How to Increase Brightness of Near-Infrared Fluorescent Proteins in Mammalian Cells

Shemetov, Anton A.; Oliinyk, Olena; Verkhusha, Vladislav V.

doi:10.1016/j.chembiol.2017.05.018

Cited by 63 publications

(79 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To date, several series of NIR FPs have been published [15][16][17][18][19][20][21][22] . For BphP-based NIR FPs, the apparent brightness of a FP in mammalian cells is one of the most important properties to be considered 23 . Because apoprotein NIR FPs incorporate BV with different affinities and specificities, and their expression levels and protein stabilities vary, their effective brightness varies too and does not correlate with the molecular brightness.…”

mentioning

confidence: 99%

A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales

et al. 2020

Self Cite

View full text Add to dashboard Cite

Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve~40 nm resolution in live cells. In living mice we detect~10 5 fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670 nm to 720 nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities.

show abstract

mentioning

confidence: 99%

A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…For this reason, biosynthesis of PCB has also been engineered into eukaryotic "chassis" organisms that naturally do not produce these compounds (75)(76)(77)(78). Increased expression of heme oxygenases has been used to enhance the Cyanobacterial photoactivatable adenylyl cyclases synthesis of BV in bacterial and mammalian cells (79,80). Indeed, bilin availability remains a critical issue to resolve for optogenetic applications because, in all cases tested, chromophore binding is critical for optimizing the dynamic range of two-color regulation by phytochrome and CBCR systems (39,54,81).…”

Section: Applications Beyond Photosynthetic Speciesmentioning

confidence: 99%

Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells

Blain-Hartung

Rockwell

Moreno

et al. 2018

Journal of Biological Chemistry

View full text Add to dashboard Cite

Class III adenylyl cyclases generate the ubiquitous second messenger cAMP from ATP often in response to environmental or cellular cues. During evolution, soluble adenylyl cyclase catalytic domains have been repeatedly juxtaposed with signal-input domains to place cAMP synthesis under the control of a wide variety of these environmental and endogenous signals. Adenylyl cyclases with light-sensing domains have proliferated in photosynthetic species depending on light as an energy source, yet are also widespread in nonphotosynthetic species. Among such naturally occurring light sensors, several flavin-based photoactivated adenylyl cyclases (PACs) have been adopted as optogenetic tools to manipulate cellular processes with blue light. In this report, we report the discovery of a cyanobacteriochrome-based photoswitchable adenylyl cyclase (cPAC) from the cyanobacterium sp. Unlike flavin-dependent PACs, which must thermally decay to be deactivated, cPAC exhibits a bistable photocycle whose adenylyl cyclase could be reversibly activated and inactivated by blue and green light, respectively. Through domain exchange experiments, we also document the ability to extend the wavelength-sensing specificity of cPAC into the near IR. In summary, our work has uncovered a cyanobacteriochrome-based adenylyl cyclase that holds great potential for the design of bistable photoswitchable adenylyl cyclases to fine-tune cAMP-regulated processes in cells, tissues, and whole organisms with light across the visible spectrum and into the near IR.

show abstract

“…The molecular brightness of biliprotein labels in solution and effective brightness in mammalian cells can differ dramatically, possibly owing to various factors under different conditions . As reported previously, we tested these new FPs as biolabels mainly in HEK293T cells (Table ).…”

Section: Resultsmentioning

confidence: 99%

“…Bacteriophytochromes, which covalently bind the heme‐derived linear tetrapyrrole compound biliverdin (BV), have broken out of the visible range and extended their spectra into the far‐red (FR; λ =650–700 nm) and near‐infrared (NIR; λ =700–770 nm) regions, where most deep tissues show maximum transmittance during in vivo imaging . Due to the effective binding of endogenous BV in mammalian cells, bacteriophytochromes have been successively evolved as FR and/or NIR fluorescence biomarkers, which are applied to label eukaryotic cells and whole animals . It remains a challenge to increase the effective brightness for deep‐tissue in vivo imaging and spectral multiplexing .…”

Section: Introductionmentioning

confidence: 97%

Very Bright Phycoerythrobilin Chromophore for Fluorescence Biolabeling

Hou

Ding

et al. 2019

ChemBioChem

View full text Add to dashboard Cite

Biliproteins have extended the spectral range of fluorescent proteins into the far‐red (FR) and near‐infrared (NIR) regions. These FR and NIR fluorescent proteins are suitable for the bioimaging of mammalian tissues and are indispensable for multiplex labeling. Their application, however, presents considerable challenges in increasing their brightness, while maintaining emission in FR regions and oligomerization of monomers. Two fluorescent biliprotein triads, termed BDFP1.2/1.6:3.3:1.2/1.6, are reported. In mammalian cells, these triads not only have extremely high brightness in the FR region, but also have monomeric oligomerization. The BDFP1.2 and BDFP1.6 domains covalently bind to biliverdin, which is accessible in most cells. The BDFP3.3 domain noncovalently binds phycoerythrobilin that is added externally. A new method of replacing phycoerythrobilin with proteolytically digested BDFP3.3 facilitates this labeling. BDFP3.3 has a very high fluorescence quantum yield of 66 %, with maximal absorbance at λ=608 nm and fluorescence at λ=619 nm. In BDFP1.2/1.6:3.3:1.2/1.6, the excitation energy that is absorbed in the red region by phycoerythrobilin in the BDFP3.3 domain is transferred to biliverdin in the two BDFP1.2 or BDFP1.6 domains and fluoresces at λ≈670 nm. The combination of BDFP3.3 and BDFP1.2/1.6:3.3:1.2/1.6 can realize dual‐color labeling. Labeling various proteins by fusion to these new fluorescent biliproteins is demonstrated in prokaryotic and mammalian cells.

show abstract

How to Increase Brightness of Near-Infrared Fluorescent Proteins in Mammalian Cells

Cited by 63 publications

References 45 publications

A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales

A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales

Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells

Very Bright Phycoerythrobilin Chromophore for Fluorescence Biolabeling

Contact Info

Product

Resources

About