of human erythroid progenitor cells. Time-dependent changes in the enucleation fraction when human D10 erythroblasts were cultured for 72 hours with (Figure 3D showing cell cycles and Fig. 3C, closed circles) or without (Figure 3A showing cell cycles and Fig. 3C, open circles) various concentrations of the dynein inhibitor EHNA are provided. Mature untreated erythroblasts differentiated and began the enucleation process (Fig. 3B). EHNA dose-dependently inhibited the increase in the enucleation fraction, and the nucleus remained in the center of cells (Fig. 3E). The absence of a nonspecific effect of EHNA (400 μmol/L) on enucleation was confirmed by the expression of dynein and α-tubulin (Supplementary Figure E6), as well as CD71 and GPA (Supplementary Figure E7). There were no significant differences in the gene expression of GATA1 and GLUT1 after culture of cells for 48 hours with or without EHNA (Supplementary Figure E8). As reported previously, monastrol did not block human erythroblast enucleation (Supplementary Figure E9A–C) [5Ubukawa K. Guo Y.M. Takahashi M. et al.Enucleation of human erythroblasts involves non-muscle myosin IIB.Blood. 2012; 119: 1036-1044Crossref PubMed Scopus (62) Google Scholar]. Figure 4 illustrates that none of the MTOC inhibitors tested in this study blocked enucleation.Figure 4Inhibitors of MOTCs do not block erythroblast enucleation. D10 cells were cultured for 3 days in the presence of EPO with or without the inhibitors Plk1 (ON-01910), aurora A (MLN8237), and aurora B (hesperadin). (A) Effects of the indicated concentrations of inhibitors or vehicle on enucleation of D11–D13 erythroblasts. Results are presented as the means ± SD of three independent experiments. (B) Cell cycle analysis of D11 cells after culture for 24 hours with (red areas) or without (black lines) 1,000 nmol/L ON-01910, 1,000 nmol/L MLN8237, or 100 nmol/L hesperadin. A representative result from three independent experiments is presented as the mean ± SD. (C) May–Grünwald–Giemsa staining of D11–D13 cells after culture for 24, 48, or 72 hours with or without 1,000 nmol/L ON-01910, 1,000 nmol/L MLN8237, or 100 nmol/L hesperadin. A representative result from three independent experiments is shown. Bar = 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next assessed the effect of MTOC and motor protein inhibitors on the positioning of the nucleus during terminal differentiation of human D10 erythroblasts (Table 1). The cells were classified as described under Methods and in Supplementary Figure E1. Among these cells, the centered and polarized cell fractions were 73.0 ± 2.7% (range, 68.5% to 76.1%) and 16.7 ± 1.6% (range, 14.6% to 19.3%), respectively. After incubation of the cells for an additional 24 hours with no inhibitors, the centered and polarized fractions had changed to 46.9 ± 4.4% (range, 43.9%–53.2%) and 21.5 ± 3.1% (range, 15.7%–24.3%), respectively. Over the same 24 h, the reticulocyte fraction increased from 7.7 ± 1.4% (range, 5.3%–9.0%) to 29.6 ± 3.7% (range, 28.0%–30.5%).Table 1Morphologic analysis of cells cultured with or without inhibitors∗Centered refers to cells in which the nucleus was localized at the center of the cell. Polarized refers to spherical cells, which contained a condensed nucleus located to one side, near the plasma membrane. Reticulocytes refer to enucleated c. Other cell types, such as multinucleate cells and cells with condensed apoptotic nuclei, were classified as “other.” Images of cells classified as described are shown in Supplementary Figure E1 (online only, available at www.exphem.org). The fractions of each cell type were calculated as described in the text. Results are presented as the mean ± SD of three independent experiments.InhibitorCenteredPolarizedOtherReticulocytesControl 0 h76.1 ± 3.316.0 ± 2.91.2 ± 0.26.7 ± 0.7 −24 h48.1 ± 6.322.9 ± 3.71.0 ± 0.628.0 ± 5.5 EHNA 400 μmol/L67.4 ± 1.9†Significantly increased, p < 0.01.21.0 ± 2.10.4 ± 0.511.1 ± 0.7‡Significantly decreased, p < 0.01.Control 0 h75.3 ± 1.716.9 ± 2.62.3 ± 1.05.5 ± 1.8 −24 h43.9 ± 5.223.4 ± 2.12.3 ± 1.930.5 ± 8.0 Monastrol 100 μmol/L47.1 ± 3.721.1 ± 2.15.3 ± 3.126.6 ± 5.0Control 0 h68.5 ± 2.919.3 ± 3.513.6 ± 1.68.7 ± 3.6 −24 h46.2 ± 1.923.2 ± 3.72.4 ± 1.728.1 ± 1.0 ON-01910 1,000 nmol/L33.1 ± 5.4§Significantly decreased, p < 0.05.15.3 ± 2.7§Significantly decreased, p < 0.05.19.9 ± 1.7†Significantly increased, p < 0.01.31.7 ± 5.5Control 0 h73.3 ± 2.318.2 ± 1.73.1 ± 0.55.3 ± 1.5 −24 h47.4 ± 2.721.5 ± 4.32.6 ± 1.528.5 ± 1.3 MLN8237 1,000 nmol/L52.6 ± 5.315.9 ± 0.73.6 ± 0.828.0 ± 4.1 Hesperadin 100 nmol/L33.6 ± 2.5‡Significantly decreased, p < 0.01.20.7 ± 2.917.7 ± 2.0†Significantly increased, p < 0.01.28.1 ± 3.6Control 0 h73.0 ± 2.218.4 ± 2.71.4 ± 1.07.3 ± 0.7 −24 h46.0 ± 1.224.3 ± 3.81.1 ± 0.928.7 ± 3.9 LY294002 100 μmol/L48.4 ± 6.326.0 ± 3.10.6 ± 0.225.1 ± 3.7EHNA = erythro-9-[3-(2-hydroxynonyl)]adenine.∗ Centered refers to cells in which the nucleus was localized at the center of the cell. Polarized refers to spherical cells, which contained a condensed nucleus located to one side, near the plasma membrane. Reticulocytes refer to enucleated c. Other cell types, such as multinucleate cells and cells with condensed apoptotic nuclei, were classified as “other.” Images of cells classified as described are shown in Supplementary Figure E1 (online only, available at www.exphem.org). The fractions of each cell type were calculated as described in the text. Results are presented as the mean ± SD of three independent experiments.† Significantly increased, p < 0.01.‡ Significantly decreased, p < 0.01.§ Significantly decreased, p < 0.05. Open table in a new tab EHNA = erythro-9-[3-(2-hydroxynonyl)]adenine. Inhibiting dynein using EHNA significantly inhibited the decline in the centered cell fraction and the increase in the reticulocyte fraction that were seen in untreated control cells over the 24-h period from D10 to D11 (Table 1 and Supplementary Figure E10). By contrast, inhibition of kinesin Eg5 using monastrol had no effect on nuclear positioning, nor did inhibition of PI3K. Treatment with the aforementioned inhibitors of Plk1, aurora A, or aurora B led to reductions in the centered cell fraction (Table 1 and Supplementary Figure E10). Because the dynein inhibitor EHNA increased the centered cell fraction in day 10–11 cells and blocked erythroblast enucleation, we investigated the expression of MTOC-related proteins during terminal differentiation of human erythroblasts. γ-Tubulin was immunochemically detected in the cytoplasm in erythroblasts from D7 (CFU-E) to D13 (late-stage erythroblasts and reticulocytes) (Fig. 5), and the expression of γ-tubulin did not to change during that period. Western blotting revealed that dynein was expressed during terminal erythroblast differentiation (Fig. 5). Little or no Eg5, Plk1, aurora A or aurora B was expressed in D13 (Fig. 5). These results suggest dynein is a key regulator of nuclear positioning and enucleation in terminally differentiated erythroblasts. In D7 CFU-Es, γ-tubulin was immunochemically detected around the periphery of the nucleus and in the cleavage furrow, and α-tubulin (microtubules) was localized around the γ-tubulin (Fig. 6A). In D11 cells, the nucleus was displaced to one side of the cell, and γ-tubulin was located in the center of cell (Supplementary Figure E11). Although dynein was broadly distributed in the cytoplasm and did not co-localize with α-tubulin in D8 cells, in D11 cells, it was distributed in aggregates on the periphery of the nucleus and co-localized with α-tubulin at or near the MTOCs (Fig. 6B and Supplementary Figure E12 containing high-magnification images). Treating the D10 cells with 400 μmol/L EHNA blocked dynein aggregation such that it remained diffuse in the cytoplasm (Fig. 6B). In contrast to dynein, Eg5 co-localized with α-tubulin at the cleavage furrow in mitotic CFU-Es (Supplementary Figure E13A). In D7 and D8 CFU-Es, Plk1 and aurora B also co-localized with α-tubulin at the cleavage furrow (Supplementary Figure E13B and D), and aurora A localized around the spindles in D7 cells (Supplementary Figure E13C). But little if any Eg5, Plk1, aurora A, or aurora B was detected in D11 cells (Supplementary Figure E13A–D). Taken together, these results suggest that accumulation of dynein around the MTOCs is necessary for polarization and subsequent enucleation of erythroblasts. In the present study, we found that among the motor proteins and MTOC regulatory proteins tested, only dynein was necessary for cell division and nuclear polarization during terminal differentiation and enucleation. Kinesin Eg5 and the MTOC regulators Plk1, aurora A, and aurora B were absent or dispensable. Our findings thus indicate that enucleation is a process apparently independent of MTOC-related proteins, but dependent on dynein. During differentiation of CFU-Es into mature erythroblasts lacking the ability to divide, the" @default.
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• W2214952942 title "Erythroblast enucleation is a dynein-dependent process" @default.
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