Mechanisms Regulating Megakaryocyte Endomitosis

描述（由申请人提供）：多倍体，即每个细胞中二倍体DNA含量的增加，发生在多种细胞中，包括巨核细胞（MK），其中它是通过细胞内有丝分裂细胞周期实现的。MK倍体的程度影响血小板的水平和质量。在寻找调节MK有丝分裂，我们发现，细胞周期蛋白D3是主要的D-型细胞周期蛋白在这个谱系和它的上调，无论是异位或血小板生成素治疗，增加MK倍性水平在体内。最近，据报道，在体内敲除细胞周期蛋白E，细胞周期蛋白D3的一个已知的目标，显着降低MK倍性水平。这与这些小鼠中其他谱系和器官的正常发育截然不同。基于已知的细胞周期蛋白E从静息GO期拯救细胞的能力和我们的相关研究结果，我们提出了新的论点，即MK遵循几个内有丝分裂细胞周期过渡到静息期，并且细胞周期蛋白E独特地允许细胞周期重新进入。多倍化MK也被编程为跳过后期和胞质分裂。我们推测，纺锤体中间区在后期的配置和染色体乘客蛋白（我们最近的发现），这一功能和相关的变化是功能的意义。为了加强对相关机制的探索，我们打算开发一种新的小鼠模型，其中含有标记的染色体和微管，这将允许细胞的活体成像。通过这种方法和表达实验，我们将研究染色体和微管动力学以及有丝分裂周期的连续性程度。提出了四个具体的研究目标：1。研究细胞周期蛋白E（cyclin E）对MK细胞增殖的影响。探讨细胞周期蛋白E对MK细胞多倍化的分子机制; 3.建立标记染色体和微管的巨噬细胞体内模型，研究该细胞系内有丝分裂的动力学;研究MK细胞内有丝分裂过程中染色体乘客蛋白的再分布过程及其对MK倍性水平的影响。两者合计，追求这些研究目标，应该提高我们的巨核细胞多倍化的分子机制的理解，影响血小板生物合成的过程，因此，血液止血。

DESCRIPTION (provided by applicant): Polyploidy, the increase of diploid DNA content per cell, occurs in a variety of cells, including megakaryocytes (MKs), where it is achieved by an endomitotic cell cycle. The degree of MK ploidy influences platelet level and quality. In the search for regulators of MK endomitosis, we found that cyclin D3 is the predominant D-type cyclin in this lineage and that its upregulation, either ectopically or by thrombopoietin treatment, increases MK ploidy level in vivo. Recently, it was reported that an in vivo knock out of cyclin E, a known target of cyclin D3, significantly diminishes MK ploidy level. This is strikingly different from the normal development of other lineages and organs in these mice. Based on the known ability of cyclin E to rescue cells from a resting GO phase and on our related findings, we propose the novel contention that MKs follow few endomitotic cell cycles with transition into a resting phase, and that cyclin E uniquely allows cell cycle re-entry. Polyploidizing MKs are also programmed to skip late anaphase and cytokinesis. We hypothesize that the spindle midzone at late anaphase is atypically configured and that this feature and associated changes in chromosome passenger proteins (our recent finding) are of functional significance. To enhance exploration of related mechanisms, we intend to develop a novel mouse model with MKs containing marked chromosomes and microtubules that will allow live imaging of cells. Via this approach and expression experiments, we will study chromosome and microtubule dynamics as well as the degree of continuity of endomitotic cycles. Four Specific Aims of research are proposed: 1. To examine the ability of elevated cyclin E to promote MK ploidy in vivo; 2. To explore the molecular mechanism of cyclin E requirement for MK polyploidization; 3. To generate an in vivo model of MKs with labeled chromosomes and microtubules and to study the dynamics of endomitosis in this lineage; 4. To study the process of redistribution of chromosome passenger proteins during MK endomitosis and its effect on MK ploidy level. Taken together, pursuing these aims of research should enhance our understanding of the molecular mechanisms of megakaryocyte polyploidization, a process that impacts platelet biogenesis and hence, blood hemostasis.