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Formin mDia Functions in Mitosis

Formin mDia 在有丝分裂中的功能

基本信息

批准号：
8917249
负责人：
Yinghui Mao
金额：
$ 30.35万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-30 至 2016-12-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Accurate delivery of one copy of each chromosome in mitosis is essential for faithful transmission of genetic information during each cell division. Errors in this process result in abnormal numbers of chromosomes (a condition described as aneuploidy), which early in development lead to lethal developmental defects and later are hallmarks of human tumor progression. Proper chromosome segregation requires that each chromosome is bi-oriented with one kinetochore attaches to microtubules from one pole of a bipolar spindle while the other sister kinetochore is attached to microtubules from the opposite pole. Improper chromosome attachments, such as synthetic and merotelic attachments, frequently occur in mitosis. A long standing question in the mitosis field is how cells achieve proper stable end-on kinetochore microtubule attachment. Using mammalian cultured cells and purified components in vitro, we intend to determine how formin mDia3 plays a novel role at kinetochores in contribute to stable microtubule attachment. We have shown that knockdown of mDia3 in mammalian cultured cells using siRNA results in defects in metaphase chromosome alignment and stable kinetochore microtubule attachment. These defects can be rescued by a siRNA-resistant wild-type mDia3 construct and mDia3 mutants that are defective in actin nucleation, but not in microtubule stabilization. We have further shown that mDia3 is phosphorylated by Aurora B kinase in vitro and expression of a non- phosphorylatable mDia3 mutant in cells had a chromosome misalignment phenotype. Using microtubule- binding- and EB1-binding-deficient mDia3 mutants for rescue of mDia3 siRNA depletion phenotypes, we will determine whether mDia3 at the kinetochore could act directly by its microtubule activity or indirectly via its interaction partners, EB1 and APC, in contributing to stable kinetochore microtubule attachment. We will use microtubule co-sedimentation analysis with purified components to determine whether stably binding of mDia3, along with EB1 and APC, to the microtubule lattice influences the microtubule binding affinity of the Ndc80 complex, which has been implicated as a core microtubule binding force at the kinetochore. Using immunofluorescence analysis and live cell imaging, we will continue to determine whether Aurora B phosphorylation of mDia3 may represent part of the mechanism for correction of microtubule attachment errors. We will examine how Aurora B phosphorylation affects mDia3 microtubule binding and stabilization activities in vitro using purified components. We will also examine the mitotic phenotypes resulted from the expression of either the non-phosphorylatable or the phosphomimetic mDia3 mutants.

描述(由申请人提供)：在有丝分裂中准确传递每个染色体的一个副本对于在每个细胞分裂过程中忠实地传递遗传信息是必不可少的。这一过程中的错误会导致染色体数量异常(这种情况被描述为非整倍体)，这种情况在发育早期会导致致命的发育缺陷，后来成为人类肿瘤进展的标志。适当的染色体分离要求每条染色体都是双向的，一个着丝粒从两极纺锤体的一极附着于微管，而另一姊妹着丝粒从相反的极附着于微管。在有丝分裂中，经常会出现不正确的染色体附着，如合成的和分生组织的附着。有丝分裂领域的一个长期存在的问题是，细胞如何获得适当稳定的端接着动粒微管。利用哺乳动物体外培养的细胞和纯化的成分，我们打算确定Forin mDia3如何在运动中枢发挥新的作用，促进稳定的微管附着。我们已经证明，使用siRNA敲除哺乳动物培养细胞中的mDia3会导致中期染色体排列和稳定的动粒微管附着的缺陷。这些缺陷可以通过siRNA抗性野生型mDia3构建和mDia3突变体来挽救，这些突变体在肌动蛋白核化方面存在缺陷，但在微管稳定性方面没有缺陷。我们进一步证明，mDia3在体外被Aurora B激酶磷酸化，并且在细胞中表达的一个非磷酸化的mDia3突变体具有染色体错位表型。利用微管结合和EB1结合缺陷的mDia3突变体拯救mDia3 siRNA耗竭表型，我们将确定动粒上的mDia3是否直接通过其微管活性或通过其相互作用伙伴EB1和APC间接作用于动粒微管的稳定附着。我们将使用纯化组分的微管共沉淀分析来确定mDia3与EB1和APC稳定地与微管晶格结合是否影响NDC80复合体的微管结合亲和力，这已被认为是动粒上的核心微管结合力。利用免疫荧光分析和活细胞成像，我们将继续确定mDia3的Aurora B磷酸化是否可能是纠正微管附着错误的部分机制。我们将使用纯化的成分在体外检测Aurora B磷酸化如何影响mDia3微管结合和稳定活性。我们还将研究非磷酸化或拟磷酸化mDia3突变体的表达所导致的有丝分裂表型。