MECHANISMS OF MICTOTIC SPINDLE ASSEMBLY AND FUNCTION

下一阶段的重点将是纺锤体、着丝粒和着丝粒蛋白。 实现精确染色体分离的机制。准确性是至关重要的，因为失误- 即使分离一条染色体也会产生非整倍体，这可能会导致癌症或发育缺陷。 动点在确保准确分离方面至少有五个关键作用：1)它们产生一个可扩散的信号 对于主轴检查点，延迟后期操作，直到MTS将姊妹动轴正确连接到 相反的两极并排列在中期板块上；2)它们提供稳定但动态的对MT的附着 Plus Ends用于关闭主轴检查点活动并防止MT连接中出现错误；3)它们充当一种力量- 与正端解聚偶联的产生解聚酶用于染色体的极向移动 4)它们提供了一个张力敏感的滑动离合器，从极点产生张力。 在动粒期间保持与MTS聚合正端附着的KMT的通量 远离磁极的运动，以及5)它们纠正MT附着中的错误，从而使KMT形成 相反的两极(分生组织方向)不会导致落后的染色体和错误的分离 后期。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KMT附着和MT依赖的细胞质分裂皮质部位的信号。多数 我们的研究主要集中在哺乳动物组织细胞的蛋白质功能上，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。 我们计划的一大优势一直是，并将继续是新的开发和应用 测量活细胞和重组制剂中蛋白质功能的显微技术。

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 mis- segregation 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.