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Vol. 15, Issue 10, 4658-4668, October 2004

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Visualization of Regulated Exocytosis with a Granule-Membrane Probe Using Total Internal Reflection Microscopy

Miriam W. Allersma ^*, Li Wang , Daniel Axelrod ^*, and Ronald W. Holz  

Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109; ^* Department of Physics and Biophysics Research Division, University of Michigan, Ann Arbor, MI 48109

Submitted February 23, 2004; Revised July 15, 2004; Accepted July 20, 2004
Monitoring Editor: Jennifer Lippincott-Schwartz

Secretory granules labeled with Vamp-green fluorescent protein (GFP) showed distinct signatures upon exocytosis when viewed by total internal reflection fluorescence microscopy. 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.

Abbreviations used: DMPP, 1,1 dimethyl-4-phenylpiperzinium; GFP, green fluorescent protein; hGH, human growth hormone; PSS, physiological salt solution; TIRFM, total internal reflection fluorescence microscopy.

The online version of this article contains supplemental material accessible through http://www.molbiolcell.org.

Corresponding author. E-mail address: holz{at}umich.edu.

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