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

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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.