DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the molecular mechanisms underlying mitotic spindle orientation in mammalian cells. The position of the mitotic spindle determines the direction of chromosome segregation and subsequent cleavage plane of the mother cell. Coordinated cell polarization and spindle orientation plays critical roles during morphogenesis in regulating organ size and shape. It is also the foundation for asymmetric cell division, which allows the daughter cells to inherit different cell fate determinants, a process that is crucial for stem cell function. Perturbation of stem cell function is linked to a wide range of human diseases including neurodegeneration and cancer. Spindle positioning involves the interaction between astral microtubules (MTs) and the cell cortex. Recent studies have revealed the central role played by Pins (LGN and AGS3 in mammals) and the heterotrimeric G protein alpha subunit (Ga) in regulating spindle orientation during asymmetric cell division, but how they are targeted to and maintained at the cell cortex and how they direct spindle orientation remain largely unknown. Recently, we identified a cortical NuMA/LGN/Ga protein complex which can exert forces on astral MTs during mitosis. How is this complex coupled to actin cytoskeleton which is required for the cortical localization of LGN? What forms the phisical link between this complex and astral MTs? We found that filamin-A, an actin binding protein, binds to NuMA, and that cytoplasmic dynein, the minus-end-directed microtubule-based motor protein, interacts with LGN. Based on these preliminary results, we plan to further study the targeting and force generating mechanisms for the corical NuMA/LGN/Ga complex and propose the following specific aims: 1) analyze the dynamics of cortical LGN and NuMA;2) test the hypothesis that filamih-A is required for cortical targeting or maintenance of LGN during mitosis;3) test the hypothesis that LGN exerts forces on astral MTs through a direct interaction with cytoplasmic dynein, and that this interaction is regulated by Ga;4) Analyze the effects of cortical LGN/Ga on microtubule dynamics and test whether dynein mediates these effects. Results from the proposed studies will advance our understanding of the molecular mechanisms by which spindle orientation and asymmetric cell division are achieved in mammalian cells.
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