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Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biophysics".

Deadline for manuscript submissions: closed (30 July 2020) | Viewed by 78678

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Dr. Kiryl D. Piatkevich

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McGovern Institute for Brain Research, Cambridge, MA, USA
Interests: fluorescent biosensors; optogenetics; expension microscop; neuroengineering
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

We would like to kindly invite you to contribute to the Special Issue "Advanced Molecular Probes for Structural and Functional Imaging of Biological Systems" of International Journal of Molecular Sciences (Impact Factor 4.183). The Special Issue will elucidate the engineering, validation, and application of cutting-edge molecular probes for functional and structural imaging, from cells to intact animals. Novel imaging modalities enabling greater utility of such probes, as well as new biological findings gleaned from experiments performed using these probes, are also within the scope of the Special Issue.

Research articles may present the design and development of novel fluorescent proteins and biosensors as well as biochemical, spectroscopic, structural, and biophysical studies that help elucidate the molecular mechanisms of fluorescence and the biosensing of such probes. Application articles should demonstrate the use of novel molecular probes in complex samples or present new imaging modalities that expand the application of currently available probes. Papers may also focus on addressing important biological questions using advanced molecular probes. In addition, the Special Issue will provide a forum for sharing and discussing perspectives on the development of the field in the next several years. Perspectives should present the authors’ opinions on important new directions in molecular probe design and application. Reviews can cover recent advances in the field of fluorescent proteins and biosensors, or novel or emerging class of fluorescent proteins or biosensors.

Dr. Kiryl D. Piatkevich
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

fluorescent proteins
fluorescent biosensors
in vivo microscopy
super-resolution microscopy
protein engineering

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Published Papers (11 papers)

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Research

Jump to: Review

27 pages, 6792 KiB

Open AccessArticle

Gephyrin-Lacking PV Synapses on Neocortical Pyramidal Neurons

by Dika A. Kuljis, Kristina D. Micheva, Ajit Ray, Waja Wegner, Ryan Bowman, Daniel V. Madison, Katrin I. Willig and Alison L. Barth

Int. J. Mol. Sci. 2021, 22(18), 10032; https://doi.org/10.3390/ijms221810032 - 17 Sep 2021

Cited by 3 | Viewed by 5590

Abstract

Gephyrin has long been thought of as a master regulator for inhibitory synapses, acting as a scaffold to organize γ-aminobutyric acid type A receptors (GABA_ARs) at the post-synaptic density. Accordingly, gephyrin immunostaining has been used as an indicator of inhibitory synapses; despite this, the pan-synaptic localization of gephyrin to specific classes of inhibitory synapses has not been demonstrated. Genetically encoded fibronectin intrabodies generated with mRNA display (FingRs) against gephyrin (Gephyrin.FingR) reliably label endogenous gephyrin, and can be tagged with fluorophores for comprehensive synaptic quantitation and monitoring. Here we investigated input- and target-specific localization of gephyrin at a defined class of inhibitory synapse, using Gephyrin.FingR proteins tagged with EGFP in brain tissue from transgenic mice. Parvalbumin-expressing (PV) neuron presynaptic boutons labeled using Cre- dependent synaptophysin-tdTomato were aligned with postsynaptic Gephyrin.FingR puncta. We discovered that more than one-third of PV boutons adjacent to neocortical pyramidal (Pyr) cell somas lack postsynaptic gephyrin labeling. This finding was confirmed using correlative fluorescence and electron microscopy. Our findings suggest some inhibitory synapses may lack gephyrin. Gephyrin-lacking synapses may play an important role in dynamically regulating cell activity under different physiological conditions. Full article

(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems)

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12 pages, 6600 KiB

Open AccessArticle

Genetically Encoded Red Photosensitizers with Enhanced Phototoxicity

by Dmitry A. Gorbachev, Dmitry B. Staroverov, Konstantin A. Lukyanov and Karen S. Sarkisyan

Int. J. Mol. Sci. 2020, 21(22), 8800; https://doi.org/10.3390/ijms21228800 - 20 Nov 2020

Cited by 10 | Viewed by 5034

Abstract

Genetically encoded photosensitizers are increasingly used as optogenetic tools to control cell fate or trigger intracellular processes. A monomeric red fluorescent protein called SuperNova has been recently developed, however, it demonstrates suboptimal characteristics in most phototoxicity-based applications. Here, we applied directed evolution to this protein and identified SuperNova2, a protein with S10R substitution that results in enhanced brightness, chromophore maturation and phototoxicity in bacterial and mammalian cell cultures. Full article

(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems)

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15 pages, 4707 KiB

Open AccessArticle

The Cruciality of Single Amino Acid Replacement for the Spectral Tuning of Biliverdin-Binding Cyanobacteriochromes

by Keiji Fushimi, Hiroki Hoshino, Naeko Shinozaki-Narikawa, Yuto Kuwasaki, Keita Miyake, Takahiro Nakajima, Moritoshi Sato, Fumi Kano and Rei Narikawa

Int. J. Mol. Sci. 2020, 21(17), 6278; https://doi.org/10.3390/ijms21176278 - 30 Aug 2020

Cited by 4 | Viewed by 3774

Abstract

Cyanobacteriochromes (CBCRs), which are known as linear tetrapyrrole-binding photoreceptors, to date can only be detected from cyanobacteria. They can perceive light only in a small unit, which is categorized into various lineages in correlation with their spectral and structural characteristics. Recently, we have succeeded in identifying specific molecules, which can incorporate mammalian intrinsic biliverdin (BV), from the expanded red/green (XRG) CBCR lineage and in converting BV-rejective molecules into BV-acceptable ones with the elucidation of the structural basis. Among the BV-acceptable molecules, AM1_1870g3_BV4 shows a spectral red-shift in comparison with other molecules, while NpF2164g5_BV4 does not show photoconversion but stably shows a near-infrared (NIR) fluorescence. In this study, we found that AM1_1870g3_BV4 had a specific Tyr residue near the d-ring of the chromophore, while others had a highly conserved Leu residue. The replacement of this Tyr residue with Leu in AM1_1870g3_BV4 resulted in a blue-shift of absorption peak. In contrast, reverse replacement in NpF2164g5_BV4 resulted in a red-shift of absorption and fluorescence peaks, which applies to fluorescence bio-imaging in mammalian cells. Notably, the same Tyr/Leu-dependent color-tuning is also observed for the CBCRs belonging to the other lineage, which indicates common molecular mechanisms. Full article

(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems)

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12 pages, 1296 KiB

Open AccessArticle

Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins

by Claire Siebert, Corinne Mercier, Donald K. Martin, Patricia Renesto and Beatrice Schaack

Int. J. Mol. Sci. 2020, 21(15), 5496; https://doi.org/10.3390/ijms21155496 - 31 Jul 2020

Cited by 5 | Viewed by 3284

Abstract

Responsible for tularemia, Francisella tularensis bacteria are highly infectious Gram-negative, category A bioterrorism agents. The molecular mechanisms for their virulence and resistance to antibiotics remain largely unknown. FupA (Fer Utilization Protein), a protein mediating high-affinity transport of ferrous iron across the outer membrane, is associated with both. Recent studies demonstrated that fupA deletion contributed to lower F. tularensis susceptibility towards fluoroquinolones, by increasing the production of outer membrane vesicles. Although the paralogous FupB protein lacks such activity, iron transport capacity and a role in membrane stability were reported for the FupA/B chimera, a protein found in some F. tularensis strains, including the live vaccine strain (LVS). To investigate the mode of action of these proteins, we purified recombinant FupA, FupB and FupA/B proteins expressed in Escherichia coli and incorporated them into mixed lipid bilayers. We examined the porin-forming activity of the FupA/B proteoliposomes using a fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) probe. Using electrophysiology on tethered bilayer lipid membranes, we confirmed that the FupA/B fusion protein exhibits pore-forming activity with large ionic conductance, a property shared with both FupA and FupB. This demonstration opens up new avenues for identifying functional genes, and novel therapeutic strategies against F. tularensis infections. Full article

(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems)

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14 pages, 2008 KiB

Open AccessArticle

Mitochondrial pH Nanosensors for Metabolic Profiling of Breast Cancer Cell Lines

by Consuelo Ripoll, Mar Roldan, Rafael Contreras-Montoya, Juan J. Diaz-Mochon, Miguel Martin, Maria J. Ruedas-Rama and Angel Orte

Int. J. Mol. Sci. 2020, 21(10), 3731; https://doi.org/10.3390/ijms21103731 - 25 May 2020

Cited by 12 | Viewed by 3724

Abstract

The main role of mitochondria, as pivotal organelles for cellular metabolism, is the production of energy (ATP) through an oxidative phosphorylation system. During this process, the electron transport chain creates a proton gradient that drives the synthesis of ATP. One of the main features of tumoral cells is their altered metabolism, providing alternative routes to enhance proliferation and survival. Hence, it is of utmost importance to understand the relationship between mitochondrial pH, tumoral metabolism, and cancer. In this manuscript, we develop a highly specific nanosensor to accurately measure the intramitochondrial pH using fluorescence lifetime imaging microscopy (FLIM). Importantly, we have applied this nanosensor to establish differences that may be hallmarks of different metabolic pathways in breast cancer cell models, leading to the characterization of different metabophenotypes. Full article

(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems)

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33 pages, 10478 KiB

Open AccessArticle

FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity

by Natalia V. Barykina, Vladimir P. Sotskov, Anna M. Gruzdeva, You Kure Wu, Ruben Portugues, Oksana M. Subach, Elizaveta S. Chefanova, Viktor V. Plusnin, Olga I. Ivashkina, Konstantin V. Anokhin, Anna V. Vlaskina, Dmitry A. Korzhenevskiy, Alena Y. Nikolaeva, Konstantin M. Boyko, Tatiana V. Rakitina, Anna M. Varizhuk, Galina E. Pozmogova and Fedor V. Subach

Int. J. Mol. Sci. 2020, 21(8), 3012; https://doi.org/10.3390/ijms21083012 - 24 Apr 2020

Cited by 20 | Viewed by 7382

Abstract

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging. Full article

(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems)

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24 pages, 4004 KiB

Open AccessArticle

Novel Genetically Encoded Bright Positive Calcium Indicator NCaMP7 Based on the mNeonGreen Fluorescent Protein

by Oksana M. Subach, Vladimir P. Sotskov, Viktor V. Plusnin, Anna M. Gruzdeva, Natalia V. Barykina, Olga I. Ivashkina, Konstantin V. Anokhin, Alena Y. Nikolaeva, Dmitry A. Korzhenevskiy, Anna V. Vlaskina, Vladimir A. Lazarenko, Konstantin M. Boyko, Tatiana V. Rakitina, Anna M. Varizhuk, Galina E. Pozmogova, Oleg V. Podgorny, Kiryl D. Piatkevich, Edward S. Boyden and Fedor V. Subach

Int. J. Mol. Sci. 2020, 21(5), 1644; https://doi.org/10.3390/ijms21051644 - 28 Feb 2020

Cited by 42 | Viewed by 9727

Abstract

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an ΔF/F of 27 and high affinity (K_d of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 Å resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively. Full article

(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems)

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Review

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99 pages, 14390 KiB

Open AccessReview

In Vivo Imaging with Genetically Encoded Redox Biosensors

by Alexander I. Kostyuk, Anastasiya S. Panova, Aleksandra D. Kokova, Daria A. Kotova, Dmitry I. Maltsev, Oleg V. Podgorny, Vsevolod V. Belousov and Dmitry S. Bilan

Int. J. Mol. Sci. 2020, 21(21), 8164; https://doi.org/10.3390/ijms21218164 - 31 Oct 2020

Cited by 36 | Viewed by 8928

Abstract

Redox reactions are of high fundamental and practical interest since they are involved in both normal physiology and the pathogenesis of various diseases. However, this area of research has always been a relatively problematic field in the context of analytical approaches, mostly because of the unstable nature of the compounds that are measured. Genetically encoded sensors allow for the registration of highly reactive molecules in real-time mode and, therefore, they began a new era in redox biology. Their strongest points manifest most brightly in in vivo experiments and pave the way for the non-invasive investigation of biochemical pathways that proceed in organisms from different systematic groups. In the first part of the review, we briefly describe the redox sensors that were used in vivo as well as summarize the model systems to which they were applied. Next, we thoroughly discuss the biological results obtained in these studies in regard to animals, plants, as well as unicellular eukaryotes and prokaryotes. We hope that this work reflects the amazing power of this technology and can serve as a useful guide for biologists and chemists who work in the field of redox processes. Full article

(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems)

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41 pages, 7464 KiB

Open AccessReview

GPCR-Based Dopamine Sensors—A Detailed Guide to Inform Sensor Choice for In Vivo Imaging

by Marie A. Labouesse, Reto B. Cola and Tommaso Patriarchi

Int. J. Mol. Sci. 2020, 21(21), 8048; https://doi.org/10.3390/ijms21218048 - 28 Oct 2020

Cited by 30 | Viewed by 13167

Abstract

Understanding how dopamine (DA) encodes behavior depends on technologies that can reliably monitor DA release in freely-behaving animals. Recently, red and green genetically encoded sensors for DA (dLight, GRAB-DA) were developed and now provide the ability to track release dynamics at a subsecond resolution, with submicromolar affinity and high molecular specificity. Combined with rapid developments in in vivo imaging, these sensors have the potential to transform the field of DA sensing and DA-based drug discovery. When implementing these tools in the laboratory, it is important to consider there is not a ‘one-size-fits-all’ sensor. Sensor properties, most importantly their affinity and dynamic range, must be carefully chosen to match local DA levels. Molecular specificity, sensor kinetics, spectral properties, brightness, sensor scaffold and pharmacology can further influence sensor choice depending on the experimental question. In this review, we use DA as an example; we briefly summarize old and new techniques to monitor DA release, including DA biosensors. We then outline a map of DA heterogeneity across the brain and provide a guide for optimal sensor choice and implementation based on local DA levels and other experimental parameters. Altogether this review should act as a tool to guide DA sensor choice for end-users. Full article

(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems)

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25 pages, 2940 KiB

Open AccessReview

Engineering Photosensory Modules of Non-Opsin-Based Optogenetic Actuators

by Xiaocen Lu, Yi Shen and Robert E. Campbell

Int. J. Mol. Sci. 2020, 21(18), 6522; https://doi.org/10.3390/ijms21186522 - 7 Sep 2020

Cited by 19 | Viewed by 6339

Abstract

Optogenetic (photo-responsive) actuators engineered from photoreceptors are widely used in various applications to study cell biology and tissue physiology. In the toolkit of optogenetic actuators, the key building blocks are genetically encodable light-sensitive proteins. Currently, most optogenetic photosensory modules are engineered from naturally-occurring photoreceptor proteins from bacteria, fungi, and plants. There is a growing demand for novel photosensory domains with improved optical properties and light-induced responses to satisfy the needs of a wider variety of studies in biological sciences. In this review, we focus on progress towards engineering of non-opsin-based photosensory domains, and their representative applications in cell biology and physiology. We summarize current knowledge of engineering of light-sensitive proteins including light-oxygen-voltage-sensing domain (LOV), cryptochrome (CRY2), phytochrome (PhyB and BphP), and fluorescent protein (FP)-based photosensitive domains (Dronpa and PhoCl). Full article

(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems)

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36 pages, 1041 KiB

Open AccessReview

The Role of Amino Acids in Neurotransmission and Fluorescent Tools for Their Detection

by Rochelin Dalangin, Anna Kim and Robert E. Campbell

Int. J. Mol. Sci. 2020, 21(17), 6197; https://doi.org/10.3390/ijms21176197 - 27 Aug 2020

Cited by 95 | Viewed by 10631

Abstract

Neurotransmission between neurons, which can occur over the span of a few milliseconds, relies on the controlled release of small molecule neurotransmitters, many of which are amino acids. Fluorescence imaging provides the necessary speed to follow these events and has emerged as a powerful technique for investigating neurotransmission. In this review, we highlight some of the roles of the 20 canonical amino acids, GABA and β-alanine in neurotransmission. We also discuss available fluorescence-based probes for amino acids that have been shown to be compatible for live cell imaging, namely those based on synthetic dyes, nanostructures (quantum dots and nanotubes), and genetically encoded components. We aim to provide tool developers with information that may guide future engineering efforts and tool users with information regarding existing indicators to facilitate studies of amino acid dynamics. Full article

(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems)

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