Mechanisms of Mitotic Spindle Assembly

描述(申请人提供)：生物体的繁殖、发育和存活依赖于遗传物质被平等和准确地分离到两个子细胞中。这种细胞通过适当地组装一种称为有丝分裂纺锤体的大分子结构来排列和分离染色体，从而确保了DNA的命运。纺锤体由微管和许多调节微管动力的相关蛋白组成，以组织和塑造纺锤体。关于纺锤体的组装有很多模型，然而，中心体、动粒和染色质在这一过程中的作用还没有明确的定义。虽然中心体是体细胞MT成核的主要来源，但也存在染色质介导的MT成核。染色质介导的纺锤体组装是由RanGTP和染色体乘客复合体(CPC)梯度介导的，这些梯度的下游蛋白调节MT的动力学，对纺锤体组织至关重要。许多与细胞周期相关的治疗技术被用来改变参与纺锤体组装的MT动力学或马达蛋白。因此，了解纺锤体最初是如何组织的将有助于表征作为治疗药物靶标的MT相关蛋白的活性。在本提案中，我将：1)确定在没有染色质和动粒的情况下纺锤体是如何组织的，其中我将确定在两个模型系统中，在没有这些成分的情况下，MT是如何在纺锤体中成核和组织的。2)定义RanGTP和CPC梯度用于纺锤体组装的机制，以检验在正常细胞中这两个梯度重叠并协同形成适当的纺锤体的假设。我将使用CPC的基于FRET的传感器来测试这个模型，以检测当RanGTP梯度被抑制时梯度的分布。综上所述，这些实验将有助于进一步了解这些MT成核因素如何协调合适的纺锤体组织。

DESCRIPTION (provided by applicant): Organism reproduction, development, and survival are dependent on the equal and accurate segregation of genetic material into two daughter cells. The cell ensures the fate of DNA by properly assembling a macromolecular structure called the mitotic spindle to align and segregate chromosomes. The spindle is composed of microtubules (MTs) and many associated proteins that regulate MT dynamics to organize and shape the spindle. There are many proposed models for how the spindle assembles, however, the contributions of centrosomes, kinetochores, and chromatin to this process are not clearly defined. Although centrosomes are a primary source of MT nucleation in somatic cells, chromatin-mediated MT nucleation also occurs. Chromatin-mediated spindle assembly is mediated by the RanGTP and Chromosome Passenger Complex (CPC) gradients in which downstream proteins of these gradients regulate MT dynamics and are important for spindle organization. Many cell cycle related therapeutic techniques are used to alter MT dynamics or motor proteins that are involved in spindle assembly. Therefore, understanding how the spindle is initially organized will be beneficial in characterizing the activity of MT associated proteins that are the targets of therapeutic agents. In this present proposal I will: 1) Determine how the spindle is organized in the absence of chromatin and kinetochores in which I will determine how MTs are nucleated and organized in spindles formed in the absence of these components in two model systems. 2) Define the mechanisms utilized by the RanGTP and CPC gradients for spindle assembly to test the hypothesis that in a normal cell these two gradients overlap and cooperate for proper spindle formation. I will test this model using a FRET-based sensor of the CPC to detect the distribution of the gradient when the RanGTP gradient has been suppressed. Together these experiments will allow for further understanding of how these MT nucleating factors coordinate for proper spindle organization.