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A more recent version of this article appeared on October 1, 2004
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Submitted on February 23, 2004
Revised on July 15, 2004
Accepted on July 20, 2004
, 
Department of Pharmacology and *Department of Physics and Biophysics Research Division, University of Michigan, Ann Arbor, MI 48109
Monitoring Editor: Jennifer Lippincott-Schwartz
Secretory granules labeled with Vamp-GFP showed distinct signatures upon exocytosis when viewed by total internal reflection fluorescence microscopy (TIRFM). In 
90% of fusion events, we observed a large increase in fluorescence intensity coupled with a transition from a small punctate appearance to a larger, spreading cloud with free diffusion of the Vamp-GFP into the plasma membrane. Quantitation suggests that these events reflect the progression of an initially fused and spherical granule flattening into the plane of the plasma membrane as the Vamp-GFP simultaneously diffuses through the fusion junction. Approximately 10% of the events showed a transition from puncta to ring-like structures coupled with little or no spreading. The ring-like images correspond quantitatively to granules fusing and retaining concavity (recess of 200 nm). A majority of fusion events involved granules that were present in the evanescent field for at least 12 s. However, 20% of the events involved granules that were present in the evanescent field for no more than 0.3 s, indicating that the interaction of the granule with the plasma membrane that leads to exocytosis can occur within that time. In addition, 10% of the exocytotic sites were much more likely to occur within a granule diameter of a previous event than can be accounted for by chance, suggestive of sequential (piggy-back) exocytosis that has been observed in other cells. Overall granule behavior before and during fusion is strikingly similar to exocytosis previously described in the constitutive secretory pathway.
Corresponding author.
E-mail: holz{at}umich.edu
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