Dynamin-dependent Membrane Drift Recruits AMPA Receptors to Dendritic Spines

The surface expression and localization of AMPA receptors (AMPARs) at dendritic spines are tightly controlled to regulate synaptic transmission. Here we show that de novo exocytosis of the GluR2 AMPAR subunit occurs at the dendritic shaft and that new AMPARs diffuse into spines by lateral diffusion in the membrane. However, membrane topology restricts this lateral diffusion. We therefore investigated which mechanisms recruit AMPARs to spines from the shaft and demonstrated that inhibition of dynamin GTPase activity reduced lateral diffusion of membrane-anchored green fluorescent protein and super-ecliptic pHluorin (SEP)-GluR2 into spines. In addition, the activation of synaptic N-methyl-d-aspartate (NMDA) receptors enhanced lateral diffusion of SEP-GluR2 and increased the number of endogenous AMPARs in spines. The NMDA-invoked effects were prevented by dynamin inhibition, suggesting that activity-dependent dynamin-mediated endocytosis within spines generates a net inward membrane drift that overrides lateral diffusion barriers to enhance membrane protein delivery into spines. These results provide a novel mechanistic explanation of how AMPARs and other membrane proteins are recruited to spines by synaptic activity.AMPA³ receptors (AMPARs) are of fundamental importance because they mediate the majority of fast excitatory synaptic transmission in the mammalian central nervous system (1). Most excitatory synapses are characterized morphologically by dendritic spines that contain an electron-dense postsynaptic density (PSD) at their head (2, 3). PSD is highly enriched in AMPARs and associated proteins equired for synaptic transmission and signal transduction (4-6). Activity-evoked changes in functional postsynaptic AMPARs mediate the two main forms of synaptic plasticity believed to underlie learning and memory in the hippocampus (7). Long term potentiation involves the activity-dependent recruitment of AMPARs to the postsynaptic membrane and a concurrent increase in AMPA-mediated transmission, whereas long term depression is a decrease in synaptic AMPAR function (8).The number and subunit composition of synaptic AMPARs are stringently regulated, but despite intense investigation, the processes by which AMPARs are delivered to and retained at the PSD remain controversial. Using photoreactive antagonists and electrophysiology, it has been proposed that AMPARs are only inserted in the plasma membrane at the cell body and laterally diffuse long distances to synapses (9). In direct contrast, approaches using real-time imaging have suggested that AMPARs are inserted in the plasma membrane of the dendritic shaft close to, but not at, dendritic spines (10). It has also been suggested that AMPARs could be inserted directly into the plasma membrane of the PSD (11).Independent of the route of delivery for new AMPARs to synapses, it is well established that lateral diffusion in the plasma membrane allows the exchange of receptors in and out of the PSD (12-14). Using palmitoylated membrane-anchored GFP (mGFP), which partitions to the inner leaflet of the plasma membrane, it has also been reported that diffusion is significantly retarded within spines compared with the shaft and that AMPAR activation increases the rates of mGFP diffusion in spines (15). In addition, we have shown previously that membrane protein movement into and out of spines is slow compared with lateral diffusion on non-spiny membrane (16), and modeling studies have predicted that spine length is a major determinant of the time a protein takes to reach the PSD (17). More recently, it has been proposed that endocytosis at specialized endocytic zones close to the PSD within spines is required to maintain the steady state complement of synaptic AMPARs (18).Taken together these findings suggest that endocytosis and exocytosis as well as lateral diffusion and membrane topology may all play important roles in regulating membrane protein mobility in spines. The interrelationships between these processes, however, remain unclear. Here we used FRAP (fluorescence recovery after photobleaching) and multisite FLIP (fluorescence loss in photobleaching) to visualize super-ecliptic pHluorin-tagged GluR2 surface expression and AMPAR movement in real time. We examined how lateral diffusion is regulated in spines both by blocking dynamin GTPase activity and stimulating NMDARs. Combined with Monte Carlo simulations on lattices fitting theoretical spines, our data indicate that the membrane topology of spines alone is sufficient to constrain lateral diffusion. NMDAR activation facilitates AMPAR recruitment to spines by a process that involves the recruitment of plasma membrane, together with the constituent membrane proteins, from adjacent regions of the dendritic shaft being drawn into the spine to replace membrane that is internalized during endocytosis. In other words, our results suggest a mode of lateral diffusion that is neither free nor anomalous. Rather, we show the directional diffusion of membrane-embedded proteins toward the postsynapse driven by the endocytosis within the spine. These results provide a new mechanistic explanation of how synaptic activity can overcome topology-induced diffusion barriers to recruit new membrane proteins to the spine.