The Kinetochore-Microtubule Interface in Vertebrate Cells

描述（由申请人提供）：当细胞经历基因变化，使其能够在正常生长限制下存活和繁殖时，癌症就会发生。当这些异常细胞侵入并殖民到通常为其他细胞保留的外来区域时，癌症就会发生进展。非整倍性，即少量完整染色体的丢失或获得，现在被认为是人类癌症发生和发展的主要原因。事实上，所有人类实体瘤最常见的特征是存在非整倍体细胞。我们必须了解细胞发展的机制，以防止非整倍性，这是由不准确的染色体分离产生的。准确性主要取决于有丝分裂染色体通过着丝点附着到有丝分裂纺锤体上。我们刚刚开始了解这种界面的分子性质，并在确定着丝点与微管（MT）连接所必需的关键着丝点复合体方面取得了相当大的进展。Ndc80复合体由4种蛋白组成：Ndc80（人类中在癌症中高表达的Hec1）、Nuf2、Spc24和Spc25。该复合体不仅在着丝点- mt附着中起作用，而且在有丝分裂检查点中起作用，有丝分裂检查点是细胞用来防止错误细胞分裂的重要抗增殖机制。在癌症研究中，Nuf2和Hec1缺失的人类细胞在有丝分裂中受阻，随后发生凋亡。Hec1是一个关键的着丝粒成分，我们将继续研究，因为最近的证据表明，RNA干扰Hec1可以抑制体内肿瘤的生长，使其成为抗癌治疗的重要靶点。我们建议通过以下方法进一步研究Hec1和Ndc80复合体：(1)描述Hec1的功能区域，重点研究其在有丝分裂检查点、凋亡细胞死亡和着丝粒- mt形成和调控中的作用；(2)鉴定和表征与Hec1相互作用的蛋白质；(3)确定Nuf2（也是Ndc80复合体的成员）在人类着丝粒功能中的作用。我将在Ted Salmon教授的指导下开始这些研究，并在第二年过渡到一个独立的实验室。这一年的指导将使我获得额外的工具，以补充我在生物化学和细胞生物学方面的技能，因此我将处于一个强有力的位置，为有丝分裂和癌症生物学领域做出重大贡献。

DESCRIPTION (provided by applicant): Cancer initiation can occur when a cell undergoes a genetic change that allows it to survive and reproduce in defiance of normal growth constraints. Cancer progression occurs when these aberrant cells invade and colonize in foreign areas that are normally reserved for other cells. Aneuploidy, the loss or gain of a small number of whole chromosomes, is now thought to be a major contribution to initiation and progression of human cancer. In fact, the most common hallmark of all human solid tumors is the presence of aneuploid cells. It is essential that we understand the mechanisms that cells have developed to prevent aneupoidy, which is produced by inaccurate chromosome segregation. Accuracy depends critically on attachment of mitotic chromosomes via their kinetochores to the mitotic spindle. We are just beginning to understand the molecular nature of this interface, and have made considerable progress in identifying a key kinetochore complex that is essential for kinetochore-microtubule (MT) attachment. The Ndc80 complex is comprised of 4 proteins: Ndc80 (Hec1 in humans for Highly Expressed in Cancer), Nuf2, Spc24, and Spc25. This complex not only has roles in kinetochore-MT attachment, but also in the mitotic checkpoint, an important anti-proliferation mechanism cells use to prevent erroneous cell division. Important for cancer research, human cells depleted of Nuf2 and Hec1 arrest in mitosis and subsequently undergo apoptosis. Hec1 is a key kinetochore component that we will continue to study as recent evidence has indicated that RNA interference against Hec1 inhibits tumor growth in vivo, making it an important target for anti-cancer therapies. We propose to further study Hec1 and the Ndc80 complex by (1) delineating the functional regions of Hec1 with emphasis on its roles in the mitotic checkpoint, in apoptotic cell death, and in kinetochore-MT formation and regulation, (2) identifying and characterizing proteins that interact with Hec1, and (3) determining the role of Nuf2 (also a member of the Ndc80 complex) in human kinetochore function. I will initiate these studies under the mentorship of Professor Ted Salmon and transition into an independent laboratory during the second year of the award. The mentored year will allow me to gain additional tools that will complement my skills in biochemistry and cell biology, so that I will be in a strong position to make significant contributions to the fields of mitosis and cancer biology.