Formin mDia Functions in Mitosis

Formin mDia 在有丝分裂中的功能

• 批准号: 8542868
• 负责人: Yinghui Mao
• 金额: $ 36.25万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8542868
• 关键词: Actins; Affect; Affinity; Aneuploidy; Binding; Binding Sites; Biochemical; Biological Assay; Cancer Biology; Cell division; Cells; Chromosome Segregation; Chromosomes; Complex; Cultured Cells; Data; Defect; Development; Filament; Genetic; Goals; Human; Immunofluorescence Immunologic; In Vitro; Individual; Kinetochores; Lead; Life; Malignant Neoplasms; Metaphase; Metaphase Plate; Microtubule Bundle; Microtubule Stabilization; Microtubules; Mitosis; Mitotic; Molecular; Mutation; N-terminal; Pharmaceutical Preparations; Phenotype; Phosphorylation; Phosphorylation Site; Play; Process; Prometaphase; Protein Isoforms; Regulation; Reporting; Resistance; Role; Sedimentation process; Sister; Site; Small Interfering RNA; Testing; Time; aurora B kinase; base; cancer therapy; cellular imaging; improved; in vitro activity; mutant; novel; segregation; transmission process; tumor progression

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）：有丝分裂中每条染色体的一份副本的准确传递对于每次细胞分裂期间遗传信息的忠实传递至关重要。这一过程中的错误会导致染色体数量异常（一种称为非整倍性的情况），这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。正确的染色体分离要求每条染色体都是双向的，一个着丝粒从双极纺锤体的一个极附着到微管，而另一个姐妹着丝粒从相反的极附着到微管。有丝分裂中经常发生不正确的染色体附着，例如合成附着和杂裂附着。有丝分裂领域长期存在的问题是细胞如何实现适当稳定的着丝粒微管末端附着。使用哺乳动物培养的细胞和体外纯化的成分，我们打算确定福尔马林mDia3如何在动粒中发挥新的作用，有助于稳定的微管附着。我们已经证明，使用 siRNA 在哺乳动物培养细胞中敲低 mDia3 会导致中期染色体排列和稳定着丝粒微管附着的缺陷。这些缺陷可以通过 siRNA 抗性野生型 mDia3 构建体和肌动蛋白成核缺陷但微管稳定性缺陷的 mDia3 突变体来弥补。我们进一步表明，mDia3 在体外被 Aurora B 激酶磷酸化，并且细胞中不可磷酸化的 mDia3 突变体的表达具有染色体错位表型。使用微管结合和 EB1 结合缺陷的 mDia3 突变体来拯救 mDia3 siRNA 耗尽表型，我们将确定着丝粒处的 mDia3 是否可以直接通过其微管活性发挥作用，或通过其相互作用伙伴 EB1 和 APC 间接发挥作用，从而有助于稳定的着丝粒微管附着。我们将使用纯化成分的微管共沉降分析来确定 mDia3 以及 EB1 和 APC 与微管晶格的稳定结合是否会影响 Ndc80 复合物的微管结合亲和力，该复合物被认为是动粒处的核心微管结合力。使用免疫荧光分析和活细胞成像，我们将继续确定 mDia3 的 Aurora B 磷酸化是否可能代表微管附着错误校正机制的一部分。我们将使用纯化的组分在体外研究 Aurora B 磷酸化如何影响 mDia3 微管结合和稳定活性。我们还将检查非磷酸化或拟磷酸化 mDia3 突变体的表达所产生的有丝分裂表型。