Opening the door to super-resolution microscopy in oxidizing cellular compartments
Various cellular compartments such as the endoplasmic reticulum or the mitochondrial intermembrane space may be considered as "hostile" environments, because particularly oxidizing. This is also the case in the bacterial periplasm, a space of major importance for the understanding of cellular respiration, biofilms formation and antibiotic resistance. When proteins of interest are fused to fluorescent proteins to allow their microscopic observation, the latter, once secreted into the oxidizing environments, are generally unable to fold correctly and thus fluoresce. There is one notable exception, Superfolder GFP, unfortunately unsuitable for super resolution microscopy. In this work, combining biochemistry, crystallography and photophysical studies, we realized the rational engineering of Superfolder-GFP in order to make this marker photoswitchable. To this purpose, we constructed a chimeric protein combining Superfolder-GFP and rsEGFP2, a GFP derivative used for super-resolution in non-oxidizing environments. The result : rsFolder is a new tool is a new tool allowing the observation of oxidizing environments such as the periplasm with resolutions of the order of 70 nm. The development of rsFolder is currently ongoing in order to obtain new generations of even more efficient markers for biologists and to access otherwise unobservable hostile cell territories in super-resolution.
El Khatib, M., Martins, A., Bourgeois, D., Colletier, J.-P. & Adam, V. Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm. Scientific Reports 6, 18459 (2016). DOI : 10.1038/srep18459