Figure 3. BcLOV4 membrane translocation dynamics depend on temperature and light exposure.

A) BcLOV-mCh membrane recruitment was quantified at various temperatures and light exposures using live cell imaging. B) Representative images of membrane recruitment at low and high temperatures. Activation at 25 °C permitted sustained membrane recruitment while recruitment at 37 °C was transient (stimulation performed at 1.45 W/cm² and 3% duty cycle). Image brightness was adjusted at each time point for clarity to account for photobleaching. C) Quantification of membrane recruitment at various temperatures (1.45 W/cm² at 3% duty cycle) reveals a temperature-dependent decay of membrane translocation. D) Quantification of membrane recruitment at various light exposures (at 36 °C and 1.45 W/cm²) shows light-dependent decay of BcLOV-mCh translocation. Each trace is the mean membrane fluorescence +/− SEM of three biologically independent samples, with each replicate representing the mean of ~100 cells. See Supplemental Figure 10 for unnormalized traces and quantification workflow. E) Schematic of a 3-state model of BcLOV4 membrane translocation. F) Fitting the model to live-cell translocation data provides parameter values for k₁, k₂, and k₃(T). G) Heat map depicts the decay rate of BcLOV4 membrane localization as a function of temperature and light exposure. Decay rates were calculated by simulating sustained illumination over a range of duty cycles and temperatures and fitting the modeled decay rate to a single exponential decay. Color indicates the decay constant λ (1 divided by the time to reach 37% of maximum signal). Larger λ indicates faster decay. See Supplementary Figure 10, 13, and Methods for imaging and model details.