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11 pages, 1808 KiB

Open AccessArticle

Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose

by Ekaterina O. Serebrovskaya, Nadezda M. Podvalnaya, Varvara V. Dudenkova, Anna S. Efremova, Nadya G. Gurskaya, Dmitry A. Gorbachev, Artem V. Luzhin, Omar L. Kantidze, Elena V. Zagaynova, Stanislav I. Shram and Konstantin A. Lukyanov

Int. J. Mol. Sci. 2020, 21(14), 5004; https://doi.org/10.3390/ijms21145004 - 15 Jul 2020

Cited by 7 | Viewed by 3518

Abstract

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based [...] Read more.

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia. Full article

(This article belongs to the Special Issue Imaging with Fluorescent Proteins)

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20 pages, 4097 KiB

Open AccessArticle

Genetically Encoded Photosensitizers as Light-Triggered Antimicrobial Agents

by Fabienne Hilgers, Nora Lisa Bitzenhofer, Yannic Ackermann, Alina Burmeister, Alexander Grünberger, Karl-Erich Jaeger and Thomas Drepper

Int. J. Mol. Sci. 2019, 20(18), 4608; https://doi.org/10.3390/ijms20184608 - 17 Sep 2019

Cited by 23 | Viewed by 4804

Abstract

Diseases caused by multi-drug resistant pathogens have become a global concern. Therefore, new approaches suitable for treating these bacteria are urgently needed. In this study, we analyzed genetically encoded photosensitizers (PS) related to the green fluorescent protein (GFP) or light-oxygen-voltage (LOV) photoreceptors for [...] Read more.

Diseases caused by multi-drug resistant pathogens have become a global concern. Therefore, new approaches suitable for treating these bacteria are urgently needed. In this study, we analyzed genetically encoded photosensitizers (PS) related to the green fluorescent protein (GFP) or light-oxygen-voltage (LOV) photoreceptors for their exogenous applicability as light-triggered antimicrobial agents. Depending on their specific photophysical properties and photochemistry, these PSs can produce different toxic ROS (reactive oxygen species) such as O₂^•− and H₂O₂ via type-I, as well as ¹O₂ via type-II reaction in response to light. By using cell viability assays and microfluidics, we could demonstrate differences in the intracellular and extracellular phototoxicity of the applied PS. While intracellular expression and exogenous supply of GFP-related PSs resulted in a slow inactivation of E. coli and pathogenic Gram-negative and Gram-positive bacteria, illumination of LOV-based PSs such as the singlet oxygen photosensitizing protein SOPP3 resulted in a fast and homogeneous killing of these microbes. Furthermore, our data indicate that the ROS type and yield as well as the localization of the applied PS protein can strongly influence the antibacterial spectrum and efficacy. These findings open up new opportunities for photodynamic inactivation of pathogenic bacteria. Full article

(This article belongs to the Special Issue Imaging with Fluorescent Proteins)

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18 pages, 5496 KiB

Open AccessArticle

Near-Infrared Genetically Encoded Positive Calcium Indicator Based on GAF-FP Bacterial Phytochrome

by Oksana M. Subach, Natalia V. Barykina, Konstantin V. Anokhin, Kiryl D. Piatkevich and Fedor V. Subach

Int. J. Mol. Sci. 2019, 20(14), 3488; https://doi.org/10.3390/ijms20143488 - 16 Jul 2019

Cited by 32 | Viewed by 4199

Abstract

A variety of genetically encoded calcium indicators are currently available for visualization of calcium dynamics in cultured cells and in vivo. Only one of them, called NIR-GECO1, exhibits fluorescence in the near-infrared region of the spectrum. NIR-GECO1 is engineered based on the near-infrared [...] Read more.

A variety of genetically encoded calcium indicators are currently available for visualization of calcium dynamics in cultured cells and in vivo. Only one of them, called NIR-GECO1, exhibits fluorescence in the near-infrared region of the spectrum. NIR-GECO1 is engineered based on the near-infrared fluorescent protein mIFP derived from bacterial phytochromes. However, NIR-GECO1 has an inverted response to calcium ions and its excitation spectrum is not optimal for the commonly used 640 nm lasers. Using small near-infrared bacterial phytochrome GAF-FP and calmodulin/M13-peptide pair, we developed a near-infrared calcium indicator called GAF-CaMP2. In vitro, GAF-CaMP2 showed a positive response of 78% and high affinity (K_d of 466 nM) to the calcium ions. It had excitation and emission maxima at 642 and 674 nm, respectively. GAF-CaMP2 had a 2.0-fold lower brightness, 5.5-fold faster maturation and lower pH stability compared to GAF-FP in vitro. GAF-CaMP2 showed 2.9-fold higher photostability than smURFP protein. The GAF-CaMP2 fusion with sfGFP demonstrated a ratiometric response with a dynamic range of 169% when expressed in the cytosol of mammalian cells in culture. Finally, we successfully applied the ratiometric version of GAF-CaMP2 for the simultaneous visualization of calcium transients in three organelles of mammalian cells using four-color fluorescence microscopy. Full article

(This article belongs to the Special Issue Imaging with Fluorescent Proteins)

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9 pages, 3002 KiB

Open AccessArticle

Novel Phototransformable Fluorescent Protein SAASoti with Unique Photochemical Properties

by Ilya D. Solovyev, Alexandra V. Gavshina and Alexander P. Savitsky

Int. J. Mol. Sci. 2019, 20(14), 3399; https://doi.org/10.3390/ijms20143399 - 11 Jul 2019

Cited by 8 | Viewed by 3132

Abstract

SAASoti is a unique fluorescent protein (FP) that combines properties of green-to-red photoconversion and reversible photoswitching (in its green state), without any amino acid substitutions in the wild type gene. In the present work, we investigated its ability to photoswitch between fluorescent red [...] Read more.

SAASoti is a unique fluorescent protein (FP) that combines properties of green-to-red photoconversion and reversible photoswitching (in its green state), without any amino acid substitutions in the wild type gene. In the present work, we investigated its ability to photoswitch between fluorescent red (‘on’) and dark (‘off’) states. Surprisingly, generated by 400 nm exposure, the red form of SAASoti (R1) does not exhibit any reversible photoswitching behavior under 550 nm illumination, while a combination of prior 470 nm and subsequent 400 nm irradiation led to the appearance of another—R2—form that can be partially photoswitched (550 nm) to the dark state, with a very fast recovery time. The phenomenon might be explained by chemical modification in the chromophore microenvironment during prior 470 nm exposure, and the resulting R2 SAASoti differs chemically from the R1 form. The suggestion is supported by the mass spectrometry analysis of the tryptic peptides before and after 470 nm light exposure, that revealed Met164 oxidation, as proceeds in another dual phototransformable FP, IrisFP. Full article

(This article belongs to the Special Issue Imaging with Fluorescent Proteins)

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11 pages, 1105 KiB

Open AccessArticle

Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy

by Kimberly Cramer, Anna-Lena Bolender, Iris Stockmar, Ralf Jungmann, Robert Kasper and Jae Yen Shin

Int. J. Mol. Sci. 2019, 20(14), 3376; https://doi.org/10.3390/ijms20143376 - 10 Jul 2019

Cited by 13 | Viewed by 6060

Abstract

In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. However, in bacterial cells, visualization of [...] Read more.

In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. However, in bacterial cells, visualization of these assemblies can be challenging because of their small size and the presence of the cell wall. Thus, although conceptually promising, successful application of SRM techniques requires careful optimization in labeling biochemistry, fluorescent dye choice, bacterial biology and microscopy to gain biological insights. Here, we apply Stimulated Emission Depletion (STED) microscopy to visualize cell division proteins in bacterial cells, specifically E. coli and B. subtilis. We applied nanobodies that specifically recognize fluorescent proteins, such as GFP, mCherry2 and PAmCherry, fused to targets for STED imaging and evaluated the effect of various organic fluorescent dyes on the performance of STED in bacterial cells. We expect this research to guide scientists for in situ macromolecular visualization using STED in bacterial systems. Full article

(This article belongs to the Special Issue Imaging with Fluorescent Proteins)

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22 pages, 3255 KiB

Open AccessArticle

EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements

by György Vámosi, Elza Friedländer-Brock, Shehu M. Ibrahim, Roland Brock, János Szöllősi and György Vereb

Int. J. Mol. Sci. 2019, 20(13), 3370; https://doi.org/10.3390/ijms20133370 - 9 Jul 2019

Cited by 9 | Viewed by 3777

Abstract

To elucidate the molecular details of the activation-associated clustering of epidermal growth factor receptors (EGFRs), the time course of the mobility and aggregation states of eGFP tagged EGFR in the membranes of Chinese hamster ovary (CHO) cells was assessed by in situ mobility [...] Read more.

To elucidate the molecular details of the activation-associated clustering of epidermal growth factor receptors (EGFRs), the time course of the mobility and aggregation states of eGFP tagged EGFR in the membranes of Chinese hamster ovary (CHO) cells was assessed by in situ mobility assays. Fluorescence correlation spectroscopy (FCS) was used to probe molecular movements of small ensembles of molecules over short distances and time scales, and to report on the state of aggregation. The diffusion of larger ensembles of molecules over longer distances (and time scales) was investigated by fluorescence recovery after photobleaching (FRAP). Autocorrelation functions could be best fitted by a two-component diffusion model corrected for triplet formation and blinking. The slow, 100–1000 ms component was attributed to membrane localized receptors moving with free Brownian diffusion, whereas the fast, ms component was assigned to cytosolic receptors or their fragments. Upon stimulation with 50 nM EGF, a significant decrease from 0.11 to 0.07 μm²/s in the diffusion coefficient of membrane-localized receptors was observed, followed by recovery to the original value in ~20 min. In contrast, the apparent brightness of diffusing species remained the same. Stripe FRAP experiments yielded a decrease in long-range molecular mobility directly after stimulation, evidenced by an increase in the recovery time of the slow component from 13 to 21.9 s. Our observations are best explained by the transient attachment of ligand-bound EGFRs to immobile or slowly moving structures such as the cytoskeleton or large, previously photobleached receptor aggregates. Full article

(This article belongs to the Special Issue Imaging with Fluorescent Proteins)

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25 pages, 3624 KiB

Open AccessArticle

Slowly Reducible Genetically Encoded Green Fluorescent Indicator for In Vivo and Ex Vivo Visualization of Hydrogen Peroxide

by Oksana M. Subach, Tatiana A. Kunitsyna, Olga A. Mineyeva, Alexander A. Lazutkin, Dmitri V. Bezryadnov, Natalia V. Barykina, Kiryl D. Piatkevich, Yulia G. Ermakova, Dmitry S. Bilan, Vsevolod V. Belousov, Konstantin V. Anokhin, Grigori N. Enikolopov and Fedor V. Subach

Int. J. Mol. Sci. 2019, 20(13), 3138; https://doi.org/10.3390/ijms20133138 - 27 Jun 2019

Cited by 22 | Viewed by 5493

Abstract

Hydrogen peroxide (H₂O₂) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of H₂O₂ dynamics in live cells within a limited field [...] Read more.

Hydrogen peroxide (H₂O₂) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of H₂O₂ dynamics in live cells within a limited field of view. The visualization of H₂O₂ within a whole organism with a single cell resolution would benefit from a slowly reducible fluorescent indicator that integrates the H₂O₂ concentration over desired time scales. This would enable post hoc optical readouts in chemically fixed samples. Herein, we report the development and characterization of NeonOxIrr, a genetically encoded green fluorescent indicator, which rapidly increases fluorescence brightness upon reaction with H₂O₂, but has a low reduction rate. NeonOxIrr is composed of circularly permutated mNeonGreen fluorescent protein fused to the truncated OxyR transcription factor isolated from E. coli. When compared in vitro to a standard in the field, HyPer3 indicator, NeonOxIrr showed 5.9-fold higher brightness, 15-fold faster oxidation rate, 5.9-fold faster chromophore maturation, similar intensiometric contrast (2.8-fold), 2-fold lower photostability, and significantly higher pH stability both in reduced (pK_a of 5.9 vs. ≥7.6) and oxidized states (pK_a of 5.9 vs.≥ 7.9). When expressed in the cytosol of HEK293T cells, NeonOxIrr demonstrated a 2.3-fold dynamic range in response to H₂O₂ and a 44 min reduction half-time, which were 1.4-fold lower and 7.6-fold longer than those for HyPer3. We also demonstrated and characterized the NeonOxIrr response to H₂O₂ when the sensor was targeted to the matrix and intermembrane space of the mitochondria, nucleus, cell membranes, peroxisomes, Golgi complex, and endoplasmic reticulum of HEK293T cells. NeonOxIrr could reveal endogenous reactive oxygen species (ROS) production in HeLa cells induced with staurosporine but not with thapsigargin or epidermal growth factor. In contrast to HyPer3, NeonOxIrr could visualize optogenetically produced ROS in HEK293T cells. In neuronal cultures, NeonOxIrr preserved its high 3.2-fold dynamic range to H₂O₂ and slow 198 min reduction half-time. We also demonstrated in HeLa cells that NeonOxIrr preserves a 1.7-fold ex vivo dynamic range to H₂O₂ upon alkylation with N-ethylmaleimide followed by paraformaldehyde fixation. The same alkylation-fixation procedure in the presence of NP-40 detergent allowed ex vivo detection of H₂O₂ with 1.5-fold contrast in neuronal cultures and in the cortex of the mouse brain. The slowly reducible H₂O₂ indicator NeonOxIrr can be used for both the in vivo and ex vivo visualization of ROS. Expanding the family of fixable indicators may be a promising strategy to visualize biological processes at a single cell resolution within an entire organism. Full article

(This article belongs to the Special Issue Imaging with Fluorescent Proteins)

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14 pages, 3349 KiB

Open AccessArticle

Red-Shifted Aminated Derivatives of GFP Chromophore for Live-Cell Protein Labeling with Lipocalins

by Nina G. Bozhanova, Mikhail S. Baranov, Nadezhda S. Baleeva, Alexey S. Gavrikov and Alexander S. Mishin

Int. J. Mol. Sci. 2018, 19(12), 3778; https://doi.org/10.3390/ijms19123778 - 28 Nov 2018

Cited by 13 | Viewed by 4601

Abstract

Fluorogens are an attractive type of dye for imaging applications, eliminating time-consuming washout steps from staining protocols. With just a handful of reported fluorogen-protein pairs, mostly in the green region of spectra, there is a need for the expansion of their spectral range. [...] Read more.

Fluorogens are an attractive type of dye for imaging applications, eliminating time-consuming washout steps from staining protocols. With just a handful of reported fluorogen-protein pairs, mostly in the green region of spectra, there is a need for the expansion of their spectral range. Still, the origins of solvatochromic and fluorogenic properties of the chromophores suitable for live-cell imaging are poorly understood. Here we report on the synthesis and labeling applications of novel red-shifted fluorogenic cell-permeable green fluorescent protein (GFP) chromophore analogs. Full article

(This article belongs to the Special Issue Imaging with Fluorescent Proteins)

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Review

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17 pages, 1017 KiB

Open AccessReview

Next-Generation Fluorogen-Based Reporters and Biosensors for Advanced Bioimaging

by Tiphaine Péresse and Arnaud Gautier

Int. J. Mol. Sci. 2019, 20(24), 6142; https://doi.org/10.3390/ijms20246142 - 5 Dec 2019

Cited by 34 | Viewed by 7130

Abstract

Our ability to observe biochemical events with high spatial and temporal resolution is essential for understanding the functioning of living systems. Intrinsically fluorescent proteins such as the green fluorescent protein (GFP) have revolutionized the way biologists study cells and organisms. The fluorescence toolbox [...] Read more.

Our ability to observe biochemical events with high spatial and temporal resolution is essential for understanding the functioning of living systems. Intrinsically fluorescent proteins such as the green fluorescent protein (GFP) have revolutionized the way biologists study cells and organisms. The fluorescence toolbox has been recently extended with new fluorescent reporters composed of a genetically encoded tag that binds endogenously present or exogenously applied fluorogenic chromophores (so-called fluorogens) and activates their fluorescence. This review presents the toolbox of fluorogen-based reporters and biosensors available to biologists. Various applications are detailed to illustrate the possible uses and opportunities offered by this new generation of fluorescent probes and sensors for advanced bioimaging. Full article

(This article belongs to the Special Issue Imaging with Fluorescent Proteins)

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18 pages, 1332 KiB

Open AccessReview

Fluorescent Protein-Based Indicators for Functional Super-Resolution Imaging of Biomolecular Activities in Living Cells

by Kai Lu, Cong Quang Vu, Tomoki Matsuda and Takeharu Nagai

Int. J. Mol. Sci. 2019, 20(22), 5784; https://doi.org/10.3390/ijms20225784 - 17 Nov 2019

Cited by 27 | Viewed by 7363

Abstract

Super-resolution light microscopy (SRM) offers a unique opportunity for diffraction-unlimited imaging of biomolecular activities in living cells. To realize such potential, genetically encoded indicators were developed recently from fluorescent proteins (FPs) that exhibit phototransformation behaviors including photoactivation, photoconversion, and photoswitching, etc. Super-resolution observations [...] Read more.

Super-resolution light microscopy (SRM) offers a unique opportunity for diffraction-unlimited imaging of biomolecular activities in living cells. To realize such potential, genetically encoded indicators were developed recently from fluorescent proteins (FPs) that exhibit phototransformation behaviors including photoactivation, photoconversion, and photoswitching, etc. Super-resolution observations of biomolecule interactions and biochemical activities have been demonstrated by exploiting the principles of bimolecular fluorescence complementation (BiFC), points accumulation for imaging nanoscale topography (PAINT), and fluorescence fluctuation increase by contact (FLINC), etc. To improve functional nanoscopy with the technology of genetically encoded indicators, it is essential to fully decipher the photo-induced chemistry of FPs and opt for innovative indicator designs that utilize not only fluorescence intensity but also multi-parametric readouts such as phototransformation kinetics. In parallel, technical improvements to both the microscopy optics and image analysis pipeline are promising avenues to increase the sensitivity and versatility of functional SRM. Full article

(This article belongs to the Special Issue Imaging with Fluorescent Proteins)

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37 pages, 1693 KiB

Open AccessReview

Circularly Permuted Fluorescent Protein-Based Indicators: History, Principles, and Classification

by Alexander I. Kostyuk, Aleksandra D. Demidovich, Daria A. Kotova, Vsevolod V. Belousov and Dmitry S. Bilan

Int. J. Mol. Sci. 2019, 20(17), 4200; https://doi.org/10.3390/ijms20174200 - 27 Aug 2019

Cited by 83 | Viewed by 15128

Abstract

Genetically encoded biosensors based on fluorescent proteins (FPs) are a reliable tool for studying the various biological processes in living systems. The circular permutation of single FPs led to the development of an extensive class of biosensors that allow the monitoring of many [...] Read more.

Genetically encoded biosensors based on fluorescent proteins (FPs) are a reliable tool for studying the various biological processes in living systems. The circular permutation of single FPs led to the development of an extensive class of biosensors that allow the monitoring of many intracellular events. In circularly permuted FPs (cpFPs), the original N- and C-termini are fused using a peptide linker, while new termini are formed near the chromophore. Such a structure imparts greater mobility to the FP than that of the native variant, allowing greater lability of the spectral characteristics. One of the common principles of creating genetically encoded biosensors is based on the integration of a cpFP into a flexible region of a sensory domain or between two interacting domains, which are selected according to certain characteristics. Conformational rearrangements of the sensory domain associated with ligand interaction or changes in the cellular parameter are transferred to the cpFP, changing the chromophore environment. In this review, we highlight the basic principles of such sensors, the history of their creation, and a complete classification of the available biosensors. Full article

(This article belongs to the Special Issue Imaging with Fluorescent Proteins)

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19 pages, 1973 KiB

Open AccessReview

Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology

by Jean-Denis Pedelacq and Stéphanie Cabantous

Int. J. Mol. Sci. 2019, 20(14), 3479; https://doi.org/10.3390/ijms20143479 - 15 Jul 2019

Cited by 46 | Viewed by 14744

Abstract

Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been [...] Read more.

Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein–protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology. Full article

(This article belongs to the Special Issue Imaging with Fluorescent Proteins)

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