The Kinetochore-Microtubule Interface in Vertebrate Cells

描述(由申请人提供)：当细胞经历了允许其在无视正常生长限制的情况下存活和繁殖的基因变化时，就会发生癌症起始。当这些异常细胞侵入并在通常为其他细胞保留的外来区域定居时，就会发生癌症进展。非整倍体，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，所有人类实体肿瘤最常见的特征是存在非整倍体细胞。我们必须了解细胞已经发展起来防止非整倍体的机制，这是由不准确的染色体分离产生的。精确度关键取决于有丝分裂染色体通过其着丝点与有丝分裂纺锤体的连接。我们刚刚开始了解这种界面的分子性质，并在识别对动粒-微管(MT)连接至关重要的关键动粒复合体方面取得了相当大的进展。Ndc80复合体由4个蛋白质组成：Ndc80(在人类中Hec1在癌症中高表达)，Nuf2，Spc24和Spc25。这个复合体不仅在动粒-MT连接中发挥作用，而且在有丝分裂检查点也有作用，有丝分裂检查点是细胞用来防止错误细胞分裂的重要的抗增殖机制。对癌症研究很重要的是，人类细胞耗尽了Nuf2和Hec1，阻止了有丝分裂，随后发生了凋亡。Hec1是一个关键的动粒成分，我们将继续研究，因为最近的证据表明，针对Hec1的RNA干扰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式进一步研究Hec1和Ndc80复合体：(1)描述Hec1的功能区，重点是它在有丝分裂检查点、细胞凋亡和动粒-MT形成和调节中的作用；(2)鉴定和鉴定与Hec1相互作用的蛋白质；(3)确定Nuf2(也是Ndc80复合体的成员)在人类动粒功能中的作用。我将在泰德·萨蒙教授的指导下启动这些研究，并在获奖的第二年过渡到一个独立的实验室。导师的这一年将使我获得更多的工具，这些工具将补充我在生物化学和细胞生物学方面的技能，这样我将处于有利地位，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.