Mechanosensitive signaling of the Spindle Assembly Checkpoint

 描述（由申请人提供）：纺锤体组装检查点（SAC）是一种信号传导机制，可确保细胞分裂期间染色体的准确分离。众所周知，SAC对每个着丝粒与纺锤体微管的附着状态非常敏感。然而，使这种“机械敏感”信号的分子机制仍然是细胞生物学中的一个核心谜团。我们建议，着丝粒编码的机械开关组成的两个蛋白质的“终端”。这些蛋白质末端的物理分离，类似于开关的打开，对于破坏纺锤体SAC的生化信号级联是必要的。这项提议将定义使动粒能够表现为机械开关的组成蛋白质的生物化学活性和结构特性。我们将首先定义简单芽殖酵母动粒（Aim 1）中的分子细节， 然后使用HeLa细胞将关键实验扩展到人类动粒（Aim 2）。这项工作将为真核生物中机械敏感SAC信号传导奠定基础。我们还将设计一种新的生化“电位计”，量化SAC的信号传导潜力（目标3）。信号传导潜力，定义为SAC诱导和维持细胞周期停滞的能力， 在非整倍体肿瘤细胞的发育、增殖和年龄相关性不孕中起关键作用。电位计将是一个急需的和多功能的工具，用于研究细胞生物学的这些关键方面。

 DESCRIPTION (provided by applicant): The Spindle Assembly Checkpoint (SAC) is a signaling mechanism that ensures accurate chromosome segregation during cell division. It is known that the SAC is exquisitely sensitive to the state of attachment of each kinetochore to spindle microtubules. However, the molecular mechanism that enables this `mechanosensitive' signaling remains a central mystery in cell biology. We propose that the kinetochore encodes a mechanical switch consisting of two protein `terminals'. The physical separation of these protein terminals, akin to the opening of a switch, is necessary for disrupting the biochemical signaling cascade of the spindle SAC. This proposal will define the biochemical activities and structural properties of the constituent proteins that enable the kinetochore to behave as a mechanical switch. We will first define the molecular details in the simple budding yeast kinetochore (Aim 1), and then extend key experiments to the human kinetochore using HeLa cells (Aim 2). This work will establish the basis of mechanosensitive SAC signaling in eukaryotes. We will also engineer a novel, biochemical `potentiometer' that quantifies the signaling potential of the SAC (Aim 3). The signaling potential, defined as its ability of the SAC to induce and sustain cell cycle arrest, plays a key role in development, proliferation of aneuploid tumor cells, and age-related infertilit. The potentiometer will be a much-needed and versatile tool for investigating these critical aspects of cell biology.