Fig. 1: Clathrin is essential for pore closure of preformed Ω-profiles and fusion-generated Ω-profiles.
From: Clathrin mediates membrane fission and budding by constricting membrane pores
a Left: setup drawing. The cell membrane, bath and vesicles are labelled with PHG (green), A655 (red) and FFN511 (cyan), respectively. ICa and capacitance (Cm) are recorded via a whole-cell pipette. Right: sampled ICa and Cm induced by depol1s. b Confocal images of PHG, A655, and FFN511 (near cell-bottom) showing pre-spot I, II, and III at the XY- (left, ring-shape) and XZ-plane (right, Ω-shape). c PHG fluorescence (FPH), A655 fluorescence (F655), FFN511 fluorescence (FFFN), Rfs+pre, and sampled confocal images showing depol1s-induced (triangle) rapid (left), slow (middle) or large-size (right) pre-spot pore closure (pre-close). A reconstructed trace reflecting pre-close (Rfs+pre, a down-step at F655 dimming onset) is also plotted. FPH, F655 and FFFN were normalized to the baseline. d, e FPH, F655, FFFN, Rfs+pre and confocal images showing rapid and slow close-fusion (d), stay- or shrink-fusion (e). Rfs+pre: a reconstructed trace reflecting fusion (up-step, at F655 rising onset) and fusion pore closure (down-step, at F655 dimming onset). f Left: sampled western blot results of CHC, dynamin 1/2 (Dyn), adaptor protein 2 α subunit (AP2), synaptotagmin 1 (Syt1), syntaxin 1 (Stx1), and actin in chromaffin cell cultures transfected with si-Ctrl or si-CHC. Right: the percentage (mean ± s.e.m., 3 transfections) of the above proteins in cultures transfected with si-CHC (data normalized to the corresponding mean of si-Ctrl). g–i The percentage of pre-spots undergoing pore closure (pre-close%, g), the percentage of close-fusion in all fusion events (close-fusion%, h), and FFN511 20%–80% decay time (i) in: (1) si-Ctrl (19 cells), si-CHC (21 cells), or si-CHC+CHC (si-CHC transfection plus CHC overexpression, 17 cells); (2) sh-Ctrl (16 cells) or sh-CHC (16 cells, FFN511 not included); and (3) Ctrl (16 cells, no pitstop 2) or pitstop 2 (PST2, 30 μM, bath, 15–30 min; 15 cells). ***P < 0.001 (t-test). j Sampled FFN511 spot fluorescence (FFFN) decay in si-Ctrl (dotted) or si-CHC (solid) showing faster release after clathrin knockdown.
