MECHANISMS OF MITOTIC SPINDLE ASSEMBLY AND FUNCTION

描述（由申请人提供）：本资助下一阶段的主要重点将是纺锤体、着丝点和着丝粒蛋白质机制，这些机制有助于实现准确的染色体分离。准确性是至关重要的，因为即使是一条染色体的错误分离也会产生非整倍体，从而导致癌症或发育缺陷。在确保准确分离方面，着丝点至少有五个关键作用：1)它们为纺锤体检查点产生扩散信号，以延迟后期，直到姊妹着丝点被mt正确地附着到相反的极点并在中期板上对齐；2)它们提供稳定但动态的附着到MT +端，以关闭主轴检查点活动并防止MT附着错误；3)它们作为一种产生力的解聚合酶，用于染色体的极向运动，并与着丝点上kmt的正端解聚合相结合；4)它们提供了一个张力敏感的滑移离合器，从kmt的极向通量中产生张力，同时在着丝点远离极点的运动中保持与MTs聚合正端的连接。5)它们纠正MT连接中的错误，使kmt在相反极点的形成（分裂方向）不会导致后期染色体滞后和错误分离。着丝粒客运蛋白位于着丝点后面的内部着丝粒上，似乎也调节细胞质分裂皮质部位的kMT附着和mt依赖信号。我们的大多数研究都集中在哺乳动物组织细胞的蛋白质功能上，但出芽酵母有丝分裂着丝点和shmoo尖端是了解动态正端附着位点蛋白质功能的有用遗传模型。我们项目的一个主要优势是，并将继续是，开发和应用新的显微镜技术来测量活细胞和重组制剂中的蛋白质功能。

DESCRIPTION (provided by applicant): The major focus for the next period of this grant will be spindle, kinetochore and centromere protein mechanisms that act to achieve accurate chromosome segregation. Accuracy is critical because the missegregation of even one chromosome produces aneuploidy that can lead to cancer or developmental defects. Kinetochores have at least five key roles in assuring accurate segregation: 1) they produce a diffusible signal for the spindle checkpoint to delay anaphase until sister kinetochores are properly attached by MTs to opposite poles and aligned on the metaphase plate; 2) they provide stable, but dynamic, attachment to MT plus ends to turn off spindle checkpoint activity and prevent errors in MT attachment; 3) they act as a force-generating depolymerase for movement of chromosomes poleward coupled to plus-end depolymerization of kMTs at the kinetochore; 4) they provide a tension-sensitive slip clutch, generating tension from the poleward flux of kMTs while maintaining attachment to polymerizing plus ends of MTs during kinetochore movements away from the pole, and 5) they correct errors in MT attachment so that the formation of kMTs to opposite poles (merotelic orientation) does not result in lagging chromosomes and mis-segregation in anaphase. Centromere passenger proteins, which are located on the inner centromere behind the kinetochore, also appear to regulate kMT attachment and MT-dependent signaling of the cortical site for cytokinesis. Most of our studies focus on protein function in mammalian tissue cells, but budding yeast mitotic kinetochores and shmoo tips are useful genetic models for understanding protein function at dynamic plus-end attachment sites. A major strength of our program has been, and will continue to be, the development and application of new microscopy techniques for measurements of protein function in living cells and reconstituted preparations.