scholarly journals Multiplexed labeling of cellular proteins with split fluorescent protein tags

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Ryo Tamura ◽  
Fangchao Jiang ◽  
Jin Xie ◽  
Daichi Kamiyama
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Cellular Proteins ◽  
Cell Protein ◽  
Adhesion Protein ◽  
Color Palette ◽  
Focal Adhesion Protein ◽  
Fluorescent Protein Tags

AbstractSelf-complementing split fluorescent proteins (split FP1-10/11) have become an important labeling tool in live-cell protein imaging. However, current split FP systems to label multiple proteins in single cells have a fundamental limitation in the number of proteins that can be simultaneously labeled. Here, we describe an approach to expand the number of orthogonal split FP systems with spectrally distinct colors. By combining rational design and cycles of directed evolution, we expand the spectral color palette of FP1-10/11. We also circularly permutate GFP and synthesize the β-strand 7, 8, or 10 system. These split GFP pairs are not only capable of labeling proteins but are also orthogonal to the current FP1-10/11 pairs, offering multiplexed labeling of cellular proteins. Our multiplexing approach, using the new orthogonal split FP systems, demonstrates simultaneous imaging of four distinct proteins in single cells; the resulting images reveal nuclear localization of focal adhesion protein Zyxin.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

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

<p>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 <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> 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.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

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

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.

scholarly journals Insights into the mechanisms of LOV domain color tuning from a set of high-resolution X-ray structures

2021 ◽  
Author(s):  
Alina Remeeva ◽  
Vera V. Nazarenko ◽  
Kirill Kovalev ◽  
Ivan Goncharov ◽  
Anna Yudenko ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Resolution ◽  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Side Chain ◽  
Lov Domain ◽  
Charged Amino Acids ◽  
C Terminus ◽  
Maximal Shift

AbstractLight-oxygen-voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All of the currently known LOV domains have absorption maxima in the range of ∼440 to ∼450 nm, and it is not clear whether they can be shifted significantly using mutations. Here, we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine amino acid of a thermostable flavin based fluorescent protein CagFbFP (Gln148) to asparagine, aspartate, glutamate, histidine, lysine and arginine. Absorption spectra of all of the mutants are blue-shifted, with the maximal shift of 8 nm observed for the Q148H variant. While CagFbFP and its Q148N/D/E variants are not sensitive to pH, Q148H/K/R reveal a moderate red shift induced by acidic pH. To gain further insight, we determined high resolution crystal structures of all of the mutants studied at the resolutions from 1.07 Å for Q148D to 1.63 Å for Q148R. Whereas in some of the variants, the amino acid 148 remains in the vicinity of the flavin, in Q148K, Q148R and partially Q148D, the C-terminus of the protein unlatches and the side chain of the residue 148 is reoriented away from the chromophore. Our results explain the absence of color shifts from replacing Gln148 with charged amino acids and pave the way for rational design of color-shifted flavin based fluorescent proteins.

scholarly journals Genetics of single-cell protein abundance variation in large yeast populations

2013 ◽  
Author(s):  
Frank Albert ◽  
Sebastian Treusch ◽  
Arthur H Shockley ◽  
Joshua S Bloom ◽  
Leonid Kruglyak
Keyword(s):  
Single Cell ◽  
Fluorescent Protein ◽  
Single Cell Protein ◽  
Cell Protein ◽  
Cell Physiology ◽  
Protein Abundance ◽  
Large Populations ◽  
Abundance Variation ◽  
Green Fluorescent ◽  
Fluorescent Protein Tags

Many DNA sequence variants influence phenotypes by altering gene expression. Our understanding of these variants is limited by sample sizes of current studies and by measurements of mRNA rather than protein abundance. We developed a powerful method for identifying genetic loci that influence protein expression in very large populations of the yeast Saccharomyes cerevisiae. The method measures single-cell protein abundance through the use of green-fluorescent-protein tags. We applied this method to 160 genes and detected many more loci per gene than previous studies. We also observed closer correspondence between loci that influence protein abundance and loci that influence mRNA abundance of a given gene. Most loci cluster at hotspot locations that influence multiple proteins—in some cases, more than half of those examined. The variants that underlie these hotspots have profound effects on the gene regulatory network and provide insights into genetic variation in cell physiology between yeast strains.

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

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

<p>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 <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> 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.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

scholarly journals Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe

2020 ◽  
Vol 10 (12) ◽  
pp. 4373-4385
Author(s):  
Gregor W. Schmidt ◽  
Andreas P. Cuny ◽  
Fabian Rudolf
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Time Lapse ◽  
Light Dose ◽  
Live Cells ◽  
Excitation Light ◽  
Minimal Impact ◽  
Imaging Conditions

Time-lapse imaging of live cells using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study. Here, we set out to develop guidelines to avoid the confounding effects of excitation light in multi-color long-term imaging. We use widefield fluorescence microscopy to measure the effect of the administered excitation light on growth rate (here called photomorbidity) in yeast. We find that photomorbidity is determined by the cumulative light dose at each wavelength, but independent of the way excitation light is applied. Importantly, photomorbidity possesses a threshold light dose below which no effect is detectable (NOEL). We found, that the suitability of fluorescent proteins for live-cell imaging at the respective excitation light NOEL is equally determined by the cellular autofluorescence and the fluorescent protein brightness. Last, we show that photomorbidity of multiple wavelengths is additive and imaging conditions absent of photomorbidity can be predicted. Our findings enable researchers to find imaging conditions with minimal impact on physiology and can provide framework for how to approach photomorbidity in other organisms.

scholarly journals Fluorescence-based whole plant imaging and phenomics

2019 ◽  
Author(s):  
Stephen B. Rigoulot ◽  
Tayler M. Schimel ◽  
Jun Hyung Lee ◽  
Holly Brabazon ◽  
Kerry A. Meier ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Low Noise ◽  
Color Palette ◽  
Plant Canopies ◽  
Whole Plant ◽  
Library Screen ◽  
Green Fluorescent

SummaryReverse genetics approaches have revolutionized plant biology and agriculture. Phenomics has the prospect of bridging plant phenotypes with genes, including transgenes, to transform agricultural fields1. Genetically-encoded fluorescent proteins (FPs) have transformed studies in gene expression, protein trafficking, and plant physiology. While the first instance of plant canopy imaging of green fluorescent protein (GFP) was performed over 20 years ago2, modern phenomics has largely ignored fluorescence as a transgene indicator despite the burgeoning FP color palette currently available to biologists3–5. Here we show a new platform for standoff imaging of plant canopies expressing a wide variety of FP genes in leaves. The platform, the fluorescence-inducing laser projector (FILP), uses a low-noise camera to image a scene illuminated by compact diode lasers of various colors and emission filters to phenotype transgenic plants expressing multiple constitutive or inducible FPs. Of the 20 FPs screened, we selected the top performing candidates for standoff phenomics at ≥ 3 m using FILP in a laboratory-based laser range. Included in demonstrated applications is the performance of an osmotic stress-inducible synthetic promoter selected from a high throughput library screen. While FILP has unprecedented versatility as a laboratory platform, we envisage future iterations of the system for use in automated greenhouse or even drone-fielded versions of the platform for crop screening.

scholarly journals Preventing photomorbidity in long-term multi-color fluorescence imaging of S. cerevisiae and S. pombe

2017 ◽  
Author(s):  
Gregor W. Schmidt ◽  
Andreas P. Cuny ◽  
Fabian Rudolf
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Time Lapse ◽  
Light Dose ◽  
Excitation Light ◽  
Minimal Impact ◽  
Multiple Wavelengths ◽  
Imaging Conditions

AbstractTime-lapse imaging using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study. Here, we set out to develop guidelines to avoid the confounding effects of excitation light in multi-color long-term imaging. We use widefield fluorescence microscopy to measure the effect of the administered excitation light on growth (here called photomorbidity) in yeast. We find that photomorbidity is determined by the cumulative light dose at each wavelength, but independent of the way excitation light is applied. Importantly, photomorbidity possesses a threshold light dose below which no effect is detectable (NOEL, no-observed-effect level). We found, that the suitability of fluorescent proteins for live-cell imaging at the respective excitation light NOEL is equally determined by the cellular autofluorescence and the fluorescent protein brightness. Last, we show that photomorbidity of multiple wavelengths is additive and imaging conditions absent of photomorbidity can be predicted. Our findings enable researchers to find imaging conditions with minimal impact on physiology and can provide a framework for how to approach photomorbidity in other organisms.

scholarly journals Estimating numbers of fluorescent molecules in single cells by analysing fluctuations in photobleaching

2018 ◽  
Author(s):  
Elco Bakker ◽  
Peter S. Swain
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Measurement Noise ◽  
Wide Field ◽  
Stochastic Fluctuations ◽  
Biochemical Measurements ◽  
Green Fluorescent ◽  
The Impact

The impact of fluorescence microscopy has been limited by the difficulties of express-ing measurements of fluorescent proteins in numbers of molecules. Absolute numbers enable the integration of results from different laboratories, empower mathematical modelling, and are the bedrock for a quantitative, predictive biology. Here we develop a general algorithm to infer numbers of molecules from fluctuations in the photobleaching of proteins tagged with Green Fluorescent Protein. To untangle measurement noise from stochastic fluctuations, we use the linear noise approximation and Kalman filtering within a framework of Bayesian inference. Not only do our results agree with biochemical measurements for multiple proteins in budding yeast, but we also provide a statistically verified model of measurement noise for fluorescence microscopes. The experiments we require are straightforward and use only a wide-field fluorescence microscope. As such, our approach has the potential to become standard for those practising quantitative fluorescence microscopy.

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