Introduction
Release of secretory-vesicle cargo into the extracellular space by the process of exocytosis involves the fusion of these secretory vesicles with the plasma membrane. As cells need to stay the same size, the membrane that was added to the plasmalemma by the vesicles must be internalized by endocytosis. How is this membrane recycling achieved? Several conflicting theories have been proposed, from the classical endocytotic cycle, which involves full-blown fusion of the secretory vesicle with the membrane and then the pinching off and internalization of a new vesicle1, to quick local recycling following reversible fusion2 or 'kiss-and-run'3. Reversible membrane fusion has been reported to occur through the transient opening of a small aqueous pore4, 5, 6, but could such a rapid process be important in secretion? Alés and colleagues, in a paper on page 40–44 of this issue7, answer this question convincingly for chomaffin cells: full discharge of the content of secretory vesicles can occur through kiss-and-run reversible fusion, and calcium favours the process. This is the first clear-cut demonstration that multiple pathways are available for the vesicle to discharge its content and recycle, with the relative importance of each path being regulated by calcium and possibly other factors (Fig. 1).
Figure 1: Two models of coupled exocytosis–endocytosis.
![Figure 1 : Two models of coupled exocytosis|[ndash]|endocytosis.](/ncb/journal/v1/n1/thumbs/ncb0599_E3.gif)
The conventional process (left) predominates at low Ca2+ concentrations. After fusion of the vesicle with the plasma membrane, an aqueous pore is formed (1), followed by flattening of the vesicle in the plasma membrane (2). Sorting of specific components (3) and retrieval of the vesicle membrane (4) are accomplished through the formation of a clathrin coat. The recycled vesicle sheds its coat upon detaching from the membrane (5) and is then refilled to become available for a new run of discharge. The average time for vesicle fusion and retrieval is several tens of seconds. Kiss-and-run fusion (right) is favoured in high Ca2+ levels. The vesicle fuses briefly with the plasma membrane, and widening of the pore — sufficient for the full discharge of vesicle content —ensues. Quick resealing and recycling of the vesicle then follow. In synapses, this type of exocytotic–endocytic cycle might take less than 1 millisecond.
Full size image (30 KB)The conventional view of vesicle recyling, proposed in the 1970s by Heuser and Reese1, holds that, at nerve terminals, exocytosis and endocytosis are two separate processes: vesicle endocytosis is initiated at the flat plasma membrane — after the full fusion and merging of the secretory-vesicle membrane with the plasma membrane —by the sorting of specific vesicle components, assisted by a protein 'cage'. The resulting protein-coated vesicles would carry the membrane to local endosomes, from which new synaptic vesicles would be generated.
Although the rates of such a recycling pathway initially appeared to be too slow for neurons, later studies have shown that the process is much faster at synapses (time constants between 20 and 30 seconds) than in other secretory cells. Moreover, synaptic vesicles can regenerate directly from the plasmalemma, the trip to the endosome being unnecessary8, 9. General opinion converged on the two-step interpretation of vesicle recycling, which is still reported in most textbooks as the only possible way in which vesicle internalization can take place at synapses.
But many years ago Ceccarelli and colleagues2 proposed that fused vesicles might simply detach from the plasma membrane after exocytosis, quickly enough to prevent their collapse and the dispersal of their specific components. Morphological and immunocytochemical evidence in support of this theory has accumulated3. Moreover, measurements of membrane capacitance in mast and chromaffin cells showed that a small aqueous pore (with a conductance measuring less than 1 nanosiemens) forms when a vesicle fuses with the membrane4, 5, 6. The pore opening often flickers for a short time before either closing or irreversibly dilating to produce vesicle collapse. In micro-amperometric assays of released amines, these events are paralleled by a small deflection (foot) in the current, indicating some 'trickling' of the amines out of the pore; the current then either aborts or evolves into a full spike, indicating full discharge4, 5, 6. Finally, heterogeneity of endocytosis has been reported in chromaffin cells, with fast components (of 5 seconds in duration) occurring during strong cellular stimulation10, 11. However, the frequency of reversible kiss-and-run openings, and their relevance to secretion, remained uncertain.
This is where the work of Alés and colleagues7 comes in. They show, in contrast with previous results, that, in chromaffin cells, the release of the whole vesicle cargo does not require full-blown fusion. In fact, the reversible fusion pore can open up to a size above 1.5 nanosiemens, enough to discharge the full contents of the vesicle, and can still close quickly. This means that kiss-and-run exocytosis–endocytosis can sustain quantal neurotransmitter release.
How did Alés et al. obtain these new results? They used the powerful approach of coupling measurements of cell-attached membrane capacitance to assays of catecholamine, a neurotransmitter, by using a carbon fibre positioned within the patch-clamp pipette; but their success is also based on their use of different extracellular Ca2+ concentrations. As they increased the Ca2+ concentration from 5 to 90 mM, the frequency of reversible fusion events rose from 5% to almost 80%; still, in most cases, the full vesicle content was discharged. Thus, these cells appear to be able to choose between at least two routes for membrane recycling: conventional endocytosis predominates when Ca2+ levels are low, and kiss-and-run fusion is the preferred method in high Ca2+ concentrations. As well as inducing kiss-and-run, high Ca2+ amounts also decrease the duration of the process. These two effects of calcium might in fact be related to the same molecular mechanism — the increased probability of pore resealing.
Alés et al.7 applied Ca2+ extracellularly. But they assumed that its regulatory effect took place intracellularly, and the very high concentrations used were aimed at mimicking the state of intensely stimulated neurons, in which massive amounts of exocytosis occur and quick recycling, as can be sustained by kiss-and-run, is needed. It remains possible, however, that regulation by Ca2+ also takes place at the external face of the membrane. This might be particularly relevant at synapses. During exocytosis, in fact, the synaptic cleft, which is often very thin, is flooded by not only the neurotransmitter but also the entire vesicle cargo, including Ca2+, which is present in very high amounts (tens of millimoles per litre) inside secretory vesicles.
Are these results, obtained in chromaffin cells, relevant to synaptic function? It is interesting that chromaffin granules, although considerably larger than clear synaptic vesicles, resemble the other type of synaptic secretory organelle, that sustaining transmission of peptide neurotransmitters. And the molecular apparatus for exocytosis, including SNARE proteins and the entire fusion machinery, is largely similar in the two cell types. Last but not least, the time barrier of 1 millisecond, which has so far somewhat demarcated synaptic from non-neuronal secretion, is closely approached by kiss-and-run fusion in chromaffin cells. If a chromaffin granule, which has a volume about ten times that of a synaptic secretory vesicle and is filled with an organized macromolecular matrix that traps the amine neurotransmitters, can be fully discharged in a few milliseconds, it is quite conceivable that a synaptic vesicle could release its soluble neurotransmitter in a fraction of a millisecond and then rapidly pinch off from the presynaptic membrane.
The most convincing argument in support of the coherence between chromaffin cells and neurons stems from the comparison of Alés et al.'s findings7 with those reported last year by Klingauf et al. 12, who studied hippocampal neurons.
Klingauf et al.12 used a completely different approach to that of Alés and colleagues: they preloaded synaptic terminals with styryl dyes, which become incorporated into membranes. By comparing three dyes that are characterized by distinct rates of departitioning, Klingauf and colleagues were able to reveal the existence of at least two components of recycling — a slow process, with a time constant in the order of 20–30 seconds, and a much faster process, which they interpreted to be kiss-and-run recycling. Most relevant, the occurrence of the latter process increased greatly with stimulus strength and higher Ca2+ amounts. Staurosporin, a nonspecific kinase blocker, speeded up the recycling, pointing to the involvement of further signalling mechanisms in the regulation of kiss-and-run.
With their approach, however, Klingauf et al.12 could estimate the kinetics of pore closure only indirectly (they calculated the lifetime of the pore to be less than 2 seconds) and, more important, they could not prove that kiss-and-run events produce functionally relevant release of neurotransmitter. This proof is now provided by the results of Alés et al.7.
Calcium has long been known to be the major factor in regulating exocytosis. Alés and colleagues7 have now shown that this ion has a central function in regulating the whole exocytotic–endocytotic cycle, by making kiss-and-run the preferred recycling path. Huge Ca2+ concentrations are needed to regulate this very rapid process, but Ca 2+ concentration is known to rise to tremendous levels at the presynaptic membrane during stimulus-evoked neurotransmitter release. So, when the action potential invades the nerve terminal and Ca2+ rushes in, the neuron under pressure has an efficient way to communicate across the synapse: a quick kiss!
