Mob1 Localization and Function During Mitosis

描述（由申请方提供）：细胞分裂期间染色体倍性的维持需要染色体分离和胞质分裂的精确协调，以便在所有复制的姐妹染色单体正确连接到有丝分裂纺锤体并且满足纺锤体组装检查点之前，收缩环不会组装。在芽殖和分裂酵母中，该过程由称为有丝分裂退出网络/分隔起始网络的信号级联促进，该信号级联协调有丝分裂退出和胞质分裂，基于纺锤体极体，并在启动肌动蛋白环的收缩中起积极作用。到目前为止，很少有功能同源物已在动物细胞中的特点，但在终端组件（Mob1和Dbf2/Sid2）的情况下，有两个基因家族的扩展。虽然肿瘤抑制功能已被归因于哺乳动物和果蝇的直系同源物Mob1和Dbf2/Sid2在G1/S期间，很少有人知道这些分子如何参与动物细胞中的有丝分裂和胞质分裂。在培养的人类细胞中的Mob1亚型的初步研究表明，本地化动力学镜，在酵母中观察到，与Mob1富集在着丝粒和纺锤体极早期有丝分裂，和纺锤体中区和中体在胞质分裂。此外，我们已经确定，Mob1和染色体乘客复合体是相互依赖的有丝分裂早期的动粒定位。最后，我们确定了大肿瘤抑制因子2（Lats2）作为Mob1A特异性结合蛋白和Dbf2/Sid2激酶的可能功能同源物。利用这些初步研究作为基础，本申请寻求将联合收割机分子、生物化学和活细胞分析相结合，以检验Mob1蛋白在有丝分裂和间期期间在维持染色体倍性中发挥调节作用的假设。形成特定目标的实验路线将：1）确定Mob1定位于动粒的分子决定因素; 2）剖析Mob1如何影响极光B在动粒的功能; 3）评估Mob1参与Lats2-p53对细胞骨架破坏的反应。这些研究将为一个基因家族提供新的见解，该基因家族在动物细胞中似乎是细胞增殖的负调节因子（其功能丧失与肿瘤形成有关），但对于完成酵母细胞分裂至关重要。预计这些研究将帮助我们调和Mob1如何能够参与这两个非常不同但绝对重要的细胞周期调控特征。 公共卫生相关性：成人组织的正常发育和维持需要每一次细胞分裂都准确地发生，并且已经进化出特殊的检查点，作为基因组的保护措施。细胞分裂中的错误对生长控制具有不稳定作用，并且被认为是肿瘤发生中的加重因素。该提案旨在研究一个高度保守的蛋白质家族，其功能在低等真核生物中得到了很好的定义，但其作用在人类细胞中仍然知之甚少。

DESCRIPTION (provided by applicant): Maintenance of chromosomal ploidy during cell division requires a precise coordination of chromosome segregation and cytokinesis such that the contractile ring does not assemble before all replicated sister chromatids are correctly attached to the mitotic spindle and the spindle assembly checkpoint is satisfied. In budding and fission yeast, this process is facilitated by a signaling cascade termed the Mitotic Exit Network/Septation Initiation Network that coordinates mitotic exit and cytokinesis, is based at the spindle pole bodies, and plays an active role in initiating constriction of the actin ring. To date, few functional homologues have been characterized in animal cells, but in the case of the terminal components (Mob1 and Dbf2/Sid2); there have been expansions in both gene families. And while tumor suppressor functions have been ascribed to the mammalian and Drosophila orthologs of Mob1 and Dbf2/Sid2 during G1/S, little is known about how these molecules participate in mitosis and cytokinesis in animal cells. Preliminary studies of Mob1 isoforms in cultured human cells indicate that the localization dynamics mirror that observed in yeast, with Mob1 enriched at the kinetochores and spindle poles early in mitosis, and the spindle midzone and midbody during cytokinesis. Moreover, we have determined that Mob1 and the chromosomal passenger complex are mutually dependent on each other kinetochore localization early in mitosis. Lastly, we identified Large Tumor Supressor 2 (Lats2) as a Mob1A-specific binding protein and possible functional homolog of the Dbf2/Sid2 kinase. Using these preliminary studies as a foundation, this application seeks to combine molecular, biochemical and live cell analyses to test the hypothesis that Mob1 proteins perform roles in regulating in maintaining chromosomal ploidy during both mitosis and interphase. The lines of experimentation that form the Specific Aims will: 1) Determine the molecular determinants of Mob1 localization to the kinetochore; 2) Dissect how Mob1 affects Aurora B function at the kinetochore; and 3) Assess the involvement of Mob1 in the Lats2-p53 response to cytoskeletal disruption. These studies will shed novel insights into a gene family that in animal cells appears to act as a negative regulator of cell proliferation (and whose loss of function is associated with tumor formation), yet is essential for completing cell division in yeast. It is anticipated that these studies will help us reconcile how Mob1 is capable of participating in both of these very different but absolutely critical features of cell cycle regulation. Public Health Relevance: Proper development and maintenance of adult tissues requires that each cell division occurs accurately, and special checkpoints have evolved that act as safeguards for the genome. Mistakes in cell division have a destabilizing effect on growth control, and are believed to be an aggravating factor in tumorigenesis. This proposal seeks to study a highly conserved family of proteins whose functions are well defined in lower eukaryotes, but whose roles remain poorly understood in human cells.