Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

Green fluorescent protein (GFP) variants are widely used as genetically encoded fluorescent fusion tags, and there is an increasing interest in engineering their structure to develop in vivo optical sensors, such as for optogenetics and force transduction. Ensemble experiments have shown that the fluorescence of GFP is quenched upon denaturation. Here we study the dependence of fluorescence on protein structure by driving single molecules of GFP into different conformational states with optical tweezers and simultaneously probing the chromophore with fluorescence. Our results show that fluorescence is lost during the earliest events in unfolding, 3.5 ms before secondary structure is disrupted. No fluorescence is observed from the unfolding intermediates or the ensemble of compact and extended states populated during refolding. We further demonstrate that GFP can be mechanically switched between emissive and dark states. These data definitively establish that complete structural integrity is necessary to observe single-molecule fluorescence of GFP.

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Single-molecule biophysics: at the interface of biology, physics and chemistry

Journal of The Royal Society Interface ◽

10.1098/rsif.2007.1021 ◽

2007 ◽

Vol 5 (18) ◽

pp. 15-45 ◽

Cited By ~ 193

Author(s):

Ashok A Deniz ◽

Samrat Mukhopadhyay ◽

Edward A Lemke

Keyword(s):

Single Molecule ◽

Biological Molecules ◽

Single Molecule Fluorescence ◽

Complex Behaviour ◽

Single Molecule Biophysics ◽

Single Molecule Studies ◽

Dynamics And Function ◽

And Function ◽

Ensemble Experiments

Single-molecule methods have matured into powerful and popular tools to probe the complex behaviour of biological molecules, due to their unique abilities to probe molecular structure, dynamics and function, unhindered by the averaging inherent in ensemble experiments. This review presents an overview of the burgeoning field of single-molecule biophysics, discussing key highlights and selected examples from its genesis to our projections for its future. Following brief introductions to a few popular single-molecule fluorescence and manipulation methods, we discuss novel insights gained from single-molecule studies in key biological areas ranging from biological folding to experiments performed in vivo .

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New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales

Molecular Biology of the Cell ◽

10.1091/mbc.e13-03-0162 ◽

2014 ◽

Vol 25 (7) ◽

pp. 1010-1024 ◽

Cited By ~ 26

Author(s):

Sawako Yamashiro ◽

Hiroaki Mizuno ◽

Matthew B. Smith ◽

Gillian L. Ryan ◽

Tai Kiuchi ◽

...

Keyword(s):

Single Molecule ◽

Fluorescent Protein ◽

Focal Adhesions ◽

Actin Network ◽

Actin Turnover ◽

Flow Velocity Measurement ◽

Flow Speeds ◽

Green Fluorescent ◽

User Friendly

Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein–actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8–8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network.

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Construction and application of a single-molecule fluorescence microscope

Canadian Journal of Chemistry ◽

10.1139/v05-053 ◽

2005 ◽

Vol 83 (5) ◽

pp. 435-442 ◽

Cited By ~ 7

Author(s):

Sangram Bagh ◽

Matthew F Paige

Keyword(s):

Single Molecule ◽

Fluorescent Protein ◽

Ccd Camera ◽

Single Molecule Fluorescence ◽

Charge Coupled Device ◽

New Type ◽

On Chip ◽

Green Fluorescent ◽

Construction Optimization ◽

Fluorescent Molecules

In this paper, we describe the construction, optimization, and testing of an epifluorescence microscope that has single-molecule (SM) resolution and sensitivity. The microscope makes use of a novel new type of wide-area charge-coupled device (CCD) photodetector with on-chip multiplier gain. Sensitivity and spatial resolution of the instrument are demonstrated by imaging individual Rhodamine 6G (R6G) molecules and characterizing their basic photophysical behaviour under a variety of imaging conditions. A simple, general method for calibrating the photodetector (correlating CCD counts with incident photons) using a highly attenuated laser beam is presented, and the performance of the photodetector is compared with that of other detectors commonly used in SM fluorescence imaging applications. We also demonstrate the versatility of the microscope system by characterizing the SM photophysical behaviour of several other fluorescent molecules, including bodipy-FL and the enhanced green fluorescent protein (EGFP). Key words: single molecule, fluorescence, microscopy, CCD camera, calibration, photobleaching.

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7. Following biochemistry within the cell

Biochemistry: A Very Short Introduction ◽

10.1093/actrade/9780198833871.003.0007 ◽

2021 ◽

pp. 93-110

Author(s):

Mark Lorch

Keyword(s):

Green Fluorescent Protein ◽

Optical Tweezers ◽

Single Molecule ◽

Motor Proteins ◽

Fluorescent Protein ◽

Patch Clamping ◽

Signal To Noise ◽

Single Molecule Studies ◽

Technical Challenges ◽

Green Fluorescent

This chapter presents key advances in the study of individual molecules within cells. When one applies this individualistic methodology to biochemicals, one enters the realms of single-molecule biophysics. There are of course formidable technical challenges associated with single-molecule studies, not least of which is the signal-to-noise problem. The chapter discusses patch clamping, nanoscopes, and optical tweezers. Patch clamping provided insights into the protein machinery that controls the flow of ions in and out of cells. The chapter also examines cytokinesis, cytoskeletons, and motor proteins, and the use of Green Fluorescent Protein (GFP).

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Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

Biochemical Journal ◽

10.1042/bj3390299 ◽

1999 ◽

Vol 339 (2) ◽

pp. 299-307 ◽

Cited By ~ 33

Author(s):

Arthur L. KRUCKEBERG ◽

Ling YE ◽

Jan A. BERDEN ◽

Karel van DAM

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Green Fluorescent Protein ◽

Glucose Transport ◽

Cellular Localization ◽

Fluorescent Protein ◽

Hexose Transporter ◽

High Concentrations ◽

Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

Microorganisms ◽

10.3390/microorganisms9020379 ◽

2021 ◽

Vol 9 (2) ◽

pp. 379

Author(s):

Breanne M. Head ◽

Christopher I. Graham ◽

Teassa MacMartin ◽

Yoav Keynan ◽

Ann Karen C. Brassinga

Keyword(s):

Growth Kinetics ◽

Legionella Pneumophila ◽

Fluorescent Protein ◽

Disease Incidence ◽

Acanthamoeba Castellanii ◽

Infection Model ◽

Intracellular Infection ◽

Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

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Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

The Plant Cell ◽

10.1093/plcell/koab122 ◽

2021 ◽

Author(s):

Noemi Ruiz-Lopez ◽

Jessica Pérez-Sancho ◽

Alicia Esteban del Valle ◽

Richard P Haslam ◽

Steffen Vanneste ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Abiotic Stress ◽

Fluorescent Protein ◽

Mechanical Damage ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact ◽

Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

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CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

Veterinary Research ◽

10.1186/s13567-021-00964-4 ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Peng-Fei Fu ◽

Xuan Cheng ◽

Bing-Qian Su ◽

Li-Fang Duan ◽

Cong-Rong Wang ◽

...

Keyword(s):

Pseudorabies Virus ◽

Fluorescent Protein ◽

Antiviral Agents ◽

Firefly Luciferase ◽

Inhibitory Effect ◽

Economic Losses ◽

Green Fluorescent ◽

Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

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Tb3+-doped fluorescent glass for biology

Science Advances ◽

10.1126/sciadv.abd2529 ◽

2021 ◽

Vol 7 (2) ◽

pp. eabd2529

Author(s):

Kazuki Okamoto ◽

Teppei Ebina ◽

Naoki Fujii ◽

Kuniaki Konishi ◽

Yu Sato ◽

...

Keyword(s):

Single Cell ◽

Fluorescent Protein ◽

Biological Research ◽

Optical Investigation ◽

Patch Clamp Recording ◽

Rare Earth Ion ◽

Cell Transcriptome ◽

Green Fluorescent ◽

Doped Glass

Optical investigation and manipulation constitute the core of biological experiments. Here, we introduce a new borosilicate glass material that contains the rare-earth ion terbium(III) (Tb3+), which emits green fluorescence upon blue light excitation, similar to green fluorescent protein (GFP), and thus is widely compatible with conventional biological research environments. Micropipettes made of Tb3+-doped glass allowed us to target GFP-labeled cells for single-cell electroporation, single-cell transcriptome analysis (Patch-seq), and patch-clamp recording under real-time fluorescence microscopic control. The glass also exhibited potent third harmonic generation upon infrared laser excitation and was usable for online optical targeting of fluorescently labeled neurons in the in vivo neocortex. Thus, Tb3+-doped glass simplifies many procedures in biological experiments.

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The Great Capacity on Promoting Melanogenesis of Three Compatible Components in Vernonia anthelmintica (L.) Willd.

International Journal of Molecular Sciences ◽

10.3390/ijms22084073 ◽

2021 ◽

Vol 22 (8) ◽

pp. 4073

Author(s):

Yifan Lai ◽

Qingyuan Feng ◽

Rui Zhang ◽

Jing Shang ◽

Hui Zhong

Keyword(s):

Herbal Medicine ◽

Caffeic Acid ◽

Fluorescent Protein ◽

Traditional Chinese Herbal Medicine ◽

Expression Of Genes ◽

Proliferation And Differentiation ◽

Green Fluorescent ◽

Vernonia Anthelmintica

To investigate a possible methodology of exploiting herbal medicine and design polytherapy for the treatment of skin depigmentation disorder, we have made use of Vernonia anthelmintica (L.) Willd., a traditional Chinese herbal medicine that has been proven to be effective in treating vitiligo. Here, we report that the extract of Vernonia anthelmintica (L.) Willd. effectively enhances melanogenesis responses in B16F10. In its compound library, we found three ingredients (butin, caffeic acid and luteolin) also have the activity of promoting melanogenesis in vivo and in vitro. They can reduce the accumulation of ROS induced by hydrogen peroxide and inflammatory response induced by sublethal concentrations of copper sulfate in wild type and green fluorescent protein (GFP)-labeled leukocytes zebrafish larvae. The overall objective of the present study aims to identify which compatibility proportions of the medicines may be more effective in promoting pigmentation. We utilized the D-optimal response surface methodology to optimize the ratio among three molecules. Combining three indicators of promoting melanogenesis, anti-inflammatory and antioxidant capacities, we get the best effect of butin, caffeic acid and luteolin at the ratio (butin:caffeic acid:luteolin = 7.38:28.30:64.32) on zebrafish. Moreover, the effect of melanin content recovery in the best combination is stronger than that of the monomer, which suggests that the three compounds have a synergistic effect on inducing melanogenesis. After simply verifying the result, we performed in situ hybridization on whole-mount zebrafish embryos to further explore the effects of multi-drugs combination on the proliferation and differentiation of melanocytes and the expression of genes (tyr, mitfa, dct, kit) related to melanin synthesis. In conclusion, the above three compatible compounds can significantly enhance melanogenesis and improve depigmentation in vivo.

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