Photoconvertible Fluorescent Protein Research Articles

The Photoconvertible Fluorescent Protein Dendra2 Tag as a Tool to Investigate Intracellular Protein Dynamics.

Fluorescence proteins changing spectral properties after exposure to light with a specific wavelength have recently become outstanding aids in the study of intracellular protein dynamics. Herein we show using Arabidopsis SYNAPTOTAGMIN 1 as a model protein that the Dendra2 green to red photoconvertible protein tag in combination with confocal scanning laser microscopy is a useful tool to study membrane protein intracellular dynamics.

Tracking Germline Stem Cell Dynamics In Vivo in C. elegans Using Photoconversion.

The maintenance of many adult tissues depends on stem cell systems, which must balance proliferation and differentiation. To understand the properties of adult stem cell systems, one powerful tool is visualization of the cell dynamics in vivo. Here we describe a protocol to track cells in the germline progenitor zone (which includes germline stem cells) in live C. elegans adult worms. Tracking is achieved by using a genetically encoded photoconvertible fluorescent protein, where photoconversion is used to mark cells of interest and their descendants. Individual worms are immobilized, the cells of interest are selected for photoconversion, and the worms are then recovered to plates and imaged at later timepoints. This allows longitudinal studies of individual worms, providing valuable information regarding germline stem cell dynamics.

High resolution measurement of membrane receptor endocytosis.

We present a new approach to quantify the half-life of membrane proteins on the cell surface, through tagging the protein with the photoconvertible fluorescent protein, Dendra2. Upon exposure to 405 nm light, Dendra2 is photoconverted from green to red emission. Total internal reflection fluorescence microscopy (TIRF) is applied to limit visualization of fluorescence to proteins located on the plasma membrane. Conversion of Dendra2 works as a pulse chase experiment through monitoring only the population of protein that has been photoconverted. As the protein is endocytosed the red emission decreases due to the protein leaving the TIRF field of view. This method is not impacted by the insertion of new protein into the plasma membrane as newly synthesized protein only exhibits green emission. We used this approach to determine the half-life of ENaC on the plasma membrane illustrating the high temporal resolution capability of this technique compared to current methods.

Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis.

Hematogenous metastasis is a multistep, selectin-regulated process whose mechanisms remain poorly understood. To investigate this biological pathway of cancer dissemination and better understand circulating cancer cells, we developed a high-throughput methodology that integrates organ-on-chip-like microfluidic and photoconvertible protein technologies. Our approach can ascribe single-cell velocity as a traceable cell property for off-chip analysis of the direct relationships between cell molecular profiles and adhesive phenotypes in the context of physiologically relevant fluid flow. We interrogate how natively expressed selectin ligands relate to colon cancer cell rolling frequencies and velocities and provide context for previously reported disparities in invitro and invivo models of selectin-mediated adhesion and metastasis. This integrated methodology represents a versatile approach for the development of anti-metastatic therapeutics as well as to generate and test mechanistic hypotheses regarding spatiotemporal processes that occur over timescales of seconds to hours with single-cell resolution.

Lifetime-based photoconversion of EGFP as a tool for FLIM

BackgroundEGFP is a fluorescent tag extensively used in biological and biomedical research. Over the years many researches have gathered collections of cell lines bearing specific EGFP-tagged proteins. Despite its popularity some photochemical properties of EGFP remain undocumented and unused. We report on so far unexplored lifetime photoconversion of EGFP usable in FLIM. MethodsFluorescence lifetime imaging and spectral FLIM has been used for characterization of the EGFP photoconversion and protein tracking. ResultOur data suggest that EGFP can be permanently photoconverted to a short-fluorescence-lifetime form (PC-EGFP) by intense blue irradiation. PC-EGFP cannot be reverted back by 405 nm light and exhibits the same spectral emission properties with blue-shifted absorption compared to the unconverted EGFP. Fluorescence of PC-EGFP is pH-independent and the photoconversion efficiency decreases with the solvent viscosity. Utilization of the EGFP photoconversion was demonstrated by tracking of a nucleophosmin mutant in live HEK-293 T cells during its cytoplasm-nuclear relocalization induced by Leptomycin B. ConclusionsBesides potential FLIM artifacts caused by an unintended EGFP photoconversion, the controlled photoconversion turns EGFP to an excellent tool for kinetic FLIM applications. Since the photoconversion occurs in the lifetime domain, PC-EGFP can be easily distinguished from the unconverted tag by time-resolved detection while all other spectral channels stay free for multicolor labeling. General significanceThe reported lifetime photoconversion lines up EGFP with other photoconvertible fluorescent proteins with special advantage for fluorescence lifetime imaging where lifetime-photoconvertible labels are scarce.

Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids

Photoconvertible fluorescent proteins (PCFPs) are widely used as markers for the visualization of intracellular processes and for sub-diffraction single-molecule localization microscopy. Although wild type of a new photoconvertible fluorescent protein SAASoti tends to aggregate, we succeeded, via rational mutagenesis, to obtain variants that formed either tetramers or monomers. We compare two approaches: one is based on the structural similarity between SAASoti and Kaede, which helped us to identify a single point mutation (V127T) at the protein’s hydrophobic interface that leads to monomerization. The other is based on a chemical modification of amino groups of SAASoti with succinic anhydride, which converts the protein aggregates into monomers. Mass-spectrometric analysis helped us to identify that the modification of a single ε-amino group of lysine K145 in the strongly charged interface AB was sufficient to convert the protein into its tetrameric form. Furthermore, site-directed mutagenesis was used to generate mutants that proved to be either monomeric or tetrameric, both capable of rapid green-to-red photoconversion. This allows SAASoti to be used as a photoconvertible fluorescent marker for in vivo cell studies.

Distinct Pathway of Human T-Cell Leukemia Virus Type 1 Gag Punctum Biogenesis Provides New Insights into Enveloped Virus Assembly.

The assembly of virus particles is a crucial aspect of virus spread. For retroviruses, the Gag polyprotein is the key driver for virus particle assembly. In order to produce progeny virus, once Gag is translated, it must translocate from the location in the cytoplasm where it is synthesized to the plasma membrane and form an oligomeric lattice that results in Gag puncta. The biogenesis of mature Gag puncta can trigger the budding process, resulting in virus particle production. While some aspects of the dynamics of Gag oligomerization and particle biogenesis have been observed with human immunodeficiency virus type 1 (HIV-1), the process of Gag punctum biogenesis remains poorly understood, particularly for other retroviruses. Here, we have conducted the most detailed studies thus far on Gag punctum biogenesis for human T-cell leukemia virus type 1 (HTLV-1). Using mEos2 photoconvertible fluorescent proteins and total internal reflection fluorescence microscopy (TIRF), we have found that HTLV-1 Gag was recruited to Gag puncta primarily from the plasma membrane. This was in stark contrast to HIV-1 Gag, which was recruited from the cytoplasm. These observations imply fundamental differences among retroviruses regarding the orchestration of Gag punctum biogenesis, which has important general implications for enveloped virus particle assembly.IMPORTANCE This report describes the results of experiments examining the pathway by which the human retroviral Gag protein is recruited to sites along the inner leaflet of the plasma membrane where Gag punctum biogenesis occurs. In particular, clever and sensitive experimental methods were devised to image in living cells fluorescently labeled Gag protein derivatives from human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) at the plasma membrane. The photoconvertible fluorescent protein mEos2 was strategically utilized, as the fluorescence emission of Gag at the plasma membrane could be differentiated from that of cytosolic Gag. This experimental strategy allowed for the determination of the Gag recruitment pathway into Gag puncta. For HTLV-1 Gag, puncta recruited Gag primarily from the plasma membrane, while HIV-1 Gag was recruited from the cytoplasm. These observations represent the first report of HTLV-1 particle biogenesis and its contrast to that of HIV-1. The observed differences in the Gag recruitment pathways used by HTLV-1 and HIV-1 Gag provide key information that is useful for informing the discovery of novel targets for antiretroviral therapies directed at eliminating virus infectivity and spread.

Bacterial Cytological Profiling Reveals the Mechanism of Action of Anticancer Metal Complexes.

Target identification and mechanistic studies of cytotoxic agents are challenging processes that are both time-consuming and costly. Here we describe an approach to mechanism of action studies for potential anticancer compounds by utilizing the simple prokaryotic system, E. coli, and we demonstrate its utility with the characterization of a ruthenium polypyridyl complex [Ru(bpy)2dmbpy2+]. Expression of the photoconvertible fluorescent protein Dendra2 facilitated both high throughput studies and single-cell imaging. This allowed for simultaneous ratiometric analysis of inhibition of protein production and phenotypic investigations. The profile of protein production, filament size and population, and nucleoid morphology revealed important differences between inorganic agents that damage DNA vs more selective inhibitors of transcription and translation. Trace metal analysis demonstrated that DNA is the preferred nucleic acid target of the ruthenium complex, but further studies in human cancer cells revealed altered cell signaling pathways compared to the commonly administrated anticancer agent cisplatin. This study demonstrates E. coli can be used to rapidly distinguish between compounds with disparate mechanisms of action and also for more subtle distinctions within in studies in mammalian cells.

Primed Conversion: The New Kid on the Block for Photoconversion.

In 2015, a novel way to convert photoconvertible fluorescent proteins was reported that uses the intercept of blue and far-red light instead of traditional violet or near-UV light illumination. This Minireview describes and contrasts this distinct two-step mechanism termed primed conversion with traditional photoconversion. We provide a comprehensive overview of what is known to date about primed conversion and focus on the molecular requirements for it to take place. We provide examples of its application to axially confined photoconversion in complex tissues as well as super-resolution microscopy. Further, we describe why and when it is useful, including its advantages and disadvantages, and give an insight into potential future development in the field.

<em>In Vivo</em> Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

Bacterial Chemoreceptor Imaging at High Spatiotemporal Resolution Using Photoconvertible Fluorescent Proteins.

We describe two methods for high-resolution fluorescence imaging of the positioning and mobility of E. coli chemoreceptors fused to photoconvertible fluorescent proteins. Chemoreceptors such as Tar and Tsr are transmembrane proteins expressed at high levels (thousands of copies per cell). Together with their cognate cytosolic signaling proteins, they form clusters on the plasma membrane. Theoretical models imply that the size of these clusters is an important parameter for signaling, and recent PALM imaging has revealed a broad distribution of cluster sizes. We describe experimental setups and protocols for PALM imaging in fixed cells with ~10 nm spatial precision, which allows analysis of cluster-size distributions, and localized-photoactivation single-particle tracking (LPA-SPT) in live cells at ~10ms temporal resolution, which allows for analysis of cluster mobility.

PML-Nuclear Bodies Regulate the Stability of the Fusion Protein Dendra2-Nrf2 in the Nucleus

Background/Aims: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine-zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. Methods: Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2-Nrf2 in single live cells. Results: Using this approach, we show that the half-life (t1/2) of Dendra2-Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG-132 or induction of oxidative stress with tert-butylhydroquinone (tBHQ) extended the half-life of Dendra2-Nrf2 by 6- and 28-fold, respectively. By inhibiting nuclear export using Leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML-NBs (Promyelocytic Leukemia-nuclear bodies). We further demonstrate that co-expression of Dendra2-Nrf2 and Crimson-PML-I lacking two PML-I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2-Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. Conclusion: Altogether, our findings provide direct evidence for degradation of Nrf2 in the nucleus and suggest that modification of Nrf2 in PML nuclear bodies contributes to its degradation in intact cells.

Axially-confined invivo single-cell labeling by primed conversion using blue and red lasers with conventional confocal microscopes.

Green-to-red photoconvertible fluorescent proteins have been found to undergo efficient photoconversion by a new method termed primed conversion that uses dual wave-length illumination with blue and red/near-infrared light. By modifying a confocal laser-scanning microscope (CLSM) such that two laser beams only meet at the focal plane, confined photoconversion at the axial dimension has been achieved. The necessity of this custom modification to the CLSM, however, has precluded the wide-spread use of this method. Here, we investigated whether spatially-restricted primed conversion could be achieved with CLSM without any hardware modification. We found that the primed conversion of Dendra2 using a conventional CLSM with two visible lasers (473nm and 635nm) and a high NA objective lens (NA, 1.30) resulted in dramatic restriction of photoconversion volume: half-width half-maximum for the axial dimension was below 5μm, which is comparable to the outcome of the original method that used the microscope modification. As a proof of this method's effectiveness, we used this technique in living zebrafish embryos and succeeded in revealing the complex anatomy of individual neurons packed between neighboring cells. Because unmodified CLSMs are widely available, this method can be widely applicable for labeling cells with single-cell resolution.

Trapping a long-lived dark state in photoconvertible fluorescent protein mEos4b

Trapping a long-lived dark state in photoconvertible fluorescent protein mEos4b

The Role of Probe Photophysics in Localization-Based Superresolution Microscopy

Fluorescent proteins are used extensively for biological imaging applications; photoactivatable and photoconvertible fluorescent proteins (PAFPs) are used widely in superresolution localization microscopy methods such as fluorescence photoactivation localization microscopy and photoactivated localization microscopy. However, their optimal use depends on knowledge of not only their bulk fluorescence properties, but also their photophysical properties at the single molecule level. We have used fluorescence correlation spectroscopy and cross-correlation spectroscopy to quantify the diffusion, photobleaching, fluorescence intermittency, and photoconversion dynamics of Dendra2, a well-known PAFP used in localization microscopy. Numerous dark states of Dendra2 are observed both in inactive (green fluorescent) and active (orange fluorescent) forms; the interconversion rates are both light- and pH-dependent, as observed for other PAFPs. The dark states limit the detected count rate per molecule, which is a crucial parameter for localization microscopy. We then developed, to our knowledge, a new mathematical estimate for the resolution in localization microscopy as a function of the measured photophysical parameters of the probe such as photobleaching quantum yield, count rate per molecule, and intensity of saturation. The model was used to predict the dependence of resolution on acquisition parameters such as illumination intensity and time per frame, demonstrating an optimal set of acquisition parameters for a given probe for a variety of measures of resolution. The best possible resolution was then compared for Dendra2 and other widely used probes, including Alexa dyes and quantum dots. This work establishes a framework for determination of the best possible resolution using a localization microscope to image a particular fluorophore, and suggests that development of probes for use in superresolution localization microscopy must consider the count rate per molecule, the saturation intensity, the photobleaching yield, and, crucially, management of bright/dark state transitions, to optimize image resolution.

Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor

Fluorescent protein Dendra2 is a monomeric GFP-like protein that belongs to the group of Kaede-like photoconvertible fluorescent proteins with irreversible photoconversion from a green- to red-emitting state when exposed to violet-blue light. In an acidic environment, photoconverted Dendra2 turns green due to protonation of the phenolic group of the chromophore with pKa of about 7.5. Thus, photoconverted form of Dendra2 can be potentially used as a ratiometric pH-sensor in the physiological pH range. However, incomplete photoconversion makes ratiometric measurements irreproducible when using standard filter sets. Here, we describe the method to detect fluorescence of only photoconverted Dendra2 form, but not nonconverted green Dendra2. We show that the 350 nm excitation light induces solely the fluorescence of photoconverted protein. By measuring the red to green fluorescence ratio, we determined intracellular pH in live CHO and HEK 293 cells. Thus, Dendra2 can be used as a novel ratiometric genetically encoded pH sensor with emission maxima in the green-red spectral region, which is suitable for application in live cells.

Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy.

Green-to-red photoconvertible fluorescent proteins (PCFPs) such as mEos2 and its derivatives are widely used in PhotoActivated Localization Microscopy (PALM). However, the complex photophysics of these genetically encoded markers complicates the quantitative analysis of PALM data. Here, we show that intense 561 nm light (∼1 kW/cm2) typically used to localize single red molecules considerably affects the green-state photophysics of mEos2 by populating at least two reversible dark states. These dark states retard green-to-red photoconversion through a shelving effect, although one of them is rapidly depopulated by 405 nm light illumination. Multiple mEos2 switching and irreversible photobleaching is thus induced by yellow/green and violet photons before green-to-red photoconversion occurs, contributing to explain the apparent limited signaling efficiency of this PCFP. Our data reveals that the photophysics of PCFPs of anthozoan origin is substantially more complex than previously thought, and suggests that intense 561 nm laser light should be used with care, notably for quantitative or fast PALM approaches.

Photoconvertible Fluorescent Proteins and the Role of Dynamics in Protein Evolution.

Photoconvertible fluorescent proteins (pcFPs) constitute a large group of fluorescent proteins related to green fluorescent protein (GFP) that, when exposed to blue light, bear the capability of irreversibly switching their emission color from green to red. Not surprisingly, this fascinating class of FPs has found numerous applications, in particular for the visualization of biological processes. A detailed understanding of the photoconversion mechanism appears indispensable in the design of improved variants for applications such as super-resolution imaging. In this article, recent work is reviewed that involves using pcFPs as a model system for studying protein dynamics. Evidence has been provided that the evolution of pcFPs from a green ancestor involved the natural selection for altered dynamical features of the beta-barrel fold. It appears that photoconversion may be the outcome of a long-range positional shift of a fold-anchoring region. A relatively stiff, rigid element appears to have migrated away from the chromophore-bearing section to the opposite edge of the barrel, thereby endowing pcFPs with increased active site flexibility while keeping the fold intact. In this way, the stage was set for the coupling of light absorption with subsequent chemical transformations. The emerging mechanistic model suggests that highly specific dynamic motions are linked to key chemical steps, preparing the system for a concerted deprotonation and β-elimination reaction that enlarges the chromophore’s π-conjugation to generate red color.

Rational Engineering of Photoconvertible Fluorescent Proteins for Dual‐Color Fluorescence Nanoscopy Enabled by a Triplet‐State Mechanism of Primed Conversion

AbstractGreen‐to‐red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super‐resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400‐nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far‐red/near‐infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate “primed” state to be a triplet dark state formed by intersystem crossing. We show that formation of this state can be influenced by the introduction of serine or threonine at sequence position 69 (Eos notation) and use this knowledge to create “pr”‐ (for primed convertible) variants of most known green‐to‐red pcFPs.