Mob1 Localization and Function During Mitosis

描述(申请人提供)：在细胞分裂过程中维持染色体倍性需要精确协调染色体分离和胞质分裂，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和纺锤体组装检查点之前，收缩环不会组装。在发芽和分裂酵母中，这一过程是由一个被称为有丝分裂退出网络/分离启动网络的信号级联来促进的，该网络协调有丝分裂退出和胞质分裂，以纺锤体为基础，在启动肌动蛋白环的收缩中发挥积极作用。到目前为止，在动物细胞中几乎没有功能同源物的特征，但在末端组件(Mob1和Dbf2/Sid2)的情况下，这两个基因家族都有扩张。虽然在G1/S期间，肿瘤抑制功能被归因于哺乳动物和果蝇的Mob1和Dbf2/Sid2的同源基因，但对于这些分子如何参与动物细胞的有丝分裂和胞质分裂知之甚少。对培养的人类细胞中Mob1异构体的初步研究表明，Mob1的定位动力学与在酵母中观察到的情况一致，在有丝分裂早期，Mob1在动轴和纺锤体极处富含，在胞质分裂中，在纺锤体中区和中体富含Mob1。此外，我们已经确定了Mob1和染色体乘客复合体在有丝分裂早期的动粒定位是相互依赖的。最后，我们鉴定了大肿瘤抑制因子2(Lats2)是一种Mob1a特异性结合蛋白，可能是Dbf2/Sid2激酶的功能同源物。以这些初步研究为基础，本应用试图结合分子、生化和活细胞分析来检验这一假设，即Mob1蛋白在有丝分裂和间期在维持染色体倍性方面发挥调节作用。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如何影响动粒上的Aurora B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带来新的见解，该基因家族在动物细胞中似乎是细胞增殖的负调节因子(其功能的丧失与肿瘤的形成有关)，但对完成酵母中的细胞分裂是必不可少的。预计这些研究将帮助我们协调Mob1如何能够参与这两个截然不同但绝对关键的细胞周期调节功能。 公共卫生相关性：成人组织的适当发育和维护需要每个细胞准确地发生分裂，并且已经进化出作为基因组保护的特殊检查点。细胞分裂中的错误会对生长控制产生不稳定的影响，并被认为是肿瘤形成的加重因素。这项提议试图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定义良好，但在人类细胞中的作用仍知之甚少。

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.