Rho GTPases in Terminal Erythroid Maturation

描述(由申请人提供)： 哺乳动物红系的终末分化以导致网织红细胞形成的戏剧性的去核过程结束。红细胞与成红细胞岛内巨噬细胞的相互作用是有效去核的关键。红细胞生成终末阶段的缺陷是慢性炎症性疾病和肿瘤以及许多初级血液学过程中贫血的常见原因，如骨髓发育不良和地中海贫血。对红系终末成熟相关机制的了解有限，阻碍了此类疾病新疗法的发展。使用一种新的多参数高速细胞血流成像分析方案，我们证明了去核是一个类似于不对称胞质分裂的多步骤过程。它需要通过微管功能建立细胞的极性，然后形成收缩的肌球蛋白环，网织红细胞和肾核细胞之间的脂筏结合。此外，我们还发现，在去核过程中，RAC GTP酶在肌动球蛋白环中组织肌动蛋白，并在新生网织红细胞和核细胞之间的沟槽中聚集脂筏。RhoA GTP酶在子代细胞的胞质分裂和脱落中起重要作用。基于红细胞去核和胞质分裂的相似性，我们假设RAC和RhoA通过与胞质分裂类似的内在分子途径动态地控制红细胞去核。根据红细胞-巨噬细胞相互作用对有效的红细胞生成的关键作用，以及RAC和RhoA在整合素启动的信号转导中的已知功能，我们假设RAC和RhoA将关键的巨噬细胞信号转导到红细胞，调节红系终末分化和红母细胞岛内的去核。为了明确RhoA信号在红细胞生成末期调节肌动蛋白和微管细胞骨架中的作用机制以及RhoA/Rac在调节微管细胞骨架中的关系，我们培育了RhoA和rac1/rac2/rac3红系特异性缺失(EpoRGFPcre/+驱动)小鼠。为了验证我们的假设，我们建议在目标1中定义RhoA/RAC调节的细胞骨架动力学促进红细胞去核的分子过程，在目标2中确定Rho GTP酶在红细胞岛内去核和红细胞成熟中的作用。本研究的目的是研究RAC和RhoA通过动态调节肌动蛋白和微管细胞骨架来调节红细胞分化和去核的细胞内和细胞间分子机制。这项研究有可能揭示终末期红系成熟缺陷所致贫血的体内治疗干预靶点，以及提高体外红细胞产生的效率。 (摘要结束)

DESCRIPTION (provided by applicant): Terminal differentiation within the erythroid lineage in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The interactions of erythroblasts with macrophages within the erythroblastic islands are critical for efficient enucleation. Defects in the final stages of erythropoiesis are common causes of anemia in chronic inflammatory diseases and neoplasia and in many primary hematological processes, like myelodysplasia and thalassemia. Limited understanding of the mechanisms involved in terminal erythroid maturation impedes development of novel therapies for such diseases. Using a novel analysis protocol of multiparameter high-speed cell imaging in flow we demonstrated evidence that enucleation is a multi-step process resembling asymmetric cytokinesis. It requires establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocytes and pyrenocytes. Moreover, we showed that Rac GTPases organize actin in the actomyosin ring and aggregate lipid rafts in the furrow between nascent reticulocyte and pyrenocyte during enucleation. RhoA GTPase is known to play a significant role in cytokinesis and abscission of the daughter cells. Based on the resemblances between erythroblast enucleation and cytokinesis we hypothesize that Rac and RhoA dynamically control erythroblast enucleation by intrinsic molecular pathways analogous to the ones conducting cytokinesis. Based on the critical role of erythroblast-macrophage interaction for efficient erythropoiesis and the known function of Rac and RhoA in signaling initiated by integrins we hypothesize that Rac and RhoA transduce critical macrophage signals to the erythroblasts, regulating terminal erythroid differentiation and enucleation within the erythroblastic islands. To define the mechanistic contribution of RhoA signaling and the relationship of RhoA/Rac in regulating actin and microtubule cytoskeleton in the final stages of erythropoiesis, we bred mice with erythroid specific deletion (EpoRGFPcre/+ driven) of RhoA and Rac1/Rac2/Rac3. To test our hypotheses, we propose in Aim 1 to define the molecular processes by which RhoA/Rac-regulated cytoskeleton dynamics contribute to erythroblast enucleation and in Aim 2 to determine the role of Rho GTPases in enucleation and erythroblast maturation within erythroblastic islands. The goal of this proposal is to investigate the intracellular and intercellular molecular mechanisms by which Rac and RhoA regulate erythroblast differentiation and enucleation via dynamic regulation of actin and microtubule cytoskeletons. This study has the potential to reveal targets for in vivo therapeutic intervention for anemias due to terminal erythroid maturation defects as well as for improving the efficiency of red blood cell production in vitro. (End of Abstract)