Extended Data Fig. 2: Live-cell imaging as well as correlative fluorescence and FIB-SEM imaging of the fusion of clathrin-associated recycling carriers with the plasma membrane.
From: Clathrin-associated carriers enable recycling through a kiss-and-run mechanism
a, Top: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of AP1S1 of AP2-670nano+/+ cells to generate the clonal double-edited cell line AP2-670nano+/+ AP1σ1-mScarlet-I+/+. Bottom: Western blot analysis of cell lysates probed with antibodies for RFP and α-actinin. b, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with AP1μ1A-mScarlet-I, and then imaged by TIRF microscopy at the bottom surface every 1 s for 180 s. Left: T-projection and kymographs of a representative time series. Middle: Montage of the event indicated by the yellow arrow in the kymograph. Right: Intensity cohorts of AP1μ1A (red), sensor (green), and AP2 (blue) of the AP2-negative sensor-positive fusion events, and the fraction of fusion events recruited AP1μ1A (n = 12 cells). c, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with two different sgRNAs to knock out the expression of AP1μ1A (KO1 and KO2). Knockout was validated by western blot analysis using antibodies against the indicated proteins. d-f, Top: Genomic PCR analysis showing the integration of mScarlet3 into the genomic locus of EXOC7, EXOC1, or EXOC2 to generate a pool of gene-edited cells expressing AP2-670nano+/+ Exo70-mScarlet3en (d), AP2-670nano+/+ Sec3-mScarlet3en (e), or AP2-670nano+/+ Sec5-mScarlet3en (f), respectively. Bottom: Knock-in was validated by western blot analysis using antibodies against GAPDH and Exo70, Sec3, or Sec5. g, Genome-edited AP2-670nano+/+ and Sec5-mScarlet3 (pool) cells stably expressing EGFP-sensor were imaged and displayed as in (b). Intensity cohorts from 11 cells. h,i, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with Sec8-mScarlet-I or Sec15-mScarlet-I, and then imaged and displayed as in (b). Intensity cohorts from 12 (h) and 12 (i) cells. j, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with control siRNA or two different siRNAs targeting Exo70 (KD1 and KD2). Elimination of Exo70 expression was confirmed by western blot analysis using antibodies against Exo70 and GAPDH. k, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with control siRNA or two different siRNAs targeting VAMP2 (KD1 and KD2). Elimination of VAMP2 expression was confirmed by western blot analysis using antibodies against VAMP2 and GAPDH. l, FRAP was conducted on CLTA-TagRFP+/+ AP2-Halo+/+ cells stably expressing EGFP-sensor using a confocal laser scanning microscope. Left: The single frame of the cells before photobleaching. The squared region was enlarged and shown on the right. Right: The white circular region on the cell was photobleached and then the cells were imaged again at 3-s intervals for 2 min. Montage of the squared region showing recovery of the fluorescence of CLTA-TagRFP in the CARP carrier (red arrow) and two CCPs (cyan arrows). m, The relative fluorescent intensity traces of CLTA-TagRFP from CARP carriers (n = 28 from 16 cells) and CCPs (n = 28 from 18 cells). n, The workflow of correlative spinning-disk confocal fluorescence imaging with FIB-SEM. AP2-TagRFP+/+ CLTA-Halo+/+ cells stably expressing EGFP-sensor were cultured on a glass-bottom dish with position markers (I), stained with the JF646-HaloTag ligand, fixed, and then imaged by spinning-disk confocal microscopy (II). The samples were post-fixed, stained with 1% aqueous uranyl acetate, dehydrated through a series of graded ethyl alcohols, and then embedded in resin (III). The cell samples were then imaged volumetrically in 3D using FIB-SEM (IV). The collected FIB-SEM slices were automatically aligned to reconstruct the 3D image (V). The fluorescence image and the FIB-SEM image were aligned (VI) and the AP2-negative sensor-positive clathrin-containing structures identified by fluorescence imaging were segmented and reconstructed (VII). See Methods for details. Experiments were repeated three times with similar results. Cells in b-m were from one experiment. Data are shown as mean ± s.e.m. in cohorts in b and g-i; mean ± 95% CI in the fraction of events in b; mean ± s.d. in m. Scale bars, 10 μm (overview) and 1 μm (magnification) in b, g-i, and l; 1 μm in kymographs (b and g-i) and montages (b, g-i, and l). Source numerical data and unprocessed blots are available in source data.
