A photoswitchable fluorescent protein for hours-time-lapse and sub-second-resolved super-resolution imaging

Abstract Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.

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Highly biocompatible super-resolution fluorescence imaging using the fast photoswitching fluorescent protein Kohinoor and SPoD-ExPAN with L p-regularized image reconstruction

Microscopy ◽

10.1093/jmicro/dfy004 ◽

2018 ◽

Vol 67 (2) ◽

pp. 89-98

Author(s):

Tetsuichi Wazawa ◽

Yoshiyuki Arai ◽

Yoshinobu Kawahara ◽

Hiroki Takauchi ◽

Takashi Washio ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Fluorescent Protein ◽

Super Resolution ◽

Time Lapse ◽

Live Cells ◽

Polarization Angle ◽

Microscopy Technique ◽

Present Technique ◽

Super Resolution Microscopy ◽

Power Densities

Abstract Far-field super-resolution fluorescence microscopy has enabled us to visualize live cells in great detail and with an unprecedented resolution. However, the techniques developed thus far have required high-power illumination (102–106 W/cm2), which leads to considerable phototoxicity to live cells and hampers time-lapse observation of the cells. In this study we show a highly biocompatible super-resolution microscopy technique that requires a very low-power illumination. The present technique combines a fast photoswitchable fluorescent protein, Kohinoor, with SPoD-ExPAN (super-resolution by polarization demodulation/excitation polarization angle narrowing). With this technique, we successfully observed Kohinoor-fusion proteins involving vimentin, paxillin, histone and clathrin expressed in HeLa cells at a spatial resolution of 70–80 nm with illumination power densities as low as ~1 W/cm2 for both excitation and photoswitching. Furthermore, although the previous SPoD-ExPAN technique used L1-regularized maximum-likelihood calculations to reconstruct super-resolved images, we devised an extension to the Lp-regularization to obtain super-resolved images that more accurately describe objects at the specimen plane. Thus, the present technique would significantly extend the applicability of super-resolution fluorescence microscopy for live-cell imaging.

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Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

10.26434/chemrxiv.9209450.v1 ◽

2019 ◽

Author(s):

Jeffrey Chang ◽

Matthew Romei ◽

Steven Boxer

Keyword(s):

Rational Design ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Super Resolution ◽

Unit Cell Size ◽

Chlorine Substituent ◽

Gfp Chromophore ◽

The One ◽

Green Fluorescent ◽

Super Resolution Microscopy

Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of cis and trans rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the trans state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.

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Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors

10.1101/500298 ◽

2018 ◽

Cited By ~ 1

Author(s):

Shama Sograte-Idrissi ◽

Nazar Oleksiievets ◽

Sebastian Isbaner ◽

Mariana Eggert-Martinez ◽

Jörg Enderlein ◽

...

Keyword(s):

Fluorescent Proteins ◽

Super Resolution ◽

Resolving Power ◽

Acquisition Time ◽

Efficient Manner ◽

Target Dna ◽

Multiplexed Imaging ◽

Dna Strands ◽

Linkage Error ◽

20 Nm

AbstractDNA-PAINT is a rapidly developing fluorescence super-resolution technique which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20-25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using 3 nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the 3 targets with 20 nm resolution, and within only 35 minutes acquisition time.

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Dynamin-related Protein Drp1 Is Required for Mitochondrial Division in Mammalian Cells

Molecular Biology of the Cell ◽

10.1091/mbc.12.8.2245 ◽

2001 ◽

Vol 12 (8) ◽

pp. 2245-2256 ◽

Cited By ~ 1052

Author(s):

Elena Smirnova ◽

Lorena Griparic ◽

Dixie-Lee Shurland ◽

Alexander M. van der Bliek

Keyword(s):

Mammalian Cells ◽

Fluorescent Protein ◽

Time Lapse ◽

Subcellular Fractionation ◽

Related Protein ◽

Direct Role ◽

Mitochondrial Division ◽

Transfected Cells ◽

Green Fluorescent ◽

Time Lapse Photography

Mutations in the human dynamin-related protein Drp1 cause mitochondria to form perinuclear clusters. We show here that these mitochondrial clusters consist of highly interconnected mitochondrial tubules. The increased connectivity between mitochondria indicates that the balance between mitochondrial division and fusion is shifted toward fusion. Such a shift is consistent with a block in mitochondrial division. Immunofluorescence and subcellular fractionation show that endogenous Drp1 is localized to mitochondria, which is also consistent with a role in mitochondrial division. A direct role in mitochondrial division is suggested by time-lapse photography of transfected cells, in which green fluorescent protein fused to Drp1 is concentrated in spots that mark actual mitochondrial division events. We find that purified human Drp1 can self-assemble into multimeric ring-like structures with dimensions similar to those of dynamin multimers. The structural and functional similarities between dynamin and Drp1 suggest that Drp1 wraps around the constriction points of dividing mitochondria, analogous to dynamin collars at the necks of budding vesicles. We conclude that Drp1 contributes to mitochondrial division in mammalian cells.

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Visualization of the Peroxisomal Compartment in Living Mammalian Cells: Dynamic Behavior and Association with Microtubules

The Journal of Cell Biology ◽

10.1083/jcb.136.1.71 ◽

1997 ◽

Vol 136 (1) ◽

pp. 71-80 ◽

Cited By ~ 112

Author(s):

Erik A.C. Wiemer ◽

Thibaut Wenzel ◽

Thomas J. Deerinck ◽

Mark H. Ellisman ◽

Suresh Subramani

Keyword(s):

Mammalian Cells ◽

Laser Scanning ◽

Fluorescent Protein ◽

Time Lapse ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Small Subset ◽

Subcellular Compartment ◽

Directional Movement ◽

Green Fluorescent

Peroxisomes in living CV1 cells were visualized by targeting the green fluorescent protein (GFP) to this subcellular compartment through the addition of a COOH-terminal peroxisomal targeting signal 1 (GFP–PTS1). The organelle dynamics were examined and analyzed using time-lapse confocal laser scanning microscopy. Two types of movement could be distinguished: a relatively slow, random, vibration-like movement displayed by the majority (∼95%) of the peroxisomes, and a saltatory, fast directional movement displayed by a small subset (∼5%) of the peroxisomes. In the latter instance, peak velocities up to 0.75 μm/s and sustained directional velocities up to 0.45 μm/s over 11.5 μm were recorded. Only the directional type of motion appeared to be energy dependent, whereas the vibrational movement continued even after the cells were depleted of energy. Treatment of cells, transiently expressing GFP–PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisome morphology and subcellular distribution and blocked the directional movement. In contrast, the microtubule-stabilizing compound paclitaxel, or the microfilament-destabilizing drugs cytochalasin B or D, did not exert these effects. High resolution confocal analysis of cells expressing GFP–PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. The GFP–PTS1–labeled peroxisomes were found to distribute themselves in a stochastic, rather than ordered, manner to daughter cells at the time of mitosis.

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C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽

10.1098/rsob.200010 ◽

2020 ◽

Vol 10 (5) ◽

pp. 200010

Author(s):

Navaneethan Palanisamy ◽

Mehmet Ali Öztürk ◽

Emir Bora Akmeriç ◽

Barbara Di Ventura

Keyword(s):

Escherichia Coli ◽

In Silico ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Time Lapse ◽

Enhanced Yellow Fluorescent Protein ◽

Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

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Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy

ACS Nano ◽

10.1021/acsnano.5b04129 ◽

2015 ◽

Vol 9 (10) ◽

pp. 9528-9541 ◽

Cited By ~ 56

Author(s):

Sam Duwé ◽

Elke De Zitter ◽

Vincent Gielen ◽

Benjamien Moeyaert ◽

Wim Vandenberg ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Super Resolution ◽

Green Fluorescent ◽

Super Resolution Microscopy

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Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

10.1101/120642 ◽

2017 ◽

Author(s):

Stephen D. Carter ◽

Shrawan K. Mageswaran ◽

Zachary J. Farino ◽

João I. Mamede ◽

Catherine M. Oikonomou ◽

...

Keyword(s):

Electron Microscopy ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Organic Dyes ◽

Fluorescent Proteins ◽

Secretory Granules ◽

Light And Electron Microscopy ◽

Low Contrast ◽

Fluorescent Protein Tags ◽

Protein Tags

AbstractCryogenic correlated light and electron microscopy (cryo-CLEM) is a valuable tool for studying biological processes in situ. In cryo-CLEM, a target protein of interest is tagged with a fluorophore and the location of the corresponding fluorescent signal is used to identify the structure in low-contrast but feature-rich cryo-EM images. To date, cryo-CLEM studies of mammalian cells have relied on very bright organic dyes or fluorescent protein tags concentrated in virus particles. Here we describe a method to expand the application of cryo-CLEM to cells harboring genetically-encoded fluorescent proteins. We discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80K). Compared to fluorescent protein tags, these sources of autofluorescence exhibit a broader spectrum of fluorescence, which we exploited to develop a simple, robust approach to discriminate between the two. We validate this method in INS-1 E cells using a mitochondrial marker, and apply it to study the ultrastructural variability of secretory granules in a near-native state within intact INS-1E pancreatic cells by high-resolution 3D electron cryotomography.

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Quantitative assessment of near-infrared fluorescent proteins

10.21203/rs.3.rs-797586/v1 ◽

2021 ◽

Author(s):

Kiryl Piatkevich ◽

Hanbin Zhang ◽

Stavrini Papadaki ◽

Xiaoting Sun ◽

Luxia Yao ◽

...

Keyword(s):

Mammalian Cells ◽

Quantitative Assessment ◽

Near Infrared ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Oligomeric State ◽

C Elegans ◽

The Right ◽

Infrared Fluorescent Protein

Abstract Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins, which are rapidly becoming popular probes for a variety of imaging applications. To assist end-users with a selection of the right near-infrared fluorescent protein for a given application, we will conduct a quantitative assessment of intracellular brightness, photostability, and oligomeric state of 19 near-infrared fluorescent proteins in cultured mammalian cells. The top-performing proteins will be further validated for in vivo imaging of neurons in C. elegans, zebrafish, and mice. We will also assess the applicability of the selected NIR FPs for expansion microscopy and two-photon imaging.

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Application of SNAP-Tag in Expansion Super-Resolution Microscopy Using DNA Oligostrands

Frontiers in Chemistry ◽

10.3389/fchem.2021.640519 ◽

2021 ◽

Vol 9 ◽

Author(s):

Longfang Yao ◽

Li Zhang ◽

Yiyan Fei ◽

Liwen Chen ◽

Lan Mi ◽

...

Keyword(s):

Sample Preparation ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

New Technology ◽

Super Resolution ◽

Target Protein ◽

Substrate Molecule ◽

Label System ◽

Actual Target ◽

Super Resolution Microscopy

Expansion super-resolution technology is a new technology developed in recent years. It anchors the dye on the hydrogel and the dye expands with the expansion of the hydrogel so that a super-resolution map can be obtained under an ordinary microscope. However, by labeling the target protein with a first antibody and secondary antibody, the distance between the fluorescent group and the actual target protein is greatly increased. Although fluorescent proteins can also be used for expansion super-resolution to reduce this effect, the fluorescent protein is often destroyed during sample preparation. To solve this problem, we developed a novel label system for expansion microscopy, based on a DNA oligostrand linked with a fluorescent dye, acrylamide group (linker), and benzoylguanine (BG, a small substrate molecule for SNAP-tag). This protocol greatly reduced the error between the position of fluorescent group and the actual target protein, and also reduced loss of the fluorescent group during sample preparation.

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