Fig. 2. Microscopic analyses of OV-treated AK13-1 cells. (A-D) Inverse fluorescence micrographs of AK13-1 cells that were treated with 5 mM OV for 2 minutes (A,B), 10 mM OV for 6 minutes (C) or not at all (D) demonstrate details of the distribution of the CK13-EGFP chimera HK13-EGFP. The overview in (A) shows multiple granular thickenings along filamentous structures and granules that are in close apposition to residual filaments. B and C present further details of granules, which are either part of filaments, in the near vicinity of filaments, or without apparent connection. C depicts, in addition, variously shaped CK formations ranging from spheroidal bodies to elongated rod-like structures. Note also the perseverance of desmosome-anchored filaments (arrowheads in B), whereas granulation has proceeded considerably in other nearby CKFs. Bars, 2 µm. (E-H) Electron microscopy (E,F) and immunoelectron microscopy (G,H) of AK 13-1 cells depicting different stages of CKF network breakdown during OV treatment (2 mM for 3 minutes in E,G,H; 10 mM for 2 minutes in F). Note the abundant dense filament bundles with local densities (+) and the multiple amorphous granules (^*) that are often in association with residual filaments (E, inset in E,G) but become separated during later stages (F). Normal-appearing microtubules are present while filament aggregation is already in progress (arrowheads in E). For immunoelectron microscopy, antibodies against EGFP were used in combination with 1 nm gold-labeled secondary antibodies and the silver amplification technique. The label is primarily detectable on the surface of dense filament bundles and aggregates, whereas comparatively little labeling is seen within these structures. Bars, 200 nm.