CHARACTERIZATION OF MOB1 DYNAMICS IN LIVING CELLS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在细胞分裂过程中维持染色体倍性需要染色体分离和细胞质分裂(胞质分裂)的精确协调，以便在后期开始之前收缩环不会组装。细胞分裂后期进入胞质分裂的进程依赖于纺锤体组装检查点，该检查点监控染色体附着在纺锤体上，并调节姐妹染色单体分离和CDK1失活(有丝分裂退出)的开始。在酵母中，有丝分裂检查点还调节一个被称为分隔启动网络(SIN)的信号级联，该信号级联调节胞质分裂的启动。在高等真核生物中，SIN的功能同源物很少，但在所有真核生物中都发现了一个名为Mob1的末端成分。四种人类Mob1嵌合体的表达表明，Mob1定位于纺锤体极点和动点，直到后期开始，胞质分裂时定位于中央纺锤体和中体。此外，我们还发现了一种新的相互作用，它可能是酵母Dbf2/Sid2蛋白在哺乳动物细胞中的同源蛋白。以这些初步研究为基础，本申请旨在评估Mob1在协调哺乳动物细胞有丝分裂和胞质分裂中所起的作用。本申请中提出的实验路线将使用共定位研究和活细胞成像来表征四个人类Mob1家族成员的定位动力学。此外，Mob1在有丝分裂和胞质分裂协调中的作用将通过特异性地干扰Mob1/Lats2在体内的相互作用或通过表达Mob1的Lats2结合突变体来评估。这些努力最终应该会导致对有丝分裂和胞质分裂是如何协调的更清楚的理解，并证实单细胞真菌用来调节隔膜的策略在高等真核生物中是否保守。

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. Maintenance of chromosomal ploidy during cell division requires a precise coordination of chromosome segregation and cytoplasmic partitioning (cytokinesis) such that the contractile ring does not assemble before the onset of anaphase. Progression of dividing cells into anaphase into cytokinesis is dependent on the spindle assembly checkpoint, which monitors chromosome attachment to the spindle and regulates the onset of sister chromatid segregation and CDK1 inactivation (mitotic exit). In yeast, the mitotic checkpoint also regulates a signaling cascade termed the Septation Initiation Network (SIN) that regulates the initiation of cytokinesis. Few functional homologues of the SIN have been identified in higher eukaryotes, but a terminal component named Mob1 has been identified in all eukaryotes. Expression of GFP chimeras of the four human Mob1s reveal that Mob1 localizes to the spindle poles and kinetochores up until anaphase onset, and the central spindle and midbody during cytokinesis. Additionally, we have identified a novel interaction between Mob1A and LATS2 kinase, which may serve as the functional homolog of the yeast Dbf2/Sid2 kinase in mammalian cells. Using these preliminary studies as a foundation, this application seeks to evaluate the roles that Mob1 plays in coordinating mitosis and cytokinesis in mammalian cells. The lines of experimentation proposed in this application will characterize the localization dynamics of the four human Mob1 family members using co-localization studies and live cell imaging. Further, the role of Mob1 in the coordination of mitosis and cytokinesis will be assessed by specifically disrupting Mob1/Lats2 interactions in vivo by siRNA knockdown of Mob1 or by expression of Lats2-binding mutants of Mob1. These efforts should ultimately lead to a clearer understanding of how mitosis and cytokinesis is coordinated, and confirm whether the strategies employed by unicellular fungi to regulate septation are conserved in higher eukaryotes.