Formin mDia Functions in Mitosis

描述（由申请人提供）：有丝分裂中每条染色体的准确传递对于每次细胞分裂中遗传信息的忠实传递至关重要。这个过程中的错误导致染色体数量异常（一种被称为非整倍体的情况），在发育早期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是双向的，一个着丝点从双极纺锤体的一端附着在微管上，而另一个姐妹着丝点从另一端附着在微管上。不正确的染色体附着，如合成的和分裂的附着，经常发生在有丝分裂中。有丝分裂领域的一个长期存在的问题是细胞如何实现适当稳定的端部着丝点微管附着。利用体外培养的哺乳动物细胞和纯化的成分，我们打算确定formin mDia3如何在着丝点中发挥新作用，以促进稳定的微管附着。我们已经证明，在哺乳动物培养的细胞中，使用siRNA敲低mDia3会导致中期染色体排列缺陷和稳定的着丝点微管附着。这些缺陷可以通过抗sirna野生型mDia3构建体和肌动蛋白成核缺陷但微管稳定缺陷的mDia3突变体来修复。我们进一步证明mDia3在体外被Aurora B激酶磷酸化，并且细胞中表达的不可磷酸化的mDia3突变体具有染色体错位表型。利用微管结合缺陷和EB1结合缺陷的mDia3突变体来挽救mDia3 siRNA耗损表型，我们将确定着丝点上的mDia3是通过其微管活性直接起作用，还是通过其相互作用伙伴EB1和APC间接起作用，以促进稳定的着丝点微管附着。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，Ndc80复合物是着丝点上的核心微管结合力。通过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。我们将使用纯化组分研究Aurora B磷酸化如何影响mDia3微管结合和体外稳定活性。我们还将研究由非磷酸化或拟磷mDia3突变体的表达引起的有丝分裂表型。

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.