Htt requires dynein interacting domain to facilitate the transport of Golgi-derived vesicles. A) HEK cells were treated with scRNA or siRNA-htt prior transfection with pARIS-mCherry-httQ23. Cellular lysates were immunoprecipitated using htt-4C8 or anti-dynein (DIC) antibodies and immunocomplexes were subjected to SDS-PAGE to detect either htt or dynein. Dynein co-precipitates with htt when htt-4C8 antibody is used to pull-down endogenous and exogenous htt (Upper panel). Conversely, immunoprecipitation of dynein (lower panel) pulls down both endogenous and exogenous htt (indicated by arrows, lower mobility band corresponding to pARIS-mCherry-httQ23). The same amount of mouse or rabbit IgG's were used as internal immunoprecipitation controls. SN stands for supernatant; IP denotes immunoprecipitation. B) A deletion mutant lacking the minimal dynein interaction domain, denoted as pARIS-mCherry-httQ23-Δdyn, is unable to bind to endogenous dynein (lane 11). (C) Golgi reassembly was monitored in HeLa cells stably expressing GFP-mannosidase II, silenced for endogenous htt and expressing pARIS-mCherry-httQ23-Δdyn as the only cellular source of htt. Most of the cells expressing pARIS-mCherry-httQ23-Δdyn failed to reassemble the GA after NZ washout, suggesting that htt-dynein interaction is required to transport retrogradely Golgi-derived vesicles. Scale bar 10 μm. (D) Quantification of the Golgi dispersion as the mean volume per Golgi particle (μm3) before and after after NZ washout for the different treatments. Results were obtained from 3 independent experiments in which 192 cells were scored. One way ANOVA followed by Fisher's Post-hoc test: ***p < 0.0001; NS non significant. All comparisons t = 0 vs t = 120; siRNA-htt 0.073 ± 0.008 vs 0.158 ± 0.06; siRNA-htt + pARIS-mCherry-httQ23: 0.222 ± 0.035 vs 3.763 ± 0.712; siRNA-htt + pARIS-mCherry-httQ23-Δdyn: 0.073 ± 0.010 vs 0.244 ± 0.072.
Pardo et al. Molecular Brain 2010 3:17 doi:10.1186/1756-6606-3-17