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

描述（由申请人提供）：我在哥伦比亚大学Richard Vallee博士的实验室学习，主修细胞生物学。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）运动蛋白动力蛋白如何附着在细胞内的货物上，如囊泡和着丝点。我对动丝点动力蛋白功能的研究吸引了我去了解这种细胞器的功能作为我的博士后工作，我选择了北卡罗来纳大学教堂山分校泰德·萨尔蒙博士的实验室进行这项研究。在Salmon实验室，我研究着丝点如何稳定地附着在纺锤体mt上，以便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博士后研究的主要工具，但我意识到我需要获得分子生物学、生物化学和遗传学方面的新技能，以便在我未来的研究中取得成功，并能够继续为有丝分裂研究界做出最高水平的贡献。美国国立卫生研究院独立之路奖将为我提供必要的基础设施和支持，以产生试剂和开发独立的工具，并对有丝分裂研究领域产生影响。在我在Salmon实验室（与Jean Cook实验室合作）进行博士后研究的初始阶段，我发现了着丝粒微管（kMT）附着中复制许可蛋白Cdt1的一种新的着丝粒功能，并使用细胞生物学工具进行了广泛的研究，以了解其在这种能力中的功能[Nature cell Biology 2012， Vol. 14(6)]。这项研究表明，Cdt1与真正的着丝点蛋白Hec1一起发挥其有丝分裂作用，其在kMT附着中的功能已被充分记录。此外，研究表明Ndc80复合体通过一个独特的环结构域将Cdt1招募到着丝点上，这对kMT附着也很重要。为了更详细地理解Cdt1和Ndc80环的作用，我需要广泛地运用一套涉及分子生物学、生物化学和遗传学的新技能。我提出的研究的具体目标是#1)阐明Cdt1和Ndc80复合物如何协调控制kMT附着，#2)探测Ndc80环结构域和/或Cdt1是否需要招募其他微管相关蛋白（MAPs）到着丝点以帮助稳定的kMT附着。我将使用生化、高级细胞生物学和遗传学手段测试Cdt1是否直接与环结构域和mt结合，作为这两个成分之间的桥梁，进一步测试这种相互作用是否对招募其他map很重要。作为共同导师，Ted Salmon和Jennifer Deluca将为高级成像和其他生化分析提供专业知识和工具。作为导师，Jean Cook将提供分子生物学、蛋白质表达和纯化方面的专业知识和指导。作为贡献者，Kevin Slep将提供蛋白质纯化和生化分析的设备和专业知识。作为第二个贡献者，Arshad Desai将提供秀丽隐杆线虫遗传学和功能分析方面的培训和专业知识。

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.