Figure 1.
OSBP dynamics regulate apicobasal cargo sorting. (A–C) hTERT-RPE1 cells were cotransfected with EGFP-GPI-expressing RUSH plasmid and mCherry-PHOSBP construct. Epifluorescence time-lapse imaging was performed the next day. Biotin was added to the cells to initiate cargo release at 5 min, and PIK93 was applied 30 min after the start of the experiment at 500 nM. Framed Golgi areas indicate regions that were used to generate kymographs shown in B. Intensity values corresponding to the indicated spots of the Golgi area over time were plotted on C. Bar = 10 μm. (D and E) MDCK cells were nucleofected to express mCherry-PHOSBP. Epifluorescence time-lapse imaging was performed on the next day. Kymograph corresponding to the indicated Golgi area (D) and Golgi fluorescence intensity values over time were plotted (E). PIK93 was added to the cells at the indicated time point at 500 nM. Bar = 10 μm. (F and G) MDCK cells were cotransfected with EGFP-GPI and mCherry-CDH1 expressing RUSH constructs, and cargo release was triggered by biotin addition on the next day. Following biotin addition, cells were kept at 19.5°C for 1.5 h to synchronize cargoes at the TGN. Cells were released from the temperature block, fixed, or kept in the presence of DMSO or 500 nM PIK93 at 37°C for 40 min followed by fixation and confocal microscopy. Line-plots indicate the fluorescence intensities of each cargo at the marked areas. Arrowheads point to post-Golgi vesicles positive for single or double cargoes (F). Distribution of each single- and double cargo-containing post-Golgi vesicle in control and PIK93-treated cells. N = number of independent experiments presented, n = number of cells/sample used for the analysis. In each cell, at least 50 vesicles were counted. Error bars show SEM, and P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. Bar = 10 μm, diameter of the insets = 8.7 μm. (H and I) MDCK cells were seeded in Matrigel and left to form cysts for 72 h in the presence of DMSO or 500 nM PIK93. Cysts were then fixed, stained, and analyzed by confocal microscopy. The arrowhead indicates Gp135 facing the extracellular matrix (H). PIK93 treatment increases the percentage of MDCK cysts with morphology defects. More cysts are shown in Fig. S1 B. N = number of independent experiments presented. Statistics were obtained by counting at least 70 cysts/condition. P value was calculated by unpaired t test. Bar = 10 μm, diameter of the insets = 14.5 μm. (J and K) MDCK cells were nucleofected with control or OSBP-silencing siRNA. 24 h later, cells were transfected again with basolateral CDH1 (J) or apical EGFP-GPI (K) RUSH model cargoes. Epifluorescence time-lapse microscopy was performed on the next day and fluorescence intensity values corresponding to Golgi areas were plotted. Bold lines with the shaded areas indicate mean ± SEM. P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. N = number of independent experiments presented. n = number of cells used to calculate mean and error. The three independent experiments are shown in Fig. S1 I. Bars = 10 μm. (L and M) MDCK cells were nucleofected with non-targeting or OSBP-silencing siRNA. 24 h later, cells were cotransfected with basolateral CDH1 and apical EGFP-GPI model cargo-expressing constructs. On the next day, biotin was added, and then cells were kept at 19.5°C for 1.5 h to synchronize cargoes at the TGN. Following this, cells were kept at 37°C for 40 min, followed by fixation and visualization by confocal microscopy. Line-plots indicate fluorescence intensities of each cargo at the corresponding areas and arrowheads point to post-Golgi vesicles positive for single or double cargoes (L). Distribution of each single- and double cargo-containing post-Golgi vesicle in control and PIK93-treated cells (M). Seven cells/condition were quantified; in each cell, at least 30 post-Golgi vesicles were counted. N = number of independent experiments presented, n = number of cells/sample used for the analysis. Error bars show SEM and P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. Bar = 10 μm, diameter of the insets = 8.7 μm. (N–P) MDCK cells were nucleofected with control siRNA or siOSBP, seeded into Matrigel 24 h later, and left to develop cysts for 72 h. Representative cysts stained with apical/basolateral markers are shown in N. More cysts are shown on Fig. S1 J. Efficacy of RNAi-based OSBP silencing was tested 4 d post-nucleofection in MDCK cells (O). OSBP silencing increases the number of MDCK cysts with morphology defects. Each experiment was done in triplicate/condition and statistics were obtained by counting at least 70 cysts/condition. P value was calculated by unpaired t test. N = number of independent experiments presented. Bar = 10 μm, diameter of the insets = 14.5 μm. Source data are available for this figure: SourceData F1.

OSBP dynamics regulate apicobasal cargo sorting. (A–C) hTERT-RPE1 cells were cotransfected with EGFP-GPI-expressing RUSH plasmid and mCherry-PHOSBP construct. Epifluorescence time-lapse imaging was performed the next day. Biotin was added to the cells to initiate cargo release at 5 min, and PIK93 was applied 30 min after the start of the experiment at 500 nM. Framed Golgi areas indicate regions that were used to generate kymographs shown in B. Intensity values corresponding to the indicated spots of the Golgi area over time were plotted on C. Bar = 10 μm. (D and E) MDCK cells were nucleofected to express mCherry-PHOSBP. Epifluorescence time-lapse imaging was performed on the next day. Kymograph corresponding to the indicated Golgi area (D) and Golgi fluorescence intensity values over time were plotted (E). PIK93 was added to the cells at the indicated time point at 500 nM. Bar = 10 μm. (F and G) MDCK cells were cotransfected with EGFP-GPI and mCherry-CDH1 expressing RUSH constructs, and cargo release was triggered by biotin addition on the next day. Following biotin addition, cells were kept at 19.5°C for 1.5 h to synchronize cargoes at the TGN. Cells were released from the temperature block, fixed, or kept in the presence of DMSO or 500 nM PIK93 at 37°C for 40 min followed by fixation and confocal microscopy. Line-plots indicate the fluorescence intensities of each cargo at the marked areas. Arrowheads point to post-Golgi vesicles positive for single or double cargoes (F). Distribution of each single- and double cargo-containing post-Golgi vesicle in control and PIK93-treated cells. N = number of independent experiments presented, n = number of cells/sample used for the analysis. In each cell, at least 50 vesicles were counted. Error bars show SEM, and P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. Bar = 10 μm, diameter of the insets = 8.7 μm. (H and I) MDCK cells were seeded in Matrigel and left to form cysts for 72 h in the presence of DMSO or 500 nM PIK93. Cysts were then fixed, stained, and analyzed by confocal microscopy. The arrowhead indicates Gp135 facing the extracellular matrix (H). PIK93 treatment increases the percentage of MDCK cysts with morphology defects. More cysts are shown in Fig. S1 B. N = number of independent experiments presented. Statistics were obtained by counting at least 70 cysts/condition. P value was calculated by unpaired t test. Bar = 10 μm, diameter of the insets = 14.5 μm. (J and K) MDCK cells were nucleofected with control or OSBP-silencing siRNA. 24 h later, cells were transfected again with basolateral CDH1 (J) or apical EGFP-GPI (K) RUSH model cargoes. Epifluorescence time-lapse microscopy was performed on the next day and fluorescence intensity values corresponding to Golgi areas were plotted. Bold lines with the shaded areas indicate mean ± SEM. P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. N = number of independent experiments presented. n = number of cells used to calculate mean and error. The three independent experiments are shown in Fig. S1 I. Bars = 10 μm. (L and M) MDCK cells were nucleofected with non-targeting or OSBP-silencing siRNA. 24 h later, cells were cotransfected with basolateral CDH1 and apical EGFP-GPI model cargo-expressing constructs. On the next day, biotin was added, and then cells were kept at 19.5°C for 1.5 h to synchronize cargoes at the TGN. Following this, cells were kept at 37°C for 40 min, followed by fixation and visualization by confocal microscopy. Line-plots indicate fluorescence intensities of each cargo at the corresponding areas and arrowheads point to post-Golgi vesicles positive for single or double cargoes (L). Distribution of each single- and double cargo-containing post-Golgi vesicle in control and PIK93-treated cells (M). Seven cells/condition were quantified; in each cell, at least 30 post-Golgi vesicles were counted. N = number of independent experiments presented, n = number of cells/sample used for the analysis. Error bars show SEM and P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. Bar = 10 μm, diameter of the insets = 8.7 μm. (N–P) MDCK cells were nucleofected with control siRNA or siOSBP, seeded into Matrigel 24 h later, and left to develop cysts for 72 h. Representative cysts stained with apical/basolateral markers are shown in N. More cysts are shown on Fig. S1 J. Efficacy of RNAi-based OSBP silencing was tested 4 d post-nucleofection in MDCK cells (O). OSBP silencing increases the number of MDCK cysts with morphology defects. Each experiment was done in triplicate/condition and statistics were obtained by counting at least 70 cysts/condition. P value was calculated by unpaired t test. N = number of independent experiments presented. Bar = 10 μm, diameter of the insets = 14.5 μm. Source data are available for this figure: SourceData F1.

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