Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion.
Confocal imaging of GFP-tagged secretory granules combined with the use of impermeant extracellular dyes permits direct observation of insulin packaged in secretory granules, trafficking of these granules to the plasma membrane, exocytotic fusion of granules with the plasma membrane, and eventually the retrieval of membranes by endocytosis. Most such studies have been done in tumor cell lines, using either confocal methods or total internal reflectance microscopy. Here we compared these methods by using GFP-syncollin or PC3-GFP plus rhodamine dextrans to study insulin granule dynamics in insulinoma cells, normal mouse islets, and primary pancreatic beta cells. We found that most apparently docked granules did not fuse with the plasma membrane after stimulation. Granules that did fuse typically fused completely, but a few dextran-filled granules lingered at the membrane. Direct recycling of granules occurred only rarely. Similar results were obtained with both confocal and total internal reflection microscopy, although each technique had advantages for particular aspects of the granule life cycle. We conclude that insulin exocytosis involves a prolonged interaction of secretory granules with the plasma membrane, and that the majority of exocytotic events occur by full, not partial, fusion.
Ma
Li
L
Department of Medicine, University of Chicago, IL 60637, USA.
Bindokas
Vytautas P
VP
Kuznetsov
Andrey
A
Rhodes
Christopher
C
Hays
Lori
L
Edwardson
J Michael
JM
Ueda
Kazuya
K
Steiner
Donald F
DF
Philipson
Louis H
LH
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D045744:Cell Line, Tumor; D005089:Exocytosis / Q000502:physiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma; D007425:Intracellular Membranes / Q000502:physiology; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008561:Membrane Fusion; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal / Q000379:methods; D010190:Pancreatic Neoplasms; D022142:Secretory Vesicles / Q000378:metabolism* / Q000502:physiology
D007328:Insulin; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins