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Fig. 1. Constitutive bulk membrane retrieval in axonal growth cones. (A) Basal uptake of FM4-64 (red in a' and b') in neurons at 2 DIV that were incubated with KRH containing the dye and imaged after a 2-minute washing. (a,a') A broad region of intense FM4-64 staining is associated with the growth cone (arrowhead), whereas few dispersed punctae appear in the cell body (asterisk). Note the lack of FM4-64 internalization along the axon shaft. (b,b') Higher-magnification views of a growth cone, showing internalization of FM4-64 in large organelles at the interface between the central and peripheral domains (T domain). Note the vacuolar organization of the FM4-64-positive compartment in b. (B) Basal uptake of FM4-64 (red) in neurons incubated with the dye for 1 minute, fixed and retrospectively stained for the axon-specific marker dephospho-Tau-1 (green). Intense FM4-64 uptake is visible in growth cones of Tau-1-positive axons (asterisks) but not in Tau-1-negative perspective dendrites (arrowheads). (C) Differential uptake of FM4-64 (red) in growth cones that are associated with distinct axonal branches of the same neuron. One of the growth cones (arrowhead) shows strong constitutive endocytosis, whereas the other (asterisk) is inactive. Scale bar: 13.5 µm (Aa,a'); 5 µm (Ab,b'); 25 µm (B); 10 µm (C).

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