Fig. 6

Mt3 deletion likely causes an abnormal distribution of clathrin and PICALM through actin disruption. a, b Confocal fluorescence micrographs of WT and Mt3 ^−/− astrocytes immunostained with an anti-PICALM antibody (A) or an anti-clathrin antibody (B). Cells from WT and Mt3 ^−/− mice were treated for 1 h with vehicle only (CTL), 100 nM CytD, 1 μM LatB or 10 μg/ml of N-terminal Mt3 peptide, and then immunostained. The plasma membrane was stained with Alexa Fluor 594-WGA, and the nuclei were identified by counterstaining with Hoechst 33342 (blue). Mt3 ^−/− cells recapitulated the CytD- and LatB-triggered alterations in the size and localization of the PICALM- and clathrin-positive signals. But, the Mt3 peptide treatment (10 μg/ml) restored just in part to the WT condition. The corresponding 3D images are shown in the lower panel. Scale bar, 20 μm. c, d Bars indicate the relative changes in the size of the PICALM (C)- and clathrin (D)-positive signals in astrocytes from WT and Mt3 ^−/− mice treated with vehicle only (CTL), 100 nM CytD, 1 μM LatB, or 10 μg/ml of N-terminal Mt3 peptide. Mt3 ^−/− and drug-treated WT cells showed more wide-necked, strong signals compared with those in WT controls, but Mt3 peptide-treated Mt3 ^−/− cells still exhibited somewhat broad and intense signal (***P < 0.001, **P < 0.01, *P < 0.05 vs. WT CTL; n = 5 experiments). e Western blotting for PICALM and clathrin. WT and Mt3 ^−/− cells were treated with vehicle, 100 nM CytD or 1 μM LatB for 1 h and immunoblotted with anti-PICALM and anti-clathrin antibodies. Bars show the density ratio of PICALM and clathrin relative to tubulin. All ratio values were normalized to the ratio in WT controls (Mt3 ^+/+ CTL), defined as 1 (***P < 0.001, **P < 0.01, *P < 0.05 vs. WT CTL; n = 5 experiments)