Mechanisms Regulating Megakaryocyte Endomitosis

描述(申请人提供)：多倍体，即每个细胞二倍体DNA含量的增加，发生在各种细胞中，包括巨核细胞(MK)，它是通过内有丝分裂细胞周期实现的。MK倍体程度影响着血小板的水平和质量。在寻找MK内丝分裂调控因子的过程中，我们发现细胞周期蛋白D3是这一谱系中最主要的D型细胞周期蛋白，它的上调，无论是异位还是通过促血小板生成素治疗，都会增加体内的MK倍体水平。最近，据报道，在体内敲除Cyclin E，一个已知的Cyclin D3的靶标，显著降低MK倍体水平。这与这些小鼠其他血统和器官的正常发育明显不同。基于已知的细胞周期蛋白E将细胞从静止期拯救出来的能力和我们的相关发现，我们提出了新的论点，即MKs遵循少数内丝分裂细胞周期并过渡到静止期，并且Cyclin E唯一允许细胞周期重新进入。多倍化的MK也被编程为跳过后期和胞质分裂。我们假设，纺锤体中区在后期是非典型的配置，并且这一特征和染色体乘客蛋白的相关变化(我们最近的发现)具有功能意义。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，该模型含有标记的染色体和微管，可以对细胞进行实时成像。通过这种方法和表达实验，我们将研究染色体和微管的动力学以及内丝分裂周期的连续性程度。提出了四个具体的研究目标：1.检测细胞周期蛋白E在体内促进MK倍性的能力；2.探索MK多倍化所需的细胞周期蛋白E的分子机制；3.建立带有标记染色体和微管的MK细胞体内模型，研究该系的内丝分裂动力学；4.研究MK细胞减数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。综上所述，追求这些研究目标应该会加强我们对巨核细胞多倍化的分子机制的理解，这一过程会影响血小板的生物生成，从而影响血液止血。

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.