Structure of AIS coat after actin filament and MT extraction. (A and B) Fluorescence microscopy of detergent-extracted DIV10 neurons stained with Alexa Fluor 647–phalloidin (F-actin) and antibodies to AnkG and β3 tubulin (MTs). (A) Control neuron. (B) Neuron treated first with cold calcium-containing buffer to depolymerize MTs and then with gelsolin to sever actin filaments. (C–E) PREM of DIV10 neurons treated with gelsolin (C), cold calcium (D), or both (E) and stained with phalloidin immunogold (C and D). The AIS coat maintains integrity in all cases. (C) Low-magnification image of the proximal axon. White outline shows axon boundary. On the bottom right side, the coat spreads away from the axon shaft, probably being stretched by an actin-based protrusion removed by gelsolin treatment. Boxes c1 and c2 are enlarged in corresponding panels. (c1) Dense AIS coat along main axon and beginning of stretched region, where transversely oriented thin fibrils (blue) can be seen. (c2) Expanded coat at the putative branch junction contains numerous fibrils. Immunogold phalloidin staining in both c1 and c2 reveals only short filaments (i.e., individual particles and small gold clusters). (D) Dense coat in the axon shaft after MT depletion. Long, thick filaments are neurofilaments. Actin filament staining is comparable to that in untreated neurons (see Fig. 5 A). (E) Unstained AIS coat after removal of both actin filaments and MTs has fibrillar–globular structure similar to that in untreated neurons. Bars: (A and B) 10 µm; (C) 1 µm; (c1 and c2) 100 nm; (D and E) 200 nm.