Saccharomyces cerevisiae microtubule and kinetochore dynamics

酿酒酵母微管和动粒动力学

• 批准号: 10623066
• 负责人: GEORJANA BARNES
• 金额: $ 40.13万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623066
• 关键词: Anaphase; Behavior; Binding; Biochemical; Biological Assay; Birth; Cell Cycle; Cell Cycle Stage; Cell Nucleus; Cells; Chromosomes; Complex; Congenital Abnormality; Coupling; Cytoplasm; Cytoskeleton; DNA; Defect; Devices; Fluorescence Microscopy; Genetic; Kinesin; Kinetochores; Laboratories; Lateral; Learning; Malignant Neoplasms; Mediating; Microtubules; Mitosis; Mitotic; Mitotic spindle; Molecular Machines; Morphologic artifacts; Plus End of the Microtubule; Process; Property; Protein Dynamics; Protein Kinase; Proteins; Regulation; Saccharomyces cerevisiae; Saccharomycetales; Source; Structure; System; Time; Tubulin; cancer prevention; chromosome replication; daughter cell; genome integrity; mutant; preservation; protein complex; segregation

Project Summary/Abstract For replicated chromosomes to be segregated to two daughter cells accurately, the microtubule (MT) cytoskeleton must be completely remodeled to form a bipolar spindle. A large, dynamic protein complex called the kinetochore attaches replicated chromosomes to microtubules emanating from opposite spindle poles. Proper kinetochore-microtubule attachment is vital for preservation of genomic integrity and prevention of cancer and birth defects. Therefore, mitotic spindle assembly and kinetochore attachment must be well coordinated. Challenging understanding of these processes and their coordination is the fact that mitotic spindles and kinetochores are extremely complex molecular machines (kinetochores contain >60 proteins), that they are targets of phosphoregulation by multiple protein kinases, and that they possess a striking range of biochemical activities. Kinetochore activities include: (1) lateral MT binding, (2) translocation along the MT lattice to the plus end, (3) conversion from lateral to end-binding, (4) association with dynamic MT ends while tubulin subunits are exchanged, and (5) serving as force-coupling devices between chromosomes and MT plus ends during anaphase A. Understanding how the kinetochore performs its various functions, the structural underpinnings of these activities, and how these activities are regulated post-translationally, is far from complete. A biochemical cell-lysate assay recently developed in the Barnes laboratory combines, for the first time, two of the most powerful approaches for studies of microtubule dynamics: biochemical extract studies and genetics. Dynamics of single microtubules and single kinetochores associated with these microtubules are revealed and quantitatively analyzed by highly sensitive Total Internal Reflection Fluorescence microscopy. Cell lysates synchronized to specific cell-cycle stages are made from budding yeast mutants of specific mitotic proteins. Quantitative analysis of MT dynamics and kinetochore activities will establish how these parameters are regulated in the cell cycle, and these studies will identify the specific proteins that carry out the specific behaviors. Since mitosis is a highly conserved process, lessons learned from these studies are expected to apply broadly. Unlike many other assays, this assay exclusively uses homologous sources of tubulin and interacting proteins, avoiding artefacts that arise from species mismatch incompatibilities. Proposed studies build upon recent unique observations of microtubule dynamics regulation and kinetochore dynamic activity in this lysate system. The objectives are: (1) To investigate biochemical activities of intact kinetochores and their regulation, and to relate kinetochore structure to function; and (2) To investigate how microtubule dynamics are regulated through the cell cycle in both the nucleus and the cytoplasm, focusing on Kar3 and Kip3 kinesins.

项目总结/摘要 为了使复制的染色体准确地分离到两个子细胞，微管（MT） 细胞骨架必须完全重塑以形成双极纺锤体。一个巨大的动态蛋白质复合体， 动粒将复制的染色体附着在从纺锤体两极发出的微管上。 正确的着丝粒-微管附着对于保持基因组的完整性和防止 癌症和先天缺陷因此，有丝分裂纺锤体组装和动粒附着必须良好 协调一致。对这些过程及其协调的理解具有挑战性的是，有丝分裂 纺锤体和动粒是极其复杂的分子机器（动粒含有>60种蛋白质）， 它们是多种蛋白激酶磷酸化调节的靶点，并且它们具有惊人的范围， 生化活动。动粒的活动包括：（1）横向MT结合，（2）沿MT沿着移位 晶格的正端，（3）从横向到末端结合的转换，（4）与动态MT末端的关联， 微管蛋白亚基交换，（5）作为染色体和MT+之间的力偶联装置 在后期A结束。了解动粒如何执行其各种功能， 这些活动的基础，以及这些活动如何在事后进行监管， 完成.最近在巴恩斯实验室开发的一种生化细胞裂解物测定法结合了 时间，研究微管动力学的两个最有力的方法：生化提取物研究 和遗传学。单个微管和与这些微管相关的单个动粒的动力学是 通过高灵敏度的全内反射荧光显微镜进行显示和定量分析。 与特定细胞周期阶段同步的细胞裂解物由特定有丝分裂相的芽殖酵母突变体制成。 proteins. MT动力学和动粒活动的定量分析将确定这些参数如何 在细胞周期中受到调节，这些研究将确定执行特定功能的特定蛋白质。 行为。由于有丝分裂是一个高度保守的过程，从这些研究中吸取的经验教训， 应用广泛。与许多其他测定法不同，该测定法仅使用微管蛋白和微管蛋白的同源来源。 相互作用的蛋白质，避免由物种错配不相容性引起的假象。拟定研究 基于最近对微管动力学调节和动粒动力学活性的独特观察， 这个裂解系统。目的是：（1）研究完整的动粒的生化活性， 调控，并将动粒结构与功能联系起来;（2）研究微管动力学是如何 在细胞核和细胞质中通过细胞周期调节，集中于Kar 3和Kip 3驱动蛋白。