CHARACTERIZATION OF MOB1 DYNAMICS IN LIVING CELLS

活细胞中 MOB1 动力学的表征

• 批准号: 7960229
• 负责人: Charles Bradley Shuster
• 金额: $ 12.87万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7960229
• 关键词: Anaphase; Binding; Biomedical Research; CDC2 Protein Kinase; Cell division; Cells; Chimera organism; Chromosome Segregation; Chromosomes; Computer Retrieval of Information on Scientific Projects Database; Cytokinesis; Eukaryota; Family member; Foundations; Funding; Grant; Homologous Gene; Human; Institution; Kinetochores; LATS2 gene; Lead; Life; Maintenance; Mammalian Cell; Mitosis; Mitotic; Mitotic Checkpoint; Monitor; Names; New Mexico; Phosphotransferases; Play; Ploidies; Research; Research Personnel; Resources; Role; Signal Transduction; Sister Chromatid; Small Interfering RNA; Source; United States National Institutes of Health; Yeasts; cellular imaging; fungus; in vivo; mutant; novel; segregation

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 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配（胞质分裂）的精确协调，以便收缩环在后期开始之前不会组装。分裂细胞进入后期进入胞质分裂取决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（有丝分裂退出）的开始。在酵母中，有丝分裂检查点还调节称为分隔启动网络 (SIN) 的信号级联，调节胞质分裂的启动。在高等真核生物中几乎没有发现 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 的末端元件。四种人类 Mob1 的 GFP 嵌合体的表达表明，Mob1 定位于纺锤体极和动粒直至后期开始，以及胞质分裂期间的中央纺锤体和中体。此外，我们还发现了 Mob1A 和 LATS2 激酶之间的一种新的相互作用，它可能作为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发挥的作用。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 家族成员的定位动态。此外，Mob1 在协调有丝分裂和胞质分裂中的作用将通过 Mob1 的 siRNA 敲低或 Mob1 的 Lats2 结合突变体的表达来特异性破坏体内 Mob1/Lats2 相互作用来评估。这些努力最终应该使人们更清楚地了解有丝分裂和胞质分裂是如何协调的，并确认单细胞真菌调节分隔的策略在高等真核生物中是否保守。