Mechanisms of Mitotic Fidelity

项目摘要 有丝分裂的主要目的是将遗传物质准确地分配到两个子细胞之间。 减数分裂或有丝分裂的缺陷导致非整倍体，这是出生缺陷的重要原因，也是 肿瘤的发生。正确的纺锤体功能依赖于微管（MT）的精确时空控制 动力学和整合的力量的马达蛋白。调节MT动力学的缺陷导致纺锤体 多极性、不适当的着丝粒-MT附着、延迟的有丝分裂进程和不适当的染色体 种族隔离尽管产生了广泛的零件清单主轴，一个主要的悬而未决的问题是 了解MT动力学和运动蛋白活性如何在空间和时间上进行调节，以确保 适当的纺锤体结构和染色体分离。这部分是由于缺乏适当的 可用于将关键监管生化事件与这些事件的控制位置联系起来的工具 在空间上位于主轴中。我们实验室的工作有助于确定驱动蛋白的成员如何 超家族参与纺锤体组织、染色体聚集、着丝粒-MT附着、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FLIM和超分辨率显微镜相结合，现在使我们能够解决 主轴电动机是活动的或不活动的。此外，我们对主轴电机的分析使我们开始 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： 主轴。在未来五年，我们会集中研究三个主要范畴： 运动调节机制，利用我们的专业知识与各种生化，生物物理和超， 分辨率成像方法，以整合蛋白质活性及其在背景下的空间和时间控制 的spindle。2)维持正常纺锤体形态发生的一个主要方面不仅来自 调节纺锤体结构和动力学的个体马达的生化活动， 了解它们的时空规律。随着我们众多FRET生物传感器的发展 我们处于理想的位置，可以使用FLIM成像来可视化何时何地激活单个电机 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， 运动活动对于遗传物质的正确分离也至关重要，从而确保有丝分裂的保真度。 我们将利用我们的能力，产生不同水平的倍性细胞，以了解如何缺陷 在纺锤体结构中增加染色体负荷影响染色体分离的保真度。 总的来说，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物 其功能具有高精度。这种整体方法使我们能够对 有丝分裂保真度的全局控制。

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.