FLIRT: fast local infrared thermogenetics for subcellular control of protein function

Abstract

FLIRT (fast local infrared thermogenetics) is a microscopy-based technology to locally and reversibly manipulate protein function while simultaneously monitoring the effects in vivo. FLIRT locally inactivates fast-acting temperature-sensitive mutant proteins. We demonstrate that FLIRT can control temperature-sensitive proteins required for cell division, Delta–Notch cell fate signaling, and germline structure in Caenorhabditis elegans with cell-specific and even subcellular precision.

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Fig. 1: FLIRT calibration and application for spatiotemporal control of temperature-sensitive protein function in vivo.
Fig. 2: FLIRT for subcellular control of temperature-sensitive protein function.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank all members of the Canman, Shirasu-Hiza, and Dumont laboratory for their support; H. Kim, I. Thomas, C. Walsh, B. Lesea-Pringle, K. Rimu, F. Jung, and E. Blake for laboratory assistance; C. Connors for comments on this manuscript; I. Greenwald, B. Bowerman, and B. Goldstein for helpful discussions; and J. Priess (Fred Hutchinson Cancer Research Center) and the Caenorhabditis Genomics Center for worm strains. We thank S. Wildfang, A. Ratz, and C. Anderson for technical assistance; A. Kummel and A. Garcia Badaracco for assistance on the thermochromatic dye analysis; and B. O’Shaughnessy, S. Wang, and S. Thiyagarajan for advice on thermal distribution. This work was funded by a Charles H. Revson Senior Fellowship in Biomedical Science (T.D.), FRM-DEQ20160334869 (J.D.); NIH-R01-GM105775 (S.M.H.); NIH-R01-AG045842 (MSH); NIH-DP2-OD008773 (J.C.C.); and NIH-R01GM117407 (J.C.C.).

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Contributions

S.M.H., S.S., T.D., and J.C.C. conceived of the experiments. S.M.H. and S.S. conducted all of the experiments with help from T.D. and Y.Z. J.C.W. and J.C.C. designed the microscope light path. S.M.H., S.S., T.D., M.S.H., J.C.W., J.D., and J.C.C. made intellectual contributions and wrote the manuscript. S.M.H., S.S., and J.C.C. made the figures.

Corresponding author

Correspondence to Julie C. Canman.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 FLIRT microscope light path and laser alignment.

a, Schematic of the FLIRT microscope optical path and microfluidic temperature control system. b, Representative images of nano545-UCP-based visualization of the FLIRT ROI21. N = 5 independent replicates.

Supplementary Figure 2 Thermochromatic dye-based temperature calibration.

a, Schematic depicting experimental design to calibrate the FLIRT microscope using a thermochromatic dye. b, Experimental timeline demonstrating the thermochromatic dye upshift and FLIRT experiments to determine chromatic transition point. c, Representative images of transmitted light through the thermochromatic dye after temperature upshift (left column) or after FLIRT using the 16-µm (center column) or 27-µm (right column) diameter masks. N = 4 independent experiments. Scale bar, 10 µm. d,e, Graphs depicting the change in transmitted light intensity with whole coverslip upshift from 8 °C d, or FLIRT targeting using the 16-µm (left) or 27-µm (right) diameter masks while e, the thermochromatic dye-coated coverslip was held at 9, 10, or 11 °C. White dashed circles indicate FLIRT-targeted ROIs. The number of experimental replicates (N) is listed or indicated on individual graphs. Error bars, mean +/− s.e.m.

Supplementary Figure 3 mCherry fluorescence-based temperature and gradient calibration.

a, Schematics and representative images of FLIRT microscope thermal calibration using mCherry::HistoneH2B fluorescence in late-stage embryos (~100–200 cells) and either whole-embryo upshift (left) or while FLIRT targeting with increasing infrared laser power using the 16-µm or 27-µm diameter masks (right). N = 7 biologically independent embryos. b, Quantification of mCherry fluorescence intensity levels37 on temperature upshift (left) or using FLIRT with increasing infrared laser power (right). Error bars, mean +/− s.d. c, Schematic of FLIRT thermal gradient measurement across an embryo. d, Characterization of FLIRT thermal gradient. A gradient of ~4–8 °C was maintained on the other end of the same embryo, depending on the infrared mask used and hold temperature set point. Dashed lines represent the mask edges; error bars, mean +/− s.e.m. e, Measurement of the effect of hold temperature on FLIRT thermal gradient when FLIRT is used to heat the mask region to ~25.5 °C with varying hold temperatures (10–16 °C) and laser powers (8.5–13.9 mW, right table). The green dashed line at x = 16 µm shows the mask edge and the black dashed line at y = 24.5 °C shows the restrictive temperature for myosin-II(ts) mutants (Fig. 1c,d and Supplementary Figs. 4 and 6a). Error bars, mean +/− s.e.m. Note: 26 °C temperature upshift and 16 °C hold data are also shown in both d and e. f, Bar graph showing cell division phenotypes for control embryos at different hold temperatures.

Supplementary Figure 4 FLIRT-mediated inhibition of temperature-sensitive protein function is local and reversible.

a, Schematic (left) and representative images (right) from P1 cell-specific FLIRT experiments in either control (top) or myosin-II(ts) (bottom) two-cell embryos. See Supplementary Video 1. b, Representative images from FLIRT reversibility experiments in control (top two rows) and myosin-II(ts)28,29 (bottom two rows) two-cell embryos where the AB (top row) or P1 (bottom row) cell is targeted with the 16-µm mask for a ~4-min window after anaphase onset then turned off. See Supplementary Video 2. Red (schematic) and white (images) dashed circles indicate FLIRT-targeted ROIs. Time is in seconds after FLIRT initiation. The number of biologically independent AB and P1 cells that completed cytokinesis is indicated below each schematic (left). Scale bar, 10 µm.

Supplementary Figure 5 FLIRT targeting cell division at lower hold temperature and temperature-sensitive mutant specificity.

a, Schematics (left) and representative images from FLIRT experiments targeting to ~25.5 °C in either control (top row) or myosin-II(ts) (bottom three rows) one-cell embryos either on one side of the cell equator or a cell pole with a hold temperature of 14 °C and 10.1 mW of laser power (see Supplementary Fig. 3e), either throughout cell division (see Supplementary Video 4) or for an 8-min window (~6 min after anaphase onset, bottom row; see Supplementary Video 6). b, Schematic (left) and representative images from FLIRT experiments targeting the cell equator in one-cell Delta(ts) embryos. White dashed circles indicate FLIRT-targeted ROIs. Time is in seconds after FLIRT initiation. The number of biologically independent experimental replicates that completed cell division is indicated below each experimental schematic (left). Scale bar, 10 µm.

Supplementary Figure 6 FLIRT-targeted control embryos maintain cell polarity and viability.

a, Schematic (left) and representative images (right) depicting an assay to monitor the effect of equatorial FLIRT on cell polarity and daughter size asymmetry in one-cell embryos expressing PAR-6 (PARtitoning defective) and PAR-2. Quantification of cell polarity and daughter cell asymmetry during cell division (bottom center) and at the two-cell stage (bottom right). N = 7 biologically independent embryos for 16 °C (0 mW), 7 for 16 °C (8.5 mW), 6 for 14 °C (0 mW), and 7 for 14 °C (10.1 mW). Unpaired two-tailed t-test; n.s., no significance, P > 0.05. Error bars, mean +/− s.d.; see Supplementary Table 1 for statistical analysis. b, Experimental timeline for post-FLIRT viability assay. The yellow arrowhead indicates embryo transfer to a worm plate, gray arrowheads indicate developmental imaging, and yellow arrows indicate embryo location on a worm plate. Images of a representative individual control embryo rescued after equatorial FLIRT (top row—and shown at the two-cell stage in the micrograph indicating the FLIRT ROI) or dissected at the one-cell stage (no FLIRT control, bottom) throughout development. Quantification of embryonic viability for isolated one-cell-stage embryos (right). Red (schematic) and white (images) dashed circles indicate FLIRT-targeted ROIs. Time is in minutes after FLIRT initiation. The number of biologically independent experimental replicates (N) is indicated on individual bar graphs. White scale bars, 10 µm; yellow scale bar, 100 µm.

Supplementary Figure 7 Delta–Notch activity transcriptional reporter characterization with whole-embryo temperature upshifts.

a, Schematic depicting Delta–Notch signaling in four-cell C. elegans embryos. b, Schematic depicting the tbx-38 Delta–Notch activity reporter18. c, Experimental timeline for whole-embryo thermal control characterization of the Delta(ts) mutants and the tbx-38p::mCherry::HistoneH1 Delta–Notch activity reporter. d, Measurement (top) and schematic (bottom) depicting an mCherry bioassay used to measure the thermal gradient generated during FLIRT experiments in Delta(ts) mutants shown in Fig. 2a. Error bars, mean +/− s.e.m.; the green dotted line at x = 16 µm shows the mask edge and the black dotted line at y = 23 °C shows the restrictive temperature for Delta(ts) mutants determined from the whole-embryo upshift experiments in e. e, Representative images from whole-embryo upshift experiments with either control (left) or Delta(ts) (right) embryos at the ~200-cell stage after development at the indicated temperature, or after a brief ~25-min upshift to 24 °C during the four-cell stage. Bottom left, dot plots depicting the percentage of total nuclei expressing the mCherry reporter at the ~50-cell stage. The number of biologically independent experimental replicates (N) is indicated under each dot plot. Tukey’s multiple-comparisons test, α = 0.05, n.s., no significance, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001. Error bars, mean +/− s.d.; see Supplementary Table 1 for statistical analysis. Scale bars, 10 µm.

Supplementary Figure 8 In situ inhibition of CYK-4 in the adult C. elegans gonad with whole-worm upshifts.

a, Schematic of an adult C. elegans showing the location of the syncytial gonad and expected membrane partition retraction phenotype in control and cyk-4(ts) worms on upshift to 26 °C. b, Representative images from whole-animal upshift experiments with control and cyk-4(ts) mutants. Time is in minutes after temperature upshift. Orange arrows indicate membrane partition retraction. c, Schematic (left) and quantification (right) of membrane partition length before and after thermal upshift after whole-animal upshift to the restrictive temperature. Control: N = 8 worms, cyk-4(ts) at 16 °C: N = 7 worms, and cyk-4(ts) at 26 °C: N = 5 worms; with four partitions measured per worm. Unpaired two-tailed t-test; n.s., no significance, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001. Error bars, mean +/− s.d.; see Supplementary Table 1 for statistical analysis. Scale bar, 10 µm.

Supplementary information

41592_2018_168_MOESM3_ESM.mov

FLIRT: cell-specific inhibition of myosin-II(ts) function: FLIRT inactivation of myosin-II(ts) function specifically in one cell (AB, anterior cell; P1, posterior cell) of a two-cell embryo. FLIRT prevents contractile ring constriction in the myosin-II(ts) FLIRT-targeted cell, but not the non-targeted cell or control embryos. 30 s per frame. Red circle, 16 µm FLIRT ROI; FLIRT is initiated at t = 0 and kept on throughout the experiment. Hold temperature, 16 °C. FLIRT laser power, 8.5 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of seven (control AB, myosin-II(ts) AB, and myosin-II(ts) P1 FLIRT) or eight (control P1 FLIRT) independent experiments.

41592_2018_168_MOESM4_ESM.mov

Reversibility of FLIRT, cell-specific inhibition of myosin-II(ts) function: brief FLIRT inactivation of myosin-II(ts) function specifically in one cell (AB, anterior cell; P1, posterior cell) of a two-cell embryo. Contractile ring constriction in myosin-II(ts) initiates after the FLIRT laser is turned off, but is not impacted in controls. 30 s per frame. Red circle, 16 µm. FLIRT ROI; FLIRT is initiated at t = 0 and turned off ~4 min after anaphase onset. Hold temperature, 16 °C. FLIRT laser power, 8.5 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of seven (control AB, myosin-II(ts) AB, and myosin-II(ts) P1 FLIRT) or eight (control P1 FLIRT) independent experiments.

41592_2018_168_MOESM5_ESM.mov

Subcellular inhibition of myosin-II(ts) function (16 °C hold): FLIRT inactivation of either an equatorial or polar region of dividing control and myosin-II(ts) one-cell embryos. FLIRT targeting one side of the equatorial region of myosin-II(ts), but not a polar region, blocks contractile ring constriction from the targeted side. FLIRT targeting control cells does not block division. 30 s per frame. Red circle, 16 µm FLIRT ROI; FLIRT is initiated during prometaphase, videos are aligned to t = 0 at metaphase before anaphase onset and kept on throughout the experiment. Hold temperature, 16 °C. FLIRT laser power, 8.5 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of 14 (control FLIRT), seven (myosin-II(ts) polar FLIRT), and eight (myosin-II(ts) equatorial FLIRT) independent experiments.

41592_2018_168_MOESM6_ESM.mov

Subcellular inhibition of myosin-II(ts) function (14 °C hold): FLIRT inactivation of either an equatorial or polar region of dividing control and myosin-II(ts) one-cell embryos. FLIRT targeting one side of the equatorial region of myosin-II(ts), but not a polar region, blocks contractile ring constriction from the targeted side. FLIRT targeting control cells does not block division. 30 s per frame. Red circle, 16 µm FLIRT ROI; FLIRT is initiated during prometaphase, videos are aligned to t = 0 at metaphase before anaphase onset and kept on throughout the experiment. Hold temperature, 14 °C. FLIRT laser power, 10.1 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of 12 (control FLIRT), 7 (myosin-II(ts) polar FLIRT), and 7 (myosin-II(ts) equatorial FLIRT) independent experiments.

41592_2018_168_MOESM7_ESM.mov

Reversibility of FLIRT, equatorial myosin-II(ts) function (16 °C hold): brief FLIRT inactivation of the equatorial region of a dividing myosin-II(ts) one-cell embryo. Contractile ring constriction in myosin-II(ts) initiates rapidly after the FLIRT laser is turned off. 30 s per frame. Red circle, 16 µm FLIRT ROI; FLIRT is initiated during prometaphase and turned off ~6 min after anaphase onset. The video shows t = 0 at metaphase before anaphase onset. Hold temperature, 16 °C. FLIRT laser power, 8.5 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of nine independent experiments.

41592_2018_168_MOESM8_ESM.mov

Reversibility of FLIRT, equatorial myosin-II(ts) function (14 °C hold): brief FLIRT inactivation of the equatorial region of a dividing myosin-II(ts) one-cell embryo. Contractile ring constriction in myosin-II(ts) initiates rapidly after the FLIRT laser is turned off. 30 s per frame. Red circle, 16 µm FLIRT ROI; FLIRT is initiated during prometaphase and turned off ~6 min after anaphase onset. The video shows t = 0 at metaphase before anaphase onset. Hold temperature, 14 °C. FLIRT laser power, 10.1 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of seven independent experiments.

41592_2018_168_MOESM9_ESM.mov

In situ FLIRT inhibition of cyk-4(ts) function in the germ line: FLIRT targeting a single germline membrane partition in cyk-4(ts) but not control worms causes shortening of the FLIRT-targeted membrane partition, but not of non-targeted adjacent membrane partitions. 60 s per frame. Red circle, 16 µm FLIRT ROI; FLIRT is initiated at t = 0 and is kept on throughout the experiment. Hold temperature, 16 °C. FLIRT laser power, 8.5 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of seven (control) or ten (cyk-4(ts)) independent experiments.

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1, and Supplementary Notes 1 and 2

Reporting Summary

Supplementary Video 1

FLIRT: cell-specific inhibition of myosin-II(ts) function: FLIRT inactivation of myosin-II(ts) function specifically in one cell (AB, anterior cell; P1, posterior cell) of a two-cell embryo. FLIRT prevents contractile ring constriction in the myosin-II(ts) FLIRT-targeted cell, but not the non-targeted cell or control embryos. 30 s per frame. Red circle, 16 µm FLIRT ROI; FLIRT is initiated at t = 0 and kept on throughout the experiment. Hold temperature, 16 °C. FLIRT laser power, 8.5 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of seven (control AB, myosin-II(ts) AB, and myosin-II(ts) P1 FLIRT) or eight (control P1 FLIRT) independent experiments.

Supplementary Video 2

Reversibility of FLIRT, cell-specific inhibition of myosin-II(ts) function: brief FLIRT inactivation of myosin-II(ts) function specifically in one cell (AB, anterior cell; P1, posterior cell) of a two-cell embryo. Contractile ring constriction in myosin-II(ts) initiates after the FLIRT laser is turned off, but is not impacted in controls. 30 s per frame. Red circle, 16 µm. FLIRT ROI; FLIRT is initiated at t = 0 and turned off ~4 min after anaphase onset. Hold temperature, 16 °C. FLIRT laser power, 8.5 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of seven (control AB, myosin-II(ts) AB, and myosin-II(ts) P1 FLIRT) or eight (control P1 FLIRT) independent experiments.

Supplementary Video 3

Subcellular inhibition of myosin-II(ts) function (16 °C hold): FLIRT inactivation of either an equatorial or polar region of dividing control and myosin-II(ts) one-cell embryos. FLIRT targeting one side of the equatorial region of myosin-II(ts), but not a polar region, blocks contractile ring constriction from the targeted side. FLIRT targeting control cells does not block division. 30 s per frame. Red circle, 16 µm FLIRT ROI; FLIRT is initiated during prometaphase, videos are aligned to t = 0 at metaphase before anaphase onset and kept on throughout the experiment. Hold temperature, 16 °C. FLIRT laser power, 8.5 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of 14 (control FLIRT), seven (myosin-II(ts) polar FLIRT), and eight (myosin-II(ts) equatorial FLIRT) independent experiments.

Supplementary Video 4

Subcellular inhibition of myosin-II(ts) function (14 °C hold): FLIRT inactivation of either an equatorial or polar region of dividing control and myosin-II(ts) one-cell embryos. FLIRT targeting one side of the equatorial region of myosin-II(ts), but not a polar region, blocks contractile ring constriction from the targeted side. FLIRT targeting control cells does not block division. 30 s per frame. Red circle, 16 µm FLIRT ROI; FLIRT is initiated during prometaphase, videos are aligned to t = 0 at metaphase before anaphase onset and kept on throughout the experiment. Hold temperature, 14 °C. FLIRT laser power, 10.1 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of 12 (control FLIRT), 7 (myosin-II(ts) polar FLIRT), and 7 (myosin-II(ts) equatorial FLIRT) independent experiments.

Supplementary Video 5

Reversibility of FLIRT, equatorial myosin-II(ts) function (16 °C hold): brief FLIRT inactivation of the equatorial region of a dividing myosin-II(ts) one-cell embryo. Contractile ring constriction in myosin-II(ts) initiates rapidly after the FLIRT laser is turned off. 30 s per frame. Red circle, 16 µm FLIRT ROI; FLIRT is initiated during prometaphase and turned off ~6 min after anaphase onset. The video shows t = 0 at metaphase before anaphase onset. Hold temperature, 16 °C. FLIRT laser power, 8.5 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of nine independent experiments.

Supplementary Video 6

Reversibility of FLIRT, equatorial myosin-II(ts) function (14 °C hold): brief FLIRT inactivation of the equatorial region of a dividing myosin-II(ts) one-cell embryo. Contractile ring constriction in myosin-II(ts) initiates rapidly after the FLIRT laser is turned off. 30 s per frame. Red circle, 16 µm FLIRT ROI; FLIRT is initiated during prometaphase and turned off ~6 min after anaphase onset. The video shows t = 0 at metaphase before anaphase onset. Hold temperature, 14 °C. FLIRT laser power, 10.1 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of seven independent experiments.

Supplementary Video 7

In situ FLIRT inhibition of cyk-4(ts) function in the germ line: FLIRT targeting a single germline membrane partition in cyk-4(ts) but not control worms causes shortening of the FLIRT-targeted membrane partition, but not of non-targeted adjacent membrane partitions. 60 s per frame. Red circle, 16 µm FLIRT ROI; FLIRT is initiated at t = 0 and is kept on throughout the experiment. Hold temperature, 16 °C. FLIRT laser power, 8.5 mW. Green, GFP::plasma membrane; magenta, mCherry::histone2B. The video is representative of seven (control) or ten (cyk-4(ts)) independent experiments.

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Hirsch, S.M., Sundaramoorthy, S., Davies, T. et al. FLIRT: fast local infrared thermogenetics for subcellular control of protein function. Nat Methods 15, 921–923 (2018). https://doi.org/10.1038/s41592-018-0168-y

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