BiFCo: Visualising cohesin assembly/disassembly cycle in living cells

Abstract

Cohesin is a ring-shaped protein complex highly conserved in evolution that is composed in all eukaryotes of at least two SMC proteins (Structural Maintenance of Chromosomes) SMC1 and SMC3 in humans (Psm1 and Psm3 in fission yeast), and the kleisin RAD21 (Rad21 in fission yeast). Mutations in its components or its regulators cause genetic syndromes (known as cohesinopathies) and several types of cancer. It has been shown in a number of organisms that only a small fraction of each subunit is assembled into complexes. Therefore, the presence of an excess of soluble components hinders dynamic chromatin loading/unloading studies using fluorescent fusions in vivo. Here, we present a system based on bimolecular fluorescent complementation in the fission yeast Schizosaccharomyces pombe, named Bi-molecular Fluorescent Cohesin (BiFCo) that selectively excludes signal from individual proteins to allow monitoring the complex assembly/disassembly within a physiological context during a whole cell cycle in living cells. This system may be expanded and diversified in different genetic backgrounds and other eukaryotic models, including human cells.