MECHANISMS OF MITOTIC SPINDLE ASSEMBLY AND FUNCTION

描述(由申请人提供)：下一阶段的主要重点将是纺锤体、着丝粒和着丝粒蛋白质机制，以实现准确的染色体分离。准确性至关重要，因为即使是一个染色体的错误分离也会产生非整倍体，这可能会导致癌症或发育缺陷。动点在保证准确分离中至少有五个关键作用：1)它们为纺锤体检查点产生一个可扩散的信号，以延迟后期，直到姐妹动点被MTS正确地连接到相对的两极并排列在中期板上；2)它们提供稳定但动态的与MT+末端的连接，以关闭纺锤体检查点的活动，防止MT附着中的错误；3)它们作为一种产生力量的解聚酶，用于染色体的极向运动，与KMT在动粒上的正端解聚连接；4)它们提供了对张力敏感的滑动离合器，在动粒离开极点的运动过程中，从KMT的极向通量产生张力，同时保持与MT的聚合正端的附着，以及5)它们纠正了MT附着中的错误，从而使KMT形成到相反的极(分生组织方向)不会导致后期染色体滞后和错误分离。着丝粒乘客蛋白位于着丝粒后面的内着丝粒上，似乎也调节着丝粒附着和细胞质分裂皮质部位的MT依赖信号。我们的大部分研究集中在哺乳动物组织细胞中的蛋白质功能上，但发芽酵母有丝分裂动点和shmoo尖端是了解动态正端连接位点蛋白质功能的有用遗传模型。我们计划的一个主要优势是，并将继续发展和应用新的显微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.