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Fig. 10. Developmental control of endocytic activities in the growth cone. (A) Growth cone (DIC, left) of a neuron at 5 DIV expressing ECFP-VAMP2 (green) that was subjected to two 15-minute-spaced applications of FM4-64 (red) for 1 minute in either KRH (first application, middle) or high K+ (second application, right). The depolarizing step induces FM4-64 loading in VAMP2-positive SVs (arrowheads) with a pattern that is markedly different from that of constitutive endocytic compartments. (B) Neurons at 7 DIV that were incubated with FM4-64 for 1 minute in KRH. At this stage, constitutive FM4-64 uptake is not associated with growth cones (asterisk), whereas it is present in punctate endocytic compartments along the neurite network. (C) Growth cone of a 7-DIV neuron expressing ECFP-VAMP2 (green) incubated with high K+ for 1 minute in the presence of FM4-64 (red). Arrowheads point to VAMP2-positive SVs that internalized the dye upon depolarization. (D) Quantification of basal FM4-64 uptake in growth cones of neurons at 3, 5 and 7 DIV incubated with FM4-64/KRH for 1 minute, rapidly washed and then exposed to high K+ for 1 minute. Bulk endocytosis is suppressed at later developmental stages (mean ± s.e.m.; n=30-40 growth cones per treatment). At all time points, FM4-64 uptake was significantly different (P<0.001, Tukey's test). Scale bar: 7 µm (A,C); 12.5 µm (B, left); 7.5 µm (B, right).
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