Rho GTPases in Terminal Erythroid Maturation

描述（由申请人提供）： 哺乳动物红系细胞的终末分化以戏剧性的去核过程结束，导致网织红细胞的形成。成红细胞岛内成红细胞与巨噬细胞的相互作用对于有效的去核至关重要。红细胞生成最后阶段的缺陷是慢性炎性疾病和肿瘤以及许多原发性血液学过程（如骨髓增生异常和地中海贫血）中贫血的常见原因。对终末红细胞成熟机制的有限理解阻碍了此类疾病新疗法的开发。使用一种新的分析协议的多参数高速细胞成像流，我们证明了去核是一个多步骤的过程，类似于不对称胞质分裂的证据。它需要通过微管功能建立细胞极性，然后形成收缩性肌动球蛋白环，以及网织红细胞和蛋白核细胞之间的脂筏聚结。此外，我们发现，Rac GTP酶组织肌动蛋白的肌动球蛋白环和聚合物脂筏之间的新生网织红细胞和蛋白核细胞在去核的沟。已知RhoA GT3在子细胞的胞质分裂和分裂中起重要作用。基于成红细胞去核和胞质分裂之间的相似性，我们假设Rac和RhoA通过类似于进行胞质分裂的内在分子途径动态地控制成红细胞去核。基于成红细胞-巨噬细胞相互作用对有效红细胞生成的关键作用以及Rac和RhoA在由整合素引发的信号传导中的已知功能，我们假设Rac和RhoA对成红细胞至关重要的巨噬细胞向成红细胞发出信号，调节成红细胞岛内的终末红细胞分化和去核。为了确定RhoA信号传导的机制贡献以及RhoA/Rac在红细胞生成的最后阶段中调节肌动蛋白和微管细胞骨架的关系，我们培育了RhoA和Rac 1/Rac 2/Rac 3的红细胞特异性缺失（EpoRGFPcre/+驱动）的小鼠。为了验证我们的假设，我们建议在目标1中定义的分子过程中，RhoA/Rac调节的细胞骨架动力学有助于成红细胞去核，并在目标2中确定的作用，Rho GTP酶的去核和成红细胞成熟内的成红细胞岛。本研究的目的是探讨Rac和RhoA通过动态调节肌动蛋白和微管细胞骨架来调节成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由于终末红细胞成熟缺陷引起的贫血的体内治疗干预的靶点，以及提高体外红细胞产生的效率。 (End摘要）

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)