Figure 6.
Liprin-α1 assembles in a presynaptic-like complex in MIN6 β cells and interacts with insulin granules via β2-syntrophin. (A) MIN6 cells expressing GFP (control) or GFP–liprin-α1 were incubated in basal (2.8 mM glucose) and stimulated (16.7 mM glucose) conditions. Cells were lysed for IP with anti-GFP nanobody-conjugated beads. Immunoprecipitates were subjected to bottom-up proteomics with DIA and protein identification and quantification using DIA-NN. Immunoprecipitates from basal and stimulated groups were pooled, and significant interactors were identified with SAINTexpress (n = 6 GFP control, n = 6 GFP–liprin-α1) and plotted with log10 average intensity on the x axis and log2 fold change of GFP–liprin-α1 over GFP control on the y axis. Each point represents an individual protein; red points represent statistical significance Bayesian false discovery rate (BFDR). (B) Box-and-whisker plots showing median LFQ intensities and 1.5 times the interquartile range for specific proteins of interest. B, basal; S, stimulated. (C) Anti-GFP immunoprecipitates were also analyzed by immunoblotting, showing pull-down of liprin-β1 and β2-syntrophin with GFP–liprin-α1. Band intensities are plotted as a bar graph, normalized to GFP control. (D) Co-IP of native liprin-α1 with liprin-β1 and β2-syntrophin in MIN6 β cells. Cells were lysed for IP with protein A/G magnetic beads, and the immunoprecipitates were analyzed by immunoblotting using anti-liprin-α1, anti–liprin-β1, and anti–β2-syntrophin antibodies. Band intensities are plotted as a bar graph, normalized to IgG. (E) Representative immunofluorescence of an islet within a pancreatic slice. Liprin-α1 (red) and β2-syntrophin (green) are both enriched at the β cell–ECM interface (laminin; blue), also seen in a histogram showing relative fluorescence intensity at the β cell basal (capillary), apical, and lateral regions (49 cells, 9 islets across 3 animals), and a line scan across a region of interest. Quantification of colocalization between liprin-α1 and β2-syntrophin using Pearson’s correlation coefficient between the two channels. 90° indicates a 90° rotation of one of the two analyzed channels before analysis. (F) Immunofluorescence staining of liprin-α1 and β2-syntrophin in isolated dispersed β cells. β2-syntrophin is present and colocalized in liprin-α1 clusters across the β cell/laminin interface. Source data are available for this figure: SourceData F6.

Liprin-α1 assembles in a presynaptic-like complex in MIN6 β cells and interacts with insulin granules via β2-syntrophin. (A) MIN6 cells expressing GFP (control) or GFP–liprin-α1 were incubated in basal (2.8 mM glucose) and stimulated (16.7 mM glucose) conditions. Cells were lysed for IP with anti-GFP nanobody-conjugated beads. Immunoprecipitates were subjected to bottom-up proteomics with DIA and protein identification and quantification using DIA-NN. Immunoprecipitates from basal and stimulated groups were pooled, and significant interactors were identified with SAINTexpress (n = 6 GFP control, n = 6 GFP–liprin-α1) and plotted with log10 average intensity on the x axis and log2 fold change of GFP–liprin-α1 over GFP control on the y axis. Each point represents an individual protein; red points represent statistical significance Bayesian false discovery rate (BFDR). (B) Box-and-whisker plots showing median LFQ intensities and 1.5 times the interquartile range for specific proteins of interest. B, basal; S, stimulated. (C) Anti-GFP immunoprecipitates were also analyzed by immunoblotting, showing pull-down of liprin-β1 and β2-syntrophin with GFP–liprin-α1. Band intensities are plotted as a bar graph, normalized to GFP control. (D) Co-IP of native liprin-α1 with liprin-β1 and β2-syntrophin in MIN6 β cells. Cells were lysed for IP with protein A/G magnetic beads, and the immunoprecipitates were analyzed by immunoblotting using anti-liprin-α1, anti–liprin-β1, and anti–β2-syntrophin antibodies. Band intensities are plotted as a bar graph, normalized to IgG. (E) Representative immunofluorescence of an islet within a pancreatic slice. Liprin-α1 (red) and β2-syntrophin (green) are both enriched at the β cell–ECM interface (laminin; blue), also seen in a histogram showing relative fluorescence intensity at the β cell basal (capillary), apical, and lateral regions (49 cells, 9 islets across 3 animals), and a line scan across a region of interest. Quantification of colocalization between liprin-α1 and β2-syntrophin using Pearson’s correlation coefficient between the two channels. 90° indicates a 90° rotation of one of the two analyzed channels before analysis. (F) Immunofluorescence staining of liprin-α1 and β2-syntrophin in isolated dispersed β cells. β2-syntrophin is present and colocalized in liprin-α1 clusters across the β cell/laminin interface. Source data are available for this figure: SourceData F6.

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