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

描述(申请人提供)：在真核细胞中，两极有丝分裂纺锤体的形成对基因组的稳定繁殖至关重要。在人类中，这一过程中的错误与癌症有关。基本上，将微管组织成双极纺锤体所需的所有蛋白质现在都是已知的。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运动蛋白，其功能丧失会导致单极纺锤体和有丝分裂停止。EG5的化学抑制剂目前正在作为癌症治疗药物进行临床试验。EG5已经被证明相对于彼此滑动微管，这是建立纺锤体的两极几何形状所需的关键微管组织功能。然而，微管滑动是如何与EG5的运动性相联系的，以及这种马达蛋白是如何被调控的，仍然是未知的。为了解决这些悬而未决的问题，单分子荧光显微镜将与光学捕获显微操作相结合，以表征单个EG5分子滑动微管相对于彼此的运动性。变构调节机制在EG5功能中的作用将被研究，EG5的突变结构将有助于分析EG5的S结构如何影响其运动性和调节。最后，开发一种与重心标记兼容的EG5荧光标记的新策略，将增加利用FIONA和FRET等强大的生物物理方法研究EG5的可及性。EG5功能的详细描述将为了解有丝分裂纺锤体组装的基本细胞过程提供重要的见解。 与公共卫生相关。EG5是脊椎动物体内的一种分子马达，是细胞分裂和遗传物质分配到新细胞所需机械的重要组成部分。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，并将有助于开发更好的癌症预防和治疗方法。

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.