Mob1 Localization and Function During Mitosis

描述（由申请人提供）：在细胞分裂过程中，染色体倍性的维持需要染色体分离和细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在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。在出芽和分裂酵母中，这一过程由一个称为有丝分裂退出网络/分裂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细胞质分裂，基于纺锤极体，并在启动肌动蛋白环的收缩中起积极作用。迄今为止，在动物细胞中鉴定的功能同源物很少，但在末端组分（Mob1和Dbf2/Sid2）；两个基因家族都有扩展。虽然在G1/S期间，Mob1和Dbf2/Sid2的同源基因在哺乳动物和果蝇中具有肿瘤抑制功能，但对于这些分子如何参与动物细胞的有丝分裂和细胞分裂却知之甚少。在培养的人细胞中对Mob1亚型的初步研究表明，Mob1的定位动态与在酵母中观察到的一致，在有丝分裂早期，Mob1富集在着丝点和纺锤极，在细胞质分裂期间，Mob1富集在纺锤体中间区和中间体。此外，我们已经确定，在有丝分裂早期，Mob1和染色体客运复合体相互依赖于彼此的着丝点定位。最后，我们确定了大肿瘤抑制因子2 （Lats2）是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，本应用程序寻求结合分子，生化和活细胞分析来验证Mob1蛋白在有丝分裂和间期期间调节维持染色体倍性的假设。形成特定目标的实验线将：1)确定Mob1定位到着丝点的分子决定因素；2)分析Mob1如何影响着丝点Aurora B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），但在酵母中完成细胞分裂是必不可少的。预计这些研究将帮助我们理解Mob1如何能够参与这两个非常不同但绝对关键的细胞周期调节特征。

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.