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

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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.