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

我在哥伦比亚大学的理查德·瓦利博士的实验室读研究生时， 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）马达蛋白动力蛋白，附着于细胞内货物如囊泡和动粒。我 对动力蛋白在动粒中的功能的研究吸引了我去理解这个细胞器的功能 我选择了位于查佩尔山的北卡罗来纳州大学的泰德·萨尔蒙博士的实验室 进行这项研究。在Salmon实验室，我研究了动粒如何稳定地附着在纺锤体MT上， 有丝分裂时染色体的可靠分离。尽管细胞生物学一直是 对于我的大部分博士后研究，我意识到我需要获得新的技能， 分子生物学，生物化学和遗传学是成功的，在我未来的研究努力，并能够 继续为有丝分裂研究界做出最高水平的贡献。NIH途径， 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，对有丝分裂研究领域产生影响。在初始 我在Salmon实验室（与Jean Cook实验室合作）的博士后研究任期的一个阶段， 在动粒微管中发现了一种新的复制许可蛋白Cdt 1的动粒功能 (kMT)依恋和我使用细胞生物学工具进行了广泛的研究，以了解其 [Nature Cell Biology 2012，Vol.14（6）]。这项研究表明，Cdt 1执行其 有丝分裂作用与真正的动粒蛋白Hec 1有关，Hec 1在kMT附着中的功能很好 记录在案。此外，已经证明Ndc 80复合物通过一种新的途径将Cdt 1募集到着丝粒中。 独特的环结构域，这也被发现是重要的kMT附件。了解Cdt 1的作用 和Ndc 80环更详细，我需要广泛地采用一套新的技能，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标是#1）阐明Cdt 1 和Ndc 80复合物协调以控制kMT附着，和#2）探测Ndc 80环结构域和/或 Cdt 1需要募集其他微管相关蛋白（MAP）到着丝粒中，以帮助稳定的kMT 附件的权限.我将使用生物化学，先进的细胞生物学和遗传学手段测试Cdt 1是否直接结合到 环结构域和MT充当这两个组件之间的桥梁，并且如果这种相互作用 招募其他地图很重要。作为共同导师，特德鲑鱼和詹妮弗德卢卡将提供专业知识， 先进的成像和其他生化分析所需的工具。作为导师，吉恩·库克将提供 分子生物学、蛋白质表达和纯化方面的专业知识和指导。作为一个贡献者，凯文 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，阿尔沙德德赛将提供培训和专业知识与C。线虫遗传学和功能测定。

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.