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

描述(由申请人提供)：我在哥伦比亚大学理查德·瓦莱博士的实验室读研究生时接受了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，研究微管(MT)马达蛋白Dynein是如何附着在细胞内的小泡和动粒等货物上的。我在运动中枢对动力蛋白功能的研究吸引了我对这种细胞器在博士后工作中的功能的理解，我选择了北卡罗来纳大学教堂山分校的特德·萨蒙博士的实验室进行这项研究。在鲑鱼实验室，我研究了动点如何稳定地附着在纺锤体MT上，以便在有丝分裂期间忠实地分离染色体。尽管细胞生物学一直是我大部分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、生物化学和遗传学形式的新技能，才能在未来的研究努力中取得成功，并能够继续为有丝分裂研究社区做出最高水平的贡献。NIH独立之路奖将为我提供必要的基础设施和支持，以产生试剂和开发工具，使其独立并对有丝分裂研究领域产生影响。在我在Salmon实验室从事博士后研究的最初阶段(与Jean Cook实验室合作)，我发现了动粒微管(KMT)附件中复制许可蛋白CDT1的一种新的动粒功能，并利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下的功能[自然细胞生物学，2012年第14卷(6)]。这项研究揭示了CDT1与一种真正的动粒蛋白Hec1有关的有丝分裂作用，Hec1在KMT附着中的作用已经得到了很好的证明。此外，还证明了Ndc80复合体通过一个独特的环结构域将CDT1招募到动点中心，这也被发现对KMT的附着是重要的。为了更详细地了解CDT1和Ndc80环的作用，我需要广泛使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的是：1)阐明CDT1和Ndc80复合体是如何协调控制KMT附着的；2)探测Ndc80环域和/或CDT1是否需要招募其他微管相关蛋白(MAP)到动点，以帮助稳定的KMT附着。我将使用生化、先进的细胞生物学和遗传学手段测试CDT1是否直接与环区和MTS结合，以作为这两个成分之间的桥梁，以及这种相互作用是否对招募其他图谱很重要。作为联合导师，Ted Salmon和Jennifer Deluca将为先进的成像和其他所需的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方面的设备和专业知识。作为第二个贡献者，Arshad Desai将提供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.