Figure 6.

ER/Golgi trafficking is inhibited in neurons expressing mhtt. Fluorescence micrographs of cultured primary striatal neurons (E17) from C57BL6 mice (Cnt) (A), 150(+/+) mice (B) and 150(+/−)/Cav1(+/−) mice (C) transfected with a plasmid encoding tsGFP-VSVG. Transfected neurons were allowed to recover for 16 h at 40°C to ensure ER/Golgi retention. The temperature was lowered to 32°C (the permissive temperature), and trafficking of tsGFP-VSVG was observed for the period of 3 h. The tsGFP-VSVG (green) was transported out of the Cnt neurons in large distinctive vesicles at the indicated times (A); tsGFP-VSVG (green) was not transported out of the 150(+/+) neurons, and remained localized near the Golgi-like perinuclear compartment for up to 180 min (B, arrows). Reduction of mhtt–Cav1 interaction in 150(+/−)/Cav1(+/−) neurons restored post-Golgi trafficking to the extent observed in Cnt mice (C). The tsGFP-VSVG is green, cis-Golgi is identified using GM130 antibody (red) and the nuclear staining by Hoechst is blue. Images were taken on LSM510 confocal microscope with ×100 DIC lens (1.4 NA). Scale bar is 5 µm. (D and E) Quantification of cells with perinuclear localization of tsGFP-VSVG at different time at permissive temperature in neurons from Cnt, 150(+/+), 150(+/−) and 150(+/−)/Cav1(+/−) animals. At least 20 cells were taken for every time point. Results are expressed as mean ± SEM. Student's t-test was applied to analyze results represented in (D); one-way ANOVA followed by the Newman–Keuls post hoc test for multiple comparisons was applied to analyze results in (E). *P < 0.01; **P < 0.0001.