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

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。 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(Microtubule，MT)运动蛋白Dynein，附着在细胞内的小泡和动粒上。我的 动力蛋白在运动中枢的功能研究吸引了我对这个细胞器的功能的理解 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泰德·萨蒙博士的实验室 进行这项研究。在鲑鱼实验室，我研究了kintochres如何稳定地附着在纺锤体MTS上 有丝分裂期间染色体的忠实分离。尽管细胞生物学一直是主要的工具 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，形式是 分子生物学、生物化学和遗传学在我未来的研究工作中取得成功，并能够 继续为有丝分裂研究社区做出最高水平的贡献。美国国立卫生研究院通往 独立奖将为我提供必要的基础设施和支持，以生产试剂 并开发出独立的、对有丝分裂研究领域有影响的工具。在最初的 我在鲑鱼实验室(与Jean Cook实验室合作)的博士后研究任期的第一阶段，我有 在动粒微管中发现了复制许可蛋白CDT1的新动粒功能 (国民党)Attach和我利用细胞生物学工具进行了广泛的研究，以了解其 在这方面发挥作用[《自然细胞生物学》，2012年第14(6)卷]。这项研究揭示了cdt1执行其 与真正的动粒蛋白Hec1相关的有丝分裂作用，Hec1在KMT附着中的作用很好 有记录在案。此外，还证明了Ndc80复合体通过一种 唯一的环状结构域，也被发现对KMT附件很重要。了解CDT1的作用 更详细地说，我需要广泛地使用一套新的技能，涉及分子 生物学、生物化学和遗传学。我提议的研究的具体目的是：1)阐明CDT1是如何 并且NDC80复合体协调以控制KMT附着物，以及#2)探测NDC80环路结构域和/或 CDT1需要招募其他微管相关蛋白(MAP)到运动中枢，以帮助稳定的KMT 附属品。我将使用生化、先进的细胞生物学和遗传学手段测试CDT1是否直接结合到 环域和MT用作这两个组件之间的桥梁，并且如果该交互是 重要的是招募其他地图。作为共同导师，Ted Salmon和Jennifer Deluca将提供专业知识和 需要用于高级成像和其他生化分析的工具。作为一名导师，让·库克将提供 在分子生物学、蛋白质表达和纯化方面的专业知识和指导。作为贡献者，凯文 SLEP将提供蛋白质纯化和生化分析方面的设备和专业知识。作为第二名 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。

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.