Photoconvertible Fluorescent Protein Research Articles

Mechanistic Investigations of Green mEos4b Reveal a Dynamic Long-Lived Dark State.

Green-to-red photoconvertible fluorescent proteins (PCFPs) are key players in advanced microscopy schemes such as photoactivated localization microscopy (PALM). Whereas photoconversion and red-state blinking in PCFPs have been studied intensively, their green-state photophysical behavior has received less attention. Yet dark states in green PCFPs can become strongly populated in PALM schemes and exert an indirect but considerable influence on the quality of data recorded in the red channel. Furthermore, green-state photoswitching in PCFPs can be used directly for PALM and has been engineered to design highly efficient reversibly switchable fluorescent proteins (RSFPs) amenable to various nanoscopy schemes. Here, we demonstrate that green mEos4b efficiently switches to a long-lived dark state through cis-trans isomerization of its chromophore, as do most RSFPs. However, by combining kinetic crystallography, molecular dynamics simulations, and Raman spectroscopy, we find that the dark state in green mEos4b is much more dynamic than that seen in switched-off green IrisFP, a biphotochromic PCFP engineered from the common EosFP parent. Our data suggest that H-bonding patterns maintained by the chromophore in green PCFPs and RSFPs in both their on- and off-states collectively control photoswitching quantum yields. The reduced number of H-bonds maintained by the dynamic dark chromophore in green mEos4b thus largely accounts for the observed lower switching contrast as compared to that of IrisFP. We also compare the long-lived dark states reached from green and red mEos4b, on the basis of their X-ray structures and Raman signatures. Altogether, these data provide a unifying picture of the complex photophysics of PCFPs and RSFPs.

Photoconvertible fluorescent proteins: a versatile tool in zebrafish skeletal imaging.

One of the most frequently applied techniques in zebrafish (Danio rerio) research is the visualisation or manipulation of specific cell populations using transgenic reporter lines. The generation of these transgenic zebrafish, displaying cell- or tissue-specific expression of frequently used fluorophores such as Green Fluorescent Protein (GFP) or mCherry, is relatively easy using modern techniques. Fluorophores with different emission wavelengths and driven by different promoters can be monitored simultaneously in the same animal. Photoconvertible fluorescent proteins (pcFPs) are different from these standard fluorophores because their emission spectrum is changed when exposed to UV light, a process called photoconversion. Here, the benefits and versatility of using pcFPs for both single and dual fluorochrome imaging in zebrafish skeletal research in a previously generated osx:Kaede transgenic line are illustrated. In this line, Kaede, which is expressed under control of the osterix, otherwise known as sp7, promoter thereby labelling immature osteoblasts, can switch from green to red fluorescence upon irradiation with UV light. First, this study demonstrates that osx:Kaede exhibits an expression pattern similar to a previously described osx:nuGFP transgenic line in both larval and adult stages, hereby validating the use of this line for the imaging of immature osteoblasts. More in-depth experiments highlight different applications for osx:Kaede, such as lineage tracing and its combined use with in vivo skeletal staining and other transgenic backgrounds. Mineral staining in combination with osx:Kaede confirms osteoblast-independent mineralisation of the notochord. Osteoblast lineage tracing reveals migration and dedifferentiation of scleroblasts during fin regeneration. Finally, this study shows that combining two transgenics, osx:Kaede and osc:GFP, with similar emission wavelengths is possible when using a pcFP such as Kaede.

CRISPR screening identifies M1AP as a new MYC regulator with a promoter-reporter system.

BackgroundMYC is one of the proto-oncogenes contributing to tumorigenesis in many human cancers. Although the mechanism of MYC regulation is still not fully understood, learning about the comprehensive mechanism controlling the transcriptional activity of MYC will lead to therapeutic targets. The CRISPR/Cas9 library system is a simple and powerful screening technique. This study aims to identify new transcriptional upstream activators of MYC using the CRISPR activation library with new promoter-reporter systems.Methods and ResultsThe MYC promoter-reporter system was developed with a photoconvertible fluorescent protein, Dendra2, and named “pMYC-promoter-Dendra2.” This MYC promoter-reporter system was designed to harbor a proximal MYC promoter at (3.1 kb). Both the CRISPR activation library and pMYC-promoter-Dendra2 were induced to HEK 293T cells, and Dendra2-positive cells, that are supposed that MYC should be upregulated, were collected individually by a cell sorter. Among the 169 cells collected, 12 clones were successfully established. Then, pMYC-promoter-Dendra2 was transfected again into these 12 clones, and two of 12 clones showed Dendra2 positivity. In this procedure, the cells with non-specific autofluorescence were correctly distinguished by utilizing the photoswitchable character of Dendra2. Using extracted genomic DNA of these two Dendra2 positive clones, polymerase chain reaction (PCR) was performed to amplify the guide RNA (gRNA) containing region, which was introduced by the CRISPR activation library. Eventually, PLEKHO2, MICU, MBTPS1, and M1AP were identified, and these gRNAs were transfected individually into HEK 293T cells again using the CRISPR activation system. Only M1AP gRNA transfected cells showed Dendra2-positive fluorescence. Then, the overexpression vector for M1AP with a doxycycline-inducible vector confirmed that M1AP induced high MYC expression by real-time quantitative PCR and western blot. Furthermore, the dual-luciferase assay showed a significant increase of promoter activity, and MYC mRNA was higher in M1AP- overexpressing cells. M1AP is highly expressed in several cancers, though, a positive correlation between M1AP and MYC was observed only in human acute myeloid leukemia.ConclusionThe present study confirmed that the experimental method using the CRISPR library technology functions effectively for the identification of molecules that activate endogenous MYC. This method will help elucidate the regulatory mechanism of MYC expression, as well as supporting further drug research against malignant tumors.

MEosBrite Are Bright Variants of mEos3.2 Developed by Semirational Protein Engineering.

mEos3.2 is a photoconvertible fluorescence protein with comparatively low brightness, which limits its application in live Super resolution microscopy. To address this issue, we have used semi-rational protein engineering to develop mEosBrite, a new class of improved brightness variants. The improvement in the brightness was confirmed by expression in E.coli as well as mammalian cell lines. Furthermore, biophysical characterization suggests that all the three mEosBrite variant proteins display higher quantum yield, truly monomeric form, less cytotoxicity and lower protein aggregation as compared to the wild type mEos3.2 protein. Most importantly, because of their high photoconversion efficiency mEosBrite variants could be an excellent tool for single-molecule and intensity fluctuation based super-resolution microscopy.

In vivo tracking: Using a photoconvertible system to probe immune cell entry and exit in the context of inflammation

Abstract Chronic inflammatory diseases such as Crohn’s disease and rheumatoid arthritis affect millions of people worldwide. During an inflammatory response, immune cells enter the inflamed tissue to mediate pathogen clearance, however, their activation and overaccumulation in the tissue can lead to chronic inflammation and autoimmunity. Little is known about the signals that regulate immune cell entry into and exit from these inflamed tissues or about the retention cues that play a role in their accumulation. Therefore, a better understanding of these signals would inform the design of therapeutics intended to alleviate chronic inflammation. To study immune cell movement between a site of inflammation and lymphoid organs, our lab has acquired a mouse model that allows for the in vivo labeling of a subset of cells in time and space. The cells of Kaede transgenic mice constitutively express the photoconvertible fluorescent Kaede protein. CFA/OVA-inflamed ears were photoconverted from Kaede green to red by violet light exposure and the trafficking of Kaede red cells out of the skin to the draining lymph node was assessed over the course of 24 hours. Based on preliminary data, we found that, of the subsets assessed, neutrophils and CD4+ T cells made up the largest percentages of immune cells exiting the inflamed tissue on day 5 of inflammation. On the other hand, far fewer conventional dendritic cells and macrophages were seen to enter the draining lymph node from the inflamed tissue. The role of these tissue-emigrant immune cells is not yet clear, but cells such as neutrophils may play a role in shaping adaptive immune responses in the draining lymph node.

Quantitative Imaging Reveals Distinct Contributions of SnRK2 and ABI3 in Plasmodesmatal Permeability in Physcomitrella patens.

Cell-to-cell communication is tightly regulated in response to environmental stimuli in plants. We previously used a photoconvertible fluorescent protein Dendra2 as a model reporter to study this process. This experiment revealed that macromolecular trafficking between protonemal cells in Physcomitrella patens is suppressed in response to abscisic acid (ABA). However, it remains unknown which ABA signaling components contribute to this suppression and how. Here, we show that ABA signaling components SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 2 (PpSnRK2) and ABA INSENSITIVE 3 (PpABI3) play roles as an essential and promotive factor, respectively, in regulating ABA-induced suppression of Dendra2 diffusion between cells (ASD). Our quantitative imaging analysis revealed that disruption of PpSnRK2 resulted in defective ASD onset itself, whereas disruption of PpABI3 caused an 81-min delay in the initiation of ASD. Live-cell imaging of callose deposition using aniline blue staining showed that, despite this onset delay, callose deposition on cross walls remained constant in the PpABI3 disruptant, suggesting that PpABI3 facilitates ASD in a callose-independent manner. Given that ABA is an important phytohormone to cope with abiotic stresses, we further explored cellular physiological responses. We found that the acquisition of salt stress tolerance is promoted by PpABI3 in a quantitative manner similar to ASD. Our results suggest that PpABI3-mediated ABA signaling may effectively coordinate cell-to-cell communication during the acquisition of salt stress tolerance. This study will accelerate the quantitative study for ABA signaling mechanism and function in response to various abiotic stresses.

Tracking the subcellular protein localization process by combining the time-gated method and photoconvertible fluorescence protein

Tracking the subcellular protein localization process by combining the time-gated method and photoconvertible fluorescence protein

Photoconvertible diazaxanthilidene dyes for live cell imaging.

Within the past two decades, photoconvertible fluorescent proteins (PC-FPs) have emerged as a class of useful proteins for the visualization and tracking of individual cells, complex cellular mechanisms, protein-protein interactions, and other dynamic processes. Despite the utility of these proteins, they are inherently limited by a number of factors including large size and inflexibility of tag location within a protein of interest. The following chapter describes the discovery and use of a small molecule photoconvertible dye based on the novel diazaxanthilidene scaffold. The diazaxanthilidene dye presented in this chapter is shown to be an effective alternative to well-known PC-FPs for spatiotemporally controlled cell labeling experiments.

Dual Illumination Enhances Transformation of an Engineered Green-to-Red Photoconvertible Fluorescent Protein.

The molecular mechanisms for the photoconversion of fluorescent proteins remain elusive owing to the challenges of monitoring chromophore structural dynamics during the light-induced processes. We implemented time-resolved electronic and stimulated Raman spectroscopies to reveal two hidden species of an engineered ancestral GFP-like protein LEA, involving semi-trapped protonated and trapped deprotonated chromophores en route to photoconversion in pH 7.9 buffer. A new dual-illumination approach was examined, using 400 and 505 nm light simultaneously to achieve faster conversion and higher color contrast. Substitution of UV irradiation with visible light benefits bioimaging, while the spectral benchmark of a trapped chromophore with characteristic ring twisting and bridge-H bending motions enables rational design of functional proteins. With the improved H-bonding network and structural motions, the photoexcited chromophore could increase the photoswitching-aided photoconversion while reducing trapped species.

Dual Illumination Enhances Transformation of an Engineered Green‐to‐Red Photoconvertible Fluorescent Protein

AbstractThe molecular mechanisms for the photoconversion of fluorescent proteins remain elusive owing to the challenges of monitoring chromophore structural dynamics during the light‐induced processes. We implemented time‐resolved electronic and stimulated Raman spectroscopies to reveal two hidden species of an engineered ancestral GFP‐like protein LEA, involving semi‐trapped protonated and trapped deprotonated chromophores en route to photoconversion in pH 7.9 buffer. A new dual‐illumination approach was examined, using 400 and 505 nm light simultaneously to achieve faster conversion and higher color contrast. Substitution of UV irradiation with visible light benefits bioimaging, while the spectral benchmark of a trapped chromophore with characteristic ring twisting and bridge‐H bending motions enables rational design of functional proteins. With the improved H‐bonding network and structural motions, the photoexcited chromophore could increase the photoswitching‐aided photoconversion while reducing trapped species.

Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes.

Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

Fast Super-Resolution Imaging Technique and Immediate Early Nanostructure Capturing by a Photoconvertible Fluorescent Protein.

Low temporal resolution and limited photocontrollable fluorescent protein probes have restricted the widespread application of single-molecule localization microscopy (SMLM). In the current study, we developed a new photoconvertible fluorescent protein (PCFP), pcStar, and quick single molecule-guided Bayesian localization microscopy (Quick-SIMBA). The combination of pcStar and Quick-SIMBA achieved the highest temporal resolution (0.1-0.25 s) with large field-of-view (76 × 9.4 μm2 -76 × 31.4 μm2) among the SMLM methods, which enabled the dynamic movements of the endoplasmic reticulum dense tubular matrix to be resolved. Moreover, pcStar extended the application of SMLM to imaging the immediate early nanostructures in Drosophila embryos and revealed a specific "parallel three-pillar" structure in the neuronal-glial cell junction, helping to elucidate glial cell "locking" and support of neurons during Drosophila embryogenesis.

Nuclear pores as versatile reference standards for quantitative superresolution microscopy.

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions.Here we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise reference standards for molecular counting.These cell lines will enable the broad community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

Relationship between relocation of phototropin to the chloroplast periphery and the initiation of chloroplast movement in Marchantia polymorpha.

The blue‐light photoreceptor kinase phototropin (phot) mediates chloroplast movement in response to light and temperature. Phot predominantly localizes at the plasma membrane, but also resides in the cytosol and the chloroplast periphery. Although the phot localized to the chloroplast periphery is thought to mediate chloroplast movement, the localization mechanism is unknown. In this study, we found that chloroplast movement does not occur in 0‐day‐old gemma cells of the liverwort Marchantia polymorpha but that the movement is induced in 1‐day‐old gemmaling cells. Along with this physiological change, the subcellular localization of phot also changed: In 0‐day‐old gemma cells, phot localized at the plasma membrane and the cytosol, but in 1‐day‐old gemmaling cells, the phot disappeared from the cytosol and appeared at the chloroplast periphery. When the relocalization was tracked using a photoconvertible fluorescent protein, the cytosolic phot relocated to the plasma membrane, and the plasma membrane‐resident phot relocated to the chloroplast periphery. The blue‐light‐dependent activation of phot kinase activity enhanced this relocalization. Mutated phot deficient in blue‐light reception or kinase activity had a severely reduced ability to localize at the chloroplast periphery. These findings suggest that photoactivated phot localizes at the chloroplast periphery to initiate chloroplast movement.

Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM.

Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of longer tracks in sptPALM with minimum effort.

Peripheral lymph nodes contain migratory and resident innate lymphoid cell populations.

Tissue residency is considered a defining feature of the innate lymphoid cell (ILC) populations located within mucosal and adipose tissues. ILCs are also present within all lymphoid tissues, but whether ILCs migrate between lymphoid and nonlymphoid sites and in what context is poorly understood. To determine whether migratory ILCs exist within peripheral lymph nodes (LNs), we labeled all cells within the brachial LN (bLN) of transgenic mice expressing a photoconvertible fluorescent protein by direct exposure to light. Tracking of cellular changes in the labeled LN revealed the gradual migration of new ILCs into the tissue, balanced by egress of ILCs dependent on sphingosine-1-phosphate receptors. Most of the migratory ILCs were ILC1s, entering LNs directly from the circulation in a CD62L- and CCR7-dependent manner and thus behaving like conventional natural killer (cNK) cells. Upon egress, both ILC1s and cNK cells were found to recirculate through peripheral LNs. A distinct population of migratory ILC2s were detected in the LN, but most of the ILC3s were tissue resident. Functionally, both migratory and resident ILC1s within LNs were able to rapidly produce IFN-γ to support the generation of robust TH1 T cell responses after immunization. Thus, migratory and resident ILC populations exist within peripheral LNs, with ILC1s, akin to cNK cells, able to traffic into these tissues where they can contribute to the initiation of adaptive immunity.

VE-PTP stabilizes VE-cadherin junctions and the endothelial barrier via a phosphatase-independent mechanism.

Vascular endothelial (VE) protein tyrosine phosphatase (PTP) is an endothelial-specific phosphatase that stabilizes VE-cadherin junctions. Although studies have focused on the role of VE-PTP in dephosphorylating VE-cadherin in the activated endothelium, little is known of VE-PTP's role in the quiescent endothelial monolayer. Here, we used the photoconvertible fluorescent protein VE-cadherin-Dendra2 to monitor VE-cadherin dynamics at adherens junctions (AJs) in confluent endothelial monolayers. We discovered that VE-PTP stabilizes VE-cadherin junctions by reducing the rate of VE-cadherin internalization independently of its phosphatase activity. VE-PTP serves as an adaptor protein that through binding and inhibiting the RhoGEF GEF-H1 modulates RhoA activity and tension across VE-cadherin junctions. Overexpression of the VE-PTP cytosolic domain mutant interacting with GEF-H1 in VE-PTP-depleted endothelial cells reduced GEF-H1 activity and restored VE-cadherin dynamics at AJs. Thus, VE-PTP stabilizes VE-cadherin junctions and restricts endothelial permeability by inhibiting GEF-H1, thereby limiting RhoA signaling at AJs and reducing the VE-cadherin internalization rate.

Imaging through the Whole Brain of Drosophila at λ/20 Super-resolution

SummaryRecently, many super-resolution technologies have been demonstrated, significantly affecting biological studies by observation of cellular structures down to nanometer precision. However, current super-resolution techniques mostly rely on wavefront engineering or wide-field imaging of signal blinking or fluctuation, and thus imaging depths are limited due to tissue scattering or aberration. Here we present a technique that is capable of imaging through an intact Drosophila brain with 20-nm lateral resolution at ∼200 μm depth. The spatial resolution is provided by molecular localization of a photoconvertible fluorescent protein Kaede, whose red form is found to exhibit blinking state. The deep-tissue observation is enabled by optical sectioning of spinning disk microscopy, as well as reduced scattering from optical clearing. Together these techniques are readily available for many biologists, providing three-dimensional resolution of densely entangled dendritic fibers in a complete Drosophila brain. The method paves the way toward whole-brain neural network studies and is applicable to other high-resolution bioimaging.

Imaging Through the Whole Brain of Drosophila at λ/20 Super-Resolution

Recently, many super-resolution technologies were demonstrated, significantly impacting biological studies by observation of cellular structures down to nanometer precision. However, current super-resolution techniques mostly rely on wavefront engineering or wide-field imaging of signal blinking/fluctuation, and thus imaging depth are limited due to tissue scattering/aberration. Here we present a technique that is capable to image through an intact Drosophila brain with 20 nm lateral resolution at ~200 μm depth. The spatial resolution is provided by molecular localization of a photoconvertible fluorescent protein Kaede, whose blinking state is reported for the first time. The deep-tissue observation is enabled by optical sectioning of spinning disk microscopy, as well as reduced scattering of optical clearing. Together these techniques are readily available for many biologists, providing three-dimensionally resolution of densely entangled dendritic fibers in a complete Drosophila brain. The method paves the way toward whole-brain neural network studies, and is applicable to other high-resolution bio-imaging.

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells.

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells and protein ensembles. Thus far, no method has been available to quantify in bulk and in live cells how many of the PA-FPs expressed are photoactivated to fluoresce. Here, we present a protocol involving internal rulers, i.e., genetically coupled spectrally distinct (photoactivatable) fluorescent proteins, to ratiometrically quantify the fraction of all PA-FPs expressed in a cell that are switched on to be fluorescent. Using this protocol, we show that different modes of photoactivation yielded different photoactivation efficiencies. Short high-power photoactivation with a confocal laser scanning microscope (CLSM) resulted in up to four times lower photoactivation efficiency than hundreds of low-level exposures applied by CLSM or a short pulse applied by widefield illumination. While the protocol has been exemplified here for (PA-)GFP and (PA-)Cherry, it can in principle be applied to any spectrally distinct photoactivatable or photoconvertible fluorescent protein pair and any experimental set-up.