FIGURE 1:
Microinjected MCD and STARD4 overexpression enhance sterol transport to ERC. (A) DHE transport kinetics to the ERC measured by FRAP. U2OS cells were labeled with DHE and incubated with 10 μg/ml Alexa Fluor 633–Tf for 15 min at 37°C in Medium A/glucose to identify the ERC. Where indicated, MCD and rhodamine-dextran were coinjected immediately before labeling cells with DHE. STARD4-overexpressing cells were transfected with GFP-STARD4 for 48 h before the experiment. An image was taken before photobleaching. DHE in the ERC was photobleached (red circles), and images were taken every 30 s. Cells were maintained at 37°C. Scale bar: 20 μm. (B and C) The fluorescence intensity ratio of the photobleached area (ERC) to the entire cell was calculated for each time point and normalized using “prebleach” (100%) and “t = 0” (0%) images. (B) Measurements for single cells. The number of MCD molecules injected was estimated by quantifying the rhodamine fluorescence (see Materials and Methods). MCD (8 × 109): t1/2 = 0.9 min; injected MCD (8 × 108): t1/2 = 2.6 min; control: t1/2 = 3.2 min. (C) FRAP recovery curves for cells transfected with GFP-STARD4 or depleted for STARD4 by shRNA transfection. Each data point is derived from an average of five experiments (± SE). The lines are single exponential fits.