The researchers used a special form of microscopy (total internal reflection fluorescence microscopy) capable of focusing solely on the narrow plane in which the vesicle and membrane merge. "It's a little like the guy looking under the streetlight for his keys because it's the only place he can see, but we've actually arranged for the streetlight to be focused on exactly where we're interested," Simon says. "We get the vesicles at the point of fusion without the background noise of everything else going on inside the cell."
For the first time, Jaiswal was able to observe individual post-Golgi vesicles as they deliver cargo in their lumen and cargo carried in the membrane shell that surrounds the vesicle. This ability revealed behaviors that were not expected. The researchers showed first that the most common delivery of post-Golgi cargo is a so-called kiss-and-run exchange in which the vesicle partially merges with the membrane and delivers some, but not all, of its contents. Some vesicles, those packing neurotransmitters, for instance, are mobilized by a flood of calcium to spill their haul, signaling nearby cells. By adjusting the levels of calcium inside the cell, the researchers definitively found that calcium did not affect constitutive exocytosis of post-Golgi vesicles. Then they successively inhibited three molecules known for their membrane-bending role in endocytosis -- clathrin, dynamin and actin. In the absence of any one of these molecules, the researchers found that the vesicles merged fully with the membrane and disgorged all their cargo, which they had shown was an aberration in exocytosis, not the rule, as had been previously assumed. Together, the experiments demonstrate that cells employ some of the same molecules for importing and exporting cargo. "They use the same machinery for both," Jaiswal says. "This blurs the line between endocytosis and exocytosis. Perhap
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