Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting

Bajar, Bryce T.; Wang, Emily S.; Lam, Amy; Kim, Bongjae B.; Jacobs, C.; Howe, Elizabeth S.; Davidson, Michael W.; Lin, Michael Z.; Chu, Jun

doi:10.1038/srep20889

Cited by 369 publications

(330 citation statements)

References 37 publications

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“…E. coli maintain tight control of cytoplasmic calcium and adjust calcium concentration in response to changing external conditions (24,25). To test if E. coli displayed calcium dynamics, we imaged single cells expressing a genetically encoded calcium sensor, GCaMP6f (20) tethered to the calciuminsensitive fluorophore mRuby3 (26) to ensure proper expression in cases with low cytoplasmic calcium concentration. Constitutive expression of GCaMP6f resulted in slower growth compared with nonexpressing cells (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

Voltage-gated calcium flux mediates Escherichia coli mechanosensation

Bruni

Weekley

Dodd

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

131

116

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Electrically excitable cells harness voltage-coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects from this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in Escherichia coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux altered mechanically induced changes in protein concentration, while inducing calcium flux reproduced these changes. Thus, voltage and calcium relay a bacterial sense of touch and alter cellular lifestyle. Although the calcium effectors remain unknown, these data open a host of new questions about E. coli, including the identity of the underlying molecular players, as well as other signals conveyed by voltage and calcium. These data also provide evidence that dynamic voltage and calcium exists as a signaling modality in the oldest domain of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings.alcium is a universal and indispensable signaling ion used in all known eukaryotes (1). Calcium concentration gradients across the plasma membrane and intracellular organelles enable highly dynamic fluxes via orchestrated channel openings to generate tightly controlled spatial and temporal patterns. The dynamics in both space and time are encoded and decoded into varying and sometimes opposing cellular signals (2, 3). In electrically excitable cells, including neurons and muscle cells, voltage-gated calcium channels (VGCCs) couple membrane depolarization to calcium influx, which can then alter cellular physiology. The myriad uses of calcium by cells highlight its crucial role in cellular maintenance.Despite calcium's ubiquity and utility across biological domains, little is known about how calcium is regulated in bacteria, especially at the single cell level (4). Fluorescent calcium dyes used in eukaryotic cells are not able to pass through the cell wall without pretreatment with the chelating agent EDTA, prohibiting studies of calcium with high temporal and spatial resolution in bacteria. Isotope labeling and luminescent probes have contributed population-level measurements of calcium, but were unable to resolve any potential cellular heterogeneity. In population measurements, cytoplasmic ...

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Section: Resultsmentioning

confidence: 99%

Voltage-gated calcium flux mediates Escherichia coli mechanosensation

Bruni

Weekley

Dodd

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

131

116

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“…For the sfCherry2 1-10/11 system, the fact that we have only observed lysosome puncta when labeling ER proteins suggests that this problem could be potentially resolved by increasing its pKa with rational designs 32 . Moreover, our engineering platform can be easily adapted to generating other red split FPs based on novel bright FPs such as TagRFP-T 34 , mRuby3 35 , or mScarlet 36 .…”

Section: Discussionmentioning

confidence: 99%

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

Feng

Sekine

Pessino

et al. 2017

Preprint

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Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact.To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen2 1-10/11 that improves the ratio of complemented signal to the background of FP 1-10 -expressing cells compared to the commonly used split GFP 1-10/11 ; as well as a 10-fold brighter red-colored split-sfCherry2 1-10/11 . Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry2 11 and GFP 11 , revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes. 5,6 , identification of cell contacts and synapses 7,8 , as well as scaffolding protein assembly 3, 9, 10 . Recently, they have also enabled the generation of large-scale human cell line libraries with fluorescently tagged endogenous proteins through CRISPR/Cas9-based gene editing 11 .So far, the most commonly used self-complementing split FP was GFP 1-10D7/11M3 OPT (which we refers to as GFP 1-10/11 ), engineered from super-folder GFP (sfGFP) 12 . With the splitting point between the tenth and eleventh β-strands, the resulting GFP 11 fragment is a 16-amino acid (a.a.) short peptide. The corresponding GFP 1-10 fragment remains almost non-fluorescent until complementation, making GFP 1-10/11 well suited for protein labeling by fusing GFP 11 to the target protein and over-expressing GFP 1-10 in the corresponding subcellular compartments. However, there lacks a second, orthogonal split FP system with comparable complementation performance for multicolor imaging and multiplexed scaffolding of protein assembly. Previously, a sfCherry 1-10/11 system 3 was derived from super-folder Cherry, an mCherry variant optimized for folding efficiency 13 . However, its overall fluorescent brightness is substantially weaker than an intact sfCherry fusion, potentially due to its limited complementation efficiency 3 . Although two-color imaging with sfCherry 1-10/11 and GFP 1-10/11 has been done using tandem sfCherry 11 to amplify the sfCherry signal for over-expressed targets, it is still too dim to detect most endogenous proteins.In this paper, we report a screening strategy for the direct engineering of self-complementing split FPs. Using this strategy, we have generated a yellow-green-colored mNeonGreen2 1-10/11 (mNG2) that has an improved ratio of complemented signal to the background of FP 1-10 -expressing cells as compared to GFP 1-10/11 , as well as a red-colored sfCherry2 1-...

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“…For pLenti-CDH5-mCitrine-IRES-puro, CAG-CDH5-mCitrine was released and ligated as SalI/NotI fragment into a custom-made and XhoI/NotI-digested pLenti-MCS-IRES-puro. pLenti-CDH5-mRuby3-IRES-puro was generated by releasing mCitrine from pLenti-CDH5-mCitrine-IRES-puro as an AgeI/NotI fragment and replacing it with mRuby3 46 , kindly provided by Michael Lin (Stanford University), by Gibson assembly 47 . For pLenti-CDH5-mEGFP, CAG-CDH5-mEGFP was first subcloned into pENTR1A-noCCDB (Addgene #17398) as a SalI/XhoI fragment, then transferred by LR recombination into pLenti-X1-puro-DEST (Addgene #17297).…”

Section: Methodsmentioning

confidence: 99%

Engulfed cadherin fingers are polarized junctional structures between collectively migrating endothelial cells

et al. 2016

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The development and maintenance of tissues requires collective cell movement, during which neighboring cells coordinate the polarity of their migration machineries. Here, we ask how polarity signals are transmitted from one cell to another across symmetrical cadherin junctions, during collective migration. We demonstrate that collectively migrating endothelial cells have polarized VE-cadherin-rich membrane protrusions, “cadherin fingers”, which leading cells extend from their rear and follower cells engulf at their front, thereby generating opposite membrane curvatures and asymmetric recruitment of curvature sensing proteins. In follower cells, engulfment of cadherin fingers occurs along with the formation of a lamellipodia-like zone with low actomyosin contractility, and requires VE-cadherin/catenin complexes and Arp2/3-driven actin polymerization. Lateral accumulation of cadherin fingers in follower cells precedes turning, and increased actomyosin contractility can initiate cadherin finger extension as well as engulfment by a neighboring cell, to promote follower behavior. We propose that cadherin fingers serve as guidance cues that direct collective cell migration.

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Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting

Cited by 369 publications

References 37 publications

Voltage-gated calcium flux mediates Escherichia coli mechanosensation

Voltage-gated calcium flux mediates Escherichia coli mechanosensation

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

Engulfed cadherin fingers are polarized junctional structures between collectively migrating endothelial cells

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