Kinesin-Like Proteins in the Mitotic Mechanism

智力优势：微管激酶样蛋白对有丝分裂机制至关重要。为了有效，它们的作用还必须暂时整合在纺锤体机制中，以确保在有丝分裂结束之前染色体分裂。Kinesin-14家族蛋白在真核生物中普遍存在，能够通过控制微管组织、动力学和纺锤体蛋白的锚定来影响有丝分裂的进程。该项目将为有丝分裂机制的三个重要方面提供见解：(1)运动蛋白-微管蛋白相互作用的分子性质；(2) Kinesin-14 plkl1控制纺锤体和微管参数以及所需的蛋白复合物；(3) Kinesin-14 plkl1p控制中期后Mad2p转变（双极到单极到赤道）及其对有丝分裂结束的重要性。关于(1)方面，Paluh博士将验证这样的假设，即驱动蛋白- γ -微管蛋白在两极的相互作用是为驱动蛋白-14家族蛋白保留的，而微管微管蛋白包含了多个驱动蛋白家族结合的额外信息。目前还没有在分子水平上定义动蛋白-微管蛋白相互作用的信息，这是该领域的关键机制组成部分。关于(2)方面，双极主轴装配的潜在机制仍然是根本未知的。Paluh博士之前的研究工作在这个过程中发现了Kinesin 14 plkl1p和γ -微管蛋白的复合物。在(3)方面，Mad2p是泛素介导的蛋白水解的关键调节因子，并通过抑制APC/C，后期促进复合体/环小体，提供有丝分裂进程的时间控制。APC/C泛素连接酶调节姐妹染色单体内聚和有丝分裂后期退出。Mad2p在着丝点上监测后期前的染色体微管附着和张力。Paluh博士之前的研究工作将Mad2p的保守运动与有丝分裂退出途径中的极点和sin样纺锤体转移联系起来。Paluh博士在分析有丝分裂机制的各个方面拥有丰富的细胞周期专业知识，并掌握了用于监测染色体分离、微管动力学、纺锤体蛋白和检查点机制的试剂。她将使用各种不同的方法，包括比较序列分析、结构分析、诱变、有丝分裂的延时视频显微镜、遗传分析和使用gst拉下、共免疫沉淀和酵母双杂交测定的分子生化方法。更广泛的影响：该研究将积极地将有丝分裂机制的新发现与本科培训联系起来，这在促进学生参与各级科学研究和争取少数民族参与方面有着良好的记录。帕鲁博士在教学和研究方面培养本科生方面有着堪称典范的记录，帕鲁博士以前培养的学生正在继续从事富有成效和公认的科学事业。迄今为止，帕鲁博士已经培训了11名本科生研究人员。在这些学生中，有六名学生能够参加国内和国际科学会议，其中一名学生在会议上作为演讲者介绍了这项工作。本科生研究人员对支持该项目的初步研究结果做出了广泛贡献。七名学生共同撰写了与本项目相关的会议摘要或手稿。培养了两名少数民族女大学生；其中之一阿德里安娜·罗德里格斯（Adrianna Rodriguez）最近获得了波士顿学院青年校友科学奖。在Paluh博士的直接指导下，学生们在发展严谨的科学方法的同时，对细胞周期控制机制有了全面的了解。本项目中使用的实验方法范围可供本科生研究人员使用，使他们能够半独立地工作。

Intellectual Merit: Microtubule kinesin-like proteins are crucial to the mitotic mechanism. To be effective, their actions must also integrate temporally in the spindle mechanism to ensure chromosome partitioning before mitotic exit. Kinesin-14 family proteins are ubiquitous in eukaryotes and able to effect mitotic progression through control of microtubule organization, dynamics and anchoring of spindle proteins. This project will provide insights into three important aspects of the mitotic mechanism: (1) the molecular nature of kinesin-tubulin interactions; (2) Kinesin-14 Pkl1 controlled spindle and microtubule parameters and required protein complexes; and (3) Kinesin-14 Pkl1p control of post-metaphase Mad2p transitions (bipolar to unipolar to equatorial) and their importance to mitotic exit. With regard to aspect (1), Dr. Paluh will test the hypothesis that kinesin-gamma-tubulin interactions at poles are reserved for Kinesin-14 family proteins, while microtubule tubulins contain additional information for binding of multiple kinesin families. No information is currently available at the molecular level that defines kinesin-tubulin interactions, which is a critical mechanistic component for the field. With regard to aspect (2), the underlying mechanism of bipolar spindle assembly remains fundamentally unknown. Dr. Paluh's prior research work identified a complex of Kinesin 14 Pkl1p and gamma-tubulin in this process. With regard to aspect (3), Mad2p is a key regulator of ubiquitin-mediated proteolysis and provides temporal control of mitotic progression by inhibiting the APC/C, anaphase promoting complex/cyclosome. The APC/C ubiquitin ligase regulates both sister chromatid cohesion and mitotic exit after anaphase. Mad2p at kinetochores monitors chromosome-microtubule attachment and tension preceding anaphase. Dr. Paluh's prior research work links Mad2p conserved movement to poles and SIN-like spindle transitions in mitotic exit pathways. Dr. Paluh has extensive cell cycle expertise in analysis of all aspects of the mitotic mechanism and reagents are in hand to monitor chromosome segregation, microtubule dynamics, spindle proteins, and checkpoint mechanisms. She will use a variety of different approaches, including comparative sequence analysis, structural analysis, mutagenesis, time-lapse video microscopy of mitosis, genetic analysis and molecular biochemical approaches that employ GST-pull downs, co-immunoprecipitation, and yeast two-hybrid assays. Broader Impact: The research will actively link new discoveries in the mitotic mechanism with undergraduate training that has a track record for promoting student participation at all levels of scientific investigation and strives for minority involvement. Dr. Paluh has an exemplary record of training undergraduates in teaching and research, and the students previously trained by Dr. Paluh are continuing in productive and recognized science careers. Eleven undergraduate researchers have been trained by Dr. Paluh to date. Of these, six students were able to attend national and international scientific meetings, including one who presented the work as a speaker at the meeting. Undergraduate researchers contributed extensively to preliminary findings that support this project. Seven students are coauthors on meeting abstracts or manuscripts related to this project. Two minority women undergraduates have been trained; one of these, Adrianna Rodriguez, was recently awarded the Boston College Young Alumni in Science Award. Under Dr. Paluh's direct tutelage, students acquire a comprehensive understanding of cell cycle control mechanisms while developing the rigors of scientific methodology. The range of experimental methodologies used in this project are accessible by undergraduate researchers, allowing them to work semi-independently.