Extended Data Fig. 4: Imaging-based screening and validation of screening results.
From: Clathrin-associated carriers enable recycling through a kiss-and-run mechanism
a, The workflow of live-cell TIRF imaging-based screening of proteins related to AP2-negative sensor-positive fusion events. AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with constructs expressing different proteins tagged with mScarlet-I, and then imaged by TIRF microscopy at the bottom surface every 1 s for 180 s. Each time series was first subjected to multi-channel detection and tracking with the EGFP-sensor channel as the 'master' channel to identify the sensor-positive AP2-negative tracks. Each time series was then subjected to multi-channel detection and tracking with the mScarlet-I channel as the 'master' channel. The AP2-negative sensor-positive tracks with significant overlap with the mScarlet-I tracks were classified as positive for the mScarlet-I-tagged protein. The fraction of AP2-negative sensor-positive tracks with positive signals from the mScarlet-I channel was calculated for each protein. The fluorescence intensity distribution heatmap reflecting the relative timing of recruitment of each protein to the EGFP-sensor was plotted. b, For each indicated mScarlet-1-tagged protein, the kymograph from a representative time series is shown. AP2-negative sensor-positive fusion events that recruited the indicated proteins are highlighted by arrows. c,d, For each indicated mScarlet-1-tagged protein, the kymograph from a representative time series is shown. AP2-negative sensor-positive fusion events lacking the recruitment of the indicated proteins are highlighted by arrows (c). The fraction of AP2-negative sensor-positive fusion events exhibiting significant signals from the indicated mScarlet-I-tagged protein is shown in (d) (mean ± 95% CI; n = 8-12 cells). The 95th percentile of the calculated fraction of related events from 133 cells (pooled from the twelve proteins) is 0.18. In this study, the mScarlet-I-tagged protein was considered to be associated with CARP carriers if the averaged fraction of related events exceeds 0.20 (dotted line). e, AP2-670nano+/+ cells stably expressing the EGFP-sensor were genome-edited again to express HIP1R-mScarlet-I+/+, mScarlet-I-SNX9+/+, mScarlet-I-EHD1 (pool), mScarlet-I-Rab7a (pool), LAMP1-mScarlet-I (pool), or flotillin-1-mScarlet-I (pool), and then imaged by TIRF microscopy at the bottom surface every 1 s for 180 s. From left to right: the T-projection and kymographs of a time series; the fraction of sensor-positive events recruited the indicated proteins (mean ± 95% CI); and the intensity cohorts (mean ± s.e.m.; n = 10, 16, 14, 12, 12, and 12 cells). Experiments were repeated three times with similar results. Cells in d and e were from one experiment. Scale bars, 1 μm in kymographs (b, c, and e); 10 μm and 1 μm (magnification) in T-projections (e). Source numerical data are available in source data.
