Influence of Fluorescent Protein Maturation on FRET Measurements in Living Cells

Zhang, Biying; Mavrova, Sara N; Berg, Jonas van den; Kristensen, Steffan K.; Mantovanelli, Luca; Veenhoff, Liesbeth M.; Poolman, Bert; Boersma, Arnold J.

doi:10.1021/acssensors.8b00473

Cited by 37 publications

(52 citation statements)

References 34 publications

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“…When placed in a crowded environment, the probe will obtain more compressed conformations, thus increasing the FRET efficiency. We utilized this FRET-based probe, named crGE, and harboring mCerulean3 as donor and mCitrine as acceptor ( Liu et al, 2018 ). We also developed a new probe, named CrGE2.3 by exchanging the donor and acceptor for mEGFP and mScarlet-I, respectively ( Figure 2A , left ).…”

Section: Resultsmentioning

confidence: 99%

A physicochemical perspective of aging from single-cell analysis of pH, macromolecular and organellar crowding in yeast

Mouton

Thaller

Crane

et al. 2020

eLife

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Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells we observe drastic changes in organellar volume, leading to crowding on the µm scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells.

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Section: Resultsmentioning

confidence: 99%

A physicochemical perspective of aging from single-cell analysis of pH, macromolecular and organellar crowding in yeast

Mouton

Thaller

Crane

et al. 2020

eLife

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“…coli BL21(DE3) cells and allowed the cells to adapt to the increased medium osmolarity. To monitor the macromolecular crowding, we expressed the crGE probe under leaky expression of the T7 promoter, which prevents maturation artifacts, as we described previously (23). To compare our results with literature data, we performed the experiments in morpholinepropanesulfonic (MOPS)-glucose medium (2, 4, 18).…”

Section: Resultsmentioning

confidence: 99%

Decreased Effective Macromolecular Crowding in Escherichia coli Adapted to Hyperosmotic Stress

et al. 2019

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Escherichia coli adapts to changing environmental osmolality to survive and maintain growth. It has been shown that the diffusion of green fluorescent protein (GFP) in cells adapted to osmotic upshifts is higher than expected from the increase in biopolymer volume fraction. To better understand the physicochemical state of the cytoplasm in adapted cells, we now follow the macromolecular crowding during adaptation with fluorescence resonance energy transfer (FRET)-based sensors. We apply an osmotic upshift and find that after an initial increase, the apparent crowding decreases over the course of hours to arrive at a value lower than that before the osmotic upshift. Crowding relates to cell volume until cell division ensues, after which a transition in the biochemical organization occurs. Analysis of single cells by microfluidics shows that changes in cell volume, elongation, and division are most likely not the cause for the transition in organization. We further show that the decrease in apparent crowding upon adaptation is similar to the apparent crowding in energy-depleted cells. Based on our findings in combination with literature data, we suggest that adapted cells have indeed an altered biochemical organization of the cytoplasm, possibly due to different effective particle size distributions and concomitant nanoscale heterogeneity. This could potentially be a general response to accommodate higher biopolymer fractions yet retaining crowding homeostasis, and it could apply to other species or conditions as well. IMPORTANCE Bacteria adapt to ever-changing environmental conditions such as osmotic stress and energy limitation. It is not well understood how biomolecules reorganize themselves inside Escherichia coli under these conditions. An altered biochemical organization would affect macromolecular crowding, which could influence reaction rates and diffusion of macromolecules. In cells adapted to osmotic upshift, protein diffusion is indeed faster than expected on the basis of the biopolymer volume fraction. We now probe the effects of macromolecular crowding in cells adapted to osmotic stress or depleted in metabolic energy with a genetically encoded fluorescence-based probe. We find that the effective macromolecular crowding in adapted and energy-depleted cells is lower than in unstressed cells, indicating major alterations in the biochemical organization of the cytoplasm.

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“…Chloramphenicol (250 g/mL) was added to the cultures to stall bacterial growth and protein production. 60 The cultures were maintained at 37ºC and 230 RPM and aliquots were taken every 30-60 minutes to measure the Optical Density (OD) and fluorescence. The t1/2 at 37ºC was calculated by calculating the time required for the FP to reach half the maximum fluorescence value.…”

Section: Chromophore Maturation Kinetics and Cytotoxicitymentioning

confidence: 99%

Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

Mukherjee

Hung

Douglas

et al. 2020

Preprint

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The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and appropriate localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, FusionRed-MQV, which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (~140,000 M -1 cm -1 ), increased radiative rate constant and reduced non-radiative rate constant with respect to its precursor. The properties of FusionRed-MQV derive from three mutations -M42Q, C159V and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the phenol and the acylimine ends of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the phenol end of the chromophore were mutated. The M42Q mutation is located near the acylimine end of the chromophore and was discovered using sitedirected mutagenesis guided by x-ray crystal structures. FusionRed-MQV exhibits 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells (based on FACS) compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells.

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Influence of Fluorescent Protein Maturation on FRET Measurements in Living Cells

Cited by 37 publications

References 34 publications

A physicochemical perspective of aging from single-cell analysis of pH, macromolecular and organellar crowding in yeast

A physicochemical perspective of aging from single-cell analysis of pH, macromolecular and organellar crowding in yeast

Decreased Effective Macromolecular Crowding in Escherichia coli Adapted to Hyperosmotic Stress

Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

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