Figure 1. Light-triggered dissociation... | Rockefeller University Press

Figure 1.

$Figure 1. Light-triggered dissociation of UVR8-tagged proteins. (A) Schematic of light-triggered dissociation experiment. UVR8 fused to prenylated GFP (UVR8-memGFP) localizes to the plasma membrane where it recruits UVR8-mCh. Dissociation of the UVR8 dimer releases UVR8-mCh to the cytosol. (B) A HEK293T cell expressing UVR8-memGFP along with UVR8-mCh. Note the strong plasma membrane association of both GFP and mCh signals before UV-B illumination. mCh signal is mostly cytosolic 15 s after a brief (3 s) exposure to UV-B. Bar, 5 µm. (C) Time course of UVR8-mCh dissociation from the plasma membrane. Left-most panel shows the UVR8-memGFP localization at the plasma membrane. After UV-B treatment, the mCh signal rapidly dissociates from the plasma membrane. Bar, 1.5 µm. The kymograph shows the complete time course of translocation after a 3-s UV-B treatment (purple bar). Bar, 1 µm. (D) Cytosolic mCh signal was quantified and fit with a single exponential equation. The average time constant (τ) for UVR8 dissociation was 1.9 ± 0.2 s (n = 4 cells from 2 independent experiments). (E) Cytosolic mCh signal was quantified after varying durations of UV-B (arrow 1), followed by a saturating (10 s) UV-B exposure (arrow 2) to determine the fraction of UV-B remaining on the plasma membrane. Shown is representative data from single trials (with each exposure duration performed on a different cell on a fresh coverslip). (F) Average results of the light titration experiment shown in E. At least four different cells were quantified for each condition from four separate experiments. Error bars represent standard deviations of the mean.$

Light-triggered dissociation of UVR8-tagged proteins. (A) Schematic of light-triggered dissociation experiment. UVR8 fused to prenylated GFP (UVR8-memGFP) localizes to the plasma membrane where it recruits UVR8-mCh. Dissociation of the UVR8 dimer releases UVR8-mCh to the cytosol. (B) A HEK293T cell expressing UVR8-memGFP along with UVR8-mCh. Note the strong plasma membrane association of both GFP and mCh signals before UV-B illumination. mCh signal is mostly cytosolic 15 s after a brief (3 s) exposure to UV-B. Bar, 5 µm. (C) Time course of UVR8-mCh dissociation from the plasma membrane. Left-most panel shows the UVR8-memGFP localization at the plasma membrane. After UV-B treatment, the mCh signal rapidly dissociates from the plasma membrane. Bar, 1.5 µm. The kymograph shows the complete time course of translocation after a 3-s UV-B treatment (purple bar). Bar, 1 µm. (D) Cytosolic mCh signal was quantified and fit with a single exponential equation. The average time constant (τ) for UVR8 dissociation was 1.9 ± 0.2 s (n = 4 cells from 2 independent experiments). (E) Cytosolic mCh signal was quantified after varying durations of UV-B (arrow 1), followed by a saturating (10 s) UV-B exposure (arrow 2) to determine the fraction of UV-B remaining on the plasma membrane. Shown is representative data from single trials (with each exposure duration performed on a different cell on a fresh coverslip). (F) Average results of the light titration experiment shown in E. At least four different cells were quantified for each condition from four separate experiments. Error bars represent standard deviations of the mean.

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