Figure 7.

Rapid dissociation of multiple scaffolding proteins by disruption of F-actin. A, Time-lapse image sequences of neurons expressing EGFP/EYFP-tagged scaffolding proteins before and after application of latrunculin A. Latrunculin A induced the rapid decrease in fluorescence in GKAP, Shank, and PSD-Zip45 clusters. PSD-95 clusters did not change their fluorescence after latrunculin A treatment. Scale bar, 5 μm. B, Time course of relative fluorescence change in scaffolding protein clusters after latrunculin A application. C, Response of endogenous scaffolding proteins to latrunculin A administration. Neurons were fixed 10 min after application of latrunculin A, and total fluorescence intensity of clusters was measured after immunostaining of scaffolding proteins. A significant decrease in immunoreactivity for GKAP, Shank, and PSD-Zip45 was observed (∗∗∗p < 0.001). D, Loss of fluorescence intensity after latrunculin A administration in neurons pretreated with 2-bromopalmitate (2-Br-Pal). Previous application of 2-bromopalmitate did not enhance dissociation of EYFP-GKAP and EGFP-Shank molecules. E, Interaction of PSD-95, GKAP, and Shank after acute disruption of F-actin by latrunculin A (Lat). Cells were extracted with radioimmunoprecipitation assay buffer and immunoprecipitated (i.p.) with either anti-GKAP or anti-Shank antibody. Reduced interactions of Shank with both PSD-95 and GKAP were observed. In contrast, immunoprecipitation with anti-GKAP antibody revealed unaltered interaction of GKAP and PSD-95. F, Immunoprecipitation of EGFP-Shank by using anti-GFP antibody. After treatment with latrunculin A, cells were extracted with NP-40 buffer and immunoprecipitated. Latrunculin A treatment reduced the interaction between EGFP-Shank and Homer. Error bars indicate SEM.