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Mammalian Cell Surface Reorganization During Cell Division

Final Report Summary - MCS (Mammalian Cell Surface Reorganization During Cell Division)

Cell division is central to life. Any mistake during cell division may result in developmental abnormalities or complex diseases such as cancer. Cell division requires the coordinated activities of different cellular components such as the microtubule and actin cytoskeleton, chromosomes and membrane. Global regulation of cell's interior is driven under the influence of the master kinase Cdk1 and these regulatory mechanisms have been extensively studied in many laboratories (Nigg, 2001; Nurse, 1990). In contrast less is known about changes in the biochemistry of cell membrane during cell division. This proposal uncovers the most understudied cellular compartment during cell division, cell surface.

Cell surface morphology undergoes dramatic reshaping at the onset of mitosis. As cells enter mitosis they transiently lose their adherence and round up. At cytokinesis the daughter cells spread back to regain their interphase morphology (Cramer and Mitchison, 1997; Mitchison, 1992). Not only cultured cells but also dividing cells in tissues round-up and de-adhere during mitosis (Jinguji and Ishikawa, 1992). These morphologic changes are thought to be necessary for the geometric requirements of cell division that facilitate organization within the mitotic cells (Cramer and Mitchison, 1993; Maddox and Burridge, 2003; Rosenblatt et al., 2001; Stewart et al., 2011). The biochemical changes on the cell surface are likely to be critical for better understanding of tissue development and cancer. In order to analyze the cell-cycle dependent changes in the cell surface biochemistry, we took advantage of the mass spectrometry and quantitative proteomic technology to examine cell cycle dependent biochemical changes in the cell membrane. By labeling cell surface using membrane-impermeable biotin, we isolated the cell surface-exposed proteins and quantified their change between interphase and mitosis. 660 cell surface and cell surface associated proteins were identified and their surface exposure was quantified at two cell cycle stages corresponding to interphase and mitotic. 64 proteins were reproducible enriched on the mitotic or interphase cell surface (Ozlu et al., 2015).

According to our analysis, one of the most prominent classes of proteins whose cell-surface exposure changes during progression through mitosis was adhesion molecules. Two members of protocadherins, PCDH 7 and PCDH1 were upregulated at the onset of mitosis. We study the molecular mechanism and the function of mitosis selective cell surface localization of protocadherin proteins. We confirmed the mitosis selective cell surface localization of PCDH7 and PCDH1 using different methods including western blotting, immunostaining of endogenous protein and live imaging of transiently expressed PCDH7::GFP fusion gene. We show that PCDH7&1 are required for development of full mitotic rounding pressure at the onset of mitosis. Knockdown of PCDH1 using RNAi slowed down the dynamics of mitotic rounding. In addition we expanded our analysis to other mitotic enriched cell surface proteins identified in our proteomic screen. Studying the regulatory mechanisms on the cell surface shed new light on the cell division and open new directions in mitosis research. Long-term career goal of this grantee is to apply quantitative proteomics and cell biology techniques to bring a better understanding of how cell division is regulated. Funding this application provided with the means necessary to the establishment of the applicant's quantitative proteomics laboratory in Turkey and accelerated exchange of knowledge and experience between Europe and the US.

References:

Cramer, L., and Mitchison, T.J. (1993). Moving and stationary actin filaments are involved in spreading of postmitotic PtK2 cells. The Journal of cell biology 122, 833-843.
Cramer, L.P. and Mitchison, T.J. (1997). Investigation of the mechanism of retraction of the cell margin and rearward flow of nodules during mitotic cell rounding. Molecular biology of the cell 8, 109-119.
Jinguji, Y., and Ishikawa, H. (1992). Electron microscopic observations on the maintenance of the tight junction during cell division in the epithelium of the mouse small intestine. Cell Struct Funct 17, 27-37.
Maddox, A.S. and Burridge, K. (2003). RhoA is required for cortical retraction and rigidity during mitotic cell rounding. The Journal of cell biology 160, 255-265.
Mitchison, T.J. (1992). Compare and contrast actin filaments and microtubules. Mol Biol Cell 3, 1309-1315.
Nigg, E.A. (2001). Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol 2, 21-32.
Nurse, P. (1990). Universal control mechanism regulating onset of M-phase. Nature 344, 503-508.
Ozlu, N., Qureshi, M.H. Toyoda, Y., Renard, B.Y. Mollaoglu, G., Ozkan, N.E. Bulbul, S., Poser, I., Timm, W., Hyman, A.A. et al. (2015). Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis. The EMBO journal 34, 251-265.
Rosenblatt, J., Raff, M.C. and Cramer, L.P. (2001). An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism. Current biology : CB 11, 1847-1857.
Stewart, M.P. Helenius, J., Toyoda, Y., Ramanathan, S.P. Muller, D.J. and Hyman, A.A. (2011). Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding. Nature 469, 226-230.