More than 30 years after the original proposals, the debate between supporters of the different models is still going strong. Using increasingly sophisticated methods, evidence for both models is continuously being published – only to be contested later. In the following paragraphs, we will summarize recent pro and con evidence for both models and discuss that not only the kiss-and-run but also the ‘classical’ pathway is capable of explaining fast, stimulus-associated endocytosis.
Collapse and clathrin-mediated endocytosis
Collapse of vesicles into the plasma membrane, at least during strong stimulation, has been well established in numerous preparations. Furthermore, there is plenty of evidence that clathrin- and dynamin-dependent retrieval of synaptic vesicles is essential for the vesicle cycle (8,9). Typical experiments supporting full fusion are those in which strongly stimulated preparations are shown to lose some of their vesicles [with or without an enlargement of the plasma membrane (10)]. Perturbing endocytosis, as in the dynamin temperature-sensitive mutant of Drosophila, shibire, also results in empty nerve terminals (11). Endocytic intermediates such as clathrin-coated vesicles and especially infoldings/cisternae have been observed in many preparations (reviewed in 5 and references therein).
There is one flaw with this type of evidence, which has been pointed out for decades. Harsh stimulation is requiredto see such intermediates, and thus they may not be representative for the processes functioning in vivo. Also, one of the most common preparations used for studies of synaptic activity, cultured hippocampal neurons, is resistant to such approaches – extensive stimulation is unable to reduce the number of vesicles significantly, and endocytic intermediates are not frequently observed (12,13; but also see 14). Even in preparations where clathrin-mediated endocytosis (CME) is well established, it is not trivial to correlate quantitatively the number of coated vesicles or pits with the number of released vesicles (15); a possible answer is that endocytic intermediates are normally short lived and thus are difficult to see with mild stimulation.
Full fusion is also supported by the uptake of bulky molecules such as antibodies into synaptic vesicles during stimulation (16,17); even sandwiches of primary and secondary antibodies can be internalized (18), which is hard to imagine to be accomplished through a narrow fusion pore. Furthermore, in an elegant set of experiments, the collapse of vesicles has recently been investigated by capacitance recording (which measures the total nerve terminal membrane area) in conjunction with interference reflection microscopy (19), which measures the size of the footprint of the nerve terminal. The two measurements paralleled each other in the large nerve terminals of goldfish bipolar cells, suggesting that every fusing vesicle also contributes to the increase in the footprint. Interestingly, some rapid endocytosis does persist in presence of clathrin-perturbing agents in this preparation (20), even though bulk endocytosis [abundant here (5)] might be called in rather than kiss-and-run [note that bulk endocytosis can be extremely rapid (7)].
At least in some synapses, molecular interference with CME can block all vesicle retrieval, suggesting that it is the main recycling mechanism. For instance, overexpression of a dominant-negative form of the clathrin adaptor protein (AP) 180, or RNA interference knock down of clathrin heavy chain, blocked all endocytosis following stimulation in hippocampal synapses (21). However, the treated nerve terminals did have vesicles ready to release at the start of stimulation – after having their CME inhibited for hours (during which spontaneous activity must have released a significant number of vesicles) – indicating that either the treatments drastically prolonged CME, but did not block it completely, or there are also other types of endocytosis.
A major problem associated with the kiss-and-run model is that because of its transient and short-lived nature, it is almost impossible to ‘capture’ such events in EM. In synapses, size and packing density of synaptic vesicles render it impossible to image individual vesicles (unless selectively labeled, see below), and EM only provides ‘frozen’ snapshots. Images of recently fused vesicles, immediately after stimulation, have been obtained in quick-freeze EM (22,23). Pores are large, around 20 nm in diameter (for synaptic vesicles of only approximately 50 nm in diameter) and tend to grow larger with time (22), suggestive of full fusion progressing after formation of the initial pore. Furthermore, observing (labeled) vesicles seemingly in the process of getting endocytosed but lacking obvious clathrin coats should not be used as kiss-and-run evidence, especially as imaging clathrin coats is not trivial (24).
Rapid endocytosis (∼1–10 seconds) has been used as evidence for kiss-and-run as CME is expected to take many seconds. Whether CME does need many seconds to proceed is debatable, especially if there are any preformed clathrin-coated vesicles on the membrane waiting to be endocytosed (7,25; see below). Nevertheless, rapid endocytosis has been observed by capacitance recording at the calyx of Held in the auditory pathway (26) with a time constant of approximately 100 milliseconds, far beyond CME ability. However, a subsequent study with the same preparation used neurotoxins to block exocytosis and indicated that the capacitance transient interpreted as kiss-and-run may not have been related to vesicle recycling (27), inducing a re-evaluation of the original data, which suggest that endocytosis is more than an order of magnitude slower than originally thought (28).
Stronger evidence for kiss-and-run was recently obtained by cell-attached capacitance recording, in the form of capacitance ‘flickers’, in which the signal increases, remains constant between a few milliseconds and about 2 seconds and then abruptly returns to baseline. This paradigm corresponds to the fusion and subsequent reinternalization of single synaptic vesicles and seems to apply for approximately 20% of the vesicles (29; see also 30 for a similar discussion of microvesicles in pituitary nerve terminals). It is highly unlikely that a single vesicle would be retrieved by CME within such a short time period following exocytosis. In these experiments, however, it cannot be fully excluded that the exocytosed and endocytosed vesicles are different – i.e. one vesicle fused and another one was subsequently retrieved (see also discussion of readily retrievable vesicles below).
The strongest (although contested) support for kiss-and-run has been derived from experiments using synaptic preparations with fluorescently labeled synaptic vesicles. Synaptic vesicles can be loaded during recycling with various fluorescent fluid-phase or membrane labels, among which styryl FM dyes are the most common. The release of the fluorescent dye upon a subsequent round of stimulation (destaining) has been used as a direct measure of exocytosis for more than a decade. Some FM dyes are more hydrophilic than others, and it was thought that they would be released preferentially in kiss-and-run events, while collapse would allow all dyes to escape the vesicle equally well. Indeed, the hydrophobic dyes FM 1–84 and FM 1–43 were released more slowly than the hydrophilic FM 2–10 from hippocampal nerve terminals (31). However, the difference between the dyes was not reproduced in a different preparation [frog neuromuscular junction (NMJ); 32], and no difference was later seen in hippocampal synapses at physiological temperature (33).
In a similar experiment, bromophenol blue (BPB) was added to the bath when vesicles preloaded with FM dyes exocytosed. Bromophenol blue quenches all FM fluorescence but is membrane impermeant; however, as a small molecule, it is expected to penetrate into the vesicles through fusion pores that otherwise are too short lived or narrow for FM dyes to escape (34). If vesicles undergo full collapse and lose all their dye, the drop in FM fluorescence during exocytosis should not be affected by BPB. The experiment indicated that a significant proportion of dye was retained in a BPB-sensitive pool, even at physiological temperature. The interpretation of the BPB-sensitive pool as ‘kiss-and-run’ vesicles, however, has been challenged because the dye remaining in the preparation may be trapped in the synaptic cleft and in the pre- and postsynaptic membranes instead of in the synaptic vesicles (21), especially as this type of background contributes significantly to the overall nerve terminal fluorescence (24).
The loss of FM fluorescence from single individual vesicles has also been investigated. In nerve terminals of the bipolar cells from goldfish retina, single vesicles appear to lose all the dye within milliseconds (35), suggestive of collapse. Different results have been obtained in hippocampal neurons when it was attempted to label only a single vesicle per bouton (36). Stimulation resulted in partial loss of dye, indicative of transient fusion, with few boutons losing all fluorescence at once. Subsequently, it was reported that the smaller destaining events were also slower, in good agreement with the loss of dye through a narrow pore (37). However, this interpretation has also been challenged recently by showing that weakly stained boutons released all of their dye within less than 100 milliseconds (38) and that partial release is only observable in more strongly labeled boutons, which would suggest that ‘partial’ release simply reflects the release of one of the few labeled vesicles.
Finally, fusion pores too transient to allow for FM uptake were also proposed. Stimulation with hypertonic sucrose resulted in little FM dye uptake but normal neurotransmitter release (39). In contrast, however, no difference in FM uptake between application of hypertonic sucrose and electrical stimulation was found in neuromuscular preparations (24).
Some of the newest tools – pHluorins – have also been employed in attempting to differentiate between collapse and kiss-and-run. PHluorins are synaptic vesicle proteins fused to a pH-sensitive variant of the green fluorescent protein (GFP). The GFP moiety is linked to an intravesicular domain of the protein, and its fluorescence is quenched by the acidic pH of the vesicle. Upon exocytosis, the fluorescence increases dramatically because of the exposure to the neutral extracellular pH. Endocytosis is studied by measuring the requenching of the pHluorins.
The protein most commonly used for such studies is synaptobrevin, the GFP chimera being termed synaptopHluorin. Gandhi and Stevens (25) analyzed the endocytosis behavior after release of single synaptic vesicles and found that a fraction of the events returned to baseline with half-times of less than a second. As this delay covers not only endocytosis but also the ensuing reacidification, the data were interpreted as evidence for kiss-and-run. However, this type of analysis, again, is not trivial. The majority of action potentials did not trigger (measurable) release of vesicles at normal calcium concentrations and were recorded as failures. Other action potentials were followed by rapid upward deflections of the signal, which returned to baseline within one or a few frames, and were recorded as kiss-and-run events (25, Fig. 4e). We agree with the suggestion of Granseth et al. (21) that it cannot be excluded that some of these events represent upward flicking noise, which coincided with stimulation. Also, lateral diffusion of synaptopHluorin after exocytosis may cause rapid loss of signal from the region of interest.
Unlike CME whose molecular details are well worked out (8), it has been difficult to identify proteins functioning in kiss-and-run. Dynamin is generally considered to be required for all endocytosis (27,40). A candidate protein with a specific function in kiss-and-run is endophilin, whose inactivation was proposed to switch vesicles to kiss-and-run (41), although later studies are more in agreement with a slowing of CME in such preparations (42,43).