MECHANISMS OF MICTOTIC SPINDLE ASSEMBLY AND FUNCTION

下一个资助期的重点将是纺锤体、动粒和着丝粒蛋白 这些机制可以实现精确的染色体分离。准确性至关重要，因为错误- 即使一条染色体的分离也会产生非整倍性，从而导致癌症或发育缺陷。 动粒在保证精确分离方面至少有五个关键作用：1）它们产生可扩散的信号 纺锤体检查点延迟后期，直到姐妹动粒被MT正确地附着到 两极相反，并在中期板上对齐; 2）它们提供稳定的，但动态的，附着到MT 加端关闭纺锤体检查点活性并防止MT附着错误; 3）它们充当力- 产生用于染色体向极移动的解聚酶， 动粒处的kMT; 4）它们提供张力敏感的滑动离合器，从极向产生张力 kMT的通量，同时在动粒过程中保持与MT的聚合正末端的连接 运动远离极点，和5）他们纠正错误的MT附件，使形成的kMT， 相反的两极（部分末端取向）不会导致落后的染色体和错误分离， 后期着丝粒过客蛋白，位于动粒后面的内着丝粒上， 也似乎调节kMT附着和MT依赖性信号皮质部位的胞质分裂。最 我们的研究集中在哺乳动物组织细胞中的蛋白质功能，但芽殖酵母有丝分裂动粒和 Shmoo tips是用于理解蛋白质在动态正末端附着位点的功能的有用的遗传模型。 我们计划的一个主要优势一直是，并将继续是，新的开发和应用。 显微镜技术用于测量活细胞和重组制剂中的蛋白质功能。

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.