Mechanisms of Mitotic Fidelity

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Project Summary The major goal of mitosis is to distribute the genetic material accurately between the two daughter cells. Defects in meiosis or mitosis lead to aneuploidy, which is a significant cause of birth defects and is a hallmark of tumorigenesis. Proper spindle function relies on precise spatial and temporal control of microtubule (MT) dynamics and the integration of forces of motor proteins. Defects in regulated MT dynamics lead to spindle multi-polarity, improper kinetochore-MT attachments, delayed mitotic progression, and improper chromosome segregation. Despite the generation of an extensive parts list for the spindle, a major unanswered question is to understand how MT dynamics and motor protein activity are spatially and temporally regulated to ensure proper spindle architecture and chromosome segregation. This has been due, in part, to a lack of appropriate tools that could be used to relate key regulatory biochemical events to where those events are controlled spatially in the spindle. Our lab's work has been instrumental in defining how members of the kinesin superfamily contribute to spindle organization, chromosome congression, kinetochore-MT attachments, error correction, chromosome segregation, and cytokinesis. Our recent implementation of new FRET-based biosensors combined with FLIM and super resolution microscopy is now enabling us to address where in the spindle motors are active or inactive. In addition, our analysis of spindle motors has allowed us to begin to understand how spatial gradients, such as the Ran-GTP gradient, modulate motor activity in the context of the spindle. Over the next 5 years we will focus our studies on three major areas: 1) We will examine the mechanisms of motor regulation by using our expertise with a variety of biochemical, biophysical and super- resolution imaging approaches to integrate protein activity with its spatial and temporal control in the context of the spindle. 2) A major aspect of maintaining proper spindle morphogenesis comes not only from the biochemical activities of the individual motors that regulate spindle structure and dynamics but also from understanding their spatial and temporal regulation. With the development of our numerous FRET biosensors we are ideally positioned to use FLIM imaging to visualize when and where individual motors are activated and to understand their interactions with key regulatory molecules. 3) Proper regulation of MT dynamics and motor activity are also critical to the proper segregation of the genetic material, thus ensuring mitotic fidelity. We will take advantage of our ability to generate cells with different levels of ploidy to understand how defects in spindle architecture under increased chromosome load affect the fidelity of chromosome segregation. Overall, our studies will elucidate how cells use spatial information to assemble macromolecular complexes that function with high precision. This holistic approach allows us to make significant new insight into the global control of mitotic fidelity.