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，是动物细胞的主要微管组织中心。通过对微管的影响，中心体参与了许多基本的细胞过程，尤其是细胞分裂。尽管中心体对细胞生物学很重要，经过一个多世纪的研究，中心体的结构、功能和组成的许多方面仍然未知。例如，关于中心体蛋白是如何组装成功能微管组织中心的，迄今知之甚少。越来越多的证据表明，中心体的功能需要TACC-TOG复合体。TACC-TOG复合体被认为可以稳定新形成的微管，但来自威斯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交付。关于TACC-TOG复合体仍然存在的重要问题是，它们与中心体的联系是如何调节的，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复合体如何对中心体功能做出贡献。利用非洲爪哇TACC蛋白Maskin的初步研究表明：(I)Maskin在纺锤体组装中既有中心体依赖的作用，也有中心体独立的作用，(Ii)Maskin的磷酸化受Importin的调节，(Iii)Maskin与纺锤体组装因子和参与翻译调控的蛋白质相互作用，以及(Iv)放线菌亚胺，一种常用的蛋白质翻译抑制剂，破坏非洲爪哇卵提取物中的纺锤体组装。体外实验和非洲爪蛙卵提取液将作为实验系统继续深入分析Maskin的分子调控机制，并解决以下基本问题：(1)TACC-TOG复合体如何与中心体相互作用？(2)TACC-TOG复合体如何稳定远离中心体的微管？以及(3)翻译过程和纺锤体组装之间的联系是什么？智力价值：这项研究的完成将提供比目前可用的更详细的TACC蛋白如何影响微管形成的机制图。更广泛地说，这项工作最终将有助于阐明动物中心体如何参与微管的形成和组织。由于中心体缺陷可能导致人类和动物的细胞周期停滞、细胞死亡、胚胎死亡和男性不育，这项研究的影响远远超出细胞和发育生物学，对动物生物学的许多分支具有重要影响，包括对农业重要物种的研究。广泛影响：这一职业项目包括通过合作加强科学基础设施的活动，涉及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。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族裔和妇女)进行现代细胞生物学技术培训将是拟议的研究和教育计划的一个组成部分，并将包括更多高级研究受训人员获得宝贵的教学和指导技能的机会。本科生将从拟议的研究中受益，因为他们将获得实践经验，并体验到更好的教学技能。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计划来吸引和培训未来的教师，该计划专门旨在培训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工作者。该教育计划的其他目标是开发新的“主动学习”教学材料，并为当地和全国范围内的其他人树立榜样。该实习计划由“教学即研究”的理念推动，并将建立在NSF资助的研究、教学和学习一体化中心(CIRTL)、其Delta研究、教学和学习计划以及威斯康星大学麦迪逊分校的科学教学计划提供的基础设施和支持的基础上。

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.