Journal: Brain
Article Title: Deficiencies in vesicular transport mediated by TRAPPC4 are associated with severe syndromic intellectual disability
doi: 10.1093/brain/awz374
Figure Lengend Snippet: Impaired Golgi trafficking in fibroblasts from an affected subject is rescued by lentiviral transduction of wild-type TRAPPC4. Control fibroblasts and fibroblasts derived from Subject 1:II-1 were either untransduced (Untx) or stably transduced with wild-type TRAPPC4 lentivirus (TRAPP). Cells were then transfected with a temperature-sensitive vesicular stomatitis virus glycoprotein VSVG–GFP ts045, then incubated at the non-permissive temperature of 40°C overnight. Cells were then shifted to the permissive temperature of 32°C for the time intervals indicated, fixed with PFA then immunostained with the Golgi marker GM130 and counterstained with the nuclear marker DAPI. VSVG-GFP ts045 co-localization with GM130 was determined for a minimum of 60 cells per time point and treatment. (A) There was no difference in trafficking in naïve control cells or those transduced with TRAPPC4 lentivirus. However, in fibroblasts from the affected subject there was a significant delay in VSVG-GFP ts045 entering the Golgi, and a significant delay in exiting the Golgi compared to control cells. Golgi trafficking in fibroblasts from the affected subject was restored back to levels of control upon transduction with wild-type TRAPPC4 lentivirus. (B) Representative confocal images of intracellular trafficking (blue, DAPI; green, VSVG-GFP ts045; red, GM130). More representative figures can be found in Supplementary Figs 1 and 2. Data are average ± SD, n > 60 cells per treatment and time point. Significance was measured by a two-way ANOVA with Sidak multiple comparisons correction. ***P < 0.001, ****P < 0.0001. Scale bar = 20 µm.
Article Snippet: The trafficking dynamics we observed in control fibroblasts corresponded to previous findings where VSVG–GFP ts045 reached the Golgi at ∼30 min ( Koehler et al. , 2017 ; Milev et al. , 2017 , 2018 ) and secreted to the cell surface after 90–120 min ( Hirschberg et al. , 1998 ).
Techniques: Transduction, Control, Derivative Assay, Stable Transfection, Transfection, Virus, Incubation, Marker
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