Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells

doi:10.1038/s41467-021-25736-8

. 2021 Sep 14;12(1):5438.

doi: 10.1038/s41467-021-25736-8.

Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells

Cesar L Cuevas-Velazquez^{1

2

3

4}, Tamara Vellosillo^{5

6}, Karina Guadalupe^{7

8}, Hermann Broder Schmidt⁹, Feng Yu^{7

10}, David Moses^{7

8}, Jennifer A N Brophy^{5

6}, Dante Cosio-Acosta¹¹, Alakananda Das¹², Lingxin Wang¹², Alexander M Jones¹³, Alejandra A Covarrubias¹⁴, Shahar Sukenik^{15

16

17}, José R Dinneny^{18

19}

Affiliations

¹ Department of Biology, Stanford University, Stanford, CA, 94305, USA. cuevas@quimica.unam.mx.
² Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, 94305, USA. cuevas@quimica.unam.mx.
³ Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, 62210, Mexico. cuevas@quimica.unam.mx.
⁴ Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México, 04510, Mexico. cuevas@quimica.unam.mx.
⁵ Department of Biology, Stanford University, Stanford, CA, 94305, USA.
⁶ Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, 94305, USA.
⁷ Center for Cellular and Biomolecular Machines (CCBM), University of California, Merced, CA, 95343, USA.
⁸ Chemistry and Chemical Biology Program, University of California, Merced, CA, 95343, USA.
⁹ Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305, USA.
¹⁰ Quantitative Systems Biology Program, University of California, Merced, CA, 95343, USA.
¹¹ Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, 62210, Mexico.
¹² Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, 94305, USA.
¹³ Sainsbury Laboratory, Cambridge University, Cambridge, CB2 1LR, UK.
¹⁴ Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, 62210, Mexico. alejandra.covarrubias@ibt.unam.mx.
¹⁵ Center for Cellular and Biomolecular Machines (CCBM), University of California, Merced, CA, 95343, USA. ssukenik@ucmerced.edu.
¹⁶ Chemistry and Chemical Biology Program, University of California, Merced, CA, 95343, USA. ssukenik@ucmerced.edu.
¹⁷ Quantitative Systems Biology Program, University of California, Merced, CA, 95343, USA. ssukenik@ucmerced.edu.
¹⁸ Department of Biology, Stanford University, Stanford, CA, 94305, USA. dinneny@stanford.edu.
¹⁹ Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, 94305, USA. dinneny@stanford.edu.

PMID: 34521831
PMCID: PMC8440526
DOI: 10.1038/s41467-021-25736-8

Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells

Cesar L Cuevas-Velazquez et al. Nat Commun. 2021.

. 2021 Sep 14;12(1):5438.

doi: 10.1038/s41467-021-25736-8.

Authors

Affiliations

¹ Department of Biology, Stanford University, Stanford, CA, 94305, USA. cuevas@quimica.unam.mx.
² Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, 94305, USA. cuevas@quimica.unam.mx.
³ Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, 62210, Mexico. cuevas@quimica.unam.mx.
⁴ Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México, 04510, Mexico. cuevas@quimica.unam.mx.
⁵ Department of Biology, Stanford University, Stanford, CA, 94305, USA.
⁶ Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, 94305, USA.
⁷ Center for Cellular and Biomolecular Machines (CCBM), University of California, Merced, CA, 95343, USA.
⁸ Chemistry and Chemical Biology Program, University of California, Merced, CA, 95343, USA.
⁹ Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305, USA.
¹⁰ Quantitative Systems Biology Program, University of California, Merced, CA, 95343, USA.
¹¹ Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, 62210, Mexico.
¹² Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, 94305, USA.
¹³ Sainsbury Laboratory, Cambridge University, Cambridge, CB2 1LR, UK.
¹⁴ Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, 62210, Mexico. alejandra.covarrubias@ibt.unam.mx.
¹⁵ Center for Cellular and Biomolecular Machines (CCBM), University of California, Merced, CA, 95343, USA. ssukenik@ucmerced.edu.
¹⁶ Chemistry and Chemical Biology Program, University of California, Merced, CA, 95343, USA. ssukenik@ucmerced.edu.
¹⁷ Quantitative Systems Biology Program, University of California, Merced, CA, 95343, USA. ssukenik@ucmerced.edu.
¹⁸ Department of Biology, Stanford University, Stanford, CA, 94305, USA. dinneny@stanford.edu.
¹⁹ Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, 94305, USA. dinneny@stanford.edu.

PMID: 34521831
PMCID: PMC8440526
DOI: 10.1038/s41467-021-25736-8

Abstract

Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Experimental approaches for dynamically monitoring these intracellular effects are currently lacking. Here, we leverage the environmental sensitivity and structural plasticity of intrinsically disordered protein regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use as environmentally-responsive molecular tools.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. Design of a biosensor for studying the effects of osmotic stress on living cells.**
a Schematic representation of the biosensor design under low and high macromolecular crowding/osmolarity—prevalent intracellular conditions upon hypoosmotic or hyperosmotic stress, respectively. The conformations are selected from the ensemble of all-atom simulations of AtLEA4-5 in the corresponding conditions. Cyan: mCerulean3. Yellow: Citrine. Gray: AtLEA4-5. b Normalized FRET ratio (DxAm/DxDm) of live yeast cells treated with different concentrations of NaCl. Cells are expressing the biosensor construct using either AtLEA4-5, AtLEA4-2, or arabinose-binding protein (ABP) as the sensory domain. n = 9 independent measurements. Two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Boxes represent 25th–75th percentile (line at median) with whiskers at 1.5*IQR. c Fluorescence emission spectra of NaCl-treated live yeast cells expressing the biosensor construct using AtLEA4-5 as the sensory domain. Fluorescence values were normalized to the value at 515 nm. d Normalized FRET ratio (DxAm/DxDm) of live yeast cells expressing either AtLEA4-5, N-AtLEA4-5, or C-AtLEA4-5 biosensor constructs. Cells were treated with 1 M NaCl. n = 9 independent measurements. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Boxes represent 25th–75th percentile (line at median) with whiskers at 1.5*IQR. e Disorder propensity prediction of AtLEA4-5 (blue) and five different scrambled versions (red) using PONDR. Threshold at 0.5 disorder propensity is shown. f Normalized FRET ratio (DxAm/DxDm) of live yeast cells expressing AtLEA4-5 (blue) or five different scrambled versions (red). n = 9 independent measurements. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Boxes represent 25th–75th percentile (line at median) with whiskers at 1.5*IQR. Source data are provided as a Source Data file.

**Fig. 2. AtLEA4-5 is highly sensitive to the chemical composition of the solution.**
a Computational solution space scan of the normalized radius of gyration (Rg) of AtLEA4-5 (blue), five different scrambled sequences shown in Fig. 1e, f (red), and 70 different naturally occurring IDRs (gray) under different solution repulsion levels (low to high solution repulsion of the protein backbone). Mean ± SD from n = 5 independent simulations. For clarity, only AtLEA4-5 (blue) SD is shown. SD for the other proteins are provided in the Source Data file. b Experimental solution space scan of AtLEA4-5 and CS. Open circles show the normalized FRET ratio (DxAm/DxDm) for the indicated concentration of each solute, with two points (that often overlap) for each concentration taken from separate repeats, highlighting the reproducibility of the data. Background color intensity represents sensitivity to the addition of solute. Stronger colors indicate stronger sensitivity. Red: compaction; blue: expansion; white: no change. Solution concentrations are given in weight percent (0–25 or 0–12 wt%) or molar (0–1.5 M). Source data are provided as a Source Data file.

**Fig. 3. SED1 can dynamically track the effects of osmotic stress on live yeast cells.**
a Normalized FRET ratio (DxAm/DxDm) time course of live yeast cells expressing SED1, treated with different concentrations of NaCl. The arrow indicates the addition of the treatment. Mean ± SEM. One-way ANOVA. ***p < 2 ×10⁻¹⁶. b Normalized FRET ratio (DxAm/DxDm) of live yeast cells expressing SED1 (blue) and CS (green), treated with different concentrations of NaCl. One-way ANOVA. ***p < 2 ×10⁻¹⁶. Continuous lines were smoothed using R with a loess smoothing function. Shaded regions indicate 95% confidence intervals. c Normalized FRET ratio (DxAm/DxDm) of wild type BY4742 strain (blue), hog1∆::G418 mutant (gray), and pbs2∆::G418 mutant (black), live yeast cells expressing SED1, hyperosmotically shocked with different concentrations of NaCl. Measurements were done immediately after hyperosmotic shock. Two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Continuous lines were smoothed using R with a loess smoothing function. Shaded regions indicate 95% confidence intervals. d Normalized FRET ratio (DxAm/DxDm) time course of wild type BY4742 strain (blue), hog1∆::G418 mutant (gray), and pbs2∆::G418 mutant (black), live yeast cells expressing SED1, treated with 0.6 M sorbitol. The arrow indicates the addition of the treatment. Mean ± SEM. Two-way ANOVA. ***p < 2 ×10⁻¹⁶. Source data are provided as a Source Data file.

**Fig. 4. Tracking SED1 response to osmotic stress in single cells reveals vacuoles buffer against water loss.**
a Ratiometric image of live yeast cells expressing SED1 under 0 M and 0.5 M NaCl. Scale bar = 10 μm. Calibration bar represents the normalized FRET ratio (DxAm/DxDm). b Quantification of (a). n = 40 cells (0 M NaCl) and n = 67 cells (0.5 M NaCl). Two-sided Student’s t test. ***p = 1 ×10⁻¹⁵. c Phasor plots (left) and donor fluorescence lifetime images (right) of live yeast cells expressing SED1 under 0 M, 0.5 M, and 1 M NaCl. Signals shifted to the left side of the phasor plot represent longer fluorescence lifetimes, whereas signals shifted to the right side represent shorter fluorescence lifetimes. Scale bar = 10 μm. Calibration bar represents the donor fluorescence lifetime in nanoseconds (ns). d Quantification of the donor fluorescence lifetime of individual cells from images in (c). n = 100 cells per treatment. One-way ANOVA. ***p < 2 ×10⁻¹⁶. e FRET efficiencies of live yeast cells from images in (c). n = 5 images for each treatment. One-way ANOVA. ***p < 1 ×10^-11. Boxes represent 25th–75th percentile (line at median) with whiskers at 1.5*IQR. f Normalized donor fluorescence lifetime measured for single cells after 1 M NaCl treatment (shaded area) in a time course. The experiment was repeated 3 times with similar results. g Normalized area measured for single cells after 1 M NaCl treatment (shaded area) in a time course (same cells as in (f)). The same color represents the same cell for (f) and (g). The experiment was repeated 3 times with similar results. (h) Individual time frames showing the donor fluorescence lifetime of single yeast cells exposed to 1 M NaCl treatment at time 0 min. Scale bar = 10 μm. The calibration bar is the same as in (c). The experiment was repeated 3 times with similar results. i Pearson’s correlation of donor lifetime and vacuolar ratio values for single yeast cells under standard conditions (0 M NaCl). Pearson’s correlation coefficient r = 0.439, p-value = 4 ×10⁻⁶. Continuous line was smoothed using R with a linear method smoothing function. Shaded region indicates 95% confidence interval. j Pearson’s correlation of the change in donor lifetime (Δlifetime) and vacuolar ratio values for single yeast cells subjected to 1 M NaCl. Δlifetime = (final lifetime−initial lifetime)/initial lifetime. Pearson’s correlation coefficient r = −0.465, p-value = 3 ×10⁻⁷. Continuous line was smoothed using R with a linear method smoothing function. Shaded region indicates 95% confidence interval. Source data are provided as a Source Data file.

**Fig. 5. SED1 tracks changes in osmolarity of a wide set of organisms.**
a Normalized FRET ratio (DxAm/DxDm) of live SED1-expressing *Escherichia coli* cells treated with different concentrations of NaCl. n = 3 independent experiments. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Boxes represent 25th–75th percentile (line at median) with whiskers at 1.5*IQR. b Normalized FRET ratio (DxAm/DxDm) time course of *Nicotiana benthamiana* leaf discs transiently expressing SED1, treated with either water, 0.5 M NaCl, or 1 M sorbitol. n = 7–11 leaf discs. Mean ± SEM. One-way ANOVA. c Ratiometric image of live SED1-expressing U-2 OS cells at 300 mOsm (isosmotic) or 600 mOsm (hyperosmotic) treated with sorbitol. Scale bar = 50 μm. Calibration bar represents the normalized FRET ratio (DxAm/DxDm). d Normalized FRET ratio of SED1-expressing U-2 OS cells exposed to different osmotic treatments with sorbitol. n = 5 cells, 5 regions of interest per cell. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Boxes represent 25th–75th percentile (line at median) with whiskers at 1.5*IQR. e Normalized FRET ratio of SED1-expressing U-2 OS cells exposed to different osmotic treatments with NaCl. n = 5 cells, 5 regions of interest per cell. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Boxes represent 25th–75th percentile (line at median) with whiskers at 1.5*IQR. Source data are provided as a Source Data file.

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References

1. Yancey PH. Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J. Exp. Biol. 2005;208:2819–2830. doi: 10.1242/jeb.01730. - DOI - PubMed
1. Hosseiniyan Khatibi SM, et al. Osmolytes resist against harsh osmolarity: something old something new. Biochimie. 2019;158:156–164. doi: 10.1016/j.biochi.2019.01.002. - DOI - PubMed
1. Record MT, Jr, Courtenay ES, Cayley DS, Guttman HJ. Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water. Trends Biochem. Sci. 1998;23:143–148. doi: 10.1016/S0968-0004(98)01196-7. - DOI - PubMed
1. Spitzer J, Poolman B. The role of biomacromolecular crowding, ionic strength, and physicochemical gradients in the complexities of life’s emergence. Microbiol. Mol. Biol. Rev. 2009;73:371–388. doi: 10.1128/MMBR.00010-09. - DOI - PMC - PubMed
1. Sukenik S, Ren P, Gruebele M. Weak protein–protein interactions in live cells are quantified by cell-volume modulation. Proc. Natl Acad. Sci. USA. 2017;114:6776–6781. - PMC - PubMed

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[1] Yancey PH. Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J. Exp. Biol. 2005;208:2819–2830. doi: 10.1242/jeb.01730. - DOI - PubMed

[2] Yancey PH. Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J. Exp. Biol. 2005;208:2819–2830. doi: 10.1242/jeb.01730. - DOI - PubMed

[3] Hosseiniyan Khatibi SM, et al. Osmolytes resist against harsh osmolarity: something old something new. Biochimie. 2019;158:156–164. doi: 10.1016/j.biochi.2019.01.002. - DOI - PubMed

[4] Hosseiniyan Khatibi SM, et al. Osmolytes resist against harsh osmolarity: something old something new. Biochimie. 2019;158:156–164. doi: 10.1016/j.biochi.2019.01.002. - DOI - PubMed

[5] Record MT, Jr, Courtenay ES, Cayley DS, Guttman HJ. Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water. Trends Biochem. Sci. 1998;23:143–148. doi: 10.1016/S0968-0004(98)01196-7. - DOI - PubMed

[6] Record MT, Jr, Courtenay ES, Cayley DS, Guttman HJ. Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water. Trends Biochem. Sci. 1998;23:143–148. doi: 10.1016/S0968-0004(98)01196-7. - DOI - PubMed

[7] Spitzer J, Poolman B. The role of biomacromolecular crowding, ionic strength, and physicochemical gradients in the complexities of life’s emergence. Microbiol. Mol. Biol. Rev. 2009;73:371–388. doi: 10.1128/MMBR.00010-09. - DOI - PMC - PubMed

[8] Spitzer J, Poolman B. The role of biomacromolecular crowding, ionic strength, and physicochemical gradients in the complexities of life’s emergence. Microbiol. Mol. Biol. Rev. 2009;73:371–388. doi: 10.1128/MMBR.00010-09. - DOI - PMC - PubMed

[9] Sukenik S, Ren P, Gruebele M. Weak protein–protein interactions in live cells are quantified by cell-volume modulation. Proc. Natl Acad. Sci. USA. 2017;114:6776–6781. - PMC - PubMed

[10] Sukenik S, Ren P, Gruebele M. Weak protein–protein interactions in live cells are quantified by cell-volume modulation. Proc. Natl Acad. Sci. USA. 2017;114:6776–6781. - PMC - PubMed

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Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells

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Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells

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