The helical MreB cytoskeleton in Escherichia coli MC1000/pLE7 is an artifact of the N-Terminal yellow fluorescent protein tag.
Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not. In contrast, mCherry fused to an internal loop (MreB-RFP(SW)) does not induce helices. The helices are therefore an artifact of the placement of the fluorescent protein tag. YFP-MreB helices were also clearly distinguishable from the punctate, "patchy" localization patterns of MreB-RFP(SW), even by standard light microscopy. The many interpretations in the literature of such punctate patterns as helices should therefore be reconsidered.
Division of Biology, California Institute of Technology, Pasadena, California, USA.
Matthew T
MT
Swulius
Grant J
GJ
Jensen
D016477:Artifacts; D001426:Bacterial Proteins / Q000032:analysis; D001431:Bacteriological Techniques / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000032:analysis*; D008164:Luminescent Proteins / Q000032:analysis; D008856:Microscopy, Fluorescence / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013194:Staining and Labeling / Q000379:methods*; D000097573:Red Fluorescent Protein
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C054562:yellow fluorescent protein, Bacteria; C078473:MreB protein, E coli