Fig. 2: TUG regulates anterograde trafficking.
From: TUG protein acts through a disordered region to organize the early secretory pathway
a WT (top) and TUG KO (bottom) MEFs were fixed and stained using antibodies to the ERGIC marker ERGIC53 (magenta) and the cis-Golgin GM130 (green). Images are a single slice from a confocal stack. Dashed boxes in the top image denote peripheral punctate staining of ERGIC53, which is devoid of GM130. The dashed region in the bottom image denotes the Golgi, based on GM130 staining. Note the reduction in peripheral ERGIC53 puncta in TUG KO MEFs, compared to WT cells, and increased ERGIC53 distribution at the cis-Golgi. b Confocal images were segmented to generate ERGIC53 and GM130 surfaces. The graph compares independent ERGIC53 surfaces (not touching GM130 surfaces) in WT and TUG KO MEFs. A reduction in the number of independent ERGIC53 surfaces was observed in TUG KO MEFs. Data from 16 WT and 16 KO cells are shown; mean ± standard deviation (s.d.). c The graph compares average intensities of ERGIC53 staining at the Golgi after collapsing a confocal stack. Data from 16 WT and 16 KO cells are shown; mean ± s.d. d Images of TUG KO (top) and WT (bottom) MEFs containing retroviruses to express PAUF tagged with mKate2 and FM4 domains. The FM4 domains cause aggregation of PAUF in the ER (marked by PDI, in green; left), and is distinct from GM130 (green; right) signal in the absence of D/D solubilizer. The addition of D/D-solubilizer releases PAUF from the ER for anterograde traffic through the secretory pathway. Cells were fixed 10 min after the addition of D/D solubilizer and were imaged (right). In TUG KO MEFs (top), most signal is concentrated at the Golgi, but in WT MEFs there is also surrounding ER signal. e Data from replicates of the experiment in f were quantified, and enrichment of mKate2-tagged PAUF signal at the Golgi is normalized to the signal in the surrounding ER. N = 42 cells in each group, acquired from 4 independent experiments. The box indicates median and quartile ranges, and whiskers show the spread of the data from minimum to maximum. All statistical analyses used an unpaired two-tailed t test with Welch’s correction.
