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

doi:10.1083/jcb.149.1.33

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© The Rockefeller University Press, 0021-9525/2000/4/33/ $5.00
The Journal of Cell Biology, Volume 149, Number 1, April 3, 2000 33-40

Original Article

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

Derek Toomre^a,b, Jürgen A. Steyer^c, Patrick Keller^a,b, Wolfhard Almers^c, and Kai Simons^a,b
^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

Correspondence to: Kai Simons, Cell Biology/Biophysics Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Tel:49-6221-387334 Fax:49-6221-387512 E-mail:simons{at}embl-heidelberg.de.

Monitoring the fusion of constitutive traffic with the plasmamembrane has remained largely elusive. Ideally, fusion wouldbe monitored with high spatial and temporal resolution. Recently,total internal reflection (TIR) microscopy was used to studyregulated exocytosis of fluorescently labeled chromaffin granules.In this technique, only the bottom cellular surface is illuminatedby an exponentially decaying evanescent wave of light. We haveused a prism type TIR setup with a penetration depth of ~50nm to monitor constitutive fusion of vesicular stomatitis virusglycoprotein tagged with the yellow fluorescent protein. Fusionof single transport containers (TCs) was clearly observed andgave a distinct analytical signature. TCs approached the membrane,appeared to dock, and later rapidly fuse, releasing a brightfluorescent cloud into the membrane. Observation and analysisprovided insight about their dynamics, kinetics, and positionbefore and during fusion. Combining TIR and wide-field microscopyallowed us to follow constitutive cargo from the Golgi complexto the cell surface. Our observations include the following:(1) local restrained movement of TCs near the membrane beforefusion; (2) apparent anchoring near the cell surface; (3) heterogeneouslysized TCs fused either completely; or (4) occasionally largertubular-vesicular TCs partially fused at their tips.

Key Words: exocytosis, membrane fusion, green fluorescent protein, totalinternal reflection, docking

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