Formin mDia Functions in Mitosis

Formin mDia 在有丝分裂中的功能

• 批准号: 8038636
• 负责人: Yinghui Mao
• 金额: $ 30.16万
• 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/8038636
• 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. PUBLIC HEALTH RELEVANCE: Accurate delivery of one copy of each chromosome is essential every time a cell duplicates, a process that takes place millions of times every day in every individual. Defects in the misdistribution of chromosomes are frequently found in cancers. Understanding the molecular mechanisms may prove crucial for improving our understanding of cancer biology and for the development of new drugs in cancer treatment.

描述（由申请人提供）：准确地递送有丝分裂中每个染色体的一个副本对于在每个细胞分裂过程中忠实传播遗传信息至关重要。在此过程中的错误导致染色体数量异常（一种描述为非整倍性的疾病），在发育早期导致致命的发育缺陷，后来是人类肿瘤进展的标志。适当的染色体分离要求每个染色体都以双极纺锤体的一个极点固定在微管上双向，而另一个姐妹动型却附着在相对极点上的微管上。有丝分裂经常发生染色体附着（例如合成和商制附着）的不当染色体附着。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的稳定末端动力学微管附着。使用哺乳动物培养的细胞和纯化的成分体外，我们打算确定Formin MDIA3在动力学中如何在稳定的微管附着中起新颖的作用。我们已经表明，使用siRNA在哺乳动物培养的细胞中敲低MDIA3会导致中期染色体比对中的缺陷和稳定的Kinetochore微管附着。这些缺陷可以通过在肌动蛋白成核中有缺陷的抗siRNA耐siRNA野生型MDIA3构建体和MDIA3突变体挽救，但在微管稳定中却没有。我们进一步表明，MDIA3在体外被Aurora B激酶磷酸化，并且在细胞中非磷酸化的MDIA3突变体的表达具有染色体未对准表型。使用微管结合和EB1结合缺陷的MDIA3突变体来拯救MDIA3 siRNA耗竭表型，我们将确定Kinetochore的MDIA3是否可以通过其微管活性直接作用于其微管活性或通过其相互作用型eb1和APC的稳定kinetable microutcodpon contectect kinetable microutectioncocy contection contection contection sirtaction Active或Indirectionctional活性。我们将使用纯化的组件的微管共同补习分析来确定MDIA3与EB1和APC的稳定结合与微管晶格是否会影响NDC80复合物的微管结合亲和力，该亲和力已与Kinetochore的核心微管结合力牵涉。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校正微管附着误差的机制的一部分。我们将研究Aurora B磷酸化如何使用纯化的成分在体外影响MDIA3微管结合和稳定活性。我们还将检查由非磷酸化或磷酸化MDIA3突变体的表达产生的有丝分裂表型。 公共卫生相关性：每当单元重复时，必须准确地交付每个染色体的副本，这一过程每天每天发生数百万次。染色体错误分布的缺陷经常在癌症中发现。了解分子机制对于提高我们对癌症生物学的理解以及在癌症治疗中的新药物的发展可能至关重要。