Journal: iScience

Article Title: Cab45G trafficking through the insulin secretory pathway is altered in human type 2 diabetes

doi: 10.1016/j.isci.2024.111719

Figure Lengend Snippet: Ca 2+ -dependency of the cis -Golgi Cab45G localization and visualization of anterograde trafficking in INS1 cells (A) Immunofluorescent staining of Cab45G with either TGN38, GM130, insulin or proinsulin in INS1 cells ( n = 3), scale bar = 5 μm. (B) Pearson’s coefficient of correlation between Cab45G and TGN38 versus Cab45G and GM130, presented as mean ± SEM. (C and D) INS1 cells treated with ionomycin and EGTA in a Ca 2+ -free medium for 20 min ( n = 3). Cells were fixed and stained with antibodies targeting Cab45G, insulin, and GM130, scale bars = 5 μm. White arrows indicate post-Golgi Cab45G-positive vesicles. Manders' coefficient of colocalization was used to describe the fraction of cellular Cab45G localized in the cis -Golgi with GM130 (D), presented as mean ± SEM. (E–G) Live visualization of WT- and 6EQ-Cab45G tagged with green fluorescent protein (GFP) trafficking from the ER through the TGN during the application of the retention using selective hooks (RUSH) protocol in INS1 cells, scale bar = 5 μm Cab45G-GFP variants were held in the ER using a streptavidin hook prior to release via biotin addition, and trafficking through the TGN was monitored with free co-expression of TGN-resident protein Sialyltransferase fused to red fluorescent protein (RFP). Labels indicate time to reach peak TGN-localization following release of Cab45G-GFP variants from the ER. (F) Percentage of the peak ER-localized GFP signal intensity (left) and quantification of the rate of ER exit (right), and (G) Percentage of the peak TGN-localized GFP signal intensity (left) and quantification of the rate of TGN exit (right), presented as mean ± SEM. (H) Schematic representation of WT- and 6EQ-Cab45G proteins displaying sites of point mutation (orange) to render the six Ca 2+ -binding sites (blue) non-functional. Comparisons made using one-way ANOVA, paired, or unpaired t-test (∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001), with (F) and (G) using non-parametric testing.

Article Snippet: Rat proinsulin ELISA kit , Mercodia , 10-1118-01.

Techniques: Staining, Expressing, Mutagenesis, Binding Assay, Functional Assay

Journal: iScience

Article Title: Cab45G trafficking through the insulin secretory pathway is altered in human type 2 diabetes

doi: 10.1016/j.isci.2024.111719

Figure Lengend Snippet: Current working model of Cab45G localization through stages of β-cell function Key: ISG - insulin secretory granule, iISG - immature ISG, mISG - mature ISG, TGN - trans- Golgi network. During normal conditions (top), proinsulin trafficks through the TGN and into immature insulin secretory granules via bulk-flow. Since supply is in excess to demand it does so without the need for Cab45G-dependent acceleration; although this may be present to some extent, Cab45G predominantly resides in the cis -Golgi. Metabolic stress imposes pressure to synthesize and secrete insulin, and compensating β-cells (middle) respond by upregulating SDF4 mRNA production which increases Cab45G biosynthesis beyond the holding-capacity of the cis -Golgi. This pushes Cab45G out from this compartment to accelerate bulk-flow trafficking and drive granule production. Cab45G may then exit the maturing granule into non-insulin secretory granules. Progression into β-cell failure (bottom) is associated with an aberrant pattern of Cab45G trafficking. While anterograde Cab45G trafficking is maintained, its abundance in the perinuclear area is reduced and it accumulates in the mature insulin secretory granule.

Article Snippet: Rat proinsulin ELISA kit , Mercodia , 10-1118-01.

Techniques: Cell Function Assay

Journal: iScience

Article Title: Cab45G trafficking through the insulin secretory pathway is altered in human type 2 diabetes

doi: 10.1016/j.isci.2024.111719

Figure Lengend Snippet:

Article Snippet: Rat proinsulin ELISA kit , Mercodia , 10-1118-01.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Picogreen Assay, Plasmid Preparation, CRISPR, Software