Kobayashi Isuzu, Ubukawa Kumi, Sugawara Kotomi, Asanuma Ken, Guo Yong-Mei, Yamashita Junsuke, Takahashi Naoto, Sawada Kenichi, Nunomura Wataru
Department of Hematology, Nephrology, and Rheumatology, Graduate School of Medicine, Akita University, Akita, Japan.
Department of Hematology, Nephrology, and Rheumatology, Master Course of Graduate School of Medicine, Akita University, Akita, Japan.
Exp Hematol. 2016 Apr;44(4):247-56.e12. doi: 10.1016/j.exphem.2015.12.003. Epub 2015 Dec 24.
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.
哺乳动物的成红细胞通过一个被认为类似于胞质分裂的过程进行去核。微管组织中心（MTOCs）介导有丝分裂纺锤体装置的组织，该装置在有丝分裂期间分离染色体，并且已知对正确的胞质分裂至关重要。然而，MTOCs在成红细胞去核中的作用仍然未知。因此，我们使用人红细胞集落形成单位（CFU-Es）和从纯化的CD34(+)细胞产生的成熟成红细胞，研究了各种MTOC抑制剂对胞质分裂和去核的影响。我们发现，动力蛋白抑制剂erythro-9-[3-(2-羟基壬基)]腺嘌呤（EHNA）、驱动蛋白Eg5抑制剂monastrol，以及包括ON-01910（Plk-1）、MLN8237（极光激酶A）、海鞘素B（极光激酶B）和LY294002（PI3K）在内的各种MTOC调节剂抑制剂，均抑制了CFU-E的胞质分裂。然而，在这些抑制剂中，只有EHNA阻断了去核。此外，终末分化的成红细胞仅表达动力蛋白；未检测到其他测试蛋白中的少量或无表达。在人成红细胞的终末分化过程中，细胞核位于细胞中心的细胞比例下降，而细胞核移位至靠近质膜位置的极化细胞比例增加。动力蛋白抑制损害了核极化，从而阻断了去核。这些数据表明，动力蛋白不仅在胞质分裂中起重要作用，而且在去核中也起重要作用。因此，我们得出结论，人成红细胞去核是一个很大程度上独立于MTOCs，但依赖于动力蛋白的过程。
