Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

Abstract

Photoactivatable fluorescent proteins (PAFPs) have been widely used for superresolution imaging based on the switching and localization of single molecules. Several properties of PAFPs strongly influence the quality of the superresolution images. These properties include (i) the number of photons emitted per switching cycle, which affects the localization precision of individual molecules; (ii) the ratio of the on-and off-switching rate constants, which limits the achievable localization density; (iii) the dimerization tendency, which could cause undesired aggregation of target proteins; and (iv) the signaling efficiency, which determines the fraction of target-PAFP fusion proteins that is detectable in a cell. Here, we evaluated these properties for 12 commonly used PAFPs fused to both bacterial target proteins, H-NS, HU, and Tar, and mammalian target proteins, Zyxin and Vimentin. Notably, none of the existing PAFPs provided optimal performance in all four criteria, particularly in the signaling efficiency and dimerization tendency. The PAFPs with low dimerization tendencies exhibited low signaling efficiencies, whereas mMaple showed the highest signaling efficiency but also a high dimerization tendency. To address this limitation, we engineered two new PAFPs based on mMaple, which we termed mMaple2 and mMaple3. These proteins exhibited substantially reduced or undetectable dimerization tendencies compared with mMaple but maintained the high signaling efficiency of mMaple. In the meantime, these proteins provided photon numbers and on-off switching rate ratios that are comparable to the best achieved values among PAFPs.P hotoactivated localization microscopy, stochastic optical reconstruction microscopy, and related imaging methods take advantage of photoswitching and imaging of single molecules to circumvent the diffraction limit of spatial resolution in light microscopy (1-3). In these methods, only a subset of the fluorescent labels in the sample is switched on at any given time such that the positions of individual fluorophores can be localized from their images with high precision. Iteration of this process allows numerous fluorescent labels to be localized and an image with sub-diffraction-limit resolution to be reconstructed from the fluorophore localizations. Fluorescent proteins that can be activated from dark to fluorescent or converted from one color to another are widely used for such imaging approaches (4, 5). Although photoactivatable fluorescent proteins (PAFPs) are generally dimmer than photoswitchable dyes (6, 7) and hence give lower image resolution, the ease and high specificity of labeling protein targets in living cells with fluorescent proteins makes PAFPs highly appealing probes for imaging the dynamics of cellular structures (4,8).For single-molecule-based superresolution imaging methods, several properties of PAFPs are particularly important for the image quality. Here, we focus on four such key properties. (i) The first property is the photon budget, defined as the average number of ph...

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

“…These split constructs have allowed us to obtain two-color or super-resolution images of endogenous proteins and have revealed ER tubules with greatly reduced abundance of the translocon component Sec61B. Our platform can be easily extended to the engineering of other self-complmenting split FPs with distinct colors (e.g., mTurquoise2, mTagBFP2) 28,29 , good photoactivation performance (e.g., mMaple3, PATagRFP) 30,31 , or new functionalities (e.g., pH sensitivity) 32 . We note that for non-selfcomplementing split FPs, which are used to detect protein-protein interactions in bimolecular fluorescence complementation (BiFC) assays 33 , a different engineering platform is needed to ensure minimum affinity between the two FP fragments by themselves.…”

Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy

“…mGeos was created by mutating the first amino acids in the chromophore of the photoconvertable fluorescence protein (PCFP) mEos2 (17). However, mEos2 also tends to form dimers and higher order oligomers at high concentrations (17)(18)(19), which makes it unsuitable for labeling membrane proteins that can reach very high local concentrations due to confined 2D movement and limited rotation (20). To attack this problem, we mutated the interface amino acids of mEos2 to develop two true monomeric PCFPs, mEos3.1 and mEos3.2, that work well for photoactivated localization microscopy (PALM) (18).…”

Cytoplasmic sensing by the inner membrane histidine kinase EnvZ

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