Clathrin-mediated endocytosis: the physiological mechanism of vesicle retrieval at hippocampal synapses

Abstract

The maintenance of synaptic transmission requires that vesicles are recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, but the relative importance of these has been controversial. It is established that synaptic vesicles can collapse on fusion and the machinery for retrieving this membrane by clathrin-mediated endocytosis (CME) is enriched in the presynaptic terminal. But it has also been suggested that the majority of vesicles released by physiological stimulation are recycled by a second, faster mechanism called 'kiss-and-run', which operates in 1 s or less to retrieve a vesicle before it has collapsed. The most recent evidence argues against the occurrence of 'kiss-and-run' in hippocampal synapses. First, an improved fluorescent reporter of exocytosis (sypHy), indicates that only a slow mode of endocytosis (tau = 15 s) operates when vesicle fusion is triggered by a single nerve impulse or short burst. Second, this retrieval mechanism is blocked by overexpressing the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi. Third, vesicle fusion is associated with the movement of clathrin and vesicle proteins out of the synapse into the neighbouring axon. These observations indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of retrieval in small hippocampal synapses.

Figure 1. Calculating the rate of endocytosis from the decay in sypHy fluorescence

A, schematic illustration of the processes that regulate the time course of signals obtained using intravesicular pHluorins. 1: the pHluorin is quenched in the acidic interior of the vesicle. 2: fusion immediately causes the pHluorin to become fluorescent (state A), and it remains fluorescent after vesicle scission from the surface (state B). 3: the pHluorin is quenched as the vesicle interior becomes acidified (state C). The transitions A → B → C can be modelled as two consecutive reactions with first-order kinetics with time constants τ_e for endocytosis and τ_r for reacidification. The equations show the average time course of the fluorescence signal (F) at time t after vesicle fusion. B, a single exponential curve fit (red) with τ= 22 s provided a relatively good description of the decay in sypHy fluorescence after a single AP measured with a large ROI (green). A better fit for the initial phase of the decay was obtained by the model in A (black) using a measured τ_r= 4 s, yielding τ_e= 16 s. The light blue trace models the situation in which 80% of vesicles are taken up with τ_e1= 1 s, and the remainder with τ_e2= 16 s, both populations being reacidified with τ_r= 4 s. Reprinted from Granseth et al. (2006), Neuron 51, 773–786, with permission from Elsevier.

Figure 2. Clathrin, synaptobrevin and synaptophysin move out of the synapse on stimulation

A, example images obtained from neurons cotransfected with sypHy and either mRFP-synaptobrevin, synaptophysin-mRFP or LCa-mRFP (top), together with a difference image showing the change in distribution caused by a train of 40 APs (middle). Synapses were identified by sypHy responses (bottom). Synaptobrevin and clathrin leave the synapse and accumulate in neighbouring regions. Red lines indicate sites of mRFP decrease; note the colocalization of these sites with exocytic zones. B, average time course of the change in mRFP fluorescence in response to 4, 40 and 400 APs at 20 Hz. The sypHy response is in black. Reprinted from Granseth et al. (2006), Neuron 51, 773–786, with permission from Elsevier.