The roles of mDia2 in membrane remodeling and organelle clearance during reticulocyte formation

项目摘要 哺乳动物终末红细胞生成在红系定型和各种因素方面已得到充分研究 参与分化和增殖。对于终末分化的最后步骤知之甚少， 特别是从去核到网织红细胞成熟为红细胞。若干悬而未决的问题 这一领域包括什么因素调节去核的成红细胞最后一次有丝分裂的退出， 网织红细胞从挤出的细胞核中分离，以及什么信号参与调节网织红细胞的清除。 网织红细胞中的细胞器。用机械论的观点回答这些问题不仅对我们的 了解终末红细胞生成的基础生物学和许多红细胞相关疾病的病理生理学。 疾病，而且还为体外或离体产生红细胞的有效策略提供线索， 输血医学在这方面，我们一直致力于研究mDia formin在终末期肿瘤中的作用。 红细胞生成和我们最近未发表的新发现可能有助于阐明这些问题的线索。 使用mDia 2造血特异性基因敲除小鼠模型，我们发现mDia 2控制了 固缩核脱离所需的新生网织红细胞。网织红细胞（mDia 2） 缺陷型小鼠是刚性的，具有延伸的血影蛋白链，这主要是由于肌动蛋白原丝被破坏。mDia2 缺乏的网织红细胞也显示出膜重塑和细胞器清除的缺陷。我们进一步 在我们的初步数据中显示，Chmp 5是ESCRT III复合物的主要成分之一，是一种 新的下游介质mDia 2。我们的初步机制研究表明，mDia 2调节 血清反应因子（SRF）和Chmp 5的转录活性是SRF的潜在新靶点。这些 结果使我们假设mDia 2调节ESCRT III复合物以控制膜重塑 和网织红细胞形成期间的细胞器清除。在这个项目中，我们将使用体内小鼠模型， 移植试验，各种生化试验，和尖端显微镜研究1）功能 mDia 2在网织红细胞的形成和细胞器清除中的作用。2)mDia 2-SRF-的作用机制 Chmp 5信号在网织红细胞膜重塑和细胞器清除调节中的作用，以及3） mDia 2在网织红细胞成熟中的作用的翻译后修饰。成功完成我们的 提出的研究有可能在信号转导中开辟一个新的领域，调节去核， 网织红细胞形成。了解mDia 2作为晚期主要调节因子的新兴作用 终末红细胞生成作为一种范例将在这一领域产生重要影响。

PROJECT SUMMARY Mammalian terminal erythropoiesis has been well studied in erythroid lineage commitment and various factors involved in differentiation and proliferation. Relatively little is known in the final steps of terminal differentiation, specifically from enucleation to reticulocyte maturation into red blood cells. Several outstanding questions in this field include what factors regulate the exit of last mitosis of erythroblast for enucleation, how nascent reticulocyte separate from the extruded nucleus, and what signals are involved in regulating the clearance of organelles in reticulocytes. Answers to these questions with mechanistic insights are important not only for our understanding of the basic biology of terminal erythropoiesis and pathophysiology of many red cell-related diseases, but also to provide clues for efficient strategies of in vitro or ex vivo generation of red blood cells in transfusion medicine. In this respect, we have been working on the role of mDia formins in terminal erythropoiesis and our recent novel unpublished findings may help shed light on the clues to these questions. Using a mDia2 hematopoietic-specific knockout mouse model, we revealed that mDia2 controls the motility of the nascent reticulocyte that is required for the detachment of the pyknotic nucleus. Reticulocytes in mDia2 deficient mice are rigid with extended spectrin chains, primarily due to the disrupted actin profilaments. mDia2 deficient reticulocytes also show defects in membrane remodeling and organelle clearance. We further revealed in our preliminary data that Chmp5, one of the major components of the ESCRT III complex, is a novel downstream mediator of mDia2. Our preliminary mechanistic studies indicated that mDia2 regulates the transcriptional activity of serum response factor (SRF) and Chmp5 is a potential novel target of SRF. These results led us to hypothesize that mDia2 regulates the ESCRT III complex to control membrane remodeling and organelle clearance during reticulocyte formation. In this project, we will use in vivo mouse models, transplantation assays, various biochemical assays, and cutting-edge microscopy to study 1) the functional roles of mDia2 in the formation and organelle clearance of reticulocytes. 2) the mechanism of mDia2-SRF- Chmp5 signaling in the regulation of reticulocyte membrane remodeling and organelle clearance, and 3) the post-translational modifications of mDia2 in its roles in reticulocyte maturation. Successful completion of our proposed studies has the potential to open a new field in signaling transduction that modulates enucleation to reticulocyte formation. Understanding the emerging roles of mDia2 as a master regulator of the late stage terminal erythropoiesis will have an important impact in this field as a paradigm.