Fusion Pore Dynamics and Insulin Granule Exocytosis in the Pancreatic Islet

Insulin secretion from intact mouse pancreatic islets was investigated with two-photon excitation imaging. Insulin granule exocytosis occurred mainly toward the interstitial space, away from blood vessels. The fusion pore was unusually stable with a lifetime of 1.8 seconds. Opening of the 1.4-nanometer-diameter pore was preceded by unrestricted lateral diffusion of lipids along the inner wall of the pore, supporting the idea that this structure is composed of membrane lipids. When the pore dilated to 12 nanometers, the granules rapidly flattened and discharged their contents. Thus, our methodology reveals fusion pore dynamics in intact tissues at nanometer resolution.

[1]  L. Orci,et al.  Insulin Release by Emiocytosis: Demonstration with Freeze-Etching Technique , 1973, Science.

[2]  J. Heuser,et al.  Arrest of membrane fusion events in mast cells by quick-freezing , 1980, The Journal of cell biology.

[3]  P. Veld,et al.  Evidence Against the Presence of Tight Junctions in Normal Endocrine Pancreas , 1984, Diabetes.

[4]  Richard G. W. Anderson,et al.  The condensing vacuole of exocrine cells is more acidic than the mature secretory vesicle , 1987, Nature.

[5]  W. Almers,et al.  Currents through the fusion pore that forms during exocytosis of a secretory vesicle , 1987, Nature.

[6]  J. Zimmerberg,et al.  Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[7]  S. Bonner-Weir Morphological Evidence for Pancreatic Polarity of β-Cell Within Islets of Langerhans , 1988, Diabetes.

[8]  J. R. Monck,et al.  Tension in secretory granule membranes causes extensive membrane transfer through the exocytotic fusion pore. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[9]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[10]  D. Pipeleers Heterogeneity in Pancreatic β-cell Population , 1992, Diabetes.

[11]  W. Betz,et al.  Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals , 1992, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[12]  W. Almers,et al.  Membrane flux through the pore formed by a fusogenic viral envelope protein during cell fusion , 1993, The Journal of cell biology.

[13]  J. M. Fernández,et al.  Release of secretory products during transient vesicle fusion , 1993, Nature.

[14]  R. Chow,et al.  Rapid fluctuations in transmitter release from single vesicles in bovine adrenal chromaffin cells. , 1996, Biophysical journal.

[15]  J. Zimmerberg,et al.  Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[16]  E. Neher,et al.  Ultrastructural Organization of Bovine Chromaffin Cell Cortex—Analysis by Cryofixation and Morphometry of Aspects Pertinent to Exocytosis , 1997, The Journal of cell biology.

[17]  Y. Miyashita,et al.  Multiple Exocytotic Pathways in Pancreatic β Cells , 1997, The Journal of cell biology.

[18]  K. Kawasaki,et al.  Structural Features of Membrane Fusion between Influenza Virus and Liposome as Revealed by Quick-Freezing Electron Microscopy , 1997, The Journal of cell biology.

[19]  D. Steiner,et al.  The role of assembly in insulin's biosynthesis. , 1998, Current opinion in structural biology.

[20]  E. Neher Vesicle Pools and Ca2+ Microdomains: New Tools for Understanding Their Roles in Neurotransmitter Release , 1998, Neuron.

[21]  Y. Miyashita,et al.  Post-priming actions of ATP on Ca2+-dependent exocytosis in pancreatic beta cells. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[22]  R T Kennedy,et al.  Detection of secretion from single pancreatic beta-cells using extracellular fluorogenic reactions and confocal fluorescence microscopy. , 2000, Analytical chemistry.

[23]  J. Zimmerberg,et al.  How can proteolipids be central players in membrane fusion? , 2001, Trends in cell biology.

[24]  Y. Miyashita,et al.  Sequential-replenishment mechanism of exocytosis in pancreatic acini , 2001, Nature Cell Biology.

[25]  P. Rorsman,et al.  Delay between Fusion Pore Opening and Peptide Release from Large Dense-Core Vesicles in Neuroendocrine Cells , 2002, Neuron.

[26]  Akihiro Kusumi,et al.  Phospholipids undergo hop diffusion in compartmentalized cell membrane , 2002, The Journal of cell biology.