Mechanisms of Mitotic Fidelity

项目摘要 有丝分裂的主要目的是在两个子细胞之间准确地分配遗传物质。 减数分裂或有丝分裂的缺陷会导致非整倍体，这是出生缺陷的一个重要原因，也是一个标志。 关于肿瘤发生的研究。正确的纺锤体功能依赖于微管(MT)的精确时空控制 马达蛋白质的动力学和力的整合。调节MT动力学中的缺陷导致纺锤形 多极化、不正确的动粒-MT连接、有丝分裂进程延迟和染色体错误 种族隔离。尽管为主轴生成了一个广泛的部件列表，但一个主要的未解答的问题是 了解MT动力学和运动蛋白活性是如何在空间和时间上调节的，以确保 适当的纺锤体结构和染色体分离。这在一定程度上是由于缺乏适当的 可用于将关键的监管生化事件与这些事件的控制位置相关联的工具 在主轴上的空间上。我们的实验室的工作在定义动感成员如何 超家族参与纺锤体组织、染色体聚集、动粒-MT连接、错误 校正、染色体分离和胞质分裂。我们最近实施了新的基于FRET的 生物传感器与薄膜和超分辨率显微镜相结合，现在使我们能够解决 主轴电机有激活或非激活状态。此外，我们对主轴电机的分析使我们能够开始 了解空间梯度，如RAN-GTP梯度，如何在以下情况下调节运动活动 纺锤。在未来5年，我们会集中研究3个主要范畴：1)我们会研究 利用我们的专业知识，通过各种生化、生物物理和超 将蛋白质活性与其空间和时间控制相结合的分辨率成像方法 在主轴上。2)维持适当的纺锤体形态发生的一个主要方面不仅来自于 调节主轴结构和动力学的单个马达的生化活动，也来自 了解它们的空间和时间规律。随着我们众多FRET生物传感器的发展 我们的定位非常理想，可以使用电影成像技术直观地显示各个马达何时何地被激活 并了解它们与关键调控分子的相互作用。3)适当调节MT动态和 运动活动对于遗传物质的适当分离也是至关重要的，从而确保有丝分裂的保真度。 我们将利用我们的能力来产生具有不同倍性水平的细胞，以了解缺陷是如何 在纺锤体结构下，增加的染色体负荷会影响染色体分离的保真度。 总体而言，我们的研究将阐明细胞如何利用空间信息组装大分子复合体 具有高精度的功能。这种全面的方法使我们能够对 有丝分裂保真度的全球控制。

Project Summary The major goal of mitosis is to distribute the genetic material accurately between the two daughter cells. Defects in meiosis or mitosis lead to aneuploidy, which is a significant cause of birth defects and is a hallmark of tumorigenesis. Proper spindle function relies on precise spatial and temporal control of microtubule (MT) dynamics and the integration of forces of motor proteins. Defects in regulated MT dynamics lead to spindle multi-polarity, improper kinetochore-MT attachments, delayed mitotic progression, and improper chromosome segregation. Despite the generation of an extensive parts list for the spindle, a major unanswered question is to understand how MT dynamics and motor protein activity are spatially and temporally regulated to ensure proper spindle architecture and chromosome segregation. This has been due, in part, to a lack of appropriate tools that could be used to relate key regulatory biochemical events to where those events are controlled spatially in the spindle. Our lab's work has been instrumental in defining how members of the kinesin superfamily contribute to spindle organization, chromosome congression, kinetochore-MT attachments, error correction, chromosome segregation, and cytokinesis. Our recent implementation of new FRET-based biosensors combined with FLIM and super resolution microscopy is now enabling us to address where in the spindle motors are active or inactive. In addition, our analysis of spindle motors has allowed us to begin to understand how spatial gradients, such as the Ran-GTP gradient, modulate motor activity in the context of the spindle. Over the next 5 years we will focus our studies on three major areas: 1) We will examine the mechanisms of motor regulation by using our expertise with a variety of biochemical, biophysical and super- resolution imaging approaches to integrate protein activity with its spatial and temporal control in the context of the spindle. 2) A major aspect of maintaining proper spindle morphogenesis comes not only from the biochemical activities of the individual motors that regulate spindle structure and dynamics but also from understanding their spatial and temporal regulation. With the development of our numerous FRET biosensors we are ideally positioned to use FLIM imaging to visualize when and where individual motors are activated and to understand their interactions with key regulatory molecules. 3) Proper regulation of MT dynamics and motor activity are also critical to the proper segregation of the genetic material, thus ensuring mitotic fidelity. We will take advantage of our ability to generate cells with different levels of ploidy to understand how defects in spindle architecture under increased chromosome load affect the fidelity of chromosome segregation. Overall, our studies will elucidate how cells use spatial information to assemble macromolecular complexes that function with high precision. This holistic approach allows us to make significant new insight into the global control of mitotic fidelity.