Single molecule studies of microtuble siding by the conerved mitotic motor Eg5

描述（由申请人提供）：在真核细胞中，双极有丝分裂纺锤体的形成对于基因组的稳定繁殖至关重要。在人类中，这个过程中的错误与癌症有关。基本上所有的蛋白质需要组织微管成一个双极纺锤体现在是已知的。然而，大多数这些蛋白质的确切功能以及它们的功能是如何协调的仍然是个谜。Eg5是一种广泛保守的有丝分裂马达蛋白，其功能丧失导致单极纺锤体和有丝分裂停滞。Eg5的化学抑制剂目前正作为癌症治疗剂进行临床试验。Eg5已被证明可以使微管相对于彼此滑动，这是建立纺锤体双极几何形状所需的关键微管组织功能。然而，微管滑动如何与Eg5的运动性相结合，以及这种马达蛋白如何被调节仍然是未知的。为了解决这些突出的问题，单分子荧光显微镜将结合显微操作的光捕获来表征个别Eg5分子滑动微管相对于彼此的运动性。将研究Eg5功能中变构调节机制的参与，并且Eg5的突变构建体将允许分析Eg5的结构架构如何影响其运动性和调节。最后，开发一种新的策略，荧光标记Eg5兼容的质量中心标记将增加Eg5的可访问性，通过强大的生物物理方法，如FIONA和FRET研究。Eg5功能的详细表征将为有丝分裂纺锤体组装的基本细胞过程提供重要的见解。 公共卫生相关性。Eg5是脊椎动物中的分子马达，是细胞分裂和遗传物质分布到新细胞中所需的机器的重要组成部分。在人类中，这些过程中的错误与发育缺陷和癌症有关。了解这种蛋白质的工作原理将为细胞生物学的基本原理提供重要的见解，并将有助于更好地预防和治疗癌症。

DESCRIPTION (provided by applicant): In eukaryotic cells, formation of a bipolar mitotic spindle is essential for stable propagation of the genome. In humans, errors in this process are associated with cancer. Essentially all of the proteins required to organize microtubules into a bipolar spindle are now known. However, the precise functions of most of these proteins and how their functions are coordinated remain mysterious. Eg5 is a widely conserved mitotic motor protein whose loss of function leads to monopolar spindles and mitotic arrest. Chemical inhibitors of Eg5 are currently in clinical trials as cancer therapeutics. Eg5 has been shown to slide microtubules relative to one another, a key microtubule organizing function required to establish the spindle's bipolar geometry. However, how microtubule sliding is coupled to the motility of Eg5 and how this motor protein is regulated remain unknown. To address these outstanding questions, single molecule fluorescence microscopy will be combined with micromanipulation by optical trapping to characterize the motility of individual Eg5 molecules sliding microtubules relative to one another. The involvement of allosteric regulatory mechanisms in Eg5 function will be investigated, and mutated constructs of Eg5 will allow analysis of how Eg5's structural architecture influences its motility and regulation. Finally, development of a new strategy for fluorescent labeling of Eg5 compatible with center-of-mass labeling will increase the accessibility of Eg5 to study by powerful biophysical methods such as FIONA and FRET. The detailed characterization of Eg5 function will provide important insights into the fundamental cellular process of mitotic spindle assembly. PUBLIC HEALTH RELEVANCE. Eg5, a molecular motor in vertebrate animals, is an essential component of the machinery needed for cell division and the distribution of genetic material into new cells. In humans, errors in these processes are associated with developmental defects and cancer. Understanding how this protein works will provide important insights into fundamental principles of cell biology, and will contribute to the development of better cancer prevention and treatments.