CAREER: Analysis of TACC Protein Function in Centrosome Function and Mitotic Spindle Assembly

这项研究的总体目标是增加我们对中心体促进细胞组织的分子机制的理解。中心体是一种非膜结合的细胞器，是动物细胞中主要的微管组织中心。通过对微管的影响，中心体参与了许多基本的细胞过程，最显著的是细胞分裂。尽管中心体对细胞生物学的重要性和一个多世纪的研究，中心体的结构、功能和组成的许多方面仍然未知。例如，迄今为止对中心体蛋白如何组装成功能性微管组织中心知之甚少。新出现的证据表明中心体的功能需要TACC-TOG复合物。TACC-TOG复合物被认为可以稳定新形成的微管，但Wiese博士实验室的初步证据表明，它们也可能参与中心体成分的运输和递送。关于TACC-TOG复合物仍然存在的重要问题是它们如何与中心体结合以及它们如何调节微管的形成和稳定。本项目的目的是了解TACC-TOG复合物如何促进中心体功能。利用爪蟾TACC蛋白mask进行的初步研究表明：(1)mask在纺锤体组装中具有中心体依赖性和非中心体依赖性，(2)mask的磷酸化受输入蛋白调控，(3)mask与纺锤体组装因子以及参与翻译调节的蛋白质相互作用，(4)环己亚胺（一种常用的蛋白质翻译抑制剂）破坏爪蟾卵提取物中的纺锤体组装。体外实验和爪蟾卵提取物将作为实验系统，继续深入分析调节面罩的分子机制，并解决以下基本问题：(1)TACC-TOG复合物如何与中心体相互作用？(2) TACC-TOG复合物如何稳定远离中心体的微管？(3)平移过程与主轴装配之间有什么联系？智力优势：这项研究的完成将提供比目前更详细的TACC蛋白如何影响微管形成的机制图片。更广泛地说，这项工作将最终有助于阐明动物中心体如何参与微管的形成和组织。由于中心体缺陷可导致人类和动物的细胞周期阻滞、细胞死亡、胚胎致死和雄性不育，因此本研究的影响远远超出了细胞和发育生物学，对动物生物学的许多分支，包括农业上重要物种的研究具有重要意义。更广泛的影响：该职业项目包括通过与在细胞和分子生物学、生物化学和生物物理学方面受过高级培训的人员合作来加强科学基础设施的活动。对本科生、研究生和研究生研究助理（包括代表性不足的少数民族和妇女）进行现代细胞生物学技术培训将是拟议研究和教育计划的一个组成部分，并将包括为更多高级研究受训者提供获得宝贵教学和指导技能的机会。本科生将从拟议的研究中受益，通过获得实践经验和体验改进的教学技能。该项目的一个重要组成部分和更广泛的影响是通过在威斯康星大学麦迪逊分校生物化学系开发一个新的教学实习项目来吸引和培训未来的教师，该项目专门针对培养研究生和研究生水平的科学家，使其成为有效的教师和教育者。该教育计划的其他目标是开发新的“主动学习”教学材料，并作为当地和全国其他教育机构的榜样。该实习项目由“教学即研究”的概念推动，并将建立在美国国家科学基金会资助的研究、教学和学习整合中心（CIRTL）的基础设施和支持基础上，该中心的三角洲研究、教学和学习项目，以及威斯康星大学麦迪逊分校的威斯康星科学教学项目。

The overall goal of this research is to increase our understanding of the molecular mechanisms by which centrosomes contribute to cellular organization. The centrosome is a non-membrane-bound organelle that serves as the major microtubule-organizing center of animal cells. Through its influence on microtubules, the centrosome is involved in many fundamental cellular processes, most notably cell division. Despite its importance to cell biology and more than a century of scrutiny, many aspects of centrosome structure, function, and composition remain unknown. For example, little is known to date about how centrosomal proteins are assembled into functional microtubule organizing centers. Emerging evidence suggests that centrosome function requires TACC-TOG complexes. TACC-TOG complexes are thought to stabilize newly-formed microtubules, but preliminary evidence from Dr. Wiese's laboratory suggests that they may also be involved in the transport and delivery of centrosomal components. Important questions that remain regarding TACC-TOG complexes are how their association with the centrosome is regulated and how they regulate microtubule formation and stabilization. The objective of this project is to understand how TACC-TOG complexes contribute to centrosome function. Preliminary studies using the Xenopus TACC protein, maskin, show that (i) maskin has both centrosome dependent and centrosome independent roles in spindle assembly, (ii) the phosphorylation of maskin is regulated by importin, (iii) maskin interacts with spindle assembly factors as well as proteins involved in translational regulation, and (iv) cycloheximide, a commonly-used inhibitor of protein translation, disrupts spindle assembly in Xenopus egg extracts. In vitro assays and Xenopus egg extracts will be used as experimental systems to continue the in-depth analysis of the molecular mechanisms regulating maskin, and to address the following essential questions: (1) how do TACC-TOG complexes interact with the centrosome? (2) How do TACC-TOG complexes stabilize microtubules away from the centrosome? And (3) what is the connection between the processes of translation and spindle assembly?Intellectual Merit: Completion of this research will provide, in greater detail than currently available, a mechanistic picture of how TACC proteins affect microtubule formation. More broadly, this work will ultimately help elucidate how animal centrosomes participate in the formation and organization of microtubules. Because centrosome defects can result in cell cycle arrest, cell death, embryonic lethality, and male infertility in humans and animals, the impact of this research extends far beyond cell and developmental biology to have important implications for many branches of animal biology, including the study of agriculturally important species.Broader Impact: This CAREER project includes activities that enhance scientific infrastructure via collaborations that involve personnel with advanced training in cell and molecular biology, biochemistry, and biophysics. Training in modern cell biology techniques of undergraduate, graduate, and post-graduate research assistants (including underrepresented minorities and women) will be an integral aspect of the proposed research and educational plan, and will include the opportunity for more senior research trainees to obtain valuable teaching and mentoring skills. Undergraduate students will benefit from the proposed research by gaining hands-on experience and experiencing improved teaching skills. A significant component and broader impact of this project is to engage and train future faculty through the development of a new teaching internship program in the Biochemistry Department at UW-Madison that is specifically aimed at training graduate and post-graduate level scientists to become effective teachers and educators. Additional goals of this education plan are to develop new 'active learning' instructional materials, and to serve as a model for others locally and nationwide. The internship program is driven by the concept of 'teaching-as-research' and will be built on infrastructure and support available from the NSF-funded Center for the Integration of Research, Teaching, and Learning (CIRTL), its Delta Program for Research, Teaching and Learning, and the Wisconsin Program for Scientific Teaching on the UW-Madison campus.