Fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy

Monitoring the fusion of constitutive traffic with the plasma membrane has remained largely elusive. Ideally, fusion would be monitored with high spat ial and temporal resolution. Recently, total internal reflection (TIR) micr oscopy was used to study regulated exocytosis of fluorescently labeled chro maffin granules. In this technique, only the bottom cellular surface is ill uminated by an exponentially decaying evanescent wave of light. We have use d a prism type TIR setup with a penetration depth of similar to 50 nm to mo nitor 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 a pproached the membrane, appeared to dock, and later rapidly fuse, releasing a bright fluorescent cloud into the membrane. Observation and analysis pro vided insight about their dynamics, kinetics, and position before and durin g fusion, Combining TIR and wide-field microscopy allowed us to follow cons titutive cargo from the Golgi complex to the cell surface. Our observations include the following: (1) local restrained movement of TCs near the membr ane before fusion; (2) apparent anchoring near the cell surface; (3) hetero geneously sized TCs fused either completely; or (4) occasionally larger tub ular-vesicular TCs partially fused at their tips.