Extended Data Fig. 5: Dynamic association of proteins with the AP2-positive or negative carriers at the plasma membrane.
From: Clathrin-associated carriers enable recycling through a kiss-and-run mechanism
a, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with various proteins tagged with mScarlet-I and then imaged by TIRF microscopy as described in Fig. 3a. The EGFP-sensor and AP2-670nano dual-positive tracks were analysed for the dynamic association of mScarlet-I-tagged proteins. The fraction of events containing significant signals from the mScarlet-I-tagged protein was plotted for each protein (mean ± 95% CI; n = 7-15 cells from one representative experiment, with the exact numbers of cells shown in the source data). b, Genome-edited cells expressing AP2-670nano+/+ and CLTA-mEGFP (pool) were transiently transfected with the indicated mScarlet-I-tagged proteins, and then imaged by TIRF microscopy at the bottom surface every 1 s for 180 s. Representative montages and kymographs show the dynamic association of Sec3, Rab11a, Arf1, AP1, and dynamin2 (Dyn2) with the AP2-negative clathrin-associated carriers during their approach or stalling at the plasma membrane. mScarlet-I-tagged flotillin-1, Rab7a, Sec31A, and Sec23A are not recruited to the AP2-negative clathrin-associated carriers. Experiments were repeated twice (a) or three times (b) with similar results. Scale bars, 1 μm. Source numerical data are available in source data.
