Segregation of molecules at cell division reveals native protein localization

We introduce a nonintrusive method exploiting single-cell variability after cell division to validate protein localization. We found that Clp proteases, widely reported to form biologically relevant foci, were uniformly distributed in Escherichia coli cells, and that many commonly used fluorescent proteins caused severe mislocalization when fused to homo-oligomers. Retagging five other reportedly foci-forming proteins with the most monomeric fluorescent protein tested suggests that the foci were caused by the fluorescent tags.