Figure 5. VAMP4 localizes to iISGs and... | Rockefeller University Press

Figure 5.

$VAMP4 localizes to iISGs and is resorted to CCVs during granule maturation, and VAMP4-positive iISGs and resorted vesicles fuse with lysosomes. (A) Representative Western blot images showing the expression levels of proinsulin, insulin, VAMP4, PC1, PC2, and LAMP1 in different cellular fractions enriched by OptiPrep density gradient centrifugation (n = 3 biological independent experiments). (B) INS-1 cells were transfected with EGFP-tagged VAMP4 (green) and Halo-tagged (pro)insulin (red) plasmids for 48 h under live-cell imaging. Scale bars, 5 μm. Enlarged diagrams of the Golgi (G) region and peripheral (P) region are shown. Scale bars, 1 μm. (C) The percentage of VAMP4 localized on ISGs was higher in the Golgi region than in the peripheral region based on the three-dimensional live-cell imaging data shown in B (n = 6 sections of WT cells and KO cells from three independent experiments). The data are presented as the mean ± SEM. The statistical analyses were performed with two-way ANOVA. ****, P < 0.0001. (D) INS-1 cells were transfected with EGFP-tagged VAMP4 (green) and mCherry-tagged (pro)insulin (red) plasmids for 48 h under live-cell imaging. The diagram and a snapshot of a consecutive event showed that VAMP4 localized to the iISG surface and was removed from iISGs (indicated by arrows). (E) INS-1 cells were transfected with EGFP-tagged VAMP4 (green) and mCherry-tagged LAMP1 (red) plasmids for 48 h under live-cell imaging. The diagram and a snapshot of a consecutive event showed that VAMP4 fused with lysosomes and diffused on the lysosomal membrane (indicated by arrows), VPV, VAMP4-positive vesicle. (F) INS-1 cells were transfected with Halo-tagged LAMP1 (green) and mCherry-tagged (pro)insulin (red) plasmids for 48 h under live-cell imaging. The diagram and a snapshot of a consecutive event showed that (pro)insulin fused with lysosomes (indicated by arrows). Long-term live-cell imaging data were acquired using a Delta Vision OMX V3 system with 100× (NA = 1.40) in B and D–F. The data in D–F represent at least three biological independent experiments. Scale bars, 5 μm (D–F top) and 0.5 μm (D–F bottom). Source data are available for this figure: SourceData F5.$

VAMP4 localizes to iISGs and is resorted to CCVs during granule maturation, and VAMP4-positive iISGs and resorted vesicles fuse with lysosomes. (A) Representative Western blot images showing the expression levels of proinsulin, insulin, VAMP4, PC1, PC2, and LAMP1 in different cellular fractions enriched by OptiPrep density gradient centrifugation (n = 3 biological independent experiments). (B) INS-1 cells were transfected with EGFP-tagged VAMP4 (green) and Halo-tagged (pro)insulin (red) plasmids for 48 h under live-cell imaging. Scale bars, 5 μm. Enlarged diagrams of the Golgi (G) region and peripheral (P) region are shown. Scale bars, 1 μm. (C) The percentage of VAMP4 localized on ISGs was higher in the Golgi region than in the peripheral region based on the three-dimensional live-cell imaging data shown in B (n = 6 sections of WT cells and KO cells from three independent experiments). The data are presented as the mean ± SEM. The statistical analyses were performed with two-way ANOVA. ****, P < 0.0001. (D) INS-1 cells were transfected with EGFP-tagged VAMP4 (green) and mCherry-tagged (pro)insulin (red) plasmids for 48 h under live-cell imaging. The diagram and a snapshot of a consecutive event showed that VAMP4 localized to the iISG surface and was removed from iISGs (indicated by arrows). (E) INS-1 cells were transfected with EGFP-tagged VAMP4 (green) and mCherry-tagged LAMP1 (red) plasmids for 48 h under live-cell imaging. The diagram and a snapshot of a consecutive event showed that VAMP4 fused with lysosomes and diffused on the lysosomal membrane (indicated by arrows), VPV, VAMP4-positive vesicle. (F) INS-1 cells were transfected with Halo-tagged LAMP1 (green) and mCherry-tagged (pro)insulin (red) plasmids for 48 h under live-cell imaging. The diagram and a snapshot of a consecutive event showed that (pro)insulin fused with lysosomes (indicated by arrows). Long-term live-cell imaging data were acquired using a Delta Vision OMX V3 system with 100× (NA = 1.40) in B and D–F. The data in D–F represent at least three biological independent experiments. Scale bars, 5 μm (D–F top) and 0.5 μm (D–F bottom). Source data are available for this figure: SourceData F5.