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Fig. 4. The high-capacity endocytic process is clathrin-independent and distinct from the SV and endosomal recycling pathways. (A) Basal uptake of FM4-64 (red in the merged image) in a growth cone incubated with the dye for 1 minute, fixed and retrospectively labeled for VAMP2 (blue in the merged image) and syntaxin 13 (green in the merged image). The overlay between the fluorescence and DIC images is shown in the merged image. The bulk of FM4-64 fluorescence colocalizes with neither VAMP2 nor syntaxin 13, although occasional punctae of overlap are observed in more proximal regions (arrowheads). (B) Constitutive endocytosis in growth cones probed by exposure to various tracers (green) for 1 minute in KRH: (a) Lucifer yellow (4 mg/ml), (b) 200-nm beads, (c) 20-nm beads. FM4-64 applied during a subsequent incubation is internalized in bead-containing compartments (right panel in c; red). The overlay between fluorescence and DIC images is shown. (C) Depletion of the clathrin heavy chain (CHC) in mouse hippocampal neurons by RNAi. (Left) Representative images of neurons that were co-transfected with YFP and either control Stealth (upper panels) or CHC Stealth (bottom panels). After basal FM4-64 uptake (red), 3-DIV mouse neurons were fixed and retrospectively immunostained to assess clathrin downregulation (green). (Right) Quantification of the effect of CHC RNAi on bulk plasma-membrane endocytosis. A fivefold reduction of CHC immunoreactivity (green bars; mean ± s.e.m.; n=20 growth cones per treatment) in individual growth cones is observed following RNAi. FM4-64 bulk endocytosis (red bars) is unchanged (*P<0.001, Student's t-test, treated vs control). (D) A neuron exposed to Alexa-Fluor-488-conjugated transferrin (green in the merged image) for 1 hour and subsequently to FM4-64 (red in the merged image) for 1 minute in KRH is shown. Transferrin, which is primarily internalized in the cell body (asterisk), does not overlap with FM4-64, which is endocytosed in the growth cone (arrowhead). (E) Growth cones that were loaded with FM4-64 (KRH, 1 minute; red) and retrospectively stained with anti-Rabankyryn-5 (green in the merged image). Scale bar: 10 µm (A,B); 15 µm (C); 29.5 µm (D); 3.8 µm (E).
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