Mechanisms of Mitotic Spindle Assembly

描述（由申请人提供）：生物体的繁殖、发育和存活依赖于遗传物质在两个子细胞中的平等和准确的分离。细胞通过正确组装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DNA的命运。纺锤体由微管（MTs）和许多相关蛋白组成，这些蛋白调节微管动力学来组织和塑造纺锤体。有许多关于纺锤体如何组装的模型，然而，中心体、着丝点和染色质在这一过程中的作用尚未明确定义。虽然中心体是体细胞MT成核的主要来源，染色质介导的MT成核也发生。染色质介导的纺锤体组装是由RanGTP和染色体客运复合体（CPC）梯度介导的，这些梯度的下游蛋白质调节MT动力学，对纺锤体组织很重要。许多细胞周期相关的治疗技术被用来改变MT动力学或参与纺锤体组装的运动蛋白。因此，了解纺锤体最初是如何组织的将有助于表征MT相关蛋白的活性，而MT相关蛋白是治疗剂的靶标。在本提案中，我将：1)确定在没有染色质和着丝点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没有这些成分形成的纺锤体中，mt是如何成核和组织的。2)定义RanGTP和CPC梯度用于主轴装配的机制，以验证在正常细胞中这两个梯度重叠并合作以形成正确的主轴的假设。我将使用基于fret的CPC传感器来测试该模型，以检测RanGTP梯度被抑制时的梯度分布。总之，这些实验将允许进一步了解这些MT成核因子如何协调适当的主轴组织。

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. PUBLIC HEALTH RELEVANCE: Equal segregation of DNA into two daughter cells is essential for organism survival and development. This process is carried out by the mitotic spindle, which is the macromolecular machine composed of microtubules and associated proteins that act to attach chromosomes and segregate them during mitosis. Many therapeutic agents are used to target components of the spindle, therefore understanding how the spindle is assembled will be important for development of biomedical treatments.