MOLECULAR MECHANISMS CONTROLLING THE MITOTIC CHECKPOINT

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。细胞分裂是细胞生长、发育和更新的基本特征。每次细胞分裂时，都必须生成一份精确的基因组拷贝，并且每条复制的染色体均匀地分配给后代细胞。如果染色体分布不均，这个细胞就被称为非整倍体——染色体数量不正确的状态。非整倍体是癌细胞的病理标志，是多种出生缺陷的基础。防止非整倍体的细胞机制被称为有丝分裂检查点，它的作用是阻止染色体的分布，直到它们附着在有丝分裂纺锤体上。检查点活性的目标是E3泛素连接酶，称为后期促进复合体（APC/C）。当前的检查点功能模型围绕着直接抑制APC/C活性的非附着着丝点产生的蛋白质复合物。这些配合物的性质，即组成和翻译后的状态，仍然难以研究。通过使用串联亲和方法，我们从有丝分裂中捕获的细胞中纯化了许多检查点蛋白的复合物。从这样的细胞中，我们期望鉴定出由独立染色体产生的代表一个或多个物种的复合体。与此同时，我们也在使用荧光相关方法进行活细胞显微分析，以跟踪活细胞中的这些复合物。允许在体外用质谱法和体内用荧光法分析感兴趣的复合物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cell division is a essential feature of growth, development and renewal. Each time a cell divides an exact copy of the genome must be made and each replicated chromosome equally distributed to the progeny cells. If chromosomes are unequally distributed, the cell is said to be aneuploid - the state of having the incorrect number of chromosomes. Aneuploidy is a pathological hallmark of cancerous cells and underlies a variety of birth defects. The cellular machinery to prevent aneuploidy is called the mitotic checkpoint and acts to prevent distribution of the chromosomes until they are attached to the mitotic spindle. The target of the the checkpoint activity is an E3 Ubiquitin Ligase called the Anaphase-promoting complex (APC/C). Current models of checkpoint function revolve around protein complexes produced by unattached kinetochores that directly inhibit the activity of the APC/C. The nature, i.e. composition and post-translational state, of these complexes have remained difficult to study. Through the use of tandem affinity methods, we have purified complexes for many of the checkpoint proteins from cells arrested in mitosis. From such cells we expect to identify complexes that represent one or more species generated by unattached chromosomes. In parallel, we are also carrying out live cell microscopic analyses using fluorescence correlation methods to follow these complexes in living cells. Permitting the analysis of the complex of interest both in vitro by mass spectrometry and in vivo by fluorescence methods.