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The debate on the kiss-and-run fusion at synapses
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The debate on the kiss-and-run fusion at synapses Liming He and Ling-Gang Wu National Institute of Neurological Disorders and Stroke, 35 Convent Drive, Bldg 35, Rm. 2B-1012, Bethesda, MD 20892, USA
It has long been proposed that following vesicle fusion, a small pore might open and close rapidly without full dilation. Such ‘kiss-and-run’ vesicle fusion can in principle result in rapid vesicle recycling and influence the size and the kinetics of the resulting synaptic current. However, the existence of kiss-and-run remains highly controversial, as revealed by recent imaging and electrophysiological studies at several synapses, including hippocampal synapses, neuromuscular junctions and retinal bipolar synapses. Only a minor fraction of fusion events has been shown to be kiss-and-run, as determined using cell-attached capacitance recordings in endocrine cells, pituitary nerve terminals and calyx-type synapses. Further work is needed to determine whether kiss-and-run is a major mode of fusion and has a major role in controlling synaptic strength at synapses. Introduction More than three decades ago, Heuser and Reese [1,2] discovered that vesicle fusion is followed by full collapse of the vesicle membrane into the plasma membrane and vesicle retrieval at a location distal to the fusion site (Figure 1a). This form of fusion is called ‘full collapse fusion’. In addition, Ceccarelli and his co-workers [3,4] proposed that vesicle fusion involves the opening of a small pore, termed the ‘fusion pore’ [5,6], followed by its rapid closure without full dilation and collapse at the same site of fusion (Figure 1b). This model, referred to as ‘kiss-and-run’ [7] was based mainly on the electron microscopic observation of the uncoated omega membrane profile with a narrow neck connected to the plasma membrane at the active zone of the frog neuromuscular junction [3,4] (Figure 1b). Compared with full collapse fusion, kissand-run might provide two advantages. First, it enables rapid and economical vesicle recycling. Second, its narrow fusion pore could limit the rate of transmitter discharge out of the vesicle, resulting in a slower and smaller quantal response (Figure 1d,f) compared with full collapse fusion (Figure 1c,e). Switching between kiss-and-run and full collapse fusion might thus provide a mechanism to regulate synaptic strength and achieve synaptic plasticity [8]. The evidence for the existence of full collapse fusion is compelling, whereas the electron microscopic evidence for the existence of kiss-and-run is not [2] because it is unclear whether the omega membrane profile (Figure 1b) in the fixed tissue is on the way to collapsing into the plasma Corresponding author: Wu, L.-G. ([email protected]). Available online 31 August 2007. www.sciencedirect.com
membrane or to closing its neck. In the past decade, imaging and electrophysiological techniques have been developed to examine vesicle exo- and endocytosis at live synapses. Strong electrophysiological evidence supporting kiss-and-run fusion has been obtained at endocrine cells. Imaging data supporting the existence of kiss-and-run at synapses are mounting. This seems to have led to a general acceptance of kiss-and-run at synapses, as reflected in at least some of the literature. However, evidence against kiss-and-run as a major fusion mechanism has also accumulated. This has raised the question of whether kiss-andrun exists and is a major form of fusion at synapses. Here, we review both sides of the experimental evidence for live endocrine cells and four different synapses, including calyx of Held synapses, neuromuscular junctions, goldfish retinal bipolar synapses and cultured hippocampal synapses. We consider that kiss-and-run exists at synapses; however, the question remains open as to whether kiss-and-run is a major mode of fusion that can rapidly recycle vesicles and control quantal response at many synapses. Endocrine cells: capacitance measurements support kiss-and-run Membrane capacitance is linearly proportional to the membrane area [9]. When a vesicle fuses with the plasma membrane, it causes an increase of the plasma membrane area and thus a capacitance increase [10,11]. Similarly, vesicle retrieval from the plasma membrane causes a capacitance decrease [10,11]. Sequential capacitance upand down-steps with the same amplitude and separated by a short interval (less than a few seconds), referred to as capacitance flickers [12], have been observed in many endocrine cells, such as mast cells and chromaffin cells (Figure 2) [11–15]. A capacitance flicker reflects a single vesicle fusion and retrieval. It is often accompanied by a detectable fusion pore conductance, analogous to the conductance of a channel [16]. The pore conductance is 200–500 pS, which might correspond to a fusion pore with a diameter of 2–3 nm [16]. Depending on the size and the opening duration of the fusion pore, transmitter is discharged either partially or completely during a capacitance flicker, as revealed by the amperometric recording, an electrochemical detection of transmitter release using a carbon fiber electrode [13–15,17]. Although capacitance flickers are not the dominant form of fusion in most recording conditions [11–15], these results provide strong evidence indicating the existence of kiss-and-run fusion in endocrine cells.
0166-2236/$ – see front matter . Published by Elsevier Ltd. doi:10.1016/j.tins.2007.06.012
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Figure 1. Two modes of vesicle fusion. (a) (i) A schematic drawing of full collapse fusion, which involves the opening of the fusion pore and collapse of the vesicle membrane into the plasma membrane. (ii) An electron microscopic image of a frog neuromuscular junction, which was frozen at 5 ms after stimulation. Arrows show full collapse of the vesicle membrane to the plasma membrane. Adapted, with permission, from Ref. [65]. (b) (i) A schematic drawing of a kiss-and-run fusion, which involves the opening of a narrow fusion pore, followed by rapid pore closure. (ii) An electron microscopic image of a frog neuromuscular junction showing a horseradish peroxidase-darkened vesicle connected to the plasma membrane (arrow). The preparation was stimulated for two hours at 2 Hz in the presence of horseradish peroxidase. This image was interpreted as a vesicle about to pinch off from the plasma membrane. Adapted, with permission, from Ref. [5]. (c,d) Simulation of the miniature excitatory postsynaptic current (mEPSC) caused by a full collapse (c) and a kiss-and-run (d) fusion. The initial fusion pore diameter is assumed to be more than 10 nm for full collapse fusion (c) but 1 nm for kiss-and-run fusion (d). The simulation was performed with MCell 2.5, a program that models the three-dimensional random walk diffusion and reaction kinetics in complex spatial environments reflecting realistic cellular ultrastructure. The synapse in the model was a glutamatergic synapse containing postsynaptic a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The values of the initial fusion pore diameters were taken from estimates at the calyx-type synapse [31]. (e,f) Most measured mEPSCs had a rapid rise time [(e), three examples], probably caused by full collapse fusion. About 1.1% of measured mEPSCs had a slower rise time and a smaller amplitude [(f), three examples]. Kiss-and-run fusion provides an explanation for these slow and small mEPSCs. Data in (e) and (f) were from the same calyx of Held synapse. Panels (c–f) were adapted, with permission, from Ref. [31].
Figure 2. Kiss-and-run fusion in a rat chromaffin cell. (a) A schematic drawing showing the cell-attached capacitance recording combined with patch amperometry (carbon fiber electrode) to study vesicle fusion. (b) A kiss-and-run event revealed by the cell-attached capacitance recording combined with patch amperometry at a rat chromaffin cell. (i) Diagram of a vesicle diagram illustrating the processes occurring in the traces (ii) before, during and after the kiss-and-run event. Im and Re, the imaginary and the real components of the admittance, reflect the capacitance and the conductance, respectively. From the Im and Re trace, the fusion pore conductance (Gp) was calculated based on the equation Gp = (Re2 + Im2)/Re. The amperometric signal (A) was detected with a carbon fiber electrode inside the patch pipette. The capacitance flicker with a measured fusion pore conductance indicates kiss-and-run fusion. The amperometric signal suggests that kiss-and-run can release transmitter. Adapted, with permission, from Ref. [15]. www.sciencedirect.com
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In calf chromaffin cells, it has been suggested that rapid endocytosis, retrieval of vesicle membrane in a few seconds after exocytosis, is a result of clathrin-independent, kissand-run fusion and retrieval [18]. By contrast, slow endocytosis, retrieval of vesicle membrane in tens of seconds after exocytosis, is mediated by a clathrin-dependent mechanism after full collapse fusion [19,20]. Sustained stimulation, which increases the intracellular calcium concentration, shifts rapid endocytosis to slow endocytosis [21]. Raising the intracellular calcium concentration from 10 mM to 200 mM increases the incidence of full collapse fusion at the expense of kiss-and-run fusion [18]. These results suggest that increasing the intracellular calcium concentration inhibits kiss-and-run in calf chromaffin cells. However, in rat chromaffin cells, increasing the extracellular calcium concentration increases the contribution of kiss-and-run fusion instead, implying that intracellular calcium facilitates kiss-and-run fusion [15]. The species difference might explain this apparent discrepancy [15,18]. Alternatively, raising the extracellular calcium concentration might facilitate kiss-and-run by a mechanism not related to the increase in the intracellular calcium concentration. Calyx of Held synapse: cell-attached capacitance measurements support kiss-and-run The calyx of Held is a large, glutamatergic nerve terminal containing 600 conventional active zones [22]. Release at this nerve terminal is triggered by action potentials. Whole-cell recordings of the capacitance from this whole nerve terminal provide the fastest time resolution to study endocytosis at central synapses [23]. In an early study, the capacitance jump after one to two action potential-like stimuli decayed with a time constant of 100–300 ms, which was interpreted as rapid endocytosis [24]. This interpretation might have significantly overestimated rapid endocytosis because the transient capacitance decay was contaminated by a calcium-dependent, but exocytosisindependent, capacitance transient with a time constant of 200 ms [25,26]. In two later studies of endocytosis after an action potential-like stimulus, one reported a time constant of 10 s after a capacitance jump of 50–60 fF [25], whereas the other reported a time constant of 2.2 s (ranging from less than 0.5 s to 10 s) after a capacitance jump of 20 fF [26]. The discrepancy might have been partly the result of the difference in the capacitance jump because when the capacitance jump increased from 20 fF to 50 fF, the time constant increased from 2.2 s to 4.2 s [26]. Slight differences in the temperature, animal age, solution osmolarity [27] and slice condition could also have contributed to the discrepancy. In addition, by averaging many single fusion events from whole-cell recordings, an earlier study reported a time constant of 56 ms after a single vesicle fusion [24]. This estimate was recently found to be premature because, using a different method of baseline subtraction, no significant endocytosis with a time constant of 56 ms was found [28]. Regardless of the above discrepancies, a rapid capacitance decay with a time constant of 1–2 s was observed after various patterns of intense stimuli, including high frequency trains of action potential-like stimuli [26]. This www.sciencedirect.com
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rapid capacitance decay reflects rapid endocytosis, one of the features of kiss-and-run fusion and retrieval, because it can be blocked by a botulinum neurotoxin that blocks exocytosis [26] (Figure 3a). The signature of kiss-and-run is a transient fusion pore opening and closing, as detected with the cell-attached capacitance recording technique in endocrine cells [16]. Application of this technique to synapses has encountered two problems. First, the small size of vesicles makes it more difficult to resolve individual vesicle fusion. Second, the postsynaptic neuron apposing the presynaptic release site might prevent the electrode from accessing the release site. These two hurdles have been overcome [29–33]. In pituitary nerve terminals that do not form synapses, fusion of individual microvesicles with a similar size to synaptic vesicles has been resolved [30]. About 5% of fusion events are capacitance flickers with a fusion pore conductance of 19 pS, indicating the existence of kiss-and-run fusion [30]. At the calyx of Held synapse, the release site can be exposed by pulling out the postsynaptic neuron using a large pipette [31] (Figure 3b). At the exposed release sites, cell-attached recordings reveal individual capacitance up-steps, reflecting single vesicle fusion during high potassium application [31] (Figure 3c). A small fraction of fusion events are capacitance flickers [31] (Figure 3d). For most capacitance flickers, the fusion pore conductance is larger than 288 pS [31]. In a small fraction of capacitance flickers, however, a fusion pore conductance ranging from 15–288 pS, with a mean of 66 pS, is observed (Figure 3d), which might correspond to a fusion pore with a mean diameter of 1.1 nm [31]. The capacitance flicker duration ranges from 10 ms to 2 s, with a mean of 300 ms [31]. These results suggest that a minor fraction of fusion events are kiss-and-run during high potassium application. Although the cell-attached recording technique is considered to be the most powerful technique in resolving kissand-run fusion, it is technically very difficult to apply to conventional nerve terminals that are much smaller than the calyx of Held. Furthermore, it requires the formation of a tight seal over a small patch area. It is unclear whether such a procedure influences the incidence of kiss-and-run fusion. Neuromuscular junction: kiss-and-run is debated Kiss-and-run was first proposed at the neuromuscular junction [6]. Recent studies of kiss-and-run at this synapse mainly rely on the FM dye imaging technique. FM dyes are amphipathic molecules that can bind to the membrane but cannot penetrate it [34]. When FM dyes are applied to the extracellular solution during nerve stimulation, dye molecules bind to the membrane of fused vesicles. Subsequent retrieval of the fused vesicle membrane results in loading of the dye. After washout of the extracellular FM dyes, nerve stimulation triggers fusion of dye-loaded vesicles, enabling diffusion of the dye into the bath solution, a process called unloading [34]. Incomplete FM1-43 unloading in the presence of the protein kinase inhibitor staurosporine was first interpreted as kiss-and-run [35] but was later found to be caused by block of exocytosis at frog neuromuscular junctions [36]. In a
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Figure 3. Evidence in support of kiss-and-run at calyx-type synapses. (a) Rapid endocytosis at a calyx. The calcium current (ICa), membrane capacitance (Cm) and membrane conductance (Gm) at a calyx induced by ten pulses of 20 ms depolarization at 10 Hz at 2 min (black) and 14 min (gray) after break-in with a pipette containing 1 mM botulinum neurotoxin C at 33–34 8C. The block of the capacitance jump by botulinum neurotoxin C, which blocks exocytosis by cleaving soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) proteins, indicates that the capacitance jump is caused by exocytosis. Thus, the rapid capacitance decay after the jump reflects rapid endocytosis. Adapted, with permission, from Ref. [26]. (b) The procedure to perform cell-attached recordings at the release face of a calyx. A calyx associated with a postsynaptic neuron was first identified (i). The postsynaptic neuron was sucked and pulled away by a pipette (ii, iii), shown in (ii). Another pipette was positioned at the release face of the calyx membrane (iv) for cell-attached recordings, from which individual vesicle fusions, such as those shown in (c) and (d), were obtained. (c) Im (imaginary component of the admittance, reflecting capacitance) and Re (real component of the admittance, reflecting conductance) traces from a cell-attached recording during application of 25 mM KCl. The inset shows discrete up-steps reflecting single vesicle fusion. (d) Im, Re and the fusion pore conductance (Gp) during a capacitance flicker. The capacitance flicker with a detectable fusion pore conductance (i) indicates kiss-and-run fusion. A non-flicker up-step (ii) occurring 10 s later was not accompanied by detectable Re changes, suggesting a full collapse fusion with the fusion pore that is too fast and/or too large to resolve. Panels (b–d) are adapted, with permission, from Ref. [31].
mutant Drosophila neuromuscular junction lacking endophilin, a protein involved in clathrin-mediated endocytosis, no significant FM1-43 loading was found during stimulation [37] (Figure 4a). This defect was accompanied by depletion of 87% of vesicles in the nerve terminal. If there is no endocytosis, the remaining vesicles could only support release for 10 s during 10 Hz nerve stimulation [37]. However, release was maintained for 600 s at 10 Hz stimulation [37]. These results led to the suggestion that neurotransmission is maintained in endophilin mutants by rapid vesicle recycling through a kiss-and-run mode that prevents FM1-43 uptake but supports transmitter release [37]. A recent study of the same endophilin mutant [38] did not concur with the above results. In this study [38], FM143 loading was detected during stimulation (Figure 4b), and the quantal size was increased after stimulation, which would not be expected from a kiss-and-run fusion [38]. Although this study argued against kiss-and-run fusion, it did not exclude the possibility that kiss-andrun coexists with a slow form of endocytosis in endophilin mutants. Furthermore, it is unclear whether a slow form of endocytosis, with a small number of vesicles (13% of control), in endophilin mutants is sufficient to account for transmitter release for 600 s during 10 Hz stimulation, as reported in the first study [37]. In summary, although two studies disagree on the existence of kiss-and-run, both agree that kiss-and-run is not the dominant form of fusion [37,38]. www.sciencedirect.com
Goldfish retinal bipolar synapse: is rapid endocytosis caused by kiss-and-run fusion? Rapid endocytosis at synapses was first reported at a ribbon-type synapse, the goldfish retinal bipolar synapse, where release is triggered by graded potentials [39]. Rapid endocytosis at this synapse was originally thought to reflect kiss-and-run fusion. However, recent studies have challenged this idea. Evanescent field fluorescence microscopy has revealed that FM1-43 dye molecules loaded in single vesicles leave the vesicle within a few milliseconds completely during most or all exocytic events [40,41]. This result is consistent with full collapse exocytosis. The stimulation used to evoke exocytosis was a 6–500 ms depolarization [40,41], which has been shown to induce rapid endocytosis in other studies [42,43]. Thus, rapid endocytosis might follow full collapse exocytosis, and thereby is not a result of kiss-and-run. However, rapid diffusion of FM1-43 could also be the result of kiss-and-run with a fusion pore that enables rapid lipid exchange between the vesicle membrane and the plasma membrane. Testing this possibility in the future will therefore be important to determine whether rapid endocytosis follows full collapse or kiss-and-run fusion. With the use of interference reflection microscopy to image surface membrane in close contact with a glass coverslip, it has been suggested that vesicles rapidly collapse into the surface membrane during exocytosis evoked by a 500 ms depolarization [44]. In another study of
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Figure 4. Evidence in support of or against kiss-and-run at Drosophila neuromuscular junctions. (a) The nerve terminal of the neuromuscular junction in the wild type (w) (i), but not the mutant lacking endophilin [(ii), endo1], could be loaded with FM1-43 during high potassium-induced depolarization. The labeling intensity of boutons in the endo1 mutant did not exceed labeling of unstimulated endo1 synapses in FM1-43 for 15 min (iii). This result is in line with kiss-and-run fusion, which is too fast to enable FM dye uptake. Adapted, with permission, from Ref. [37]. (b) Both the nerve terminal of the neuromuscular junction in the wild type (wt) (i) and the mutant lacking endophilin [(ii), endo] could be loaded with FM1-43 during high potassium-induced depolarization. The insets show some labeled boutons on a larger scale. Labeled boutons can be unloaded by stimulation (not shown). These results are contrary to the results shown in (a). Adapted, with permission, from Ref. [38].
the same preparation, whole-cell capacitance recordings showed that a prolonged depolarization for several hundred milliseconds could induce rapid endocytosis [43]. Taken together, these results imply that rapid endocytosis follows full collapse fusion [44]. However, the imaging was taken only from the surface membrane in contact with the glass [44], whereas rapid endocytosis was detected from whole-cell capacitance recordings. Whether rapid endocytosis can also take place at the surface membranes in contact with a glass coverslip is unclear. Testing this possibility would be a key to ascertaining whether rapid endocytosis follows full collapse fusion. An electron microscopic study revealed that during spontaneous calcium action potentials or high potassium application, the uptake of the electron-dense marker ferritin was mostly found in large endosomes [45]. It is unclear whether these stimulation protocols induce rapid endocytosis. Nevertheless, this study raised the possibility that rapid endocytosis could be a result of bulk endocytosis, that is, retrieval of a piece of membrane larger than the size of a vesicle [45]. In summary, whether rapid endocytosis reflects kiss-and-run fusion remains an open question at goldfish retinal bipolar synapses. www.sciencedirect.com
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Cultured hippocampal synapses: kiss-and-run is highly debated Two sets of evidence support the existence of kiss-and-run at cultured hippocampal synapses. The first set is composed of three pieces of evidence, obtained by FM dye imaging [46–50]. First, vesicles loaded with FM2-10, a dye that dissociates from the membrane to the solution faster than FM1-43, were unloaded faster than FM1-43 during 10 Hz but not 1 Hz stimulation [46,51]. This result implied endocytosis that was too fast for FM1-43 to escape to the bath solution at 10 Hz stimulation. Second, single vesicles loaded with FM1-43 showed either partial (Figure 5a) or complete dye loss during stimulation [49]. The kinetics of partial dye loss was slower than that of complete dye loss [52]. Partial dye loss is consistent with a kiss-and-run mode of fusion that prevents lateral dye diffusion and is too fast for FM1-43 to escape completely to the bath solution. The duration of kiss-and-run has been estimated to be less than 1.4 s [49]. Third, partial dye loss predicts partial dye retention in vesicles. This prediction has been confirmed using a hydrophilic FM1-43 quencher, bromophenol blue [50] (Figure 5b). FM1-43 retention increased as the stimulation frequency decreased [50]. Based on the degree of dye retention, it was estimated that the contribution of kiss-and-run fusion ranges from more than 80% at 0.3 Hz to 50% at 30 Hz stimulation [50]. This conclusion is different from that of an earlier study from the same laboratory, which suggested kiss-and-run at 10 Hz but not at 1 Hz stimulation [46]. The reason for this discrepancy is unclear (but see later for possible explanations). The second set of evidence was obtained by imaging with synaptopHluorin, a pH-sensitive green fluorescence protein (pHluorin) fused with the luminal portion of the integral vesicular protein vesicle-associated membrane protein 2 (VAMP-2) [53]. SynaptopHluorin fluorescence is quenched inside the acidic environment of vesicles [53] but becomes unquenched (increases) following vesicle fusion that exposes synaptopHluorin to the alkaline environment [53]. The subsequent decay of the fluorescence signal reflects both the time course of endocytosis and reacidification of the endocytosed vesicle. Single vesicles labeled with synaptopHluorin show a transient fluorescence increase and decay after an action potential [54] (Figure 5c). When fluorescence is averaged, a fast fluorescence transient with a time constant of less than 1 s is evident [54] (Figure 5c). The pH buffer Tris, but not HEPES, which has a molecular size larger than Tris, slows vesicular reacidification and thus the transient fluorescence decay. These results suggest that kiss-and-run fusion might occur after single action potentials [54]. In addition, without using FM dyes or synaptopHluorin, a recent study suggested rapid vesicle reuse consistent with kiss-and-run fusion at hippocampal synapses [55]. This study is based on the principle that transmitter refilling depends on the vacuolar ATPase and vesicular reacidification [55]. Pharmacological block of the vacuolar ATPase or vesicular reacidification results in faster synaptic depression, as detected within 1 s during 20 Hz stimulation, implying rapid vesicle reuse [55]. However, more work is needed to demonstrate that rapid reuse is a result of kiss-and-run fusion.
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Figure 5. Imaging evidence in support of kiss-and-run at cultured hippocampal synapses. (a) (i) The images of a single FM1-43-labeled vesicle taken just before the third (1) and fourth (2) stimuli and just after the 620th (3) stimulus. Twenty stimuli were applied at 0.125 Hz, followed by 600 stimuli at 10 Hz. A fusion event after the third stimulus was considered to be followed by retrieval rapid enough to leave FM1-43 trapped in the vesicle. The remaining fluorescence was retained for 17 action potentials and then released with 600 action potentials. (ii) Corresponding fluorescence signal. DFmax is the total fluorescence of the labeled vesicle. Adapted, with permission, from Ref. [49]. (b) FM1-43 was retained in vesicles during 1 Hz (i) and 30 Hz (ii) stimulation. During repetitive stimulation, the fluorescence intensity of FM1-43-loaded vesicles decreased faster in the presence of 2 mM bromophenol blue (red symbols) than in its absence (black symbols). Bromophenol blue might enter synaptic vesicles during fusion and quenche FM1-43 if it remains there after one round of exo- and endocytosis. These results suggest rapid closure of the fusion pore and thus the presence of kiss-and-run. Abbreviations: BPB, Bromophenol blue; TRP, total recycling pool. Adapted, with permission, from Ref. [50]. (c) (i) The fluorescence signal of three individual vesicles labeled with synaptopHluorin in response to a single action potential. The transient signal is interpreted as kiss-and-run fusion and retrieval. (ii) The average fluorescence of 72 individual vesicles expressed with synaptopHluorin in response to a single action potential. The fitted curve (in black) has three decay components: (1) fast monoexponential process (t = 860 210 ms); (2) a slow, compensatory Gaussian process (t = 10.7 0.35 s); and (3) stranded t > 45 s. The fast component is interpreted as kissand-run. The size of the imaging area is not specified in this study. Adapted, with permission, from Ref. [54].
Although the above studies from several laboratories support the existence of kiss-and-run fusion, others have obtained evidence inconsistent with kiss-and-run using similar techniques. The evidence can be divided into two parts. First, FM2-10 preferentially stains the readily releasable pool of vesicles at frog neuromuscular junctions [56]; this provides an alternative interpretation for why unloading of FM2-10 is faster than that of FM1-43 at cultured hippocampal synapses [46]. As shown in goldfish retinal bipolar synapses, FM dyes diffuse more rapidly from the vesicle membrane to the plasma membrane than from the membrane to the bath solution [41]. Such a rapid lateral diffusion might complicate the interpretation of FM dye unloading, although whether it occurs at hippocampal synapses is unclear. Furthermore, differential unloading between FM2-10 and FM1-43, as observed in one laboratory [46], was not observed by another laboratory (Figure 6a) during similar frequencies (10–30 Hz) of stimulation [57]. This finding raises doubt on the observation of the differential unloading between the two FM dyes. The second set of evidence against kiss-and-run was obtained with pHluorin imaging. If the fast synaptopHluorin fluorescence transient (time constant less than 1 s; Figure 5c) observed after single action potentials is due to kiss-and-run, the time constant of vesicle reacidification should be less than 1 s. The time constant of vesicle reacidification was not measured after single action potentials [54] but was 3–5 s after a train of action potentials [58,59], raising the question of whether vesicle reacidification can be less than 1 s after single action potentials. More imporwww.sciencedirect.com
tantly, rapid postexocytosis lateral diffusion of synaptopHluorin occurs at hippocampal synapses [59–61]. When the imaging area is small (Figure 6b), rapid lateral diffusion beyond the imaging area results in a rapid fluorescence transient not related to kiss-and-run fusion [59] (Figure 6c). When the imaging area is large enough (Figure 6b) to include the space for lateral diffusion, the rapid fluorescence transient disappears (Figure 6c). When pHluorin is fused to another vesicle protein, synaptophysin (sypHy), only a slow fluorescence decay with a time constant of 15 s, which reflects slow endocytosis, is observed after 1–40 action potentials at 20 Hz stimulation [59] (Figure 6d). This is because sypHy has much less surface expression and lateral mobility than synaptopHluorin [59]. The slow fluorescence decay is blocked by overexpression of the C-terminal fragment of a clathrin adaptor protein, adaptor protein 180, or by knockdown of clathrin, suggesting that clathrin-mediated slow endocytosis is the major mechanism of vesicle retrieval [59]. This suggestion is further supported by two recent studies showing that vesicular proteins exocytosed and subsequently retrieved by endocytosis are not identical at hippocampal synapses [62,63]. These results argue against kiss-and-run as a major mechanism in physiological conditions, and suggest caution in interpreting the rapid synaptopHluorin transient as kiss-and-run fusion. However, they do not exclude the existence of kiss-and-run. Two examples are given below. First, if only a fraction of synaptic vesicles can undergo kiss-and-run fusion and retrieval, and if these vesicles can
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Figure 6. Imaging evidence against kiss-and-run at cultured hippocampal synapses. (a) FM2-10 and FM1-43 were destained to the same extent with a similar time constant (tFM2-10 = 21 s; tFM1-43 = 22 s) during a 900 action potential train at 10 Hz. A subsequent train of 900 action potentials at 30 Hz was applied to release any remaining dye, and fluorescence values normalized to the fluorescence lost during both stimulation rounds. These results argue against any significant kiss-and-run fusion. Adapted, with permission, from Ref. [57]. (b) SynaptopHluorin-labeled boutons showing a small (green, 1.6 mm square) and a large (red, 4.8 mm square) size of imaging area, which were used in panels (c) and (d). (c) Comparison of the average synaptopHluorin signal triggered by a single action potential in small (green) and large (red) imaging areas. Bold red trace is a scaled version to enable direct comparison of the kinetics of recovery, and can be described by a single exponential with t = 21 s. By contrast, the decay of the fluorescence signal from the small area (green) was biexponential, with tfast = 2 s (36%) and tslow = 20 s. These results suggest that the fast synaptopHluorin transient (green) is due to lateral diffusion of synaptopHluorin from a small to a large area. (d) Comparison of average sypHy signals triggered by a single action potential in small (green) and large (red) imaging areas. The bold red trace is a scaled version to enable direct comparison of the kinetics of recovery. Only a slow decay with a time constant of about 28 s was observed, arguing against any significant contribution of kiss-and-run after a single action potential. SypHy uses the same superecliptic pHluorin but fused to the second intravesicular loop of synaptophysin, instead of VAMP-2. SypHy has much less surface expression compared with synaptopHluorin attached to VAMP. Panels (b–d) were adapted, with permission, from Ref. [59].
also undergo full collapse fusion followed by clathrinmediated endocytosis, block of clathrin-mediated endocytosis for a long period will eventually block recycling of these vesicles once they are sorted to the clathrin-mediated pathway [64]. Thus, the block of endocytosis by long-term molecular manipulation of the clathrin-mediated pathway [59] might not necessarily exclude the existence of kissand-run. Second, because the decay of pHluorin fluorescence reflects both reacidification and endocytosis, and reacidification might take 3–5 s, a small number of kiss-and-run events would be difficult to detect [59]. Nevertheless, the conclusion that clathrin-mediated slow endocytosis is the dominant retrieval mechanism [59] is in sharp contrast to another recent study, which, based on FM dye retention in vesicles, suggests that kiss-and-run is the dominant retrieval mechanism under physiological conditions [50]. The discrepancy is difficult to reconcile with our current knowledge, perhaps because neither FM dye nor pHluorin imaging can provide unequivocal evidence of kiss-and-run. Concluding remarks and future research Cell-attached capacitance recordings reveal that a minor fraction of fusion events are kiss-and-run during high potassium application at the calyx of Held synapse. At this synapse, whether kiss-and-run occurs during more www.sciencedirect.com
physiological stimulation is an important question to address in the future. Studies using FM dye and pHluorin imaging are highly controversial regarding the existence and the dominance of kiss-and-run fusion at Drosophila neuromuscular junctions and cultured hippocampal synapses. Novel approaches that provide more direct evidence are needed to resolve the debate at these two synapses. Although rapid endocytosis occurs at goldfish retinal bipolar synapses, whether it is a result of kissand-run fusion remains an open question. Given the controversy of kiss-and-run at synapses, previous studies implying a role or invoking its molecular mechanism might require re-review when the controversy is resolved at the studied synapse. Once this controversy is resolved, we can begin to study the underlying molecular mechanism and the functional role of kiss-and-run. Acknowledgements We thank Ken Paradiso, David Nees and Ben McNeil for comments on the manuscript. This work was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program. The authors declare no competing financial interests.
References 1 Heuser, J.E. and Reese, T.S. (1973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 315–344
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29 Debus, K. and Lindau, M. (2000) Resolution of patch capacitance recordings and of fusion pore conductances in small vesicles. Biophys. J. 78, 2983–2997 30 Klyachko, V.A. and Jackson, M.B. (2002) Capacitance steps and fusion pores of small and large-dense-core vesicles in nerve terminals. Nature 418, 89–92 31 He, L. et al. (2006) Two modes of fusion pore opening revealed by cellattached recordings at a synapse. Nature 444, 102–105 32 Stanley, E.F. (1993) Single calcium channels and acetylcholine release at a presynaptic nerve terminal. Neuron 11, 1007–1011 33 Smith, S.M. et al. (2004) Recordings from single neocortical nerve terminals reveal a nonselective cation channel activated by decreases in extracellular calcium. Neuron 41, 243–256 34 Cochilla, A.J. et al. (1999) Monitoring secretory membrane with FM1– 43 fluorescence. Annu. Rev. Neurosci. 22, 1–10 35 Henkel, A.W. and Betz, W.J. (1995) Staurosporine blocks evoked release of FM1–43 but not acetylcholine from frog motor nerve terminals. J. Neurosci. 15, 8246–8258 36 Becherer, U. et al. (2001) Effects of staurosporine on exocytosis and endocytosis at frog motor nerve terminals. J. Neurosci. 21, 782–787 37 Verstreken, P. et al. (2002) Endophilin mutations block clathrinmediated endocytosis but not neurotransmitter release. Cell 109, 101–112 38 Dickman, D.K. et al. (2005) A slowed classical pathway rather than kiss-and-run mediates endocytosis at synapses lacking synaptojanin and endophilin. Cell 123, 521–533 39 Von Gersdorff, H. and Matthews, G. (1994) Inhibition of endocytosis by elevated internal calcium in a synaptic terminal. Nature 370, 652–655 40 Zenisek, D. et al. (2000) Transport, capture and exocytosis of single synaptic vesicles at active zones. Nature 406, 849–854 41 Zenisek, D. et al. (2002) A membrane marker leaves synaptic vesicles in milliseconds after exocytosis in retinal bipolar cells. Neuron 35, 1085–1097 42 Von Gersdorff, H. and Matthews, G. (1994) Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals. Nature 367, 735–739 43 Neves, G. et al. (2001) Calcium influx selects the fast mode of endocytosis in the synaptic terminal of retinal bipolar cells. Proc. Natl. Acad. Sci. U. S. A. 98, 15282–15287 44 Llobet, A. et al. (2003) Real-time measurement of exocytosis and endocytosis using interference of light. Neuron 40, 1075–1086 45 Paillart, C. et al. (2003) Endocytosis and vesicle recycling at a ribbon synapse. J. Neurosci. 23, 4092–4099 46 Klingauf, J. et al. (1998) Kinetics and regulation of fast endocytosis at hippocampal synapses. Nature 394, 581–585 47 Pyle, J.L. et al. (2000) Rapid reuse of readily releasable pool vesicles at hippocampal synapses. Neuron 28, 221–231 48 Sara, Y. et al. (2002) Fast vesicle recycling supports neurotransmission during sustained stimulation at hippocampal synapses. J. Neurosci. 22, 1608–1617 49 Aravanis, A.M. et al. (2003) Single synaptic vesicles fusing transiently and successively without loss of identity. Nature 423, 643–647 50 Harata, N.C. et al. (2006) Frequency-dependent kinetics and prevalence of kiss-and-run and reuse at hippocampal synapses studied with novel quenching methods. Neuron 49, 243–256 51 Ryan, T.A. et al. (1996) The timing of synaptic vesicle endocytosis. Proc. Natl. Acad. Sci. U. S. A. 93, 5567–5571 52 Richards, D.A. et al. (2005) Two modes of exocytosis at hippocampal synapses revealed by rate of FM1–43 efflux from individual vesicles. J. Cell Biol. 168, 929–939 53 Ryan, T.A. (2001) Presynaptic imaging techniques. Curr. Opin. Neurobiol. 11, 544–549 54 Gandhi, S.P. and Stevens, C.F. (2003) Three modes of synaptic vesicular recycling revealed by single-vesicle imaging. Nature 423, 607–613 55 Ertunc, M. et al. (2007) Fast synaptic vesicle reuse slows the rate of synaptic depression in the CA1 region of hippocampus. J. Neurosci. 27, 341–354 56 Richards, D.A. et al. (2000) Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals. Neuron 27, 551–559 57 Fernandez-Alfonso, T. and Ryan, T.A. (2004) The kinetics of synaptic vesicle pool depletion at CNS synaptic terminals. Neuron 41, 943–953
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58 Atluri, P.P. and Ryan, T.A. (2006) The kinetics of synaptic vesicle reacidification at hippocampal nerve terminals. J. Neurosci. 26, 2313–2320 59 Granseth, B. et al. (2006) Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51, 773–786 60 Sankaranarayanan, S. and Ryan, T.A. (2000) Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system. Nat. Cell Biol. 2, 197–204 61 Li, Z. and Murthy, V.N. (2001) Visualizing postendocytic traffic of synaptic vesicles at hippocampal synapses. Neuron 31, 593–605
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62 Wienisch, M. and Klingauf, J. (2006) Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical. Nat. Neurosci. 9, 1019–1027 63 Fernandez-Alfonso, T. et al. (2006) Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling. Neuron 51, 179–186 64 Harata, N.C. et al. (2006) Kiss-and-run and full-collapse fusion as modes of exo-cytosis in neurosecretion. J. Neurochem. 97, 1546–1570 65 Heuser, J.E. and Reese, T.S. (1981) Structural changes after transmitter release at the frog neuromuscular junction. J. Cell Biol. 88, 564–580
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The debate on the kiss-and-run fusion at synapses Liming He and Ling-Gang Wu National Institute of Neurological Disorders and Stroke, 35 Convent Drive, Bldg 35, Rm. 2B-1012, Bethesda, MD 20892, USA
It has long been proposed that following vesicle fusion, a small pore might open and close rapidly without full dilation. Such ‘kiss-and-run’ vesicle fusion can in principle result in rapid vesicle recycling and influence the size and the kinetics of the resulting synaptic current. However, the existence of kiss-and-run remains highly controversial, as revealed by recent imaging and electrophysiological studies at several synapses, including hippocampal synapses, neuromuscular junctions and retinal bipolar synapses. Only a minor fraction of fusion events has been shown to be kiss-and-run, as determined using cell-attached capacitance recordings in endocrine cells, pituitary nerve terminals and calyx-type synapses. Further work is needed to determine whether kiss-and-run is a major mode of fusion and has a major role in controlling synaptic strength at synapses. Introduction More than three decades ago, Heuser and Reese [1,2] discovered that vesicle fusion is followed by full collapse of the vesicle membrane into the plasma membrane and vesicle retrieval at a location distal to the fusion site (Figure 1a). This form of fusion is called ‘full collapse fusion’. In addition, Ceccarelli and his co-workers [3,4] proposed that vesicle fusion involves the opening of a small pore, termed the ‘fusion pore’ [5,6], followed by its rapid closure without full dilation and collapse at the same site of fusion (Figure 1b). This model, referred to as ‘kiss-and-run’ [7] was based mainly on the electron microscopic observation of the uncoated omega membrane profile with a narrow neck connected to the plasma membrane at the active zone of the frog neuromuscular junction [3,4] (Figure 1b). Compared with full collapse fusion, kissand-run might provide two advantages. First, it enables rapid and economical vesicle recycling. Second, its narrow fusion pore could limit the rate of transmitter discharge out of the vesicle, resulting in a slower and smaller quantal response (Figure 1d,f) compared with full collapse fusion (Figure 1c,e). Switching between kiss-and-run and full collapse fusion might thus provide a mechanism to regulate synaptic strength and achieve synaptic plasticity [8]. The evidence for the existence of full collapse fusion is compelling, whereas the electron microscopic evidence for the existence of kiss-and-run is not [2] because it is unclear whether the omega membrane profile (Figure 1b) in the fixed tissue is on the way to collapsing into the plasma Corresponding author: Wu, L.-G. ([email protected]). Available online 31 August 2007. www.sciencedirect.com
membrane or to closing its neck. In the past decade, imaging and electrophysiological techniques have been developed to examine vesicle exo- and endocytosis at live synapses. Strong electrophysiological evidence supporting kiss-and-run fusion has been obtained at endocrine cells. Imaging data supporting the existence of kiss-and-run at synapses are mounting. This seems to have led to a general acceptance of kiss-and-run at synapses, as reflected in at least some of the literature. However, evidence against kiss-and-run as a major fusion mechanism has also accumulated. This has raised the question of whether kiss-andrun exists and is a major form of fusion at synapses. Here, we review both sides of the experimental evidence for live endocrine cells and four different synapses, including calyx of Held synapses, neuromuscular junctions, goldfish retinal bipolar synapses and cultured hippocampal synapses. We consider that kiss-and-run exists at synapses; however, the question remains open as to whether kiss-and-run is a major mode of fusion that can rapidly recycle vesicles and control quantal response at many synapses. Endocrine cells: capacitance measurements support kiss-and-run Membrane capacitance is linearly proportional to the membrane area [9]. When a vesicle fuses with the plasma membrane, it causes an increase of the plasma membrane area and thus a capacitance increase [10,11]. Similarly, vesicle retrieval from the plasma membrane causes a capacitance decrease [10,11]. Sequential capacitance upand down-steps with the same amplitude and separated by a short interval (less than a few seconds), referred to as capacitance flickers [12], have been observed in many endocrine cells, such as mast cells and chromaffin cells (Figure 2) [11–15]. A capacitance flicker reflects a single vesicle fusion and retrieval. It is often accompanied by a detectable fusion pore conductance, analogous to the conductance of a channel [16]. The pore conductance is 200–500 pS, which might correspond to a fusion pore with a diameter of 2–3 nm [16]. Depending on the size and the opening duration of the fusion pore, transmitter is discharged either partially or completely during a capacitance flicker, as revealed by the amperometric recording, an electrochemical detection of transmitter release using a carbon fiber electrode [13–15,17]. Although capacitance flickers are not the dominant form of fusion in most recording conditions [11–15], these results provide strong evidence indicating the existence of kiss-and-run fusion in endocrine cells.
0166-2236/$ – see front matter . Published by Elsevier Ltd. doi:10.1016/j.tins.2007.06.012
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Figure 1. Two modes of vesicle fusion. (a) (i) A schematic drawing of full collapse fusion, which involves the opening of the fusion pore and collapse of the vesicle membrane into the plasma membrane. (ii) An electron microscopic image of a frog neuromuscular junction, which was frozen at 5 ms after stimulation. Arrows show full collapse of the vesicle membrane to the plasma membrane. Adapted, with permission, from Ref. [65]. (b) (i) A schematic drawing of a kiss-and-run fusion, which involves the opening of a narrow fusion pore, followed by rapid pore closure. (ii) An electron microscopic image of a frog neuromuscular junction showing a horseradish peroxidase-darkened vesicle connected to the plasma membrane (arrow). The preparation was stimulated for two hours at 2 Hz in the presence of horseradish peroxidase. This image was interpreted as a vesicle about to pinch off from the plasma membrane. Adapted, with permission, from Ref. [5]. (c,d) Simulation of the miniature excitatory postsynaptic current (mEPSC) caused by a full collapse (c) and a kiss-and-run (d) fusion. The initial fusion pore diameter is assumed to be more than 10 nm for full collapse fusion (c) but 1 nm for kiss-and-run fusion (d). The simulation was performed with MCell 2.5, a program that models the three-dimensional random walk diffusion and reaction kinetics in complex spatial environments reflecting realistic cellular ultrastructure. The synapse in the model was a glutamatergic synapse containing postsynaptic a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The values of the initial fusion pore diameters were taken from estimates at the calyx-type synapse [31]. (e,f) Most measured mEPSCs had a rapid rise time [(e), three examples], probably caused by full collapse fusion. About 1.1% of measured mEPSCs had a slower rise time and a smaller amplitude [(f), three examples]. Kiss-and-run fusion provides an explanation for these slow and small mEPSCs. Data in (e) and (f) were from the same calyx of Held synapse. Panels (c–f) were adapted, with permission, from Ref. [31].
Figure 2. Kiss-and-run fusion in a rat chromaffin cell. (a) A schematic drawing showing the cell-attached capacitance recording combined with patch amperometry (carbon fiber electrode) to study vesicle fusion. (b) A kiss-and-run event revealed by the cell-attached capacitance recording combined with patch amperometry at a rat chromaffin cell. (i) Diagram of a vesicle diagram illustrating the processes occurring in the traces (ii) before, during and after the kiss-and-run event. Im and Re, the imaginary and the real components of the admittance, reflect the capacitance and the conductance, respectively. From the Im and Re trace, the fusion pore conductance (Gp) was calculated based on the equation Gp = (Re2 + Im2)/Re. The amperometric signal (A) was detected with a carbon fiber electrode inside the patch pipette. The capacitance flicker with a measured fusion pore conductance indicates kiss-and-run fusion. The amperometric signal suggests that kiss-and-run can release transmitter. Adapted, with permission, from Ref. [15]. www.sciencedirect.com
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In calf chromaffin cells, it has been suggested that rapid endocytosis, retrieval of vesicle membrane in a few seconds after exocytosis, is a result of clathrin-independent, kissand-run fusion and retrieval [18]. By contrast, slow endocytosis, retrieval of vesicle membrane in tens of seconds after exocytosis, is mediated by a clathrin-dependent mechanism after full collapse fusion [19,20]. Sustained stimulation, which increases the intracellular calcium concentration, shifts rapid endocytosis to slow endocytosis [21]. Raising the intracellular calcium concentration from 10 mM to 200 mM increases the incidence of full collapse fusion at the expense of kiss-and-run fusion [18]. These results suggest that increasing the intracellular calcium concentration inhibits kiss-and-run in calf chromaffin cells. However, in rat chromaffin cells, increasing the extracellular calcium concentration increases the contribution of kiss-and-run fusion instead, implying that intracellular calcium facilitates kiss-and-run fusion [15]. The species difference might explain this apparent discrepancy [15,18]. Alternatively, raising the extracellular calcium concentration might facilitate kiss-and-run by a mechanism not related to the increase in the intracellular calcium concentration. Calyx of Held synapse: cell-attached capacitance measurements support kiss-and-run The calyx of Held is a large, glutamatergic nerve terminal containing 600 conventional active zones [22]. Release at this nerve terminal is triggered by action potentials. Whole-cell recordings of the capacitance from this whole nerve terminal provide the fastest time resolution to study endocytosis at central synapses [23]. In an early study, the capacitance jump after one to two action potential-like stimuli decayed with a time constant of 100–300 ms, which was interpreted as rapid endocytosis [24]. This interpretation might have significantly overestimated rapid endocytosis because the transient capacitance decay was contaminated by a calcium-dependent, but exocytosisindependent, capacitance transient with a time constant of 200 ms [25,26]. In two later studies of endocytosis after an action potential-like stimulus, one reported a time constant of 10 s after a capacitance jump of 50–60 fF [25], whereas the other reported a time constant of 2.2 s (ranging from less than 0.5 s to 10 s) after a capacitance jump of 20 fF [26]. The discrepancy might have been partly the result of the difference in the capacitance jump because when the capacitance jump increased from 20 fF to 50 fF, the time constant increased from 2.2 s to 4.2 s [26]. Slight differences in the temperature, animal age, solution osmolarity [27] and slice condition could also have contributed to the discrepancy. In addition, by averaging many single fusion events from whole-cell recordings, an earlier study reported a time constant of 56 ms after a single vesicle fusion [24]. This estimate was recently found to be premature because, using a different method of baseline subtraction, no significant endocytosis with a time constant of 56 ms was found [28]. Regardless of the above discrepancies, a rapid capacitance decay with a time constant of 1–2 s was observed after various patterns of intense stimuli, including high frequency trains of action potential-like stimuli [26]. This www.sciencedirect.com
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rapid capacitance decay reflects rapid endocytosis, one of the features of kiss-and-run fusion and retrieval, because it can be blocked by a botulinum neurotoxin that blocks exocytosis [26] (Figure 3a). The signature of kiss-and-run is a transient fusion pore opening and closing, as detected with the cell-attached capacitance recording technique in endocrine cells [16]. Application of this technique to synapses has encountered two problems. First, the small size of vesicles makes it more difficult to resolve individual vesicle fusion. Second, the postsynaptic neuron apposing the presynaptic release site might prevent the electrode from accessing the release site. These two hurdles have been overcome [29–33]. In pituitary nerve terminals that do not form synapses, fusion of individual microvesicles with a similar size to synaptic vesicles has been resolved [30]. About 5% of fusion events are capacitance flickers with a fusion pore conductance of 19 pS, indicating the existence of kiss-and-run fusion [30]. At the calyx of Held synapse, the release site can be exposed by pulling out the postsynaptic neuron using a large pipette [31] (Figure 3b). At the exposed release sites, cell-attached recordings reveal individual capacitance up-steps, reflecting single vesicle fusion during high potassium application [31] (Figure 3c). A small fraction of fusion events are capacitance flickers [31] (Figure 3d). For most capacitance flickers, the fusion pore conductance is larger than 288 pS [31]. In a small fraction of capacitance flickers, however, a fusion pore conductance ranging from 15–288 pS, with a mean of 66 pS, is observed (Figure 3d), which might correspond to a fusion pore with a mean diameter of 1.1 nm [31]. The capacitance flicker duration ranges from 10 ms to 2 s, with a mean of 300 ms [31]. These results suggest that a minor fraction of fusion events are kiss-and-run during high potassium application. Although the cell-attached recording technique is considered to be the most powerful technique in resolving kissand-run fusion, it is technically very difficult to apply to conventional nerve terminals that are much smaller than the calyx of Held. Furthermore, it requires the formation of a tight seal over a small patch area. It is unclear whether such a procedure influences the incidence of kiss-and-run fusion. Neuromuscular junction: kiss-and-run is debated Kiss-and-run was first proposed at the neuromuscular junction [6]. Recent studies of kiss-and-run at this synapse mainly rely on the FM dye imaging technique. FM dyes are amphipathic molecules that can bind to the membrane but cannot penetrate it [34]. When FM dyes are applied to the extracellular solution during nerve stimulation, dye molecules bind to the membrane of fused vesicles. Subsequent retrieval of the fused vesicle membrane results in loading of the dye. After washout of the extracellular FM dyes, nerve stimulation triggers fusion of dye-loaded vesicles, enabling diffusion of the dye into the bath solution, a process called unloading [34]. Incomplete FM1-43 unloading in the presence of the protein kinase inhibitor staurosporine was first interpreted as kiss-and-run [35] but was later found to be caused by block of exocytosis at frog neuromuscular junctions [36]. In a
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Figure 3. Evidence in support of kiss-and-run at calyx-type synapses. (a) Rapid endocytosis at a calyx. The calcium current (ICa), membrane capacitance (Cm) and membrane conductance (Gm) at a calyx induced by ten pulses of 20 ms depolarization at 10 Hz at 2 min (black) and 14 min (gray) after break-in with a pipette containing 1 mM botulinum neurotoxin C at 33–34 8C. The block of the capacitance jump by botulinum neurotoxin C, which blocks exocytosis by cleaving soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) proteins, indicates that the capacitance jump is caused by exocytosis. Thus, the rapid capacitance decay after the jump reflects rapid endocytosis. Adapted, with permission, from Ref. [26]. (b) The procedure to perform cell-attached recordings at the release face of a calyx. A calyx associated with a postsynaptic neuron was first identified (i). The postsynaptic neuron was sucked and pulled away by a pipette (ii, iii), shown in (ii). Another pipette was positioned at the release face of the calyx membrane (iv) for cell-attached recordings, from which individual vesicle fusions, such as those shown in (c) and (d), were obtained. (c) Im (imaginary component of the admittance, reflecting capacitance) and Re (real component of the admittance, reflecting conductance) traces from a cell-attached recording during application of 25 mM KCl. The inset shows discrete up-steps reflecting single vesicle fusion. (d) Im, Re and the fusion pore conductance (Gp) during a capacitance flicker. The capacitance flicker with a detectable fusion pore conductance (i) indicates kiss-and-run fusion. A non-flicker up-step (ii) occurring 10 s later was not accompanied by detectable Re changes, suggesting a full collapse fusion with the fusion pore that is too fast and/or too large to resolve. Panels (b–d) are adapted, with permission, from Ref. [31].
mutant Drosophila neuromuscular junction lacking endophilin, a protein involved in clathrin-mediated endocytosis, no significant FM1-43 loading was found during stimulation [37] (Figure 4a). This defect was accompanied by depletion of 87% of vesicles in the nerve terminal. If there is no endocytosis, the remaining vesicles could only support release for 10 s during 10 Hz nerve stimulation [37]. However, release was maintained for 600 s at 10 Hz stimulation [37]. These results led to the suggestion that neurotransmission is maintained in endophilin mutants by rapid vesicle recycling through a kiss-and-run mode that prevents FM1-43 uptake but supports transmitter release [37]. A recent study of the same endophilin mutant [38] did not concur with the above results. In this study [38], FM143 loading was detected during stimulation (Figure 4b), and the quantal size was increased after stimulation, which would not be expected from a kiss-and-run fusion [38]. Although this study argued against kiss-and-run fusion, it did not exclude the possibility that kiss-andrun coexists with a slow form of endocytosis in endophilin mutants. Furthermore, it is unclear whether a slow form of endocytosis, with a small number of vesicles (13% of control), in endophilin mutants is sufficient to account for transmitter release for 600 s during 10 Hz stimulation, as reported in the first study [37]. In summary, although two studies disagree on the existence of kiss-and-run, both agree that kiss-and-run is not the dominant form of fusion [37,38]. www.sciencedirect.com
Goldfish retinal bipolar synapse: is rapid endocytosis caused by kiss-and-run fusion? Rapid endocytosis at synapses was first reported at a ribbon-type synapse, the goldfish retinal bipolar synapse, where release is triggered by graded potentials [39]. Rapid endocytosis at this synapse was originally thought to reflect kiss-and-run fusion. However, recent studies have challenged this idea. Evanescent field fluorescence microscopy has revealed that FM1-43 dye molecules loaded in single vesicles leave the vesicle within a few milliseconds completely during most or all exocytic events [40,41]. This result is consistent with full collapse exocytosis. The stimulation used to evoke exocytosis was a 6–500 ms depolarization [40,41], which has been shown to induce rapid endocytosis in other studies [42,43]. Thus, rapid endocytosis might follow full collapse exocytosis, and thereby is not a result of kiss-and-run. However, rapid diffusion of FM1-43 could also be the result of kiss-and-run with a fusion pore that enables rapid lipid exchange between the vesicle membrane and the plasma membrane. Testing this possibility in the future will therefore be important to determine whether rapid endocytosis follows full collapse or kiss-and-run fusion. With the use of interference reflection microscopy to image surface membrane in close contact with a glass coverslip, it has been suggested that vesicles rapidly collapse into the surface membrane during exocytosis evoked by a 500 ms depolarization [44]. In another study of
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Figure 4. Evidence in support of or against kiss-and-run at Drosophila neuromuscular junctions. (a) The nerve terminal of the neuromuscular junction in the wild type (w) (i), but not the mutant lacking endophilin [(ii), endo1], could be loaded with FM1-43 during high potassium-induced depolarization. The labeling intensity of boutons in the endo1 mutant did not exceed labeling of unstimulated endo1 synapses in FM1-43 for 15 min (iii). This result is in line with kiss-and-run fusion, which is too fast to enable FM dye uptake. Adapted, with permission, from Ref. [37]. (b) Both the nerve terminal of the neuromuscular junction in the wild type (wt) (i) and the mutant lacking endophilin [(ii), endo] could be loaded with FM1-43 during high potassium-induced depolarization. The insets show some labeled boutons on a larger scale. Labeled boutons can be unloaded by stimulation (not shown). These results are contrary to the results shown in (a). Adapted, with permission, from Ref. [38].
the same preparation, whole-cell capacitance recordings showed that a prolonged depolarization for several hundred milliseconds could induce rapid endocytosis [43]. Taken together, these results imply that rapid endocytosis follows full collapse fusion [44]. However, the imaging was taken only from the surface membrane in contact with the glass [44], whereas rapid endocytosis was detected from whole-cell capacitance recordings. Whether rapid endocytosis can also take place at the surface membranes in contact with a glass coverslip is unclear. Testing this possibility would be a key to ascertaining whether rapid endocytosis follows full collapse fusion. An electron microscopic study revealed that during spontaneous calcium action potentials or high potassium application, the uptake of the electron-dense marker ferritin was mostly found in large endosomes [45]. It is unclear whether these stimulation protocols induce rapid endocytosis. Nevertheless, this study raised the possibility that rapid endocytosis could be a result of bulk endocytosis, that is, retrieval of a piece of membrane larger than the size of a vesicle [45]. In summary, whether rapid endocytosis reflects kiss-and-run fusion remains an open question at goldfish retinal bipolar synapses. www.sciencedirect.com
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Cultured hippocampal synapses: kiss-and-run is highly debated Two sets of evidence support the existence of kiss-and-run at cultured hippocampal synapses. The first set is composed of three pieces of evidence, obtained by FM dye imaging [46–50]. First, vesicles loaded with FM2-10, a dye that dissociates from the membrane to the solution faster than FM1-43, were unloaded faster than FM1-43 during 10 Hz but not 1 Hz stimulation [46,51]. This result implied endocytosis that was too fast for FM1-43 to escape to the bath solution at 10 Hz stimulation. Second, single vesicles loaded with FM1-43 showed either partial (Figure 5a) or complete dye loss during stimulation [49]. The kinetics of partial dye loss was slower than that of complete dye loss [52]. Partial dye loss is consistent with a kiss-and-run mode of fusion that prevents lateral dye diffusion and is too fast for FM1-43 to escape completely to the bath solution. The duration of kiss-and-run has been estimated to be less than 1.4 s [49]. Third, partial dye loss predicts partial dye retention in vesicles. This prediction has been confirmed using a hydrophilic FM1-43 quencher, bromophenol blue [50] (Figure 5b). FM1-43 retention increased as the stimulation frequency decreased [50]. Based on the degree of dye retention, it was estimated that the contribution of kiss-and-run fusion ranges from more than 80% at 0.3 Hz to 50% at 30 Hz stimulation [50]. This conclusion is different from that of an earlier study from the same laboratory, which suggested kiss-and-run at 10 Hz but not at 1 Hz stimulation [46]. The reason for this discrepancy is unclear (but see later for possible explanations). The second set of evidence was obtained by imaging with synaptopHluorin, a pH-sensitive green fluorescence protein (pHluorin) fused with the luminal portion of the integral vesicular protein vesicle-associated membrane protein 2 (VAMP-2) [53]. SynaptopHluorin fluorescence is quenched inside the acidic environment of vesicles [53] but becomes unquenched (increases) following vesicle fusion that exposes synaptopHluorin to the alkaline environment [53]. The subsequent decay of the fluorescence signal reflects both the time course of endocytosis and reacidification of the endocytosed vesicle. Single vesicles labeled with synaptopHluorin show a transient fluorescence increase and decay after an action potential [54] (Figure 5c). When fluorescence is averaged, a fast fluorescence transient with a time constant of less than 1 s is evident [54] (Figure 5c). The pH buffer Tris, but not HEPES, which has a molecular size larger than Tris, slows vesicular reacidification and thus the transient fluorescence decay. These results suggest that kiss-and-run fusion might occur after single action potentials [54]. In addition, without using FM dyes or synaptopHluorin, a recent study suggested rapid vesicle reuse consistent with kiss-and-run fusion at hippocampal synapses [55]. This study is based on the principle that transmitter refilling depends on the vacuolar ATPase and vesicular reacidification [55]. Pharmacological block of the vacuolar ATPase or vesicular reacidification results in faster synaptic depression, as detected within 1 s during 20 Hz stimulation, implying rapid vesicle reuse [55]. However, more work is needed to demonstrate that rapid reuse is a result of kiss-and-run fusion.
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Figure 5. Imaging evidence in support of kiss-and-run at cultured hippocampal synapses. (a) (i) The images of a single FM1-43-labeled vesicle taken just before the third (1) and fourth (2) stimuli and just after the 620th (3) stimulus. Twenty stimuli were applied at 0.125 Hz, followed by 600 stimuli at 10 Hz. A fusion event after the third stimulus was considered to be followed by retrieval rapid enough to leave FM1-43 trapped in the vesicle. The remaining fluorescence was retained for 17 action potentials and then released with 600 action potentials. (ii) Corresponding fluorescence signal. DFmax is the total fluorescence of the labeled vesicle. Adapted, with permission, from Ref. [49]. (b) FM1-43 was retained in vesicles during 1 Hz (i) and 30 Hz (ii) stimulation. During repetitive stimulation, the fluorescence intensity of FM1-43-loaded vesicles decreased faster in the presence of 2 mM bromophenol blue (red symbols) than in its absence (black symbols). Bromophenol blue might enter synaptic vesicles during fusion and quenche FM1-43 if it remains there after one round of exo- and endocytosis. These results suggest rapid closure of the fusion pore and thus the presence of kiss-and-run. Abbreviations: BPB, Bromophenol blue; TRP, total recycling pool. Adapted, with permission, from Ref. [50]. (c) (i) The fluorescence signal of three individual vesicles labeled with synaptopHluorin in response to a single action potential. The transient signal is interpreted as kiss-and-run fusion and retrieval. (ii) The average fluorescence of 72 individual vesicles expressed with synaptopHluorin in response to a single action potential. The fitted curve (in black) has three decay components: (1) fast monoexponential process (t = 860 210 ms); (2) a slow, compensatory Gaussian process (t = 10.7 0.35 s); and (3) stranded t > 45 s. The fast component is interpreted as kissand-run. The size of the imaging area is not specified in this study. Adapted, with permission, from Ref. [54].
Although the above studies from several laboratories support the existence of kiss-and-run fusion, others have obtained evidence inconsistent with kiss-and-run using similar techniques. The evidence can be divided into two parts. First, FM2-10 preferentially stains the readily releasable pool of vesicles at frog neuromuscular junctions [56]; this provides an alternative interpretation for why unloading of FM2-10 is faster than that of FM1-43 at cultured hippocampal synapses [46]. As shown in goldfish retinal bipolar synapses, FM dyes diffuse more rapidly from the vesicle membrane to the plasma membrane than from the membrane to the bath solution [41]. Such a rapid lateral diffusion might complicate the interpretation of FM dye unloading, although whether it occurs at hippocampal synapses is unclear. Furthermore, differential unloading between FM2-10 and FM1-43, as observed in one laboratory [46], was not observed by another laboratory (Figure 6a) during similar frequencies (10–30 Hz) of stimulation [57]. This finding raises doubt on the observation of the differential unloading between the two FM dyes. The second set of evidence against kiss-and-run was obtained with pHluorin imaging. If the fast synaptopHluorin fluorescence transient (time constant less than 1 s; Figure 5c) observed after single action potentials is due to kiss-and-run, the time constant of vesicle reacidification should be less than 1 s. The time constant of vesicle reacidification was not measured after single action potentials [54] but was 3–5 s after a train of action potentials [58,59], raising the question of whether vesicle reacidification can be less than 1 s after single action potentials. More imporwww.sciencedirect.com
tantly, rapid postexocytosis lateral diffusion of synaptopHluorin occurs at hippocampal synapses [59–61]. When the imaging area is small (Figure 6b), rapid lateral diffusion beyond the imaging area results in a rapid fluorescence transient not related to kiss-and-run fusion [59] (Figure 6c). When the imaging area is large enough (Figure 6b) to include the space for lateral diffusion, the rapid fluorescence transient disappears (Figure 6c). When pHluorin is fused to another vesicle protein, synaptophysin (sypHy), only a slow fluorescence decay with a time constant of 15 s, which reflects slow endocytosis, is observed after 1–40 action potentials at 20 Hz stimulation [59] (Figure 6d). This is because sypHy has much less surface expression and lateral mobility than synaptopHluorin [59]. The slow fluorescence decay is blocked by overexpression of the C-terminal fragment of a clathrin adaptor protein, adaptor protein 180, or by knockdown of clathrin, suggesting that clathrin-mediated slow endocytosis is the major mechanism of vesicle retrieval [59]. This suggestion is further supported by two recent studies showing that vesicular proteins exocytosed and subsequently retrieved by endocytosis are not identical at hippocampal synapses [62,63]. These results argue against kiss-and-run as a major mechanism in physiological conditions, and suggest caution in interpreting the rapid synaptopHluorin transient as kiss-and-run fusion. However, they do not exclude the existence of kiss-and-run. Two examples are given below. First, if only a fraction of synaptic vesicles can undergo kiss-and-run fusion and retrieval, and if these vesicles can
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Figure 6. Imaging evidence against kiss-and-run at cultured hippocampal synapses. (a) FM2-10 and FM1-43 were destained to the same extent with a similar time constant (tFM2-10 = 21 s; tFM1-43 = 22 s) during a 900 action potential train at 10 Hz. A subsequent train of 900 action potentials at 30 Hz was applied to release any remaining dye, and fluorescence values normalized to the fluorescence lost during both stimulation rounds. These results argue against any significant kiss-and-run fusion. Adapted, with permission, from Ref. [57]. (b) SynaptopHluorin-labeled boutons showing a small (green, 1.6 mm square) and a large (red, 4.8 mm square) size of imaging area, which were used in panels (c) and (d). (c) Comparison of the average synaptopHluorin signal triggered by a single action potential in small (green) and large (red) imaging areas. Bold red trace is a scaled version to enable direct comparison of the kinetics of recovery, and can be described by a single exponential with t = 21 s. By contrast, the decay of the fluorescence signal from the small area (green) was biexponential, with tfast = 2 s (36%) and tslow = 20 s. These results suggest that the fast synaptopHluorin transient (green) is due to lateral diffusion of synaptopHluorin from a small to a large area. (d) Comparison of average sypHy signals triggered by a single action potential in small (green) and large (red) imaging areas. The bold red trace is a scaled version to enable direct comparison of the kinetics of recovery. Only a slow decay with a time constant of about 28 s was observed, arguing against any significant contribution of kiss-and-run after a single action potential. SypHy uses the same superecliptic pHluorin but fused to the second intravesicular loop of synaptophysin, instead of VAMP-2. SypHy has much less surface expression compared with synaptopHluorin attached to VAMP. Panels (b–d) were adapted, with permission, from Ref. [59].
also undergo full collapse fusion followed by clathrinmediated endocytosis, block of clathrin-mediated endocytosis for a long period will eventually block recycling of these vesicles once they are sorted to the clathrin-mediated pathway [64]. Thus, the block of endocytosis by long-term molecular manipulation of the clathrin-mediated pathway [59] might not necessarily exclude the existence of kissand-run. Second, because the decay of pHluorin fluorescence reflects both reacidification and endocytosis, and reacidification might take 3–5 s, a small number of kiss-and-run events would be difficult to detect [59]. Nevertheless, the conclusion that clathrin-mediated slow endocytosis is the dominant retrieval mechanism [59] is in sharp contrast to another recent study, which, based on FM dye retention in vesicles, suggests that kiss-and-run is the dominant retrieval mechanism under physiological conditions [50]. The discrepancy is difficult to reconcile with our current knowledge, perhaps because neither FM dye nor pHluorin imaging can provide unequivocal evidence of kiss-and-run. Concluding remarks and future research Cell-attached capacitance recordings reveal that a minor fraction of fusion events are kiss-and-run during high potassium application at the calyx of Held synapse. At this synapse, whether kiss-and-run occurs during more www.sciencedirect.com
physiological stimulation is an important question to address in the future. Studies using FM dye and pHluorin imaging are highly controversial regarding the existence and the dominance of kiss-and-run fusion at Drosophila neuromuscular junctions and cultured hippocampal synapses. Novel approaches that provide more direct evidence are needed to resolve the debate at these two synapses. Although rapid endocytosis occurs at goldfish retinal bipolar synapses, whether it is a result of kissand-run fusion remains an open question. Given the controversy of kiss-and-run at synapses, previous studies implying a role or invoking its molecular mechanism might require re-review when the controversy is resolved at the studied synapse. Once this controversy is resolved, we can begin to study the underlying molecular mechanism and the functional role of kiss-and-run. Acknowledgements We thank Ken Paradiso, David Nees and Ben McNeil for comments on the manuscript. This work was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program. The authors declare no competing financial interests.
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