Role of the Ndc80 Loop Domain and Cdt1 in Kinetochore Microtubule Attachments

Ndc80 环结构域和 Cdt1 在动粒微管附着中的作用

• 批准号: 8989975
• 负责人: Dileep Varma
• 金额: $ 19.64万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-06 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8989975
• 关键词: Anaphase; Aneuploidy; Award; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biological Assay; C-terminal; Caenorhabditis elegans; Cell division; Cells; Cellular biology; Chromosomal Instability; Chromosome Segregation; Chromosomes; Collaborations; Communities; Complex; Congenital Abnormality; Development; Dynein ATPase; Embryo; Equipment; Generations; Genetic; Goals; Homologous Gene; Image; Immunofluorescence Immunologic; In Vitro; Kinetochores; Laboratories; Lead; Length; Malignant Neoplasms; Maps; Mentors; Metaphase Plate; Microscopy; Microtubule-Associated Proteins; Microtubules; Mitosis; Mitotic; Molecular; Molecular Biology; Motor; Mutation; N-terminal; Nature; North Carolina; Organelles; Pathway interactions; Phase; Postdoctoral Fellow; Property; Proteins; Publishing; RNA Interference; Reagent; Recruitment Activity; Replication Licensing; Reporting; Research; Research Infrastructure; Role; Salmon; Site; Structure; System; Testing; Therapeutic; Tissues; Training; United States National Institutes of Health; Universities; Vertebrates; Vesicle; Work; Yeasts; cooking; daughter cell; in vivo; insight; man; mutant; novel; protein complex; protein expression; protein function; protein purification; research study; skills; tool; yeast two hybrid system

I was trained as a cell biologist during my graduate studies in the laboratory of Dr. Richard Vallee at Columbia University. I used modern cell biological tools like microscopy and RNA interference to study how the microtubule (MT) motor protein Dynein, attaches to intracellular cargo like vesicles and kinetochores. My research on dynein function at kinetochores attracted me towards understanding the function of this organelle for my post-doctoral work and I choose Dr. Ted Salmon’s lab at the University of North Carolina at Chapel Hill for carrying out this research. In the Salmon lab, I study how kinetochores attach stably to spindle MTs for faithful segregation of chromosomes during mitosis. Even though Cell Biology has been the predominant tool for the majority of my post-doctoral research, I realize that I need to acquire novel skills in the form of molecular biology, biochemistry and genetics to be successful in my future research endeavors and to be able to continually contribute at the highest level to the mitosis research community. The NIH pathway to independence award will provide me with the necessary infrastructure and support to generate the reagents and develop the tools to be independent and have an impact on the field of mitotic research. During the initial phase of my post-doctoral research tenure in the Salmon lab (in collaboration with Jean Cook lab), I have discovered a novel kinetochore function for the replication licensing protein Cdt1 in kinetochore microtubule (kMT) attachment and I have carried out extensive research using cell biological tools to understand its function in this capacity [Nature Cell Biology 2012, Vol. 14(6)]. This study has revealed that Cdt1 performs its mitotic roles in association with a bonafide kinetochore protein, Hec1, whose function in kMT attachment is well documented. Further, it was demonstrated that the Ndc80 complex recruits Cdt1 to kinetochores through a unique loop domain which was also found to be important for kMT attachments. To understand the role of Cdt1 and the Ndc80 loop in greater detail, I need to employ extensively a novel set of skills involving molecular biology, biochemistry and genetics. The specific aims for my proposed research are to #1) elucidate how Cdt1 and the Ndc80 complex coordinate to control kMT attachments, and #2) probe if the Ndc80 loop domain and/or Cdt1 is required to recruit other microtubule-associated proteins (MAPs) to kinetochores to aid in stable kMT attachments. I will test using biochemical, advanced cell biological and genetic means if Cdt1 directly binds to the loop domain and MTs to serve as a bridge between these two components and further if this interaction is important to recruit other MAPs. As co-mentors, Ted Salmon and Jennifer Deluca will provide expertise and tools for advanced imaging and other biochemical assays required. As a mentor, Jean Cook will provide expertise and guidance with molecular biology, protein expression and purification. As a contributor, Kevin Slep will provide equipment and expertise with protein purification and biochemical assays. As a 2nd contributor, Arshad Desai will provide training and expertise with C. elegans genetics and functional assays.

在我在哥伦比亚理查德·瓦利（Richard Vallee）实验室的研究生学习期间，我接受了细胞生物学家的培训。 大学。我使用现代细胞生物学工具（如显微镜和RNA干扰）来研究 微管（MT）运动蛋白动力蛋白，附着在细胞内货物（如蔬菜和动物）上。我的 Kinechores的动力蛋白功能的研究吸引了我了解该细胞器的功能 对于我的博士后工作，我选择了北卡罗来纳大学教堂山分校的Ted Salmon博士实验室 进行这项研究。在鲑鱼实验室中，我研究了运动学如何稳定地连接到螺旋MTS 有丝分裂期间染色体的忠实分离。即使细胞生物学一直是主要工具 对于我的大多数博士后研究，我意识到我需要以 分子生物学，生物化学和遗传学将在我的未来研究中取得成功，并能够 不断为有丝分裂研究界做出最高水平。 NIH通往 独立奖将为我提供必要的基础设施和支持以产生试剂 并开发独立的工具，并对有丝分裂研究领域产生影响。在最初 我在鲑鱼实验室（Jean Cook Lab）合作的博士后研究任期的阶段，我有 在动元微管中发现了复制许可蛋白CDT1的新型动力学函数 （KMT）依恋和我已经使用细胞生物学工具进行了广泛的研究以了解其 以这种能力的功能[自然细胞生物学2012，第1卷。 14（6）]。这项研究表明，CDT1执行 有丝分裂作用与真正的动力学蛋白Hec1相关，其在KMT附件中的功能很好 记录。此外，证明NDC80复合物通过A将CDT1募集到动力学 独特的循环域也被认为对KMT附件很重要。了解CDT1的作用 NDC80循环更详细地，我需要广泛采用一组新颖的技能，涉及分子 生物学，生物化学和遗传学。我提出的研究的具体目的是＃1）阐明CDT1的方式 NDC80复合物坐标以控制KMT附件，以及＃2）探测如果NDC80循环域和/或 需要CDT1募集其他微管相关蛋白（MAP）到动力学以帮助稳定的KMT 附件。如果CDT1直接结合到 循环域和MT是这两个组件之间的桥梁，并且此相互作用是否为 招募其他地图很重要。作为联合会，TED Salmon和Jennifer DeLuca将提供专业知识， 需要用于高级成像和其他生化测定的工具。作为精神，让·库克（Jean Cook）将提供 分子生物学，蛋白质表达和纯化的专业知识和指导。作为贡献者，凯文 SLEP将提供蛋白质纯化和生化测定的设备和专业知识。作为第二 Arshad Desai撰稿人将为训练和专家提供秀丽隐杆线虫遗传学和功能测定法。