COMBINED SINGLE MOLECULE AND IN-CELL APPROACHES TO UNDERSTAND DYNEIN-MEDIATED MITOTIC CHECKPOINT SILENCING

结合单分子和细胞内方法来了解动力蛋白介导的有丝分裂检查点沉默

• 批准号: 1518083
• 负责人: Jennifer DeLuca
• 金额: $ 71.55万
• 依托单位: Colorado State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-15 至 2019-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1518083&HistoricalAwards=false
• 关键词: COMBINED; SINGLE; MOLECULE; CELL; APPROACHES

Cell division is one of the most fundamental processes of all life. Various molecular processes converge during cell division to ensure that it takes place with a high level of accuracy and fidelity. For instance, the genetic information contained with the chromosomes is faithfully divided between mother and daughter cells during each round of cell division or mitosis such that mistakes are very rarely made. The machinery that ensures high-fidelity chromosome inheritance from mother to daughter cells includes a highly elaborate assemblage of filamentous structures called microtubules, and large complex structures assembled upon the chromosomes called kinetochores. Proper division of the genetic material requires that each kinetochore make a proper and stable attachment to microtubules prior to the end of mitosis. Interestingly, kinetochores monitor and regulate their own attachment status; however, how the attachment status of each kinetochore is relayed to the machinery that initiates exit from mitosis is unknown. This project aims to determine how a molecular motor protein called dynein affects and facilitates proper division of the genetic material during mitosis. The results from this project will have significant impact on our understanding of cell division, and the various underlying processes that are tightly regulated and highly orchestrated to perform this critical biological function reproducibly and with high fidelity. In addition, this project will impact the scientific literacy of the community through the implementation of an undergraduate course, and also by teaching elementary students about cell division, and the scientific method.In this project, the investigator will examine how cells accurately and reliably segregate their genetic material during cell division. To prevent errors during chromatid separation during mitosis, cells employ a fail-safe mechanism called the spindle assembly checkpoint (SAC), which prevents mitotic progression until all chromosomes have established proper kinetochore-microtubule attachments. In this project, investigators will probe the role of the microtubule motor protein dynein in the transport (SAC) proteins away from kinetochores, thereby silencing the inhibitory signal, and allowing the initiation of anaphase and progression through mitosis. A combination of in vitro and in-cell approaches will be employed to understand the role for dynein in this process, and to understand how microtubule attachment status is translated into activation of dynein-mediated SAC protein eviction from kinetochores. These studies will be transformative because they will answer key questions regarding the SAC by implementing a newly developed single molecule approach in which kinetochore dynein activity is reconstituted in vitro. By combining this in vitro approach with in-cell high- and super-resolution fluorescence microscopy, the following key unanswered questions in cell biology will be addressed: (1) What is the role of dynein in eviction of SAC effectors from attached kinetochores? (2) What is the biological signal that initiates dyneinmediated eviction of SAC effectors from attached kinetochores? This project will lead to a greater understanding of the control of cell division while training diverse students in basic sciences and providing outreach to K-12 students.

细胞分裂是所有生命最基本的过程之一。在细胞分裂过程中，各种分子过程会聚在一起，以确保它以高水平的准确性和保真度发生。 例如，染色体所包含的遗传信息在每一轮细胞分裂或有丝分裂期间在母细胞和子细胞之间忠实地分配，因此很少发生错误。确保从母体细胞到子细胞的高保真染色体遗传的机制包括高度精细的丝状结构（称为微管）和组装在染色体上的大型复杂结构（称为动粒）。遗传物质的正确分裂要求每个动粒在有丝分裂结束前与微管正确稳定地连接。有趣的是，动粒监测和调节它们自己的附着状态;然而，每个动粒的附着状态如何被传递到启动退出有丝分裂的机制尚不清楚。该项目旨在确定一种称为动力蛋白的分子马达蛋白如何影响和促进有丝分裂过程中遗传物质的正确分裂。该项目的结果将对我们理解细胞分裂以及各种受到严格调控和高度协调的潜在过程产生重大影响，以可重复和高保真地执行这一关键生物功能。此外，本项目还将通过开设本科生课程，以及向小学生传授细胞分裂知识和科学方法，对社会的科学素养产生影响。在本项目中，研究者将研究细胞在细胞分裂过程中如何准确可靠地分离其遗传物质。为了防止在有丝分裂期间染色单体分离期间的错误，细胞采用称为纺锤体组装检查点（SAC）的故障安全机制，该机制阻止有丝分裂进程，直到所有染色体都建立了适当的着丝粒微管附着。 在这个项目中，研究人员将探索微管运动蛋白动力蛋白在运输（SAC）蛋白远离动粒中的作用，从而沉默抑制信号，并允许后期的启动和有丝分裂的进展。将采用体外和细胞内方法的组合来了解动力蛋白在此过程中的作用，并了解微管附着状态如何转化为动力蛋白介导的SAC蛋白从着丝粒驱逐的激活。这些研究将是变革性的，因为它们将通过实施新开发的单分子方法来回答关于SAC的关键问题，其中动粒动力蛋白活性在体外重建。通过将这种体外方法与细胞内高分辨率和超分辨率荧光显微镜相结合，将解决细胞生物学中以下关键的未回答的问题：（1）动力蛋白在从附着的动粒驱逐SAC效应物中的作用是什么？(2)什么是启动动力蛋白介导的SAC效应器从附着的动粒驱逐的生物信号？该项目将导致更好地了解细胞分裂的控制，同时培训基础科学的不同学生，并为K-12学生提供外展服务。