PMC

The presynaptic scaffold proteins Rim2 (a) and piccolo (b) are specifically enriched at the beta cell membrane that borders the vasculature. This enrichment is quantified in the histograms of the average fluorescence intensities either along a line drawn around the perimeter of the cells (indicated by arrows) or along a linescan (shown by the dotted line). Scale bars, 10 μm

The SNARE proteins syntaxin 1A and SNAP25 are relatively uniformly distributed around the beta cell membrane. (a, b) Low and high power images of the immunofluorescence for laminin and counter immunofluorescence of syntaxin 1A (a) and SNAP25 (b). The histograms of the average fluorescence intensity along a line drawn around the perimeter of the cells (indicated by an arrow) show no enrichment at the vascular face of the beta cell. Scale bars, 10 μm

Presynaptic scaffold protein liprin is present in beta cells and is enriched at the vascular face. (a) Immunofluorescence image of islets shows laminin (green) as a marker of the basement membrane from the vascular endothelial cells and shown as low power, large images of a mouse whole islet (scale bar, 20 μm) and enlarged images of the regions bordered by the boxes (scale bar, 10 μm). Immunofluorescence of liprin (red) shows enrichment along the vasculature using a linescan around the cell (indicated by arrow) perimeter. (b, c) GLUT2 (red) and laminin (green) (b) and insulin (red) and laminin (green) (c) immunostaining show that GLUT2 and insulin are on the membrane of all cells in the islet core proving they are beta cells. (d) An isolated beta cell, with insulin immunostaining (red) and liprin (green) at the cell membrane. (e) Quadruple immunostaining shows that ELKS and liprin are enriched along the laminin-stained vasculature. Scale bar, 10 μm

Two-photon imaging at two planes shows an uneven distribution of exocytic events across single cells. Islets were stimulated for 20 min with 15 mmol/l glucose and the location of each exocytic event was marked with a coloured circle (a). Plane 1 (P1) was recorded for the first 10 min and then the focus was changed (by 5.5 μm) and plane 2 (P2) was recorded for the next 10 min. Some cells were observed only in one plane; those observed in both planes are numbered 1–6 and all cells, particularly cell 5 in this record, show asymmetric numbers of fusion events in the different planes. (b) Histogram of the average number of exocytic events where, for each cell, the most responsive planes and least responsive planes are grouped together. The dotted line shows the number of exocytic event averaged across both planes, which would be the expected observation if the events were evenly distributed. (b). Data from four islets and 65 cells. Scale bar, 10 μm

Live-cell imaging of insulin granule fusion. (a) The images show a single cell within an isolated islet. The extracellular fluorescent dye SRB outlines the cell and when insulin granule fusion occurs this dye enters the granule, shown as time-sequence images taken at 1 s intervals. The average fluorescence changes within a region of interest placed over the fusion granule show a characteristic rapid increase in fluorescence after fusion and then typically show a slow decay; scale bars 5 μm. (b) Beta cells, within the two-photon cross-section show a non-random distribution of secretory responses to high glucose. Insulin granule exocytosis was induced in islets by exposure to high glucose (8–15 mmol/l) for 20 min. The location of each exocytic event is marked by a yellow circle and shows an uneven distribution across the cells; some cells show many events and some have none; scale bar, 5 μm. The frequency histogram (c) shows that the majority of the cells have no exocytic events but that some cells have a high number of exocytic events when compared with the Poisson distribution (line on histogram)

Two-photon live-cell imaging reveals targeting of insulin granule exocytosis to the vascular pole of beta cells. (a, c) Two-photon imaging shows clustering of sites of exocytosis (recorded over 20 min, in response to 15 mmol/l glucose) towards the fissures that run through the islets (stained brightly with extracellular fluorescent dye) (a) and analysed as a histogram (c). The histogram shows the proportion of exocytic events in the half of the cell close to the blood vessel compared to the half of the cell that is distant from the blood vessel. The dotted line on the histogram is the expected 50:50 proportion, if exocytosis was evenly spread across the cell. Scale bar, 10 μm. (b) Two-photon image capture while sequentially stepping image planes through a single cell enabled identification of the exocytosis of individual insulin granules (shown in the reconstruction as green dots) in space, in response to high-K⁺ stimulation. These exocytic fusion events cluster at the cell membrane in a region adjoining the vasculature (stained with isolectin B4 and shown in red on the reconstruction). (d) A histogram of the frequency of exocytic fusion events in relation to the distance from the vasculature shows an asymmetric response with a bias towards the vascular face of the beta cells