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11 pages, 1647 KiB

Open AccessArticle

Water-Soluble Polymer Polyethylene Glycol: Effect on the Bioluminescent Reaction of the Marine Coelenterate Obelia and Coelenteramide-Containing Fluorescent Protein

by Tatiana S. Siniakova, Alexander V. Raikov and Nadezhda S. Kudryasheva

Int. J. Mol. Sci. 2023, 24(7), 6345; https://doi.org/10.3390/ijms24076345 - 28 Mar 2023

Cited by 3 | Viewed by 967

Abstract

The current paper considers the effects of a water-soluble polymer (polyethylene glycol (PEG)) on the bioluminescent reaction of the photoprotein obelin from the marine coelenterate Obelia longissima and the product of this bioluminescent reaction: a coelenteramide-containing fluorescent protein (CCFP). We varied PEG concentrations [...] Read more.

The current paper considers the effects of a water-soluble polymer (polyethylene glycol (PEG)) on the bioluminescent reaction of the photoprotein obelin from the marine coelenterate Obelia longissima and the product of this bioluminescent reaction: a coelenteramide-containing fluorescent protein (CCFP). We varied PEG concentrations (0–1.44 mg/mL) and molecular weights (1000, 8000, and 35,000 a.u.). The presence of PEG significantly increased the bioluminescent intensity of obelin but decreased the photoluminescence intensity of CCFP; the effects did not depend on the PEG concentration or the molecular weight. The photoluminescence spectra of CCFP did not change, while the bioluminescence spectra changed in the course of the bioluminescent reaction. The changes can be explained by different rigidity of the media in the polymer solutions affecting the stability of the photoprotein complex and the efficiency of the proton transfer in the bioluminescent reaction. The results predict and explain the change in the luminescence intensity and color of the marine coelenterates in the presence of water-soluble polymers. The CCFP appeared to be a proper tool for the toxicity monitoring of water-soluble polymers (e.g., PEGs). Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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9 pages, 3925 KiB

Open AccessArticle

Ca²⁺-Triggered Coelenterazine-Binding Protein Renilla: Expected and Unexpected Features

by Alexander N. Kudryavtsev, Vasilisa V. Krasitskaya, Maxim K. Efremov, Sayana V. Zangeeva, Anastasia V. Rogova, Felix N. Tomilin and Ludmila A. Frank

Int. J. Mol. Sci. 2023, 24(3), 2144; https://doi.org/10.3390/ijms24032144 - 21 Jan 2023

Cited by 2 | Viewed by 1564

Abstract

Ca²⁺-triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a [...] Read more.

Ca²⁺-triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a significantly higher bioluminescent signal. This makes CBP a convenient substrate for luciferase-based in vitro assay. In search of a similar substrate form for the luciferase NanoLuc, a furimazine-apoCBP complex was prepared and verified against furimazine, coelenterazine, and CBP. Furimazine-apoCBP is relatively stable in solution and in a frozen or lyophilized state, but as distinct from CBP, its bioluminescence reaction with NanoLuc is independent of Ca²⁺. NanoLuc turned out to utilize all the four substrates under consideration. The pairs of CBP-NanoLuc and coelenterazine-NanoLuc generate bioluminescence with close efficiency. As for furimazine-apoCBP-NanoLuc pair, the efficiency with which it generates bioluminescence is almost twice lower than that of the furimazine-NanoLuc. The integral signal of the CBP-NanoLuc pair is only 22% lower than that of furimazine-NanoLuc. Thus, along with furimazine as the most effective NanoLuc substrate, CBP can also be recommended as a substrate for in vitro analytical application in view of its water solubility, stability, and Ca²⁺-triggering “character”. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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16 pages, 1963 KiB

Open AccessArticle

Near-Infrared Imaging of Steroid Hormone Activities Using Bright BRET Templates

by Sung-Bae Kim, Ryo Nishihara and Ramasamy Paulmurugan

Int. J. Mol. Sci. 2023, 24(1), 677; https://doi.org/10.3390/ijms24010677 - 30 Dec 2022

Viewed by 1500

Abstract

Bioluminescence (BL) is an excellent optical readout for bioassays and molecular imaging. Herein, we accomplished new near infrared bioluminescence resonance energy transfer (NIR-BRET) templates for monitoring molecular events in cells with higher sensitivity. We first identified the best resonance energy donor for the [...] Read more.

Bioluminescence (BL) is an excellent optical readout for bioassays and molecular imaging. Herein, we accomplished new near infrared bioluminescence resonance energy transfer (NIR-BRET) templates for monitoring molecular events in cells with higher sensitivity. We first identified the best resonance energy donor for the NIR-BRET templates through the characterization of many coelenterazine (CTZ)–marine luciferase combinations. As a result, we found that NLuc–DBlueC and ALuc47–nCTZ combinations showed luminescence in the blue emission wavelength with excellent BL intensity and stability, for example, the NLuc–DBlueC and ALuc47–nCTZ combinations were 17-fold and 22-fold brighter than their second highest combinations, respectively, and were stably bright in living mammalian cells for at least 10 min. To harness the excellent BL properties to the NIR-BRET systems, NLuc and ALuc47 were genetically fused to fluorescent proteins (FPs), allowing large “blue-to-red” shifts, such as LSSmChe, LSSmKate2, and LSSmNep (where LSS means Large Stokes Shift). The excellent LSSmNep–NLuc combination showed approximately 170 nm large resonance energy shift from blue to red. The established templates were further utilized in the development of new NIR-BRET systems for imaging steroid hormone activities by sandwiching the ligand-binding domain of a nuclear receptor (NR-LBD) between the luciferase and the FP of the template. The NIR-BRET systems showed a specific luminescence signal upon exposure to steroid hormones, such as androgen, estrogen, and cortisol. The present NIR-BRET templates are important additions for utilizing their advantageous imaging of various molecular events with high efficiency and brightness in physiological samples. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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9 pages, 1454 KiB

Open AccessCommunication

The Role of the 145 Residue in Photochemical Properties of the Biphotochromic Protein mSAASoti: Brightness versus Photoconversion

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

Int. J. Mol. Sci. 2022, 23(24), 16058; https://doi.org/10.3390/ijms232416058 - 16 Dec 2022

Viewed by 873

Abstract

Photoswitchable fluorescent proteins (FPs) have become indispensable tools for studying life sciences. mSAASoti FP, a biphotochromic FP, is an important representative of this protein family. We created a series of mSAASoti mutants in order to obtain fast photoswitchable variants with high brightness. K145P [...] Read more.

Photoswitchable fluorescent proteins (FPs) have become indispensable tools for studying life sciences. mSAASoti FP, a biphotochromic FP, is an important representative of this protein family. We created a series of mSAASoti mutants in order to obtain fast photoswitchable variants with high brightness. K145P mSAASoti has the highest molar extinction coefficient of all SAASoti mutants studied; C21N/K145P/M163A switches to the dark state 36 times faster than mSAASoti, but it lost its ability to undergo green-to-red photoconversion. Finally, the C21N/K145P/F177S and C21N/K145P/M163A/F177S variants demonstrated a high photoswitching rate between both green and red forms. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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22 pages, 6716 KiB

Open AccessArticle

The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer

by Oksana M. Subach, Aleksandr Tashkeev, Anna V. Vlaskina, Dmitry E. Petrenko, Filipp A. Gaivoronskii, Alena Y. Nikolaeva, Olga I. Ivashkina, Konstantin V. Anokhin, Vladimir O. Popov, Konstantin M. Boyko and Fedor V. Subach

Int. J. Mol. Sci. 2022, 23(6), 3208; https://doi.org/10.3390/ijms23063208 - 16 Mar 2022

Cited by 4 | Viewed by 2826

Abstract

Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms [...] Read more.

Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2–3- and 5–7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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17 pages, 2343 KiB

Open AccessArticle

Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy

by Elena V. Nemtseva, Dmitry V. Gulnov, Marina A. Gerasimova, Lev A. Sukovatyi, Ludmila P. Burakova, Natalya E. Karuzina, Bogdan S. Melnik and Valentina A. Kratasyuk

Int. J. Mol. Sci. 2021, 22(19), 10449; https://doi.org/10.3390/ijms221910449 - 28 Sep 2021

Cited by 3 | Viewed by 1704

Abstract

Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as [...] Read more.

Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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15 pages, 955 KiB

Open AccessArticle

Altered Induction of Reactive Oxygen Species by X-rays in Hematopoietic Cells of C57BL/6-Tg (CAG-EGFP) Mice

by Cuihua Liu, Hirokazu Hirakawa, Takanori Katsube, Yaqun Fang, Kaoru Tanaka, Mitsuru Nenoi, Akira Fujimori and Bing Wang

Int. J. Mol. Sci. 2021, 22(13), 6929; https://doi.org/10.3390/ijms22136929 - 28 Jun 2021

Cited by 2 | Viewed by 1961

Abstract

Previous work pointed to a critical role of excessive production of reactive oxygen species (ROS) in increased radiation hematopoietic death in GFP mice. Meanwhile, enhanced antioxidant capability was not demonstrated in the mouse model of radio-induced adaptive response (RAR) using rescue of radiation [...] Read more.

Previous work pointed to a critical role of excessive production of reactive oxygen species (ROS) in increased radiation hematopoietic death in GFP mice. Meanwhile, enhanced antioxidant capability was not demonstrated in the mouse model of radio-induced adaptive response (RAR) using rescue of radiation hematopoietic death as the endpoint. ROS induction by ex vivo X-irradiation at a dose ranging from 0.1 to 7.5 Gy in the nucleated bone marrow cells was comparatively studied using GFP and wild type (WT) mice. ROS induction was also investigated in the cells collected from mice receiving a priming dose (0.5 Gy) efficient for RAR induction in WT mice. Significantly elevated background and increased induction of ROS in the cells from GFP mice were observed compared to those from WT mice. Markedly lower background and decreased induction of ROS were observed in the cells collected from WT mice but not GFP mice, both receiving the priming dose. GFP overexpression could alter background and induction of ROS by X-irradiation in hematopoietic cells. The results provide a reasonable explanation to the previous study on the fate of cells and mice after X-irradiation and confirm enhanced antioxidant capability in RAR. Investigations involving GFP overexpression should be carefully interpreted. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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14 pages, 2025 KiB

Open AccessArticle

Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids

by Maša Kenda, Jan Vegelj, Barbara Herlah, Andrej Perdih, Přemysl Mladěnka and Marija Sollner Dolenc

Int. J. Mol. Sci. 2021, 22(13), 6927; https://doi.org/10.3390/ijms22136927 - 28 Jun 2021

Cited by 8 | Viewed by 5765

Abstract

Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated [...] Read more.

Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure–activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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17 pages, 4130 KiB

Open AccessArticle

A Versatile Toolkit for Semi-Automated Production of Fluorescent Chemokines to Study CCR7 Expression and Functions

by Marc Artinger, Christoph Matti, Oliver J. Gerken, Christopher T. Veldkamp and Daniel F. Legler

Int. J. Mol. Sci. 2021, 22(8), 4158; https://doi.org/10.3390/ijms22084158 - 16 Apr 2021

Cited by 10 | Viewed by 2560

Abstract

Chemokines guide leukocyte migration in different contexts, including homeostasis, immune surveillance and immunity. The chemokines CCL19 and CCL21 control lymphocyte and dendritic cell migration and homing to lymphoid organs. Thereby they orchestrate adaptive immunity in a chemokine receptor CCR7-dependent manner. Likewise, cancer cells [...] Read more.

Chemokines guide leukocyte migration in different contexts, including homeostasis, immune surveillance and immunity. The chemokines CCL19 and CCL21 control lymphocyte and dendritic cell migration and homing to lymphoid organs. Thereby they orchestrate adaptive immunity in a chemokine receptor CCR7-dependent manner. Likewise, cancer cells that upregulate CCR7 expression are attracted by these chemokines and metastasize to lymphoid organs. In-depth investigation of CCR7 expression and chemokine-mediated signaling is pivotal to understand their role in health and disease. Appropriate fluorescent probes to track these events are increasingly in demand. Here, we present an approach to cost-effectively produce and fluorescently label CCL19 and CCL21 in a semi-automated process. We established a versatile protocol for the production of recombinant chemokines harboring a small C-terminal S6-tag for efficient and site-specific enzymatic labelling with an inorganic fluorescent dye of choice. We demonstrate that the fluorescently labeled chemokines CCL19-S6^Dy649P1 and CCL21-S6^Dy649P1 retain their full biological function as assessed by their abilities to mobilize intracellular calcium, to recruit β-arrestin to engaged receptors and to attract CCR7-expressing leukocytes. Moreover, we show that CCL19-S6^Dy649P1 serves as powerful reagent to monitor CCR7 internalization by time-lapse confocal video microscopy and to stain CCR7-positive primary human and mouse T cell sub-populations. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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14 pages, 3910 KiB

Open AccessArticle

Bioluminescence Measurement of Time-Dependent Dynamic Changes of CYP-Mediated Cytotoxicity in CYP-Expressing Luminescent HepG2 Cells

by Satoru Iwado, Satoshi Abe, Mitsuo Oshimura, Yasuhiro Kazuki and Yoshihiro Nakajima

Int. J. Mol. Sci. 2021, 22(6), 2843; https://doi.org/10.3390/ijms22062843 - 11 Mar 2021

Cited by 2 | Viewed by 2074

Abstract

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or [...] Read more.

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC₅₀) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC₅₀ value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC₅₀ values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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29 pages, 9605 KiB

Open AccessArticle

Advanced Spectroscopy and APBS Modeling for Determination of the Role of His190 and Trp103 in Mouse Thymidylate Synthase Interaction with Selected dUMP Analogues

by Małgorzata Prokopowicz, Adam Jarmuła, Yannick Casamayou-Boucau, Fiona Gordon, Alan Ryder, Justyna Sobich, Piotr Maj, Joanna Cieśla, Zbigniew Zieliński, Piotr Fita and Wojciech Rode

Int. J. Mol. Sci. 2021, 22(5), 2661; https://doi.org/10.3390/ijms22052661 - 06 Mar 2021

Viewed by 4431

Abstract

A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. [...] Read more.

A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson–Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N

^{4}

-OH-dCMP, suggests its weaker influence on the enzyme–ligand interactions in N

^{4}

OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the ”abortive reaction” inhibitory mechanism of N

^{4}

OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N

^{4}

-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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Review

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14 pages, 783 KiB

Open AccessReview

Bioluminescent Dinoflagellates as a Bioassay for Toxicity Assessment

by Luíza S. Perin, Gabriela V. Moraes, Gabriela A. Galeazzo and Anderson G. Oliveira

Int. J. Mol. Sci. 2022, 23(21), 13012; https://doi.org/10.3390/ijms232113012 - 27 Oct 2022

Cited by 5 | Viewed by 3313

Abstract

Dinoflagellates bioluminescence mechanism depends upon a luciferin–luciferase reaction that promotes blue light emission (480 nm) in specialized luminogenic organelles called scintillons. The scintillons contain luciferin, luciferase and, in some cases, a luciferin-binding protein (LBP), which prevents luciferin from non-enzymatic oxidation in vivo. Even [...] Read more.

Dinoflagellates bioluminescence mechanism depends upon a luciferin–luciferase reaction that promotes blue light emission (480 nm) in specialized luminogenic organelles called scintillons. The scintillons contain luciferin, luciferase and, in some cases, a luciferin-binding protein (LBP), which prevents luciferin from non-enzymatic oxidation in vivo. Even though dinoflagellate bioluminescence has been studied since the 1950s, there is still a lack of mechanistic understanding on whether the light emission process involves a peroxidic intermediate or not. Still, bioassays employing luminous dinoflagellates, usually from Gonyaulax or Pyrocystis genus, can be used to assess the toxicity of metals or organic compounds. In these dinoflagellates, the response to toxicity is observed as a change in luminescence, which is linked to cellular respiration. As a result, these changes can be used to calculate a percentage of light inhibition that correlates directly with toxicity. This current approach, which lies in between fast bacterial assays and more complex toxicity tests involving vertebrates and invertebrates, can provide a valuable tool for detecting certain pollutants, e.g., metals, in marine sediment and seawater. Thus, the present review focuses on how the dinoflagellates bioluminescence can be applied to evaluate the risks caused by contaminants in the marine environment. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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31 pages, 1559 KiB

Open AccessReview

Bioluminescence and Photoreception in Unicellular Organisms: Light-Signalling in a Bio-Communication Perspective

by Youri Timsit, Magali Lescot, Martha Valiadi and Fabrice Not

Int. J. Mol. Sci. 2021, 22(21), 11311; https://doi.org/10.3390/ijms222111311 - 20 Oct 2021

Cited by 7 | Viewed by 7269

Abstract

Bioluminescence, the emission of light catalysed by luciferases, has evolved in many taxa from bacteria to vertebrates and is predominant in the marine environment. It is now well established that in animals possessing a nervous system capable of integrating light stimuli, bioluminescence triggers [...] Read more.

Bioluminescence, the emission of light catalysed by luciferases, has evolved in many taxa from bacteria to vertebrates and is predominant in the marine environment. It is now well established that in animals possessing a nervous system capable of integrating light stimuli, bioluminescence triggers various behavioural responses and plays a role in intra- or interspecific visual communication. The function of light emission in unicellular organisms is less clear and it is currently thought that it has evolved in an ecological framework, to be perceived by visual animals. For example, while it is thought that bioluminescence allows bacteria to be ingested by zooplankton or fish, providing them with favourable conditions for growth and dispersal, the luminous flashes emitted by dinoflagellates may have evolved as an anti-predation system against copepods. In this short review, we re-examine this paradigm in light of recent findings in microorganism photoreception, signal integration and complex behaviours. Numerous studies show that on the one hand, bacteria and protists, whether autotrophs or heterotrophs, possess a variety of photoreceptors capable of perceiving and integrating light stimuli of different wavelengths. Single-cell light-perception produces responses ranging from phototaxis to more complex behaviours. On the other hand, there is growing evidence that unicellular prokaryotes and eukaryotes can perform complex tasks ranging from habituation and decision-making to associative learning, despite lacking a nervous system. Here, we focus our analysis on two taxa, bacteria and dinoflagellates, whose bioluminescence is well studied. We propose the hypothesis that similar to visual animals, the interplay between light-emission and reception could play multiple roles in intra- and interspecific communication and participate in complex behaviour in the unicellular world. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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21 pages, 2711 KiB

Open AccessReview

Application of Fluorescent Proteins for Functional Dissection of the Drosophila Visual System

by Thomas K. Smylla, Krystina Wagner and Armin Huber

Int. J. Mol. Sci. 2021, 22(16), 8930; https://doi.org/10.3390/ijms22168930 - 19 Aug 2021

Cited by 3 | Viewed by 2760

Abstract

The Drosophila eye has been used extensively to study numerous aspects of biological systems, for example, spatio-temporal regulation of differentiation, visual signal transduction, protein trafficking and neurodegeneration. Right from the advent of fluorescent proteins (FPs) near the end of the millennium, heterologously expressed [...] Read more.

The Drosophila eye has been used extensively to study numerous aspects of biological systems, for example, spatio-temporal regulation of differentiation, visual signal transduction, protein trafficking and neurodegeneration. Right from the advent of fluorescent proteins (FPs) near the end of the millennium, heterologously expressed fusion proteins comprising FPs have been applied in Drosophila vision research not only for subcellular localization of proteins but also for genetic screens and analysis of photoreceptor function. Here, we summarize applications for FPs used in the Drosophila eye as part of genetic screens, to study rhodopsin expression patterns, subcellular protein localization, membrane protein transport or as genetically encoded biosensors for Ca²⁺ and phospholipids in vivo. We also discuss recently developed FPs that are suitable for super-resolution or correlative light and electron microscopy (CLEM) approaches. Illustrating the possibilities provided by using FPs in Drosophila photoreceptors may aid research in other sensory or neuronal systems that have not yet been studied as well as the Drosophila eye. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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14 pages, 41757 KiB

Open AccessReview

How to Select Firefly Luciferin Analogues for In Vivo Imaging

by Ryohei Saito-Moriya, Jun Nakayama, Genta Kamiya, Nobuo Kitada, Rika Obata, Shojiro A. Maki and Hiroshi Aoyama

Int. J. Mol. Sci. 2021, 22(4), 1848; https://doi.org/10.3390/ijms22041848 - 12 Feb 2021

Cited by 14 | Viewed by 4847

Abstract

Bioluminescence reactions are widely applied in optical in vivo imaging in the life science and medical fields. Such reactions produce light upon the oxidation of a luciferin (substrate) catalyzed by a luciferase (enzyme), and this bioluminescence enables the quantification of tumor cells and [...] Read more.

Bioluminescence reactions are widely applied in optical in vivo imaging in the life science and medical fields. Such reactions produce light upon the oxidation of a luciferin (substrate) catalyzed by a luciferase (enzyme), and this bioluminescence enables the quantification of tumor cells and gene expression in animal models. Many researchers have developed single-color or multicolor bioluminescence systems based on artificial luciferin analogues and/or luciferase mutants, for application in vivo bioluminescence imaging (BLI). In the current review, we focus on the characteristics of firefly BLI technology and discuss the development of luciferin analogues for high-resolution in vivo BLI. In addition, we discuss the novel luciferin analogues TokeOni and seMpai, which show potential as high-sensitivity in vivo BLI reagents. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 2.0)

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