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The centromere is located at the primary constriction of condensed chromosomes where it acts as a platform regulating chromosome segregation. The histone H3 variant CENP-A is the foundation for kinetochore formation. CENP-A directs formation of a highly dynamic molecular neighbourhood whose temporal characterisation during mitosis remains a challenge due to limitations in available techniques. BioID is a method that exploits a "promiscuous" biotin ligase (BirA118R or BirA*) to identify proteins within close proximity to a fusion-protein of interest (Roux et al., 2012, J Cell Biol, 196, 801-10). As originally described, cells expressing BirA* fusions were exposed to high biotin concentrations for 24 hours during which the ligase transferred activated biotin (BioAmp) to other proteins within the immediate vicinity. The protein neighbourhood could then be characterised by streptavidin-based purification and mass spectrometry. Here we describe a further development to this technique, allowing CENP-A interactors to be characterised within only a few minutes, in an in vitro reaction in lysed cells whose physiological progression is "frozen." This approach, termed in vitro BioID (ivBioID), has the potential to study the molecular neighbourhood of any structural protein whose interactions change either during the cell cycle or in response to other changes in cell physiology.
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- 3 Finished
1/10/11 → 30/04/17
1/01/11 → 30/09/16