Fig. 2: Clathrin at Ω-profile’s base/pore region constricts the pore.

a Left: clathrin surrounds pre-Ω’s visible pore: STED XZ-plane images of PH_G and CLC-mTFP1 for a pre-Ω with a visible pore (pre-Ω_vp) along Y-axis every 100 nm as labelled. Right: XY-plane images at two Z-planes (z1, z2) across the pore region. b STED XY-plane images of PH_G and SNAP-CLC-SiR (see Materials and methods) showing different sizes of pre-Ω’s pore surrounded by clathrin (left: pore visible; middle and right: pore too small to resolve). c Clathrin at pre-Ω’s non-visible pore and base region: STED XZ-plane images of PH_G, CLC-mTFP1 and A532 for a pre-Ω with a non-visible pore (pre-Ω_nvp, permeable to A532). d CLC-mTFP1 puncta flanked and moved with the constricting pore of a pre-Ω_vp: F_PH, F₅₃₂, CLC-mTFP1 fluorescence (F_CLC), Ω_p’s pore diameter (d_p), the distance between two CLC-mTFP1 puncta flanking the pore (d_CLC), and sampled STED XZ-plane images of PH_G/CLC-mTFP1/A532 taken at times indicated sticks. Gray circles: < 60 nm (STED resolution); triangle: depol_1s. e CLC-mTFP1 puncta at the base/pore region of a pre-Ω_nvp: F_PH, F₅₃₂, F_CLC and sampled STED XZ-plane images of PH_G/CLC-mTFP1/A532. F₅₃₂ decay (A532 was strongly excited) reflected pre-Ω pore closure. Triangle: depol_1s. f STED XZ-plane images of PH_G, FFN206, and CLC-mTFP1 (merge at the bottom) showing CLC-mTFP1 puncta at vesicle docking sites (triangles, contact between FFN511-labelled vesicles and the PH_G-labelled PM). PM and cytosol (Cyto) locations are labelled. g F_PH, F₅₃₂, F_CLC (F) and sampled XZ/Y_fix images of PH_G/A532/CLC-mTFP1 showing that PH_G/A532-labelled fs-Ω co-localized with pre-existing CLC-mTFP1 puncta at fs-Ω’s pore region (8 out of 8 fs-Ω). h Confocal images of PH-mScarlet showing the visible (I) or non-visible pore (II–IV) of 4 Ω-profiles at the XY-plane (upper) and 2D MINFLUX images of CLC-SNAP-A647 at the same XY-plane region (middle, upper and middle images merged at the bottom).