Panel A shows fluorescence images of Paxillin enhanced Green Fluorescent Protein with regions 1 and 2, time-lapse fluorescence images at 0, 5, 10, 15, 20, and 25 seconds, and line graphs of PAUF fluorescence intensity (arbitrary units) versus time (seconds) for FA positive and FA negative regions. Panel B shows a Western blot of ELKS and alpha tubulin in Control small interfering RNA and ELKS small interfering RNA cells. Panel C shows fluorescence images of PAUF MycHis in Control small interfering RNA and ELKS small interfering RNA cells. Panel D shows fluorescence images of PAUF MycHis, Paxillin, and merged images in Control small interfering RNA and ELKS small interfering RNA cells, with enlarged views of selected regions. Panel E shows a treatment timeline at 37 degrees Celsius with D/D plus CHX and fixation after delta time, followed by fluorescence images of Paxillin enhanced Green Fluorescent Protein and mKate2 FM4 PAUF at delta time equals 20 minutes and delta time equals 40 minutes. Panel F shows heat maps with histograms of Intensity (arbitrary units) versus Nearest neighbor distance (micrometers) at delta time equals 20 minutes and delta time equals 40 minutes.
CARTS are delivered close to FAs. (A) TIRF image of a live HeLa cell expressing paxillin-eGFP (green) and mKate2-FM4-PAUF (magenta). White squares highlight (i) FA-enriched (FA+) and (ii) FA-non-enriched (FA-) regions within the PM of the cell. Zoom-ins of the highlighted regions show a frame time sequence of the two merged channels. White circles highlight the vesicle disappearance along the frame sequence (i), and a random position of equal size in the FA region of the cell. Plots on the right correspond to the fluorescence intensity profiles of mKate2-FM4-PAUF signal measured within the highlighted boxed regions show in the respective zoom-ins. Scale bars are 10 µm (main image) and 1 µm (zoom-ins). (B) Knockdown efficiency of ELKS in HeLa cells stably expressing PAUF-MycHis at 72 h after siRNA transfection. (C) Distribution of CARTS upon control and ELKS knockdown in HeLa cells stably expressing PAUF-MycHis. The cells plated on FN-coated coverslips were fixed and visualized with an anti-Myc monoclonal antibody. Scale bar, 10 μm. (D) Accumulation of CARTS near FAs. The cells plated on FN-coated coverslips were fixed and visualized with an anti-Myc polyclonal antibody and an anti-paxillin monoclonal antibody (clone 349). High magnifications of the boxed areas are shown in the right of each panel. Scale bars, 10 μm. (E) HeLa cell expressing paxillin-eGFP (green) and mKate2-FM4-PAUF (magenta), subjected to an SPI assay (see Materials and methods), fixed at the indicated times after cargo release from the ER, and imaged by confocal fluorescence microscopy. Schematic of the experiment is shown on top. D/D is D/D solubilizer. Scale bar, 10 µm. (F) Quantification of E, showing a 2D histogram (and corresponding 1D projections on each cartesian axis) of mKate2-FM4-PAUF fluorescence intensity per pixel as a function of the signed distance from that pixel to the closest FA area (nearest neighbor distance, see Materials and methods for details), at the indicated time points. Source data are available for this figure: SourceData FS1.