Fedor V. Subach

Photoactivatable mCherry for high-resolution two-color fluorescence microscopy

The reliance of modern microscopy techniques on photoactivatable fluorescent proteins prompted development of mCherry variants that are initially dark but become red fluorescent after violet-light irradiation. Using ensemble and single-molecule characteristics as selection criteria, we developed PAmCherry1 with excitation/emission maxima at 564/595 nm. Compared to other monomeric red photoactivatable proteins, it has faster maturation, better pH stability, faster photoactivation, higher photoactivation contrast and better photostability. Lack of green fluorescence and single-molecule behavior make monomeric PAmCherry1 a preferred tag for two-color diffraction-limited photoactivation imaging and for super-resolution techniques such as one- and two-color photoactivated localization microscopy (PALM). We performed PALM imaging using PAmCherry1-tagged transferrin receptor expressed alone or with photoactivatable GFP–tagged clathrin light chain. Pair correlation and cluster analyses of the resulting PALM images identified ≤200 nm clusters of transferrin receptor and clathrin light chain at ≤25 nm resolution and confirmed the utility of PAmCherry1 as an intracellular probe.

Conversion of red fluorescent protein into a bright blue probe.

We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Förster resonance energy transfer applications.

Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

Green fluorescent proteins are light-induced electron donors

Proteins of the green fluorescent protein (GFP) family are well known due to their unique biochemistry and extensive use as in vivo markers. Here, we discovered a new feature of GFPs of diverse origins to act as the light-induced electron donors in photochemical reactions with various electron acceptors, including biologically relevant ones. Moreover, this process accompanying with green-to-red GFP photoconversion can be observed in living cells without additional treatment.

Red Fluorescent Protein with Reversibly Photoswitchable Absorbance for Photochromic FRET

We have developed the first red fluorescent protein, named rsTagRFP, which possesses reversibly photoswitchable absorbance spectra. Illumination with blue and yellow light switches rsTagRFP into a red fluorescent state (ON state) or nonfluorescent state (OFF state), respectively. The ON and OFF states exhibit absorbance maxima at 567 and 440 nm, respectively. Due to the photoswitchable absorbance, rsTagRFP can be used as an acceptor for a photochromic Förster resonance energy transfer (pcFRET). The photochromic acceptor facilitates determination of a protein-protein interaction by providing an internal control for FRET. Using pcFRET with EYFP as a donor, we observed an interaction between epidermal growth factor receptor and growth factor receptor-binding protein 2 in live cells by detecting the modulation of both the fluorescence intensity and lifetime of the EYFP donor upon the ON-OFF photoswitching of the rsTagRFP acceptor.

Monomeric fluorescent timers that change color from blue to red report on cellular trafficking

Based on the mechanism for chromophore formation in red fluorescent proteins, we developed three mCherry-derived monomeric variants, called fluorescent timers (FTs), that change their fluorescence from the blue to red over time. These variants exhibit distinctive fast, medium and slow blue-to-red chromophore maturation rates that depend on the temperature. At 37 degrees C, the maxima of the blue fluorescence are observed at 0.25, 1.2 and 9.8 h for the purified fast-FT, medium-FT and slow-FT, respectively. The half-maxima of the red fluorescence are reached at 7.1, 3.9 and 28 h, respectively. The FTs show similar timing behavior in bacteria, insect and mammalian cells. Medium-FT allowed for tracking of the intracellular dynamics of the lysosome-associated membrane protein type 2A (LAMP-2A) and determination of its age in the targeted compartments. The results indicate that LAMP-2A transport through the plasma membrane and early or recycling endosomes to lysosomes is a major pathway for LAMP-2A trafficking.

Chromophore Transformations in Red Fluorescent Proteins

The discovery of Anthozoa homologs of the green fluorescent protein (GFP) from jellyfish Aequorea victoria, which emit not only green but also yellow, orange, and red fluorescence, provided a powerful boost for in vivo labeling due to the colors and biochemical features never before encountered in GFP variants.1,2 GFP and several Anthozoa fluorescent proteins (FPs) have been developed into monomers suitable for protein tagging, such as: (i) permanently fluorescent conventional blue FP, yellow FPs, orange FPs, red FPs (RFPs), (ii) permanently fluorescent GFPs and RFPs with a large Stokes shift fluorescence emission (LSS-FPs), (iii) irreversibly photoactivatable/photoswitchable GFPs and RFPs,3,4 and (iv) fluorescent timers (FTs).5,6 Among various fluorescent probes, the most valuable for deep-tissue and whole-body imaging are the red-shifted FPs because of reduced autofluorescence, low light-scattering, and minimal absorbance at longer imaging wavelengths. The mechanisms of formation of the GFP-like chromophore and its transformations were studied and described very well, however only a few reviews of those for RFPs are available 2,7. Several mechanisms of the autocatalytic and photoinduced formation of the red chromophores have been proposed.7 Because of the complexity of the red chromophore transformations, no general integrating scheme has been suggested. Despite the numerous data available, there are long lasting contradictions about the formation of the red chromophore. From the discovery of a DsRed FP, the formation of a DsRed-like chromophore was commonly suggested to occur through a green GFP-like intermediate form,8 and only several recent publications uncovered that the formation of the DsRed-like chromophore occurs via a TagBFP-like blue intermediate form, not via the GFP-like one. More than 140 crystal structures are currently available for FPs of different classes, and some of them are for the same FP in a different state or containing different mutations. An overview of the structural data together with FP spectral and photochemical properties illustrate the relationship between the FPs’ structure and function. Here, we focus on a description of the chromophores in RFPs, suggest the mechanisms of the red chromophores formation and its further modifications, and attempt to discover general postulates for this complex chemistry. We also provide insights into how the red chromophore chemistry and the RFP crystal structures are translated into RFPs function. Lastly, we descuss major applications of RFPs in the modern imaging techniques.

Superresolution Imaging of Multiple Fluorescent Proteins with Highly Overlapping Emission Spectra in Living Cells

Localization-based superresolution optical imaging is rapidly gaining popularity, yet limited availability of genetically encoded photoactivatable fluorescent probes with distinct emission spectra impedes simultaneous visualization of multiple molecular species in living cells. We introduce PAmKate, a monomeric photoactivatable far-red fluorescent protein, which facilitates simultaneous imaging of three photoactivatable proteins in mammalian cells using fluorescence photoactivation localization microscopy (FPALM). Successful probe identification was achieved by measuring the fluorescence emission intensity in two distinct spectral channels spanning only ~100 nm of the visible spectrum. Raft-, non-raft-, and cytoskeleton-associated proteins were simultaneously imaged in both live and fixed fibroblasts coexpressing Dendra2-hemagglutinin, PAmKate-transferrin receptor, and PAmCherry1-β-actin fusion constructs, revealing correlations between the membrane proteins and membrane-associated actin structures.

Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide

Reactive oxygen species (ROS) are conserved regulators of numerous cellular functions, and overproduction of ROS is a hallmark of various pathological processes. Genetically encoded fluorescent probes are unique tools to study ROS production in living systems of different scale and complexity. However, the currently available recombinant redox sensors have green emission, which overlaps with the spectra of many other probes. Expanding the spectral range of recombinant in vivo ROS probes would enable multiparametric in vivo ROS detection. Here we present the first genetically encoded red fluorescent sensor for hydrogen peroxide detection, HyPerRed. The performance of this sensor is similar to its green analogues. We demonstrate the utility of the sensor by tracing low concentrations of H2O2 produced in the cytoplasm of cultured cells upon growth factor stimulation. Moreover, using HyPerRed we detect local and transient H2O2 production in the mitochondrial matrix upon inhibition of the endoplasmic reticulum Ca(2+) uptake.

Engineering of bacterial phytochromes for near-infrared imaging, sensing, and light-control in mammals

Near-infrared light is favourable for imaging in mammalian tissues due to low absorbance of hemoglobin, melanin, and water. Therefore, fluorescent proteins, biosensors and optogenetic constructs for optimal imaging, optical readout and light manipulation in mammals should have fluorescence and action spectra within the near-infrared window. Interestingly, natural Bacterial Phytochrome Photoreceptors (BphPs) utilize the low molecular weight biliverdin, found in most mammalian tissues, as a photoreactive chromophore. Due to their near-infrared absorbance BphPs are preferred templates for designing optical molecular tools for applications in mammals. Moreover, BphPs spectrally complement existing genetically-encoded probes. Several BphPs were already developed into the near-infrared fluorescent variants. Based on the analysis of the photochemistry and structure of BphPs we suggest a variety of possible BphP-based fluorescent proteins, biosensors, and optogenetic tools. Putative design strategies and experimental considerations for such probes are discussed.