Genetic code expansion, click chemistry, and light-activated PI3K reveal details of membrane protein trafficking downstream of receptor tyrosine kinases
- University of Washington, Department of Physiology & Biophysics, United States
- Department of Biochemistry and Biophysics, Oregon State University, United States
- Department of Neuroscience, University of Texas at Austin, United States
Figures
Figure 1 with 2 supplements
Using the PhyB/PIF system to activate phosphoinositide 3-kinases (PI3K) with light.
The OptoPI3K system reversibly activates PI3K to generate phosphoinositide 3,4,5-trisphosphate (PI(3,4,5)P3) at the plasma membrane (PM). (A) Diagram of PI3K subunits and domains illustrating the …
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Figure 1—source data 1
Excel data for the time courses (Figure 1D and E).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
PIF-YFP translocates to plasma membrane (PM) in response to 650 nm light and back to the cytosol in response to 750 nm light.
The PhyB-PIF light-inducible interaction is rapid (a few tens of seconds) and fully reversible (within ~30 s) and can be repeated multiple times. (A) Schematic diagram for optogenetic PhyB-PIF …
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Figure 1—figure supplement 1—source data 1
Confocal images (.lsm) generated by Zeiss 710 confocal microscope (Figure 1—figure supplement 1B).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Excel data for the time course (Figure 1—figure supplement 1C).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig1-figsupp1-data2-v1.xlsx
Figure 1—figure supplement 2
Detection of phosphoinositide 3,4,5-trisphosphate (PI(3,4,5)P3) generated by PIF-iSH2-YFP at the plasma membrane (PM) using GRP1-PH-CFP.
(A) Total internal fluorescence (TIRF) measurements of F-11 cells expressing PhyB-mCherry, PIF-iSH2-YFP, and GRP1-PH-CFP (n=9). Upon 650 nm illumination, PIF PIF-iSH2-YFP translocated to the PM …
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Figure 1—figure supplement 2—source data 1
Excel data for the time courses (Figure 1—figure supplement 2A and B).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig1-figsupp2-data1-v1.xlsx
Figure 2 with 3 supplements
Activation of phosphoinositide 3-kinases (PI3K) with light is sufficient to induce trafficking of TRPV1 to the plasma membrane (PM).
Simultaneous total internal fluorescence (TIRF) measurement of phosphoinositide 3,4,5-trisphosphate (PI(3,4,5)P3) (cyan) and TRPV1 (yellow) in the PM in response to either (A) nerve growth factor …
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Figure 2—source data 1
These data are reproduced from Stratiievska et al., 2018 (Figure 2A and B).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig2-data1-v1.xlsx
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Figure 2—source data 2
Excel data for time courses and scatter plots (Figure 2C and D).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig2-data2-v1.xlsx
Figure 2—figure supplement 1
750 nm light fails to cause inter-SH2 (iSH2) dissociation from the plasma membrane (PM) in TRPV1-expressing cells.
(A) Total internal fluorescence (TIRF) measurements of PIF-iSH2-YFP from F-11 and HEK293T/17 cells expressing TRPV1-CFP (n=5–17). The color code and lines/envelopes have the same meaning as in Figure…
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Figure 2—figure supplement 1—source data 1
Excel data for time courses and scatter plots in F-11 and HEK cells.
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
PIF-YFP dissociates from the plasma membrane (PM) in response to 750 nm light even in TRPV1-expressing cells.
TRPV1 does not retain PIF lacking inter-SH2 (iSH2) at the PM of F-11 cells. (A) Normalized total internal fluorescence (TIRF) fluorescence was recorded in F-11 cells transfected with …
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Figure 2—figure supplement 2—source data 1
Excel data for time course and scatter plot.
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig2-figsupp2-data1-v1.xlsx
Figure 2—figure supplement 3
750 nm light succeeds in causing inter-SH2 (iSH2) dissociation from the plasma membrane (PM) in TRPM4-expressing cells.
(A) Total internal fluorescence (TIRF) measurements of PIF-iSH2-YFP from F-11 and HEK293T/17 cells expressing TRPM4-CFP (n=7–17). Inset cartoons depict the model for no retention of iSH2 at the PM …
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Figure 2—figure supplement 3—source data 1
Excel data for time courses and scatter plots in F-11 and HEK cells.
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig2-figsupp3-data1-v1.xlsx
Figure 3 with 4 supplements
Labeling the TRPV1 and InsR with membrane-impermeant sTCO-Cy5.
Confocal imaging illustrates the labeling of membrane proteins incorporating the noncanonical amino acid (ncAA) Tet3-Bu with sTCO-sulfo-Cy5 in HEK293T/17 cells. The membrane-impermeable dye labeled …
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Figure 3—source data 1
Original confocal microscopic images for TRPV1 labeling (Figure 3C).
Figure 3—source data 1_BF_TRPV1.lsm: the images contain GFP (green) and bright field (black-white). Figure 3—source data 1_GFP_Cy5_ TRPV1.lsm: the images contain GFP (green) and Cy5 (red). The last images were shown.
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig3-data1-v1.zip
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Figure 3—source data 2
Original confocal microscopic images for InsR labeling (Figure 3E).
Figure 3—source data 1_BF_InsR.lsm: the images contain GFP (blue) and bright field (black-white). Figure 3—source data 1_GFP_Cy5_InsR.lsm: the images contain GFP (blue) and Cy5 (red). The last images were shown.
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig3-data2-v1.zip
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Figure 3—source data 3
Excel data for scatter plots for TRPV1 and InsR labeling (Figure 3D and F).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig3-data3-v1.xlsx
Figure 3—figure supplement 1
Incorporation of Tet3-Bu into TRPV1 and InsR.
(A) Whole-cell patch clamp recording demonstrates that TRPV1 incorporating Tet3-Bu remains functional. Application of 1 μM capsaicin to HEK-293T/17 cells transfected with either TRPV1-GFP (top) or …
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Figure 3—figure supplement 1—source data 1
Representative whole-cell patch clamp recordings.
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig3-figsupp1-data1-v1.xlsx
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Figure 3—figure supplement 1—source data 2
Whole-cell electrophysiology data from all cells with peak capsaicin-activated currents.
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig3-figsupp1-data2-v1.xlsx
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Figure 3—figure supplement 1—source data 3
Original gel image with Coomassie blue staining, original fluorescent gel image, and labeled gel images.
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig3-figsupp1-data3-v1.zip
Figure 3—figure supplement 2
Activity of InsR-K676TAG measure as its recycling upon insulin treatment.
F-11 cells were transfected with the genetically modified receptor and inspected with a confocal microscope. (A) After labeling with membrane-impermeable sTCO-Cy3B dyes, the receptor was visible in …
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Figure 3—figure supplement 2—source data 1
Original confocal microscopic images for InsR endocytosis before (9th image) and after insulin (22nd image) treatment.
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig3-figsupp2-data1-v1.zip
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Figure 3—figure supplement 2—source data 2
Excel data for scatter plot for InsR endocytosis (Figure 3—figure supplement 2B).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig3-figsupp2-data2-v1.xlsx
Figure 3—figure supplement 3
Labeling of cells with sTCO conjugates of TAMRA, JF 646, fluorescein, and sulfo-Cy5.
HEK293T/17 cells expressing InsR-Tet3-Bu-GFP were labeled with sTCO dyes. For all dyes, shown are (left) confocal images of a representative field of HEK293T/17 cells incubated with the indicated …
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Figure 3—figure supplement 3—source data 1
Original confocal microscopic images for TAMRA (90th image, plus GFP and bright field), JF 646 (72nd image, plus GFP and bright field), fluorescein (126th image, plus GFP and bright field), and Cy5 (115th image, plus GFP and bright field).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig3-figsupp3-data1-v1.zip
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Figure 3—figure supplement 3—source data 2
Excel data for labeling kinetics of sTCO dyes (Figure 3—figure supplement 3A–D).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig3-figsupp3-data2-v1.xlsx
Figure 3—video 1
Activity of InsR-K676TAG measure as its recycling upon insulin treatment.
The movie reflects the same measurements as Figure 3—figure supplement 2A. Images were recorded every 10 s. The time stamp is indicated in the upper right corner and the time of insulin application …
Figure 4
Click chemistry labeling of TRPV1-468Tet3-Bu-GFP with sTCO-Cy5 to measure nerve growth factor (NGF)-induced trafficking of TRPV1 to the plasma membrane (PM).
HEK293T/17 cells expressing TRPV1-Tet3-Bu-GFP and NGF receptor were labeled with extracellular sTCO-sulfo-Cy5 and inspected with confocal microscopy. (A) Experimental protocol. Cells were incubated …
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Figure 4—source data 1
Original confocal microscopic images for TRPV1 trafficking.
F0: 15th image, F1: 32nd image, GFP (green) and Cy5 (red) (Figure 4B and C).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig4-data1-v1.zip
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Figure 4—source data 2
Excel data for scatter plot for TRPV1 trafficking (Figure 4D and E).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig4-data2-v1.xlsx
Figure 5 with 1 supplement
Measuring light-activated phosphoinositide 3-kinases (PI3K)-induced TRPV1 and InsR trafficking to the plasma membrane (PM) using click chemistry.
(A) Illustration of the experimental protocol. HEK293T/17 cells expressing TRPV1-468Tet3-Bu-GFP and InsR-676Tet3-Bu-GFP. (B) Confocal images of sulfo-Cy5 obtained at different stages as depicted in …
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Figure 5—source data 1
Original confocal microscopic images for TRPV1 trafficking (Figure 5B and C).
Figure 5—source data 1_TRPV1_Cy5_750_1.lsm, Figure 5—source data 1_TRPV1_Cy5_750_2.lsm, Figure 5—source data 1_TRPV1_Cy5_650.lsm: the mages contain GFP (green) and Cy5 (red). Figure 5—source data 1_TRPV1_ BF_PhyB.lsm: the mages contain bright field and PhyB (magenta).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig5-data1-v1.zip
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Figure 5—source data 2
Scatter plot for TRPV1 trafficking (Figure 5D and E).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig5-data2-v1.xlsx
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Figure 5—source data 3
Original confocal microscopic images for InsR trafficking (Figure 5F and G).
Figure 5—source data 1_InsR_750_1.lsm, Figure 5—source data 1_InsR_750_2.lsm, Figure 5—source data 1_InsR_650.lsm: the mages contain GFP (green) and Cy5 (red). Figure 5—source data 1_BF_PhyB.lsm: the images contain bright field and PhyB (magenta).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig5-data3-v1.zip
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Figure 5—source data 4
Excel data for scatter plot for InsR trafficking (Figure 5H and I).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig5-data4-v1.xlsx
Figure 5—figure supplement 1
Control experiment for light-activated phosphoinositide 3-kinases (PI3K)-induced TRPV1 and InsR trafficking to the plasma membrane (PM).
The same experiment as Figure 5 without 650 nm illumination. The cells were exposed to 750 nm throughout the recording (n=11 and 10 for TRPV1-Tet3-Bu-GFP and InsR-676Tet3-Bu-GFP, respectively).
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Figure 5—figure supplement 1—source data 1
Original confocal microscopic images for TRPV1 trafficking (Figure 5—figure supplement 1B and C).
Figure 5—figure supplement 1—source data 1_TRPV1_750_1.lsm, Figure 5—figure supplement 1—source data 1_TRPV1_750_2.lsm, Figure 5—figure supplement 1—source data 1_TRPV1_750_3.lsm: the images contain GFP (green) and Cy5 (red). Figure 5—figure supplement 1—source data 1_TRPV1_BF_PhyB.lsm: the images contain bright field and PhyB (magenta).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Excel data for scatter plot for TRPV1 trafficking (Figure 5—figure supplement 1D and E).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig5-figsupp1-data2-v1.xlsx
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Figure 5—figure supplement 1—source data 3
Original confocal microscopic images for InsR trafficking (Figure 5—figure supplement 1F and G).
Figure 5—figure supplement 1—source data 1_InsR_750_1.lsm, Figure 5—figure supplement 1—source data 1_InsR_750_2.lsm, Figure 5—figure supplement 1—source data 1_InsR_750_3.lsm: the images contain GFP (green) and Cy5 (red). Figure 5—figure supplement 1—source data 1_InsR_BF.lsm: the image contains bright field. Figure 5—figure supplement 1—source data 1_InsR_PhyB.lsm: the image contains PhyB (magenta).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig5-figsupp1-data3-v1.zip
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Figure 5—figure supplement 1—source data 4
Excel data for scatter plot for InsR trafficking (Figure 5—figure supplement 1H and I).
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig5-figsupp1-data4-v1.xlsx
Figure 6 with 1 supplement
Synthesis of (A) sTCO-fluorescein and (B) sTCO-sulfo-Cy5.
Schemes illustrating the synthesis pathways for the indicated dyes.
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Figure 6—source data 1
Two NMR data files processed with TopSpin 4.1.4 software.
- https://cdn.elifesciences.org/articles/91012/elife-91012-fig6-data1-v1.zip
Figure 6—figure supplement 1
