Mob1 Localization and Function During Mitosis

描述（由申请人提供）：细胞分裂过程中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附着到有丝分裂纺锤体上并且满足纺锤体组装检查点之前，收缩环不会组装。在出芽和裂殖酵母中，这一过程由称为有丝分裂退出网络/分隔启动网络的信号级联促进，该网络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收缩中发挥积极作用。迄今为止，动物细胞中很少有功能同源物被表征，但以末端成分（Mob1 和 Dbf2/Sid2）为例；两个基因家族都出现了扩展。虽然肿瘤抑制功能被归因于哺乳动物和果蝇 G1/S 期间的 Mob1 和 Dbf2/Sid2 直系同源物，但人们对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 亚型的初步研究表明，其定位动力学与在酵母中观察到的情况相似，Mob1 在有丝分裂早期富集于动粒和纺锤体极，以及胞质分裂期间纺锤体中区和中体。此外，我们已经确定 Mob1 和染色体乘客复合体在有丝分裂早期相互依赖于彼此的动粒定位。最后，我们确定大肿瘤抑制蛋白 2 (Lats2) 是 Mob1A 特异性结合蛋白，并且可能是 Dbf2/Sid2 激酶的功能同源物。以这些初步研究为基础，本申请旨在结合分子、生化和活细胞分析来检验 Mob1 蛋白在有丝分裂和分裂间期维持染色体倍性方面发挥调节作用的假设。形成具体目标的实验线将： 1) 确定 Mob1 定位到着丝粒的分子决定因素； 2）剖析Mob1如何影响Aurora B在动粒处的功能； 3) 评估 Mob1 在 Lats2-p53 对细胞骨架破坏反应中的参与情况。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充当细胞增殖的负调节因子（其功能丧失与肿瘤形成有关），但对于酵母中完成细胞分裂至关重要。预计这些研究将帮助我们了解 Mob1 如何能够参与细胞周期调节的这两个非常不同但绝对关键的特征。 公共健康相关性：成人组织的正确发育和维护需要每次细胞分裂准确发生，并且已经进化出特殊的检查点来充当基因组的保障。细胞分裂中的错误会对生长控制产生不稳定影响，并被认为是肿瘤发生的加重因素。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已被明确定义，但其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.