Vesicles fusing with plasma membrane in adrenal chromaffin cells show up as bright spots with interference reflection microscopy.
The phrase “kiss and run” is used to refer to the retrieval of a whole vesicle back into the cell after fusion with the plasma membrane. Although kiss and run occurs in fast fusion vesicles, where the pore opening is very small, slow fusion vesicles, which open wide, were thought to collapse into the plasma membrane. To get a closer look at vesicle dynamics, the authors turned to interference reflection microscopy (IRM), which allows direct visualization of membrane deflections of as little as ten nanometers. They attached adrenal chromaffin cells—a favorite model cell type for vesicle release studies—to a coverslip, and watched as individual vesicles fused with the plasma membrane. Each fusion event caused a deformation of the membrane, visible as a bright spot due to a change in the distance between membrane and coverslip.
The authors showed that each spot on the surface quickly brightened, in keeping with a dilation of the vesicle mouth after fusion, and then gradually dimmed. “We call it ‘roaring,’” PI Leon Lagnado says, “since its behavior is reminiscent of the MGM lion.” The team's movies of vesicle opening dynamics confirmed previous results that both the speed of opening and the ultimate size of the pore were calcium dependent, with lower concentrations causing faster and smaller openings.
When they treated vesicles with strontium to prevent them from closing, the bright spots remained, rather than disappearing as would be expected if the vesicle collapsed into the membrane. And when they inhibited dynamin, which promotes vesicle scission, vesicles could still close even though their departure from the membrane was blocked, indicating that vesicle closure is regulated independently from scission.
“I suspect this process is common to other hormone-secreting cells,” Lagnado says, which in general use similar large vesicles, but it is unlikely to be seen in neurotransmitter-releasing vesicles, which are much smaller and which are known to collapse into the plasma membrane. The direct visualization allowed by IRM should be useful for answering a wide variety of other outstanding questions about vesicle fusion. RR
