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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM079506-05
Application #
8133358
Study Section
Cell Structure and Function (CSF)
Program Officer
Deatherage, James F
Project Start
2007-09-01
Project End
2013-08-31
Budget Start
2011-09-01
Budget End
2013-08-31
Support Year
5
Fiscal Year
2011
Total Cost
$259,334
Indirect Cost
Name
Georgia Regents University
Department
Neurology
Type
Schools of Medicine
DUNS #
966668691
City
Augusta
State
GA
Country
United States
Zip Code
30912
Chu, Xiaogang; Chen, Xuanyu; Wan, Qingwen et al. (2016) Nuclear Mitotic Apparatus (NuMA) Interacts with and Regulates Astrin at the Mitotic Spindle. J Biol Chem 291:20055-67
Zhu, Jinwei; Shang, Yuan; Wan, Qingwen et al. (2014) Phosphorylation-dependent interaction between tumor suppressors Dlg and Lgl. Cell Res 24:451-63
Zheng, Zhen; Wan, Qingwen; Meixiong, Gerry et al. (2014) Cell cycle-regulated membrane binding of NuMA contributes to efficient anaphase chromosome separation. Mol Biol Cell 25:606-19
Li, Youjun; Wei, Zhiyi; Yan, Yan et al. (2014) Structure of Crumbs tail in complex with the PALS1 PDZ-SH3-GK tandem reveals a highly specific assembly mechanism for the apical Crumbs complex. Proc Natl Acad Sci U S A 111:17444-9
Slim, Christiaan L; Lázaro-Diéguez, Francisco; Bijlard, Marjolein et al. (2013) Par1b induces asymmetric inheritance of plasma membrane domains via LGN-dependent mitotic spindle orientation in proliferating hepatocytes. PLoS Biol 11:e1001739
Zheng, Zhen; Wan, Qingwen; Liu, Jing et al. (2013) Evidence for dynein and astral microtubule-mediated cortical release and transport of Gýýi/LGN/NuMA complex in mitotic cells. Mol Biol Cell 24:901-13
Xiao, Zhuoni; Wan, Qingwen; Du, Quansheng et al. (2012) Galpha/LGN-mediated asymmetric spindle positioning does not lead to unequal cleavage of the mother cell in 3-D cultured MDCK cells. Biochem Biophys Res Commun 420:888-94
Wan, Qingwen; Liu, Jing; Zheng, Zhen et al. (2012) Regulation of myosin activation during cell-cell contact formation by Par3-Lgl antagonism: entosis without matrix detachment. Mol Biol Cell 23:2076-91
Zhu, Jinwei; Wen, Wenyu; Zheng, Zhen et al. (2011) LGN/mInsc and LGN/NuMA complex structures suggest distinct functions in asymmetric cell division for the Par3/mInsc/LGN and G?i/LGN/NuMA pathways. Mol Cell 43:418-31
Hao, Yi; Du, Quansheng; Chen, Xinyu et al. (2010) Par3 controls epithelial spindle orientation by aPKC-mediated phosphorylation of apical Pins. Curr Biol 20:1809-18

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