How receptors insert into cells
Scientists have observed a new mechanism of insertion for receptors in hippocampal neurons they term 'kiss and wait,' in which receptors emerge at the surface of the cell and appear to pause for up to thirty seconds before spreading laterally, according to a study appearing this week in Nature Neuroscience. The images produced in the study are the first to show individual trafficking events of receptor insertion into the plasma membrane of any cell type.
"We're proposing, although this remains for further study, that these events then represent a distinct mode of membrane protein insertion that can occur in the plasma membrane," lead author Mark von Zastrow at the University of California, San Francisco, told The Scientist.
Until now, scientists have struggled to elucidate the details of receptor insertion, because fluorescence techniques often have too high a signal-to-noise ratio to image single insertions. To overcome this technical obstacle, von Zastrow's team used total internal reflection fluorescence (TIRF) microscopy, along with a pH-sensitive variant of GFP that could fluoresce at the plasma membrane, but became silenced when exposed to an acidic environment during endocytosis. The researchers fused this GFP variant to the extracellular domain of the human ß2 adrenergic receptor, and monitored receptor insertions in pyramidal neurons exposed to an adrenergic agonist.
They observed that the receptor appeared on the membrane via two different mechanisms -- a transient mechanism in which the receptor remains on the surface for several seconds, and a persistent or mode, which lasts for tens of seconds before the receptors spread laterally throughout the membrane. Receptor activation inhibited the transient insertion events, but enhanced the persistent events. In addition, the frequency of transient events increased in response to a protein kinase A inhibitor, whereas the persistent events did not.
The benefit of TIRF is that it enables researchers to observe events very close to the cell surface, without interferences from light, von Zastrow noted -- a "critical" feature, he added, given that light from a single, inserting vesicle is very low. The fact that the two modes of insertion respond differently to receptor activation could be a basis for activity-dependent control, he noted, and the cell could be "switching" the signal from one pathway to another in response to prolonged receptor activation. Although the idea remains speculative, the theory could mean a "completely new level of plasticity in neural signaling."
Tim Ryan of Weill Medical College of Cornell University, who did not participate in the study, said he agreed that the researchers' method was critical to finding their result. "The techniques are now allowing people to look at things directly happening at the membrane?This is the first time someone has been able to visualize this class of receptor dynamics." Still, Ryan noted that it is difficult to gauge the implications of the new mode of insertion without knowing if the cell signaled differently during the persistent state.
Roberto Malinow of Cold Spring Harbor Laboratory in New York, also not a co-author, also cautioned against taking too much stock in data from dissociated cells. Specifically, "there is an issue about how much the mechanisms that are identified in dissociated culture cells are applicable to neurons in the brain," he told The Scientist.
Von Zastrow said the fact that protein kinase A inhibitor and receptor activation affect insertion into the membrane suggests this is a tightly regulated process, a new idea in receptor research. "What we've learned over the past ten years is that receptors are actually very dynamic proteins."
Links within this article
G. Yudowski et al., "Distinct modes of regulated receptor insertion to the somatodendritic plasma membrane," Nature Neuroscience, April 9, 2006.
Mark von Zastrow
"How it works: Optical trap," The Scientist, August 29, 2005.
Timothy A. Ryan