Liprin-α1 knockdown in β cells impairs localization of granule fusion. (A) Immunofluorescence staining of presynaptic scaffold proteins liprin-α1 (green) and ELKS (red) in isolated mouse β cells (insulin; blue) grown on coverslips coated with ECM (laminin-511). A line scan plotting fluorescence intensity across an orthogonal section (XZ) shows local enrichment of liprin-α1 and ELKS, but not insulin, at the laminin-cell interface. (B) When stimulated, isolated β cells bathed in an extracellular dye (SRB) and imaged with two-photon microscopy show short-lasting flashes of fluorescence as individual granules fuse with the membrane and SRB enters each fusing granule. 3D live-cell two-photon imaging with z stacks (2-µm apart) was used to record β cell exocytotic events in time and space, as shown in the cartoon. (C) Continuous recording over 15 min during stimulation with 16.7 mM glucose led to the identification of many exocytotic events, marked with yellow dots. An exemplar image of one cell shows three image planes (bottom, middle, and top) as well as a 3D projection image of the whole cell and highlights a strong bias of events at the ECM-cell interface (bottom). (D) Stimulation with 40 mM K⁺ for 15 min also induced many exocytic events, again in the exemplar images showing a strong bias to the ECM-cell interface (bottom). (E) β cells were loaded with the photolabile Ca²⁺ chelator nitrophenyl EGTA (NP-EGTA) and Fura-2, AM, enabling the UV flash photolysis-catalyzed global intracellular uncaging of Ca²⁺. (F) Ca²⁺ uncaging by a 100-ms UV flash triggered a rapid transient increase in intracellular [Ca²⁺], tracked with Fura-2, and (F) induced many exocytotic events, again with a bias of events to the ECM-cell interface (bottom). (G) Histogram of exocytotic density at the cell bottom versus upper planes (the average of planes 2–6) shows granule fusion is significantly biased toward the ECM-cell interface (bottom) for all three stimulation conditions (n ≥8 β cells obtained from ≥3 animals, two-way ANOVA followed by Bonferroni’s multiple comparisons test). (H) Western blot showing liprin-α1 expression in mouse islets infected with adenovirus encoding GFP-scrambled shRNA (control) and GFP–liprin-α1 shRNA. Quantification of liprin-α1 protein expression normalized to β-actin is shown as a histogram (n = 3; paired two-tailed Student’s t test). (I) Control β cells and cells after liprin-α1 knockdown were stimulated with 40 mM K⁺ for 30 min. Liprin-α1 knockdown had no effect on high K⁺-induced insulin secretion, normalized to total cellular insulin content (for each condition, n = 3 animals; two-way ANOVA followed by Tukey’s multiple comparisons test). (J–M) High K⁺-induced granule fusion events were recorded using 3D live-cell two-photon microscopy. (J) An exemplar of a control cell (scrambled shRNA) showed a significant bias of granule fusion events at the ECM-cell interface (bottom) compared with upper regions. A schematic diagram summarizes the dataset (n = 3 animals, 8 cells) by showing the average distribution of granule fusion events at each image plane, each dot representing an exocytic fusion density of 0.001 events µm². (K) In contrast, liprin-α1 knockdown cells had a relatively even distribution of granule fusion events around the whole cell, as shown in the example images and in the schematic diagram (n = 3 animals, 9 cells). (L) Re-expression of human GFP–liprin-α1 after knockdown partially rescued granule targeting to the ECM-cell interface (bottom), as shown in the example images and in the schematic diagram (n = 4 animals, 11 cells). (M) A histogram of the data in J–L show significant differences in the high K⁺-induced exocytic density between the upper sections and the ECM-cell interface (bottom) for control cells that was abolished with liprin-α1 shRNA and partially, but significantly, restored with the human liprin-α1 re-expression (for each condition, n = 3–4 animals; two-way ANOVA followed by Bonferroni’s multiple comparisons test). All data are shown as mean ± SEM. Source data are available for this figure: SourceData F1.