Investigating monomer-dimer switching of the cancer-associated protein, survivin: A Bimolecular Fluorescence Complementation Assay (BiFC) Approach

Session

Poster Session

Authors

Duaa Zahim (1), Sophie Rochette (1)

Affiliations

1. University of Nottingham

Keywords

Survivin, Survivin dimerization, BiFC, CD7 microscope, dimerization mutant 

Abstract text

The ability of the cancer-associated protein, survivin to switch between being monomer and dimer is critical in modulating cell division. In this study, we employed a Bimolecular Fluorescence Complementation assay (BiFC) to assay survivin dimerization. In brief, wild-type survivin and a constitutive monomer were fused at the genetic level to complementary non-fluorescent fragments of the cDNA of the fluorescent protein, Venus. In BiFC assays, once the relevant plasmids are transfected into cells, the ability of the two separate fragments to combine and fluoresce reports the ability of the two proteins or peptides to which they are fused to interact. Here Hela cells were transfected, fixed, and observed using the GFP channel and a CD7 microscope. Our results demonstrate that dimerization of wild-type survivin can be reported using this system, while a constitutive monomer provides an excellent negative control. As survivin dimerization has implications in determining fate during division, the combination of BiFC with advanced microscopy techniques provides a powerful platform for screening agents that can interfere with cell division and has the potential to identify novel anti-cancer agents.

References

Jeyaprakash, A.A., Klein, U.R., Lindner, D., Ebert, J., Nigg, E.A. and Conti, E., 2007.  Structure of a Survivin–Borealin–INCENP core complex reveals how chromosomal passengers travel together. Cell, 131(2), pp.271-285.

Truong, K. and Ikura, M., 2001. The use of FRET imaging microscopy to detect protein–protein interactions and protein conformational changes in vivo. Current opinion in structural biology, 11(5), pp.573-578.