Extended Data Fig. 8: Dynamin catalysers the scission of CARP carriers from the plasma membrane.
From: Clathrin-associated carriers enable recycling through a kiss-and-run mechanism
a, Cells stably expressing EGFP-sensor were transiently transfected with dynamin2-670nano (Dyn2-670nano) and mScarlet-I-Sec8, and then imaged at 1-s intervals by TIRF microscopy. Montage and kymographs showing the recruitment of dynamin2 during the late stage of sensor recruitment in a Sec8-positive fusion event (arrows). b, Left: Genomic PCR analysis showing the integration of mScarlet-I into the genomic locus of DNM2 to generate a pool of gene-edited cells expressing AP2-670nano+/+ Dyn2-mScarlet-Ien. Right: Western blot analysis of cell lysates probed with antibodies for RFP and GAPDH. c, U2OS cells stably expressing EGFP-sensor were transiently transfected with Dyn2-mScarlet-I and AP2-670nano, and then imaged at 1-s intervals by TIRF microscopy. Representative montage, kymographs, and relative fluorescence intensity traces with estimated uncertainties (s.d.) show the recruitment of dynamin2 during the late stage of sensor recruitment to an AP2-negative carrier (arrows). d, COS7 cells stably expressing EGFP-sensor and AP2-670nano were transiently transfected with Dyn2-mScarlet-I, and then imaged and displayed as in (c). e, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with endophilin-A2-mScarlet-I (EndoA2-mScarlet-I), and then imaged and displayed as in (c). f, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with control siRNA or two different siRNAs targeting dynamin2 (KD1 and KD2). The depletion efficiency of dynamin2 mRNA by siRNA was measured by qPCR (mean ± s.d.; n = 3 independent experiments). g, Left: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of ARPC3 to generate the clonal double-edited cell line AP2-670nano+/+ ARPC3-mScarlet-I+/+. Right: Western blot analysis of cell lysates probed with antibodies for RFP and α-actinin. h, Left: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of DBNL to generate the clonal double-edited cell line AP2-670nano+/+ mScarlet-I-mAbp1+/+. Right: Western blot analysis of cell lysates probed with antibodies for RFP and GAPDH. i, AP2-670nano+/+ mScarlet-I-mAbp1+/+ cells stably expressing EGFP-sensor were imaged at 1-s intervals by TIRF microscopy. Left: T-projections of a time series. Middle: The montage and kymographs show that mAbp1 is recruited during the late stage of sensor recruitment to an AP2-negative carrier and remains associated with the carrier after the loss of sensor signal (arrows). Right: The fraction of fusion events recruited mAbp1 (mean ± 95% CI; n = 10 cells from one experiment). Experiments were repeated three times with similar results. Scale bars, 1 μm in kymographs and montages (a, c-e, and i); 10 μm and 1 μm (magnification) in T-projection (i). Source numerical data and unprocessed blots are available in source data.
