The Kinetochore-Microtubule Interface in Vertebrate Cells

描述（由申请人提供）：当细胞发生遗传变化，使其能够在无视正常生长限制的情况下生存和繁殖时，可能会发生癌症。当这些异常细胞侵入并定居在通常为其他细胞保留的外来区域时，癌症进展就会发生。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，所有人类实体瘤最常见的标志是存在非整倍体细胞。我们必须了解细胞为防止非整倍体而发展的机制，非整倍体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其动粒与有丝分裂纺锤体的连接。我们刚刚开始了解这个接口的分子性质，并已取得了相当大的进展，确定一个关键的动粒复合物，是必不可少的动粒微管（MT）的附件。Ndc 80复合物由4种蛋白质组成：Ndc 80（Hec 1在人类癌症中高度表达），Nuf 2，Spc 24和Spc 25。这种复合物不仅在运动舞蹈-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于防止错误细胞分裂的重要抗增殖机制。对于癌症研究重要的是，人类细胞耗尽Nuf 2和Hec 1在有丝分裂中停滞，随后经历凋亡。Hec 1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，RNA干扰Hec 1抑制体内肿瘤生长，使其成为抗癌治疗的重要靶点。我们建议进一步研究Hec 1和Ndc 80复合物，通过（1）描绘Hec 1的功能区域，重点是它在有丝分裂检查点，凋亡细胞死亡，以及在运动舞蹈MT形成和调节中的作用，（2）鉴定和表征与Hec 1相互作用的蛋白质，和（3）确定Nuf 2（也是Ndc 80复合物的成员）在人动粒功能中的作用。我将在Ted Salmon教授的指导下开始这些研究，并在获奖的第二年过渡到一个独立的实验室。指导的一年将使我获得额外的工具，这将补充我在生物化学和细胞生物学的技能，使我将处于有利地位，使有丝分裂和癌症生物学领域的重大贡献。

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.