Extended Data Fig. 6: Regulation of CARP by Rab11 and Rab1 but not Rab4.
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 RAB11A to generate the clonal double-edited cell line AP2-670nano+/+ mScarlet-I-Rab11a+/+. Right: Western blot analysis of cell lysates probed with antibodies for Rab11a and α-actinin. b, Left: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of RAB11B to generate the clonal double-edited cell line AP2-670nano+/+ mScarlet-I-Rab11b+/+. Right: Western blot analysis of cell lysates probed with antibodies for Rab11b and α-actinin. c, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with mScarlet-I-SH3BP5L (left) or mScarlet-I-Rab11FIP2 (right), and then imaged by TIRF microscopy. Montage and kymograph showing EGFP-sensor recruitment to the SH3BP5L- or Rab11FIP2-positive carrier. d, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with either control siRNA or siRNA targeting Rab11a, Rab11b, or both Rab11a and Rab11b (Rab11a/Rab11b-KD1). Elimination of Rab11a and/or Rab11b expression was confirmed by western blot analysis using antibodies against Rab11a, Rab11b, and α-actinin. e, AP2-670nano+/+ mScarlet-I-Rab11b+/+ cells stably expressing EGFP-sensor were subjected to knockout of Rab11b, and then treated with control siRNA (Rab11b-KO) or two different siRNAs targeting Rab11a. Knockout of Rab11b and knockdown of Rab11a was confirmed by western blot analysis using antibodies against Rab11a, Rab11b, and α-actinin. f, Left: Western blot analysis of SUM159 cells without or with EGFP-Rab4b transfection, probed with antibodies for Rab4b and α-actinin. The expression of endogenous Rab4b in SUM159 cells is negligible. Right: AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with either control siRNA or siRNA targeting Rab4a, Rab4b, or both Rab4a and Rab4b. Elimination of Rab4a expression was confirmed by western blot analysis using antibodies against Rab4a and α-actinin. g, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with either control siRNA or siRNA targeting Rab4a, Rab4b, or both Rab4a and Rab4b, and then imaged by TIRF microscopy. The frequency of AP2-negative sensor-positive fusion events from siRNA-treated cells is shown (mean ± 95% CI; n = 14, 14, 14, and 15 cells). h, Left: Genomic PCR analysis showing single allelic integration of mScarlet-I into the genomic locus of RAB1A to generate the clonal double-edited cell line AP2-670nano+/+ mScarlet-I-Rab1a+/-. Right: Western blot analysis of cell lysates probed with antibodies for RFP and α-actinin. i, Left: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of RAB1B to generate the clonal double-edited cell line AP2-670nano+/+ mScarlet-I-Rab1b+/+. Right: Western blot analysis of cell lysates probed with antibodies for RFP and α-actinin. j, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with either control siRNA or siRNA targeting Rab1a, Rab1b, or both Rab1a and Rab1b (Rab1a/Rab1b-KD1). The knockdown efficiency was measured by real-time quantitative PCR (qPCR; mean ± s.d.; n = 5 independent experiments). The siRNA-treated cells were imaged at 1-s intervals by TIRF microscopy, and the imaging results are shown in Fig. 4m. k, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with either control siRNA or a second set of siRNAs targeting Rab1a, Rab1b, or both Rab1a and Rab1b (Rab1a/Rab1b-KD2). Left: Knockdown efficiency measured by qPCR (mean ± s.d.; n = 3 independent experiments). Right: siRNA-treated cells were imaged by TIRF microscopy. Plots showing the frequency of fusion events (mean ± 95% CI; n = 19, 18, 17, and 18 cells). l, mScarlet-I-Rab1b+/+ or mScarlet-I-Rab1a+/- cells were transiently transfected with the indicated proteins and then imaged by spinning-disk confocal microscopy. Intracellular distribution and colocalization of Rab1b or Rab1a with the indicated proteins are shown. m, EGFP-Rab11a+/+ cells were treated with control siRNA or siRNA targeting Rab1a, Rab1b, or both Rab1a and Rab1b, and then imaged by TIRF microscopy. The first frame and T-projection of EGFP-Rab11a from a time series in each treatment are shown. n, AP2-670nano+/+ mScarlet-I-Rab11a+/+cells stably expressing EGFP-sensor were treated with control siRNA or siRNA targeting both Rab1a and Rab1b, and then imaged in 3D using spinning-disk confocal microscopy (15 imaging planes spaced at 0.35 μm). Simultaneous knockdown of Rab1a and Rab1b expression caused the loss of mScarlet-I-Rab11a-positive vesicles around the bottom surface of the cell, while only having a minor effect on the mScarlet-I-Rab11a-positive vesicles near the perinuclear region. Experiments were repeated three (a-i and k-n) or five times (j) with similar results. Cells in g and k (right) were from one experiment. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test in g and k. Scale bars, 1 μm in c; 10 μm and 1 μm (magnification) in l; 10 μm in m and n. Source numerical data and unprocessed blots are available in source data.
