The reliance of modern microscopy techniques on photoactivatable fluorescent proteins prompted development of mCherry variants that are initially dark but become red fluorescent after violetlight 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 twocolor diffraction-limited photoactivation imaging and for super-resolution techniques such as oneand two-color photoactivated localization microscopy (PALM). We performed PALM imaging using PAmCherry1-tagged transferrin receptor expressed alone or with photoactivatable GFPtagged 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.Genetically encoded `photoactivatable' fluorescent proteins (PAFPs) make up a small category of fluorescent proteins 1 , but are beginning to find uses far and above those of normal' fluorescent proteins 2 . With initially little or no fluorescence within their associated spectral detection window, photoactivatable proteins can be switched on by irradiation with violet light. Thus they are useful for spatially pulse-labeling subpopulations of molecules in cells in complement to photobleaching applications and can provide other useful features such as a high contrast over background in the photoactivated region and circumvention of fluorescence contributions from newly synthesized, nonactivated PAFPs. PAFPs and photoswitchable dyes also provide probes necessary for high-resolution optical techniques, such as photoactivated localization microscopy (PALM) 3 , fluorescence photoactivated localization microscopy (FPALM) 4 , stochastic reconstruction microscopy (STORM) 5 , PALM with independent running acquisition (PALMIRA) 6 RESULTS Development of photoactivatable mCherry variantsWe analyzed data on color transitions of red fluorescent proteins to the respective nonfluorescent chromoproteins that had been generated by mutagenesis 1 and identified the corresponding crucial amino acid positions on the basis of the mCherry structure 18 . Positions spatially close to the chromophore, such as 148, 165, 167 and 203 (numbering is in accordance with GFP alignment; Supplementary Fig. 1 online), appear to be major molecular determinants of color 10,19 . We hypothesized that mutagenesis of mCherry at these positions might convert it to a photoactivatable red probe and performed saturating mutagenesis at these positions using the overlap extension approach.We screened the resulting bacterial library of the site-specific mCherry mutants by fluorescence-activated ...

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.

SUMMARY We utilized a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a novel 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 background such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which were converted into blue probes. Further improvement of a blue variant of TagRFP using random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by substantially higher brightness, faster chromophore maturation and higher pH stability than blue fluorescence proteins with a histidine in the chromophore. Detailed biochemical and photochemical analysis indicates mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging as well as the outstanding donor for green fluorescent proteins in FRET applications.

Commonly used monomeric blue fluorescent proteins suffer from moderate brightness. The brightest of them, mTagBFP, has a notably low chemical stability over time. Prolonged incubation of mTagBFP leads to its transition from a blue fluorescent state with absorbance at 401 nm to a non-fluorescent state with absorbance at 330 nm. Here, we have determined the chemical structure of the degraded product of the blue mTagBFP-like chromophore. On the basis of mTagBFP we have developed an improved variant, named mTagBFP2. mTagBFP2 exhibits 2-fold greater chemical stability and substantially higher brightness in live cells than mTagBFP. mTagBFP2 is also 1.2-fold and 1.7-fold more photostable than mTagBFP in widefield and confocal microscopy setups, respectively. mTagBFP2 maintains all other beneficial properties of the parental mTagBFP including the high pH stability and fast chromophore formation. The enhanced photostability and chromophore chemical stability of mTagBFP2 make it a superior protein tag. mTagBFP2 performs well in the numerous protein fusions and surpasses mTagBFP as a donor in Förster resonance energy transfer with several green fluorescent protein acceptors.

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 °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.Monomeric fluorescent proteins of various emission wavelengths have become invaluable tools for studying the spatial behavior of intracellular molecules, including their localization and interaction 1 . To visualize temporal and spatial molecular events, FTs 2 , which change their emission wavelengths over time, could be especially valuable. The only currently available FT is DsRed-Timer FT (also known as DsRed-E5) 3 ; however, it is a tetramer, which prevents its application as a protein fusion tag. Nevertheless, the tetrameric state of the DsRed-Timer does not limit its use to study gene activities 4 , relative age of organelles 5 and cell differentiation 3 .It has been suggested that a red DsRed-like chromophore in the red fluorescent proteins (RFPs) results from an oxidation of a protonated blue form of the GFP-like chromophore, not from the green anionic form, which is a dead-end product 6 . This suggested scheme for red chromophore maturation provides a basis for developing monomeric FTs that change their color from blue to red. The most suitable templates for this appear to be the monomeric variants of DsRed 7 . One of these variants, mCherry, was chosen for a directed molecular evolution to develop three monomeric FTs with different maturation rates between the protonated blue GFP-like and the anionic red DsRed-like chromophore states.FTs can be used as molecular genetically encoded tools to study trafficking of different cellular proteins and to provide accurate insight into the timing of intracellular processes. The sequence NIH Public Access Author ManuscriptNat Chem Biol. Author manuscript; available in PMC 2010 February 1. Published in final edited form as:Nat Chem Biol. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript of events during trafficking of different cellular proteins before they reach their final compartment has often been the subject of contradictory investigations. An example of a longstanding dilemma is the contribution of different pathways to trafficking a...

CONTENTS 1. Introduction 4308 2. Chromophore Diversity in Red Fluorescent Proteins 4309 2.1. Common Blue Intermediate in the Formation of Red Chromophores 4310 2.2. Chemical Transitions in the Formed Red Chromophores 4311 2.3. Transformation of Red Chromophores into Orange Chromophores 4313 3. Photoinduced Transformations in Red Chromophores 4313 3.1. Light Adds More Options to Chromophore Transitions 4314 3.2. Diversity of Phototransformations in DsRedlike Chromophore 4314 3.3. Chromophore Formation in Kaede-like Proteins Requires Light 4315 4. How General Are the Principles of Chromophore Transformations? 4316 4.1. Different Fluorescent Proteins Made on the Basis of DsRed and Its Derivatives 4316 4.2. Various Fluorescent Probes Derived from TagRFP 4316 5. Structure−Function Relationship in the Fluorescent Proteins 4317 5.1. Chromophore Structure Is the Major Player 4317 5.2. Support from an Immediate Environment of the Chromophore 4317 5.3. Role of the Structural Elements Distant from the Chromophore 4318 6. Red Fluorescent Proteins in Advanced Imaging 4321 6.1. Far-Red-Shifted FPs in STED Nanoscopy and Whole-Body Imaging 4321 6.2. Photoactivatable RFPs in Multicolor PALM Microscopy 4323 6.3. Application of Reversibly Switchable RFPs to Super-Resolution Techniques 4323 6.4. RFPs with Large Stokes Shifts in Multicolor Fluorescence Microscopy 4323 7. Conclusions 4324 Author Information 4324 Corresponding Author 4324 Notes 4324 Biographies 4324 Acknowledgments 4324 References 4324

Two-photon microscopy has advanced fluorescence imaging of cellular processes in living animals. Fluorescent proteins in the blue-green wavelength range are widely used in two-photon microscopy; however, the use of red fluorescent proteins is limited by the low power output of Ti-Sapphire lasers above 1,000 nm. To overcome this limitation we have developed two red fluorescent proteins, LSS-mKate1 and LSS-mKate2, which possess large Stokes shifts with excitation/emission maxima at 463∕624 and 460∕605 nm, respectively. These LSS-mKates are characterized by high pH stability, photostability, rapid chromophore maturation, and monomeric behavior. They lack absorbance in the green region, providing an additional red color to the commonly used red fluorescent proteins. Substantial overlap between the two-photon excitation spectra of the LSS-mKates and blue-green fluorophores enables multicolor imaging using a single laser. We applied this approach to a mouse xenograft model of breast cancer to intravitally study the motility and Golgi-nucleus alignment of tumor cells as a function of their distance from blood vessels. Our data indicate that within 40 μm the breast cancer cells show significant polarization towards vessels in living mice.cell polarity | intravital imaging | Keima | two-photon microscopy | mKate

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 Ca2+ uptake.