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SYMPOSIUM REPORT |
1 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK
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Abstract |
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= 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.
(Received 19 June 2007;
accepted after revision 26 June 2007;
first published online 28 June 2007)
Corresponding author L. Lagnado: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK. Email: ll1{at}mrc-lmb.cam.ac.uk
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Endocytosis in hippocampal synapses |
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Optical techniques applied to hippocampal neurons in culture have been interpreted as indicating that both fast and slow modes of endocytosis occur at synaptic boutons, but the molecular basis of these kinetically distinct mechanisms has been difficult to establish (Klingauf et al. 1998; Pyle et al. 2000; Aravanis et al. 2003b; Gandhi & Stevens, 2003; Royle & Lagnado, 2003). It is known that synaptic vesicles can collapse on fusion and the machinery for retrieving this collapsed membrane by clathrin-mediated endocytosis (CME) is enriched at many synapses (Takei et al. 1996). What has been less clear is the speed at which CME operates and its importance compared to other mechanisms of vesicle retrieval. In fact, it has been suggested that the large 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 (Aravanis et al. 2003b; Harata et al. 2006). A key feature of the ‘kiss-and-run’ model is that the vesicle is retrieved at the site of fusion before it has collapsed into the surface membrane (Fesce et al. 1994; Klingauf et al. 1998; Klyachko & Jackson, 2002; Aravanis et al. 2003b; Gandhi & Stevens, 2003; Richards et al. 2005; Harata et al. 2006).
The strongest evidence for the ‘kiss-and-run’ mode of recycling in hippocampal boutons derives from experiments investigating the behaviour of single vesicles, either by observing the loss of a membrane dye from the synaptic bouton (Aravanis et al. 2003b; Richards et al. 2005), or by using synaptopHluorin, a fluorescent protein that reports fusion and retrieval of vesicles (Gandhi & Stevens, 2003). The most commonly used membrane dye is FM1-43, which enters recycled vesicles from the extracellular medium when the synapse is stimulated. After washing the dye off, applying single action potential stimuli occasionally triggers the release of a small amount of FM1-43 from the synapse, but this amount varies from stimulus to stimulus (Aravanis et al. 2003a,b), as does the speed of loss (Richards et al. 2005). The larger events have been attributed to full collapse of the vesicle and the smaller events to partial loss of the dye through a fusion pore, envisioned to be less than 1 nm in diameter, which closes before all the dye has a chance to diffuse out of the vesicle (Aravanis et al. 2003a). Of course, another interpretation might be that all vesicles collapse but the amplitude of the signal varies because different vesicles contain different amounts of dye.
A second approach for investigating endocytosis in hippocampal synapses is to use synaptopHluorin, a genetically encoded reporter made by fusing pH-sensitive GFP to the synaptic vesicle protein synaptobrevin (Miesenbock et al. 1998). The fluorescence of synaptopHluorin increases when vesicles fuse, and then declines when they are re-acidified after endocytosis. SynaptopHluorin signals triggered by long trains of action potentials recover relatively slowly, with time constants of 15 s or more, but the responses to single action potentials are more variable (Atluri & Ryan, 2006; Granseth et al. 2006). A consistent feature of the fluorescence signal triggered by an action potential reported by Gandhi & Stevens (2003) was a fast decline occurring in less than 1 s, which they attributed to vesicle retrieval by ‘kiss-and-run’. This interpretation does not easily square with the speed of vesicle reacidification, which occurs with a time constant of 3–4 s (Atluri & Ryan, 2006; Granseth et al. 2006), and recent work using pHluorin-based reporters indicates that the fast decline in the signal is not attributable to endocytosis, but to the movement of the reporter out of the vesicle on fusion (Li & Murthy, 2001; Granseth et al. 2006).
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SypHy, an improved reporter of exocytosis and endocytosis, only detects a slow mode of endocytosis |
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We used sypHy to measure synaptic vesicle retrieval after single action potentials (APs), because this is the condition when fast kiss-and-run has been reported to be the predominant mode of fusion. To our surprise, we found that the fluorescence signal triggered by an action potential recovered in just one phase, which occurred with a time constant of 15–20 s at room temperature (Fig. 1 ; Granseth et al. 2006). Further, the speed of endocytosis was the same at synapses of high and low release probability and constant for a range of stimulus strengths up to 40 APs at 20 Hz, indicating the existence of just one, relatively slow, mode of vesicle retrieval over a range of conditions spanning the physiological activity of hippocampal synapses.
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r) is 3–4 s (10 mM Hepes; Atluri & Ryan, 2006). This information allowed us to compare our measurements with a model in which quenching of sypHy fluorescence was controlled by two consecutive and irreversible reactions with first-order kinetics: endocytosis (ke = 1/e) followed by reacidification (kr = 1/r). The black trace in Fig. 1B is a best-fit to this model with r = 4 s, which yields an estimate of e = 15.5 s. |
Endocytosis is clathrin dependent |
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15 s? The slow mode of endocytosis in retinal bipolar cells operates at a similar speed and has been shown to be dependent on clathrin and its accessory proteins (Jockusch et al. 2005). Is retrieval in hippocampal synapses also clathrin dependent? To investigate this question we inhibited CME using two different methods. The first was a dominant-negative approach entailing over-expression of the C-terminal fragment of AP180 – an accessory protein involved in CME (Ford et al. 2001). The second method was to deplete clathrin heavy chain (CHC) using RNA interference (Royle et al. 2005). Both manouveres strongly inhibited endocytosis of synaptic vesicles released by four APs (Granseth et al. 2006). The picture that emerges from these experiments is therefore relatively simple: after physiological levels of activity, CME with a time constant of about 15 s retrieves most, if not all, vesicles at small hippocampal synapses. |
Exocytosis is associated with the movement of vesicle proteins out of the synapse |
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Endocytosis is associated with the movement of clathrin out of the synapse |
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Two observations suggested that clathrin effectively ‘tracks’ the vesicle proteins leaving the synapse. First, the amount of LCa-mRFP leaving the synapse was proportional to the number of vesicles released by the stimulus, as assessed by the amplitude of the sypHy signal. Second, the movement of LCa-mRFP began without a significant delay and peaked with the sypHy signal. The movement of clathrin out of the synapse together with synaptophysin and synaptobrevin is most easily explained as representing CME of vesicles at sites removed from the active zone. This interpretation is consistent with studies showing that the machinery for CME is not at the active zone, but in the surrounding regions of membrane (Heuser & Reese, 1973; Ringstad et al. 1999; Qualmann et al. 2000; Teng & Wilkinson, 2000).
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Conclusions |
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Author's present address |
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Footnotes |
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B. Granseth and B. Odermatt contributed equally to this work.
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References |
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Abstract
Endocytosis in hippocampal...
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