Extended Data Fig. 7: Dynamics and functions of Arf1 in CARP carrier formation.
From: Clathrin-associated carriers enable recycling through a kiss-and-run mechanism
a, Left: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of ARF1 to generate the clonal double-edited cell line AP2-670nano+/+ Arf1-mScarlet-I+/+. Right: Western blot analysis of cell lysates probed with antibodies for Arf1 and α-actinin. b, Left: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of ARF3 to generate the clonal double-edited cell line AP2-670nano+/+ Arf3-mScarlet-I+/+. Right: Western blot analysis of cell lysates probed with antibodies for RFP and α-actinin. c, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with control siRNA or siRNA targeting Arf1, Arf3, Arf4, both Arf1 and Arf3 (Arf1/Arf3-KD), or both Arf1 and Arf4 (Arf1/Arf4-KD). The knockdown efficiency was measured by qPCR (mean ± s.d.; n = 6 independent experiments). d, Arf1 expression was knocked out (Arf1-KO) in AP2-670nano+/+ Arf1-mScarlet-I+/+ cells stably expressing EGFP-sensor. The cells were transiently transfected with mScarlet-I-tagged wild-type Arf1, constitutively active Arf1(Q71L), or dominant negative Arf1(T31N), and then imaged at 1-s intervals by TIRF microscopy. Plots showing the frequency of AP2-negative sensor-positive fusion events (mean ± 95% CI; n = 19, 14, 19, 20, and 20 cells). e, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with control siRNA or siRNA targeting either BIG1, BIG2, or GBF1. The knockdown efficiency was measured by qPCR (mean ± s.d.; n = 3 independent experiments). The siRNA-treated cells were imaged at 1-s intervals by TIRF microscopy, and the imaging results are shown in Fig. 5e. f, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with control siRNA or a second set of siRNAs targeting either BIG1, BIG2, or GBF1. Left: Knockdown efficiency measured by qPCR (mean ± s.d.; n = 3 independent experiments). Right: siRNA-treated cells were imaged at 1-s intervals by TIRF microscopy. Plots showing the frequency of fusion events (mean ± 95% CI; n = 12, 12, 12, and 12 cells). g,h, Genome-edited Arf1-mEGFP cells (pool) were transiently transfected with mScarlet-I-Rab11b (g) or mScarlet-I-Rab1b (h), and then imaged by spinning-disk confocal microscopy at the plane near the bottom surface. i, CLTA-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with Arf1-mScarlet-I, and then imaged at 0.3-s intervals by TIRF microscopy. Montage showing the dynamic association of clathrin at the rear part of a highly motile Arf1-labelled tubule before its fusion and the recruitment of the sensor at the plasma membrane (arrows). j, Genome-edited Arf1-mEGFP cells (pool) were transiently transfected with CLTA-mScarlet-I, and then imaged by SIM. Examples illustrate the distribution of clathrin at subregions of tubular Arf1 carriers. k, AP2-670nano+/+ cells stably expressing EGFP-sensor and genome-edited for mScarlet-I-Rab1a+/-, mScarlet-I-Rab1b+/+, mScarlet-I-Rab11a+/+, mScarlet-I-Rab11b+/+ or AP1σ1-mScarlet-I+/+ were treated with control siRNA or siRNA targeting Arf1, then imaged by spinning-disk confocal microscopy. Experiments were repeated six (c) or three times (a-b and d-k) with similar results. Cells in d and f (right) were from one experiment. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. Scale bars, 10 μm and 1 μm (magnification) in g and h; 1 μm in i; 0.5 μm in j; 10 μm in k. Source numerical data and unprocessed blots are available in source data.
