CHARACTERIZATION OF MOB1 DYNAMICS IN LIVING CELLS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。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（细胞质分裂）的精确协调，这样在后期开始之前，收缩环不会组装。细胞分裂进入后期进入细胞质分裂依赖于纺锤体组装检查点，该检查点监测染色体与纺锤体的附着，并调节姐妹染色单体分离和CDK1失活的开始（有丝分裂退出）。在酵母菌中，有丝分裂检查点也调节被称为分离起始网络（SIN）的信号级联，该信号级联调节细胞质分裂的起始。在高等真核生物中很少发现SIN的功能同源物，但在所有真核生物中都发现了一个名为Mob1的末端组分。四种人类Mob1基因的GFP嵌合体的表达表明，Mob1在后期开始前定位于纺锤体极点和着丝点，在细胞分裂期间定位于纺锤体中央和体中部。此外，我们还发现了Mob1A和LATS2激酶之间的一种新的相互作用，这可能是哺乳动物细胞中酵母Dbf2/Sid2激酶的功能同源物。以这些初步研究为基础，本应用程序旨在评估Mob1在哺乳动物细胞中协调有丝分裂和细胞分裂中的作用。本应用程序中提出的实验线将利用共定位研究和活细胞成像来表征四种人类Mob1家族成员的定位动态。此外，Mob1在协调有丝分裂和细胞分裂中的作用将通过siRNA敲低Mob1或通过表达Mob1的Lats2结合突变体来特异性地破坏体内Mob1/Lats2相互作用来评估。这些努力最终将使我们更清楚地了解有丝分裂和细胞分裂是如何协调的，并确认单细胞真菌调节分裂的策略是否在高等真核生物中保守。

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.