Segregation of molecules at cell division reveals native protein localization

We introduce a non-intrusive method exploiting post-division single-cell variability to validate protein localization. The results show that Clp proteases, widely reported to form biologically relevant foci, are in fact uniformly distributed inside Escherichia coli cells, and that many commonly used fluorescent proteins (FPs) cause severe mislocalization when fused to homo-oligomers. Re-tagging five other reportedly foci-forming proteins with the most monomeric FP tested suggests the foci were caused by the FPs.