The Price of Tags in Protein Localization Studies

dynamics of fluorescently labeled proteins has had a huge impact in understanding the organization of DNA, RNA, and protein complexes in all cells, particularly in bacteria, which do not have many features visible by electron microscopic methods (17). Yet the fluorescent protein tags used for live cell imaging, as well as small epitope tags used for purification and/or immunolocalization, are a double-edged sword. There have always been suspicions that tags could cause localization artifacts, but these suspicions usually remain just that, as publishing a paper that claims a previous localization pattern as an artifact requires an extra measure of proof, not just negative data. Swulius and Jensen, in an article in this issue (26), have obtainedpreciselythestrongconfirmatoryevidenceneededtorefute somepreviouspublishedlocalizationpatterns.Byusingcryo-electron tomography (cryo-ET) on Escherichia coli cells producing a tagged and untagged protein, they show that MreB, the bacterial actin homolog required for cylindrical cell wall growth, does not localizeasahelixunlessitistaggedwithyellowfluorescentprotein (YFP) at its N terminus. This is important because several papers had shown beautiful helical patterns of fluorescence using YFPMreB in E. coli (21, 33‐35). Images from immunofluorescence microscopy of native MreB proteins using anti-MreB did not differ significantly from this helical pattern, so the live cell data were not questioned. Finally, it was very satisfying esthetically that a relatively stable actin cable network was the organized scaffold needed to direct and orient cell wall biosynthesis. It was hard to believethatthesestrongpatternswerenotreal,andconsequently, these patterns, including MreB rings that flanked the E. coli septal ring, were assumed to be physiologically relevant by many. In Bacillus subtilis, functional green fluorescent protein (GFP)tagged MreB homologs also formed helix-like filaments and moved along helical tracks (4), consistent with immunofluorescence data (13), in support of the general helical model. MreB’s helical facade began to crack last year with the publica