Abstract

Proximity labelling by a biotin ligase combined with mass spectrometry of biotin-affinity purified proteins (BioID) has become a powerful tool to investigate protein interactions in vivo in a range of organisms, including trypanosomes. We recently discovered an interesting off-label application of BioID for protein imaging by fluorescent streptavidin. We found streptavidin imaging superior to classical antibody labelling because it (i) provides a stronger signal with no loss in resolution (ii) can image proteins in phase-separated regions that are not accessible to antibodies and (iii) provides information on localization dynamics, since “historic” interactions are preserved. We have used the method, in combination with expansion microscopy, classical BioID, neural-network based in silico predictions and reverse genetics to reinvestigate the trypanosome nuclear pore complex.