CAREER:Elucidating the Mechanism of Microtubule Dynamics through Cold-stable Tubulin Mutants

这个CAREER项目的目标是了解微管聚合物组装的分子机制，以及蛋白质构象的变化如何导致微管拆卸。微管聚合物在所有高等生物的运输和信号传递过程中起着至关重要的细胞作用。这些过程取决于微管在组装和拆卸状态之间切换的能力。为了获得对微管动力学（组装和拆卸）机制的新见解，该研究计划将产生模仿冷适应生物序列差异的微管蛋白突变体，这些突变体表现出高度稳定的微管，并确定这些突变如何影响微管动力学。这些实验的结果将解决我们对微管结构变化如何决定其组装或拆卸倾向的理解中的关键空白，从而为微管领域提供重大进展。此外，该项目将影响我们对冷适应分子机制的更广泛理解。为了进一步扩大该项目的影响，首席研究员的实验室将与丹佛公立高中的学生和教师合作，开展一项合作研究-教育计划，这些学校有少数民族人口。该计划将推进该项目的研究目标，同时培训高中教师和学生在假设驱动科学，分子生物学技术，沟通技巧的过程中，并获得更广泛的研究社区，从而激发和授权未来的科学家。该项目将为微管蛋白和微管的结构/功能研究做出独特贡献。尽管晶体学方法提供了微管蛋白稳定构象的详细分子快照，但该项目的遗传方法特别适合于定义微管蛋白动态区域的作用，而这些区域是晶体学无法获得的。首席研究员将结合来自耐寒生物的序列数据和无偏遗传筛选来确定增强微管稳定性的氨基酸变化，并确定增强稳定性的基础，使用硅蛋白模型和细胞中微管活性的定量分析以及纯化蛋白质的最小系统。

The goal of this CAREER project is to understand the molecular mechanism by which microtubule polymers assemble and how changes in the protein conformation lead to microtubule disassembly. Microtubule polymers play vital cellular roles in transport and signaling processes in all higher organisms. These processes depend on the ability of microtubules to switch between states of assembly and disassembly. To gain new insight into the mechanism of microtubule dynamics (assembly and disassembly), the research program will generate tubulin mutants that mimic sequence differences from cold-adapted organisms, which exhibit highly stable microtubules, and determine how these mutations impact microtubule dynamics. The results of these experiments will address a critical gap in our understanding of how changes in tubulin structure determine its propensity to assemble or disassemble, thereby providing a major advance for the microtubule field. In addition, this project will impact our broader understanding of molecular mechanisms of cold adaptation. To further broaden the impact of the project, the Principal Investigator's laboratory will partner with students and teachers from Denver public high schools with underrepresented minority populations in a collaborative research-education program. This program will advance the research goals of the project while training high school teachers and students in the process of hypothesis-driven science, techniques in molecular biology, communication skills, and gaining exposure to the broader research community, thereby exciting and empowering future scientists. This project will uniquely contribute to ongoing structural/function studies of tubulin proteins and microtubules. Whereas crystallographic approaches provide detailed molecular snapshots of stable conformations of the tubulin proteins, the project's genetic approach is uniquely suited to define the roles of dynamic regions of tubulins that are not accessible to crystallography. The Principal Investigator will use a combination of sequence data from cold tolerant organisms and unbiased genetic screens to identify amino acid changes that enhance microtubule stability, and to determine the basis of enhanced stability using in silico protein modeling and quantitative assays of microtubule activity in cells and in a minimal system with purified proteins.