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© The Rockefeller University Press, 0021-9525/2000//33 $5.00
The Journal of Cell Biology, Volume 149, Number 1, , 2000 33-40

Fusion of Constitutive Membrane Traffic with the Cell Surface Observed by Evanescent Wave Microscopy

^a Cell Biology/Biophysics Programme, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany
^b Max Planck Institute of Molecular Cell Biology and Genetics, D-01307 Dresden, Germany
^c Vollum Institute, Portland, Oregon 97210
Cell Biology/Biophysics Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.49-6221-38751249-6221-387334

simons{at}embl-heidelberg.de

Monitoring the fusion of constitutive traffic with the plasma membrane has remained largely elusive. Ideally, fusion would be monitored with high spatial and temporal resolution. Recently, total internal reflection (TIR) microscopy was used to study regulated exocytosis of fluorescently labeled chromaffin granules. In this technique, only the bottom cellular surface is illuminated by an exponentially decaying evanescent wave of light. We have used a prism type TIR setup with a penetration depth of 50 nm to monitor constitutive fusion of vesicular stomatitis virus glycoprotein tagged with the yellow fluorescent protein. Fusion of single transport containers (TCs) was clearly observed and gave a distinct analytical signature. TCs approached the membrane, appeared to dock, and later rapidly fuse, releasing a bright fluorescent cloud into the membrane. Observation and analysis provided insight about their dynamics, kinetics, and position before and during fusion. Combining TIR and wide-field microscopy allowed us to follow constitutive cargo from the Golgi complex to the cell surface. Our observations include the following: (1) local restrained movement of TCs near the membrane before fusion; (2) apparent anchoring near the cell surface; (3) heterogeneously sized TCs fused either completely; or (4) occasionally larger tubular-vesicular TCs partially fused at their tips.

Key Words: exocytosis • membrane fusion • green fluorescent protein • total internal reflection • docking

© 2000 The Rockefeller University Press

Abbreviations used in this paper: EPI, epi-illuminated; GFP, green fluorescent protein; TCs, transport containers; TIR, total internal reflection; VSVG3-SP-YFP, YFP-tagged vesicular stomatitis virus glycoprotein; YFP, yellow fluorescent protein.

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