Efficient switching of mCherry fluorescence using chemical caging

Abstract

Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantummechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores. fluorescent proteins | mCherry | localization microscopy | β-mercaptoethanol | photoactivation

Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations

“…Hence, next we investigated the recruitment of TDP-43 to DNA damage (53BP1) foci in more detail by super-resolution microscopy (SR), using an assay previously developed for probing individual DSB sites [ 51 ]. In cells expressing mCherry-tagged TDP-43 proteins treated with etoposide as above, we combined mCherry-caging for single molecule localization microscopy [ 41 ] in order to visualize the distribution of TDP-43 with conventional d STORM SR imaging of immunolabelled 53BP1 [ 52 ]. We analysed the resulting two-colour SR images using the ‘Interaction Factor’ ImageJ plugin, an analytical tool developed specifically to assess multicolour SR images of dense distributions within the nucleus [ 44 ].…”

“…To generate optimal blinking, Alexa Fluor 488/647 samples were imaged in PBS containing 120 mM mercaptoethylamine (MEA). mCherry was transiently caged and uncaged in a buffer containing 80 mM MEA, 5% glucose, 400 μg/mL glucose oxidase, and 35 μg/mL catalase, similar to methods recently reported by others for SR of mCherry [ 41 ] and minimizing Alexa Fluor 647 photoconversion [ 42 ].…”

“…Fixed NSC-34 cells prepared as described above on coverglass were imaged using a custom home-built SR microscope as previously described [39,40] [41] and minimizing Alexa Fluor 647 photoconversion [42]. Output TIFF stacks were processed using the ImageJ plugin ThunderSTORM with default settings and then refined further as detailed by the plugin authors [43].…”

Feedback-Driven Assembly of the Axon Initial Segment

“…The following constructs were already described: TRIM46-mCherry, TRIM46-GFP, TRIM36-mCherry and PRC1-mCherry (van Beuningen et al., 2015), 480AnkG-GFP, 480AnkG-NN-GFP, 270AnkG-GFP, 480AnkGtail-GFP (Fréal et al., 2016), myc-Kv-Nav (Bréchet et al., 2008), Rab5-GFP and Rab11-GFP (Hoogenraad et al., 2010), Rab6-GFP and NPY-GFP (Schlager et al., 2010), HA-NF186-mRFP-FKBP (Kuijpers et al., 2016), EB3-RFP (Stepanova et al., 2003) and mEOS-tubulin (Cloin et al., 2017).…”

Fluorescent Protein-Based Indicators for Functional Super-Resolution Imaging of Biomolecular Activities in Living Cells

“…An excellent introduction of SRM can be found in a timely review by Schermelleh et al [42], thus out of the scope of this paper. Although photochromism may also be induced in certain constitutive FPs in the presence of chemical cofactors, e.g., incubating mCherry with thiol or β-mercaptoethanol leads to reversible fluorescence off or red-to-blue emission [43,44], the most non-invasive and convenient approach to introduce blinking in living cells is still by using phototransformable FPs [45,46,47]. Light-induced transformation phenomena that are commonly exploited in SRM include photoactivation [48,49], photoconversion [50,51], and reversible photoswitching [52,53,54].…”