Macroscopic control of cell electrophysiology through ion channel expression
- Centro de Biotecnologıa y Genomica de Plantas (Universidad Politecnica de Madrid – Instituto Nacional de Investigacion y Tecnologıa Agraria y Alimentaria), Spain
Figures
Figure 1 with 5 supplements
Control of ion channel expression through open-loop circuit.
(A) Schematic of open-loop system driving expression of constitutively open KcsA* bacterial potassium channel. Activator IacR and repressor MarR (tagged with ODC degron; Takeuchi et al., 2008) are …
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Figure 1—source data 1
Source data for Figure 1H, I.
- https://cdn.elifesciences.org/articles/78075/elife-78075-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
The plasmids used for open-loop circuit.
Galactose-inducible activator (IacR) and repressor (MarR) constructs are shown. The potassium channel (KcsA*) plasmid contains a synthetic minimal promoter with IacR and MarR operator sites.
Figure 1—figure supplement 2
Thioflavin T (ThT) cationic dyes show linear response to external potassium applications and high stability compared to other plasma membrane potential (PMP) dyes.
Screenshots from microscopy imaging of yeast after 24 hr in different KCl concentrations, and quantification of mean ThT (A), DIBAC4(3) (B), and Dis-C3(3) (C) fluorescence (upper panels) for …
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Figure 1—figure supplement 2—source data 1
Source data for Figure 1—figure supplement 2A–C.
- https://cdn.elifesciences.org/articles/78075/elife-78075-fig1-figsupp2-data1-v2.xlsx
Figure 1—figure supplement 3
Autocorrelation and peak analysis of Thioflavin T (ThT) traces in control and open-loop system.
(A) Cumulative autocorrelation of ThT traces in control strain averaged across n = 33 (yeast communities) trapping regions show noisy, non-synchronized pattern. (B) Weak autocorrelation of ThT …
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Figure 1—figure supplement 3—source data 1
Source data for Figure 1—figure supplement 3C–E.
- https://cdn.elifesciences.org/articles/78075/elife-78075-fig1-figsupp3-data1-v2.xlsx
Figure 1—video 1
The absence of Thioflavin T (ThT) oscillations in the control strain.
Related to Figure 1D. Left panel, differential interference contrast (DIC), middle panel, ThT (cyan), and right panel, ThT color coded in heat map colors (blue [low signal], red [high signal]) of …
Figure 1—video 2
Weakly coupled Thioflavin T (ThT) oscillations in the strain integrating the open-loop synthetic circuit.
Related to Figure 1F. Left panel, differential interference contrast (DIC), middle panel, ThT (cyan), and right panel, ThT color coded in heat map colors (blue [low signal], red [high signal]) of …
Figure 2 with 9 supplements
A synthetic dual-feedback circuit coordinates ion channel expression in yeast communities.
(A) Schematic of dual-feedback circuit controlling downstream expression of bacterial potassium channel KcsA*. All components of the system are controlled by the same promoter to allow integration …
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Figure 2—source data 1
Source data for Figure 2H, I.
- https://cdn.elifesciences.org/articles/78075/elife-78075-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Computer model simulations of IacR–MarR feedback circuit.
Computer simulations of a dual-feedback circuit model (upper panel) and corresponding phase portraits with nullclines (lower panel) for the different ratio of IacR–MarR receptor turnover (1:1 (A), …
Figure 2—figure supplement 2
Plasmids used to construct dual-feedback circuit and engineered ion channels.
Activator (IacR), repressor (MarR), and destabilized reporter (dGFP) constructs are shown. All components are controlled from the same synthetic promoter containing IacR and MarR operator sites. The …
Figure 2—figure supplement 3
Excitable control of circuit output with the pair of phytohormones.
A heat map of circuit response recorder with dEGFP reporter (upper panel) to chemical gradients (lower panel). Note a precise control of circuit output is possible by using a dual phytohormone …
Figure 2—figure supplement 4
Time-lapse synthetic circuit characterization using high-throughput fluorescence assays.
dEGFP fluorescence normalized to OD600 (upper panel) and OD600 absorbance (lower panel) for 24-hr measurements using graded changes of phytohormones was used to analyze the excitable potential of …
Figure 2—figure supplement 5
The frequency of phytohormone stimuli determines the dynamics of the reporter gene under different environmental drivers.
(A–E) Left panel, dEGFP time evolutions from all traps recorded in the microfluidic device. Right panel, the heat map of dEGFP fluorescence for each of the recorded trapping regions corresponds to …
Figure 2—figure supplement 6
Analysis of response characteristics under different environments.
(A) A distribution of dGFP periods for three different frequencies of stimuli, as marked with corresponding colors. (B) Violin plot of peak amplitude distribution in all recorded yeast communities …
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Figure 2—figure supplement 6—source data 1
Source data for Figure 2—figure supplement 6A–C.
- https://cdn.elifesciences.org/articles/78075/elife-78075-fig2-figsupp6-data1-v2.xlsx
Figure 2—figure supplement 7
Tracking dynamics of KcsA* potassium channels under phytohormone rhythms.
(A) Time evolution of KcsA-EGFP reporter traces under indicated pulses of phytohormones (3-hr period). An average trace of n = 25 yeast communities is shown in black. (B) Kymograph (heat map) of …
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Figure 2—figure supplement 7—source data 1
Source data for Figure 2—figure supplement 7D–G.
- https://cdn.elifesciences.org/articles/78075/elife-78075-fig2-figsupp7-data1-v2.xlsx
Figure 2—video 1
Dual-feedback gene circuit shows coordinated oscillations of fluorescence marker.
Related to Figure 2E. Left panel, dfferential interference contrast (DIC), middle panel, dEGFP (cyan) and mCherry (cell marker), and right panel, dEGFP color coded in heat map colors (blue [low …
Figure 2—video 2
Dynamics of KcsA-EGFP fluorescent reporter in the dual-feedback circuit show coherent oscillations under 3-hour cycles of phytohormone stimuli related to Figure 2—figure supplement 7.
Left panel, differential interference contrast (DIC), middle panel, dEGFP (cyan) and mCherry (cell marker), and right panel, dEGFP color coded in heat map colors (blue [low signal], red [high …
Figure 3 with 6 supplements
Global modulation of ion channel expression and plasma membrane potential (PMP) in yeast communities through phytohormones.
(A) Representative time traces of Thioflavin T (ThT) fluorescence per community (n = 56 yeast communities) under 2-hr hormone stimuli. Average trend is shown in black. SA and IAA peaks are shown as …
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Figure 3—source data 1
Source data for Figure 3D, E.
- https://cdn.elifesciences.org/articles/78075/elife-78075-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Characterization of synchronicity of Thioflavin T (ThT) fluorescence in dynamically changing environment.
Time evolutions of ThT fluorescence across communities in 3 hr (A) and 1 hr (B) lasting phytohormone stimuli. Average traces are shown in black (C, D), kymographs related to (A) and (B) for n = 27 …
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Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1F–H.
- https://cdn.elifesciences.org/articles/78075/elife-78075-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
Synchronicity analysis of TOK1*-based circuit controlling plasma membrane potential (PMP) in yeast communities.
(A) Cumulative autocorrelation analysis of cumulative trend across all measured communities for 2 hr (black line) shows coherent oscillations in Thioflavin T (ThT) fluorescence. Peak amplitudes (B), …
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Figure 3—figure supplement 2—source data 1
Source data for Figure 3—figure supplement 2B–E.
- https://cdn.elifesciences.org/articles/78075/elife-78075-fig3-figsupp2-data1-v2.xlsx
Figure 3—video 1
Thioflavin T (ThT) oscillations controlled by phytohormone rhythms (2-hr stimuli) for yeast transformed with KcsA*-based circuit.
Related to Figure 3A. Left panel, differential interference contrast (DIC), middle panel, ThT (cyan), and right panel, ThT color coded in heat map colors (blue [low signal], red [high signal]) of …
Figure 3—video 2
Thioflavin T (ThT) oscillations controlled by phytohormone rhythms (3-hr stimuli) for yeast transformed with KcsA*-based circuit.
Related to Figure 3—figure supplement 1A. Left panel, differential interference contrast (DIC), middle panel, ThT (cyan), and right panel, ThT color coded in heat map colors (blue [low signal], red …
Figure 3—video 3
Thioflavin T (ThT) oscillations controlled by phytohormone rhythms (1-hr stimuli) for yeast transformed with KcsA*-based circuit.
Related to Figure 3—figure supplement 1B. Left panel, differential interference contrast (DIC), middle panel, ThT (cyan), and right panel, ThT color coded in heat map colors (blue [low signal], red …
Figure 3—video 4
Thioflavin T (ThT) oscillations controlled by phytohormone rhythms (2-hr stimuli) for yeast transformed with TOK1*-based circuit.
Related to Figure 3G. Left panel, differential interference contrast (DIC), middle panel, ThT (cyan), and right panel, ThT color coded in heat map colors (blue [low signal], red [high signal]) of …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 (Saccharomyces cerevisiae) | sRedM | Pérez-García et al., 2021 | BY4741 pGK1:: mCherry | Selection: KanMX (G418) |
| Strain, sRedM (Saccharomyces cerevisiae) | cLPdGFP | This study | sRedM pMarOIacO:: IacR-VP64 (pEX1004, ADDGENE_194950) pMarOIacO::MarR-RD (pEX1005, ADDGENE_ 194951) pMarOIacO::dEGFP (UbG76V-EGFP) (pEX1006, ADDGENE_194952) | Selection: KanMX (G418) -Leu, -Ura, -His |
| Strain, BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 (Saccharomyces cerevisiae) | oLPKcsA* | This study | BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pGal7:: IacR-VP64 (pEX1001, ADDGENE_ 194713) pGal7::MarR-RD (pEX1002 ADDGENE_ 194714) pMarOIacO::KcsA*(pEX1003, ADDGENE_194949) | Selection: -Leu, -Ura, -His |
| Strain, BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 (Saccharomyces cerevisiae) | sEmpty (control strain) | This study | BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Empty insert plasmids pGADT7 (Takara Bio) backbone with auxotrophic markers | Selection: -Leu, -Ura, -His |
| Strain, BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 (Saccharomyces cerevisiae) | cLPKcsA* | This study | sRedM pMarOIacO:: IacR-VP64 (pEX1004, ADDGENE_194950) pMarOIacO::MarR-RD (pEX1005, ADDGENE_ 194951) pMarOIacO::KcsA*(pEX1003, ADDGENE_194949) | Selection: -Leu, -Ura, -His |
| Strain, BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 (Saccharomyces cerevisiae) | cLPTOK1* | This study | pMarOIacO:: IacR-VP64 (pEX1004, ADDGENE_194950) pMarOIacO::MarR-RD (pEX1005, ADDGENE_ 194951) pMarOIacO::TOK1*(pEX1008, ADDGENE_194954) | Selection: -Leu, -Ura, -His |
| Strain, BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 (Saccharomyces cerevisiae) | cLPKcsA-EGFP* | This study | sRedM pMarOIacO:: IacR-VP64 (pEX1004, ADDGENE_194950) pMarOIacO::MarR-RD (pEX1005, ADDGENE_ 194951) pMarOIacO::KcsA- GFP*(pEX1007, ADDGENE_194953) | Selection: KanMX (G418) -Leu, -Ura, -His |
| Other, PMP dye: Thioflavin T | ThT | Fisher Scientific, Thermo Scientific | CAS: 2390-54-7 | |
| Other, PMP dye: Bis-(1,3-Dibutylbarbituric Acid)Trimethine Oxonol | DIBAC4(3) | VWR INTERNATIONAL EUROLAB, S.L | CAS: 70363-83-6 | |
| Other, PMP dye: 3,3'-di-n-propylthiacarbocyanine iodide | DIS-C3(3) | FISHER SCIENTIFIC | CAS: 53336-12-2 |
Additional files
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Supplementary file 1
Oligos and synthetic DNA sequences used in this study.
- https://cdn.elifesciences.org/articles/78075/elife-78075-supp1-v2.docx
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Supplementary file 2
Computer model parameters.
- https://cdn.elifesciences.org/articles/78075/elife-78075-supp2-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/78075/elife-78075-mdarchecklist1-v2.docx
