Figure 7.
SPARC traffics through alternative pathways in golgin KO cells. (A–D) SPARC RUSH assay in WT (A), GMAP210 KO (B), and Golgin-160 KO (C) cells stably expressing SPARC-SBP-mSc (magenta) and transiently transfected with an ER hook (not visible) and mannosidase II-BFP (green, outlined by white dashed line) prior to the experiment. Images are single confocal planes taken from time-lapse movies of SPARC transport after release from the ER by biotin addition at T 00:00. Time after biotin addition is indicated in the top left corner as mm:ss. White arrows indicate SPARC accumulation adjacent to the mannosidase II–positive membranes. Blue arrows highlight peripheral puncta that initiate vesicular ER–Golgi transport. Scale bars, 10 µm. (D i–iii) Quantification of (i) time to arrival at Golgi after biotin addition, (ii) Golgi transit time, measured as the time between SPARC enrichment adjacent to mannosidase II-BFP signal and the emergence of a visible post-Golgi carrier, and (iii) the number of cells in which post-Golgi carriers were identified within 45 min of imaging time, from movies represented in A–C. Individual data points represent individual cells imaged across four independent experiments, and bars show the mean and standard deviation. (i) Data were subjected to a Shapiro–Wilk test for normality (failed) and then a nested one-way ANOVA with a Kruskal–Wallis test. (ii) Data were subjected to a Shapiro–Wilk test for normality (passed) and then a nested one-way ANOVA with Tukey’s multiple comparison test. (E) Maximum projection confocal stacks of SPARC-SBP-mSc stable cell lines transfected with GM130-GFP to mark the Golgi. Scale bar, 10 µm.

SPARC traffics through alternative pathways in golgin KO cells. (A–D) SPARC RUSH assay in WT (A), GMAP210 KO (B), and Golgin-160 KO (C) cells stably expressing SPARC-SBP-mSc (magenta) and transiently transfected with an ER hook (not visible) and mannosidase II-BFP (green, outlined by white dashed line) prior to the experiment. Images are single confocal planes taken from time-lapse movies of SPARC transport after release from the ER by biotin addition at T 00:00. Time after biotin addition is indicated in the top left corner as mm:ss. White arrows indicate SPARC accumulation adjacent to the mannosidase II–positive membranes. Blue arrows highlight peripheral puncta that initiate vesicular ER–Golgi transport. Scale bars, 10 µm. (D i–iii) Quantification of (i) time to arrival at Golgi after biotin addition, (ii) Golgi transit time, measured as the time between SPARC enrichment adjacent to mannosidase II-BFP signal and the emergence of a visible post-Golgi carrier, and (iii) the number of cells in which post-Golgi carriers were identified within 45 min of imaging time, from movies represented in A–C. Individual data points represent individual cells imaged across four independent experiments, and bars show the mean and standard deviation. (i) Data were subjected to a Shapiro–Wilk test for normality (failed) and then a nested one-way ANOVA with a Kruskal–Wallis test. (ii) Data were subjected to a Shapiro–Wilk test for normality (passed) and then a nested one-way ANOVA with Tukey’s multiple comparison test. (E) Maximum projection confocal stacks of SPARC-SBP-mSc stable cell lines transfected with GM130-GFP to mark the Golgi. Scale bar, 10 µm.

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