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. 2013 Jun 14;62(7):2416–2428. doi: 10.2337/db12-1380

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© 2013 by the American Diabetes Association.

Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

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FIG. 5. — Munc18b mediates primary exocytosis and sequential SG-SG fusion in rat β-cells. A: Example of TEP imaging of single insulin SG exocytosis (single granule) in an AdGFP-transduced rat islet stimulated with 20 mmol/L glucose plus 10 nmol/L GLP-1 and 150 μm IBMX in a solution containing 0.3 mmol/L Alexa Fluor 594 hydrazide. An ω construction appeared when the SG(s) coalesced with the PM. B: Example of TEP imaging of sequential exocytosis (two granules) involving two SGs in an AdMunc18b-KR–transduced rat islet stimulated as in A. The Alexa fluorescence distinguishes several adjacent β-cells in the islet, along with small blood vessels (white arrowheads) and major blood vessels (white arrows) (i). White circle shows the position of an ω construction in a former panel (ii). An example of a single SG exocytosis shown in A with imaging interval of 1.2 s; in B, an example of 2-SG sequential events with imaging interval of 0.6 s. The numbers below each panel represent time after onset of exocytosis. Dashed circles show position of an ω construction in a former panel. Arrows point to the direction of SG fusion toward the cell interior. iii: Time courses of fluorescence at the area containing the exocytotic events. Vertical bars indicate the times when images in ii were acquired. The fluorescence value before exocytosis was set to zero. A and B: Scale bar represents 10 μm. C: Total number of exocytotic events calculated as number of exocytotic events/cell/min in β-cells from islets treated with AdGFP (8 islets, 68 cells), AdMunc18b-WT (7 islets, 44 cells), AdMunc18b-KR (6 islets, 58 cells), and AdMunc18b-E59K (5 islets, 75 cells) during a 10-min period of stimulation as in A. D: Fractions of total exocytotic events that are sequential exocytosis. The number of secondary exocytotic events considered to be sequential exocytosis is expressed as a percentage of total number of exocytotic events assessed in C. Data in C and D are shown as means ± SEM and analyzed by Steel-Dwass test for multiple comparisons using AdGFP as control. *P < 0.05, ***P < 0.001. A.U., arbitrary units.