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: 8567256
• 负责人: Dileep Varma
• 金额: $ 9.48万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8567256
• 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; Critiques; Development; Dynein ATPase; Embryo; Equipment; Generations; Genetic; Goals; Homologous Gene; Image; Immunofluorescence Immunologic; In Vitro; Individual; 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; Writing; Yeasts; cooking; daughter cell; in vivo; insight; man; meetings; mutant; novel; protein complex; protein expression; protein function; protein purification; public health relevance; research study; skills; tool; yeast two hybrid system

DESCRIPTION (provided by applicant): 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 bona fide 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.

描述（由申请人提供）：我在哥伦比亚大学理查德·瓦利（Richard Vallee）博士的研究生学习期间接受过细胞生物学家的培训。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）运动蛋白动力蛋白如何附着在细胞内货物（如囊泡和动型）上。我对动元的动力蛋白功能的研究吸引了我了解该细胞器的功能，用于我的博士后工作，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，我研究了动力学如何稳定地连接到螺旋MTS上，以在有丝分裂过程中忠实地分离染色体。尽管细胞生物学一直是我大多数博士后研究的主要工具，但我意识到，我需要以分子生物学，生物化学和遗传学的形式获得新的技能，以便在我未来的研究努力中取得成功，并能够不断为有害病研究社区做出最高水平。 NIH独立奖将为我提供必要的基础设施和支持，以生成试剂并开发独立的工具，并对有丝分裂研究领域产生影响。在我在鲑鱼实验室（Jean Cook Lab合作）中我的博士后研究任期的最初阶段，我发现了在动脉底孔（KMT）附件中复制许可蛋白CDT1的新型Kinetochore功能，并且我使用细胞生物学的工具来了解其能力[自然电池的功能[vor nydy Celliogy Intabtil Intical Intaction Inture Intife Intial Intical Incorpliogy of Tocial Intical Intical Intaction Inture Inture Inture Inture Inture Intife of Stripation Intife of Stripation Intife of Stripation of Inture 2012''的功能。 14（6）]。这项研究表明，CDT1与真正的动力学蛋白Hec1相关联，其在KMT附件中的功能已得到充分记录。此外，已经证明，NDC80复合物通过独特的循环域将CDT1募集到动力学，这对KMT附件也很重要。为了更详细地了解CDT1和NDC80循环的作用，我需要广泛采用一套涉及分子生物学，生物化学和遗传学的新颖技能。我拟议的研究的具体目的是＃1）阐明CDT1和NDC80复合物如何控制KMT附件，以及＃2）如果需要NDC80环形域和/或CDT1，则可以进行探测，以募集其他微管蛋白（MAPS）招募其他微管蛋白（MAPS），以帮助KineTochors在稳定的KINETECTEMENTMENTS中。如果CDT1直接与环域和MT结合以作为这两个组件之间的桥梁，并且此相互作用对于募集其他地图很重要，我将使用生化，晚期细胞生物学和遗传方法进行测试。作为联合官员，TED Salmon和Jennifer DeLuca将为所需的高级成像和其他生化分析提供专业知识和工具。作为导师，让·库克（Jean Cook）将通过分子生物学，蛋白质表达和纯化提供专业知识和指导。作为贡献者，Kevin Slep将提供蛋白质纯化和生化测定的设备和专业知识。作为第二贡献者，Arshad Desai将为秀丽隐杆线虫遗传学和功能测定法提供培训和专业知识。