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

Miriam W. Allersma*, Li Wang ${dagger}$ , Daniel Axelrod*, and Ronald W. Holz ${dagger}$ ${ddagger}$

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

Monitoring Editor: Jennifer Lippincott-Schwartz

Secretory granuleslabeled with Vamp-GFP showed distinct signatures upon exocytosiswhen viewed by total internal reflection fluorescence microscopy(TIRFM). In $~$ 90% of fusion events, we observed a large increasein fluorescence intensity coupled with a transition from a smallpunctate appearance to a larger, spreading cloud with free diffusionof the Vamp-GFP into the plasma membrane. Quantitation suggeststhat these events reflect the progression of an initially fusedand spherical granule flattening into the plane of the plasmamembrane as the Vamp-GFP simultaneously diffuses through thefusion junction. Approximately 10% of the events showed a transitionfrom puncta to ring-like structures coupled with little or nospreading. The ring-like images correspond quantitatively togranules fusing and retaining concavity (recess of $~$ 200 nm).A majority of fusion events involved granules that were presentin the evanescent field for at least 12 s. However, $~$ 20% of theevents involved granules that were present in the evanescentfield for no more than 0.3 s, indicating that the interactionof the granule with the plasma membrane that leads to exocytosiscan occur within that time. In addition, $~$ 10% of the exocytoticsites were much more likely to occur within a granule diameterof a previous event than can be accounted for by chance, suggestiveof sequential (piggy-back) exocytosis that has been observedin other cells. Overall granule behavior before and during fusionis strikingly similar to exocytosis previously described inthe constitutive secretory pathway.

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