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

项目总结 哺乳动物的终末红细胞生成在红系承受性和各种因素方面得到了很好的研究。 参与分化和增殖。在末端分化的最后步骤中所知的相对较少， 特别是从去核到网织红细胞成熟为红细胞。中的几个悬而未决的问题 这一领域包括什么因素调节了红细胞最后有丝分裂的退出去核，如何新生 网织红细胞从突出的核中分离出来，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。用机械的洞察力回答这些问题不仅对我们的 了解终末期红细胞生成的基本生物学和多种红细胞相关的病理生理学 也为在体外或体外产生红细胞的有效策略提供线索 输血医学。在这方面，我们一直在研究mdia Forins在终端中的作用。 红细胞生成学和我们最近未发表的新发现可能有助于揭示这些问题的线索。 利用mDia2造血特异性基因敲除小鼠模型，我们发现mDia2控制着 网织红细胞新生的网织红细胞，是脱离固缩核所必需的MDia2中的网织红细胞 缺陷小鼠是僵硬的，有延长的幽灵蛋白链，主要是由于肌动蛋白前丝被破坏。MDia2 网织红细胞缺陷还表现在膜重塑和细胞器清除方面的缺陷。我们进一步 我们的初步数据显示，作为ESCRT III复合体的主要组成部分之一的Chmp5是一种 新的mDia2下游介体。我们的初步机制研究表明，mDia2调节 血清反应因子(SRF)和Chmp5的转录活性是SRF潜在的新靶点。这些 结果使我们假设mDia2调节ESCRT III复合体以控制膜重构 以及网织红细胞形成过程中细胞器的清除。在这个项目中，我们将使用活体小鼠模型， 移植试验，各种生化分析，和尖端显微镜研究1)功能 MDia2在网织红细胞形成和细胞器清除中的作用。2)mDia2-SRF的作用机制 Chmp5信号在网织细胞膜重塑和细胞器清除中的调节作用 翻译后修饰的mDia2在网织红细胞成熟中的作用。成功完成我们的 拟议的研究有可能在信号转导中开辟一个新的领域，这种信号转导调节去核 网织红细胞形成。理解mDia2作为晚期主调节因子的新角色 作为一种范式，终末期红细胞生成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 In this respect, we have been working on the role of mDia formins in terminal erythropoiesis and our recent novel unpublished findings may help shed light on the clues to these questions.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.