Figure 7.
VAMP4 facilitates the fusion of iISGs and resorted vesicles with lysosomes for granule cargo degradation. (A) Representative EM images of pancreatic β cells from WT and KO mice were captured at 100 kV and a magnification of 4,800×. Triangle: lysosome; asterisk: ISG; Nuc: nuclei. Scale bars, 2 μm. (B) Region of interest in EM images of pancreatic β cells from WT mice showing that insulin granules and lysosomes exhibit multiple stages of interaction and fusion. These stages include the following: (Ι) ISG-lysosome contact, (II) ISG-lysosome fusion, (III) ISG-lysosome engulfment, and (IV) ISG-lysosome degradation. Images were captured at 120 kV and a magnification of 23,000×. Triangle: lysosome; asterisk: ISG. Scale bars, 0.5 μm. (C–E) The ISG density (C), density of the ISG-lysosome interaction (D), and ratio of ISG-lysosome contact events to total ISGs (E) in WT and KO mice were quantified from the EM images shown in A. Events from all stages of the ISG-lysosome interaction were included. (F) WT and KO INS-1 cells were transfected with mCherry-tagged (pro)insulin (red) and Halo-tagged LAMP1 (green) for 48 h, and live-cell imaging was performed using the Delta Vision OMX V3 system with 100× (NA = 1.40). Scale bars, 5 μm (left) and 1 μm (right). (G) The bar graph showing the quantified colocalization of LAMP1 and (pro)insulin from the data in F. 10 sections of WT cells and 12 sections of KO cells were analyzed from three biological independent experiments. (H) Representative Western blot images showing the expression levels of VAMP4, PC1, PC2, and proinsulin in WT, KO, and rescued INS-1 cells treated with 100 µM chloroquine for 0, 12, and 24 h. Actin was used as a loading control. (I) VAMP4 working model. The data used for the analysis in C–E were based on 25 β cells in two islets isolated from one WT male mouse and 24 β cells in three islets isolated from two VAMP4 KO male mice. WT and VAMP4 KO mice were 16 wk of age. The data are presented as the mean ± SEM. ****, P < 0.0001. The statistical analyses were performed with two-tailed unpaired Student’s t test (C–E and G). Source data are available for this figure: SourceData F7.

VAMP4 facilitates the fusion of iISGs and resorted vesicles with lysosomes for granule cargo degradation. (A) Representative EM images of pancreatic β cells from WT and KO mice were captured at 100 kV and a magnification of 4,800×. Triangle: lysosome; asterisk: ISG; Nuc: nuclei. Scale bars, 2 μm. (B) Region of interest in EM images of pancreatic β cells from WT mice showing that insulin granules and lysosomes exhibit multiple stages of interaction and fusion. These stages include the following: (Ι) ISG-lysosome contact, (II) ISG-lysosome fusion, (III) ISG-lysosome engulfment, and (IV) ISG-lysosome degradation. Images were captured at 120 kV and a magnification of 23,000×. Triangle: lysosome; asterisk: ISG. Scale bars, 0.5 μm. (C–E) The ISG density (C), density of the ISG-lysosome interaction (D), and ratio of ISG-lysosome contact events to total ISGs (E) in WT and KO mice were quantified from the EM images shown in A. Events from all stages of the ISG-lysosome interaction were included. (F) WT and KO INS-1 cells were transfected with mCherry-tagged (pro)insulin (red) and Halo-tagged LAMP1 (green) for 48 h, and live-cell imaging was performed using the Delta Vision OMX V3 system with 100× (NA = 1.40). Scale bars, 5 μm (left) and 1 μm (right). (G) The bar graph showing the quantified colocalization of LAMP1 and (pro)insulin from the data in F. 10 sections of WT cells and 12 sections of KO cells were analyzed from three biological independent experiments. (H) Representative Western blot images showing the expression levels of VAMP4, PC1, PC2, and proinsulin in WT, KO, and rescued INS-1 cells treated with 100 µM chloroquine for 0, 12, and 24 h. Actin was used as a loading control. (I) VAMP4 working model. The data used for the analysis in C–E were based on 25 β cells in two islets isolated from one WT male mouse and 24 β cells in three islets isolated from two VAMP4 KO male mice. WT and VAMP4 KO mice were 16 wk of age. The data are presented as the mean ± SEM. ****, P < 0.0001. The statistical analyses were performed with two-tailed unpaired Student’s t test (C–E and G). Source data are available for this figure: SourceData F7.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal