Fig. 4

Actin disruption in Mt3 ^−/− cells may underlie defects in clathrin-dependent endocytosis. a Confocal fluorescence micrographs of WT and Mt3 ^−/− astrocytes double-stained with Alexa Fluor 633-phalloidin (F-actin; red) and Hoechst 33342 (nuclei; blue). Cells from WT and Mt3 ^−/− mice were treated for 1 h with vehicle only (CTL) or with 100 nM CytD or 1 μM LatB or 10 μg/ml of N-terminal Mt3 peptide. After fixation, the cells were stained. Both CytD and LatB disrupted the actin cytoskeleton structure and polymerization (arrows). Mt3 ^−/− astrocytes (Mt3 ^−/−, CTL) exhibited fragmented actin staining similar to that in CytD- or LatB-treated WT astrocytes, but this effect was reversed in part by treatment with the Mt3 peptide (10 μg/ml). Scale bar, 20 μm. b Confocal fluorescence micrographs of WT astrocytes treated with 20 nM CtxB for 30 min at 37 °C. Cells were pretreated with vehicle, 100 nM CytD or 1 μM LatB for 1 h before and during exposure to CtxB. After fixation, cells were immunostained for the Golgi marker GM130 (red) and counterstained with the nuclear dye Hoechst 33342 (blue). CtxB uptake was disrupted by both CytD and LatB treatment. The right column of photos represents the corresponding 3D images. Scale bar, 20 μm