That has changed with this new paper. "We have taken advantage of recent advances in fluorescent 'tagging' of molecules involved in these cellular processes, as well as new microscopy technologies that give us an incredible new ability to watch all of this, up close and in real-time," says Dr. Ryan.
Specifically, Dr. Ryan used a fluorescent chemical stain called pHluorin and genetically fused it to a vesicular protein called vGlut1. "We've used this fluorescent tagging approach before, but with molecules that can exist on either the outside or the inside of the vesicle," Dr. Ryan notes.
"VGlut1 gives us a much more precise view, since it only inhabits the inside of the vesicle," he adds. "That means that when we see the green fluorescent tag move outside of the vesicle, then the vesicle itself must have ruptured in some way. This gives us a much more accurate picture of the recycling process."
At the same time, the team took advantage of new breakthroughs in optical microscopy that maximize how much of the tag's fluorescent light can be "grabbed" by the microscope. This approach allowed them, for the first time, to follow how individual synaptic molecules are delivered and retrieved from the synaptic surface.
"The result is an accurate view into this hitherto mysterious synaptic phenomenon," Dr. Ryan says.
The "resealable spout" hypothesis of vesicular recycling (also known as the "kiss-and-run" theory) may be the first casualty of this new insight.
"We observed that, although recycling appears to occur within a set but somewhat variable time-frame, it's still using the same mechanism -- the vesicle falls apart upon delivering its cargo to the cell membrane, and then enzymes go to work re-building it for the next cycle," Dr. Ryan adds. "I think this real-time observation really closes the door on the 'kiss-and-run' theory of vesicular recycling."
The new technology use
|Contact: Andrew Klein|
New York- Presbyterian Hospital/Weill Cornell Medical Center/Weill Cornell Medical College