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tra12014-sup-0001-VideoS1.movapplication/unknown2795K Video S1: G-clathrin persists after BFA treatment. Same cell imaged before (left panels) and after (right panels) BFA treatment for GFP-clathrin fluorescence. Following BFA, loss of TGN and some immobile clathrin structures are observed, as well as an increase in diffuse, soluble clathrin fluorescence. But rapidly moving G-clathrin persists. Regions in red boxes are enlarged in lower panels. Persistence of the G-clathrin/GGA1 structures is more evident when imaging GGA1, which lacks the background of stationary clathrin structures (see Video S2). Continuous image streams, 1 s in duration and ≈5.3 μm square in upper panels, were captured as described in Figure 1 and are played at real-time (30 Hz).
tra12014-sup-0002-VideoS2.movapplication/unknown3363K Video S2: G-GGA1 persists after BFA treatment. Same cell imaged before (left) and after (right) BFA treatment for YFP-GGA1 fluorescence. Following BFA, loss of TGN and some immobile GGA1 structures are observed, as well as an increase in diffuse, soluble GGA1 fluorescence. But rapidly moving G-GGA1 persists. Continuous image streams, each ≈5.3 μm square and 1 s in duration, were captured as described in Figure 1 and are played at real-time (30 Hz).
tra12014-sup-0003-VideoS3.movapplication/unknown4332K Video S3: Arf6-GTP is incorporated into G-clathrin structures. Video shows a montage of a ≈5.3-µm2 region of cell expressing Arf6(Q67L)-GFP[INT] (upper left) and mCherry-clathrin (upper right), with merge in lower left panel. Blue boxes indicate regions with colocalized G-clathrin and Arf6. Continuous image streams were captured as described in Figure 1.
tra12014-sup-0004-VideoS4.movapplication/unknown4743K Video S4: Arf6-GTP slows clathrin exchange and lengthens apparent lifetime of G-clathrin structures. Left panels show a 1-s continuous image stream of GFP-clathrin fluorescence taken immediately after photoactivation from regions of cells expressing PA-GFP-clathrin alone (upper panels) or PA-GFP-clathrin and Arf6(Q67L) (lower panels): several G-clathrin structures can be visualized in each case. After 40 s another image stream from the same regions was captured (right panels): G-clathrin structures are not detectable in cells expressing only GFP-clathrin but persist in cells expressing Arf6(Q67L), consistent with the half-times for exchange measured by FRAP and providing direct evidence of much longer G-clathrin lifetime. Each panel ≈5.3 μm square.
tra12014-sup-0005-FigureS1.docWord document115K Figure S1: Arf6(Q67L) does not affect clathrin exchange in plasma membrane coated pits. FRAP recovery curves were obtained from COS-1 cells expressing GFP-clathrin alone (open circles) or GFP-clathrin and Arf6(Q67L) (closed circles). For details, see Materials and Methods.

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