I am interested in LEM-domain proteins involved in different aspects of cellular and nuclear biology.The LEM-domain interacts specifically with the Barrier-to-Autointegration factor (BAF), one of the first or perhaps even the first protein to be relocalized from cytoplasm to chromosomes in telophase at the end of mitosis, and BAF is essential for nuclear envelope formation. I want to evaluate the importance of the different LEM-domain proteins in post-mitotic nuclear envelope reassembly and reconstruction of the nuclear lamina, by using CRISPR/Cas9 to systematically knock out these proteins in HeLa cells and other human cell lines. Proteins that are currently investigated in these experiments are LEM4/ANKLE-2, LAP2alpha and beta and emerin. The impact of deleting one or several LEM-domain proteins is assessed by examining the reassociation of GFP-tagged nuclear envelope proteins, like GFP-LaminA, to the chromosomes in telophase, using time-lapse video microscopy.
I am getting acquainted with the use of CRISPR/Cas9 to create short deletion or insertion in genomic DNA in order to knock out or knock down specific genes in commonly used cell lines like HeLa cells. I try to develop simple, easy and reproducible protocols for transfecting cells with the targeting constructs and for the screening of mutant cells.
Other expertise include molecular and cell biology techniques and microscopy, including time-lapse video microscopy.
I am interested in an general cell biology techniques, particularly those involving nuclear biology.
Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., Zhang, F., 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823.
Asencio, C., Davidson, I.F., Santarella-Mellwig, R., Ly-Hartig, T.B., Mall, M., Wallenfang, M.R., Mattaj, I.W., Gorjanacz, M., 2012. Coordination of kinase and phosphatase activities by Lem4 enables nuclear envelope reassembly during mitosis. Cell 150, 122-135.
Brachner, A., Foisner, R., 2011. Evolvement of LEM proteins as chromatin tethers at the nuclear periphery. Biochem Soc Trans 39, 1735-1741.
Haraguchi, T., Kojidani, T., Koujin, T., Shimi, T., Osakada, H., Mori, C., Yamamoto, A., Hiraoka, Y., 2008. Live cell imaging and electron microscopy reveal dynamic processes of BAF-directed nuclear envelope assembly. J Cell Sci 121, 2540-2554.
Snyers, L., Erhart, R., Laffer, S., Push, O., Weipoltshammer, K., Schöfer, C. LEM4/ANKLE-2 deficiency impairs post-mitotic re-localization of BAF, LAP2a and LaminA to the nucleus, causes nuclear envelope instability in telophase and leads to hyperploidy in HeLa cells. Eur. J. Cell Biol. 97, 63-74 (2018)
9-11 September 2019, Trnava, Slovakia