Secretion: Kiss and Run Caught on Film

Current Biology, Vol. 13, R397–R399, May 13, 2003, ©2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0960-9822(03)00320-8

Secretion: Kiss and Run Caught on Film H. Clive Palfrey1 and Cristina R. Artalejo2

Recent results have provided graphic support for the hypothesis that vesicle secretion involves a ‘kiss-andrun’ mechanism. Evanescent field microscopy has shown that, during exocytosis, intravesicular markers escape without collapse of the vesicular membrane into the surface membrane and that the empty vesicle is immediately retrieved back into the cell.

Neuroscientists have pondered the mechanism of synaptic vesicle recycling since the early observation that the number of releasable quanta far exceeds the number of synaptic vesicles estimated from micrographs (reviewed in [1]). Early electron microscopic observations of tetanically stimulated preparations were limited by the ability to visualize cycling vesicles using endocytosed probes such as peroxidase [2]. These studies suggested that vesicles fuse fully with the surface membrane, and that vesicle membrane proteins are then recaptured in coated pits at sites distant from the release zone; coated vesicles then make their way to a ‘sorting endosome’ from which new synaptic vesicles were supposed to bud. There seems little doubt that synaptic vesicle components can recycle by this slow pathway, but as suggested in 1973 by Ceccarelli and colleagues [3], perhaps it only operates under extreme conditions. According to Ceccarelli et al. [3], coated vesicles only appear after massive expansion of the presynaptic membrane concurrent with tetanization. They came up with an alternative model for physiological stimulation that envisaged recapture of intact vesicles at the sites of secretion, followed by immediate refilling with new transmitter. Unfortunately, there was little evidence to back up this ‘kiss-and-run’ hypothesis, in part because recaptured vesicles were not readily distinguished by electron microscopy from those vesicles that had not yet seen action. With the recent development of fluorescence microscopic techniques to visualize exocytosis in real time, it has become possible to test the kiss-and-run idea directly. Wolf Almers’ lab has done just that [4], and the news is good for kiss-and run fans: it seems to be the major mechanism of secretion, at least in neuroendocrine cells. Taraska et al. [4] used fluorescently tagged proteins to follow the fate of dense core granule contents and membranes after a secretory stimulus using the technique of total internal reflectance (TIR) microscopy, often referrred to as ‘evanescent field’ microscopy 1Department

of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, Illinois 60637, USA. 2Department of Pharmacology, Wayne State University, Detroit, Michigan 48201, USA.

Dispatch

(Figure 1). TIR microscopy uses the property of an incident beam of light to be reflected back when hitting a surface of lower refractive index — such as the interface between glass and aqueous solution — at a specific angle, called the ‘critical angle’ [5]. Under these conditions, light penetrates only a short distance into the aqueous layer, but if fluorophores are present they can be excited and the resultant emission analysed. The high resolution and excellent signal:noise ratio of TIR microscopy is due to the thinness of the excited slice — less than 100 nm or about one fifth the width of a typical confocal section — together with the elimination of extraneous fluorescence. Working with PC12 cells, Taraska et al. [4] expressed neuropeptide Y (NPY) or tissue plasminogen activator (tPA) as fusion proteins linked to the green fluorescent protein variant EGFP to serve as content markers, while DsRed-phogrin, a fluorescently tagged transmembrane tyrosine-phosphatase-like glycoprotein, served as a probe of the vesicular membrane. All of the markers significantly colocalized, indicating that the same pool of vesicles was being labelled. A depolarizing secretory stimulus led to the escape of vesicular contents, the rate of which varied with the size of the protein: EGFP–NPY escaped fully in less than a second, whereas the larger EGFP–tPA took several seconds. By contrast, phogrin fluorescence was not lost during stimulation and failed to disperse in the plasma membrane, a critical finding as this would be expected to occur in the full fusion model of transmitter release (Figure 1B; earlier TIR microscopy studies showed dispersion of membrane markers during constitutive secretion [6]). Subsequent dimming of phogrin fluorescence suggested that the vesicle became reacidified in the cytoplasm, possibly rendering it competent to accumulate new transmitter and be reused. In a variation of this approach, the cytosol of the same cells was non-specifically labelled with cyan fluorescent protein (CFP) and the fate of the negative image produced by unlabelled vesicles (no fluorescence) followed in the evanescent field during secretion. After stimulation, the vesicle ‘ghost’ remained intact, again suggesting no collapse into the surface membrane, as would be expected in a kiss-and-run model. A corollary of these experiments would be the demonstration of extracellular marker uptake into vesicles during the retrieval process. Such an experiment has in fact been done, this time using plasma membrane ‘lawns’ from PC12 cells [7]. Uptake of sulforhodamine at the sites of dense-core vesicle secretion was found, implying that dye uptake occurred while the fusion pore was open. Coupled with recent electrophysiological analyses of transmitter release kinetics and endocytosis, as well as genetic studies of vesicle cycling in flies and mice, the evanescent field studies affirm that transient fusion is a major regulated secretory mechanism. A prediction of the kiss-and-run model is that vesicle recapture

Dispatch R398

Figure 1. The principle behind TIR microscopy, with predicted and Complete experimental results for vesicular fusion. fusion TIR microscopy uses a modified fluoresKiss-and-run cence microscope with a laser beam steering device that orients an incident Data beam through the objective such that it strikes the aqueous interface at a critical angle that provides ‘total internal reflection’ (TIR). The incident beam can travel a certain distance into the aqueous layer, creating an ‘evanescent field’ that excites fluorophores within this range. The emitted light is collected through the A B C objective and video imaged. This provides a real-time record of the behaviour of fluEvanescent orescent particles, such as vesicles, in the field evanescent field. Taraska et al. [4] used (<100 nm) three types of vesicle labeling: (A) conPlasma tents, using EGFP–NPY for example; Coverslip membrane (B) membrane, using DsRed-phogrin; and (C) vesicle unlabelled but cytosol is filled non-specifically with a fluorescent dye. Predicted results for complete fusion and kiss-and-run models are illustrated in the upper images, with the actual data shown below. (A) Both models predict identical High behaviour for contents; note that the N. A. signal increases in intensity on release objective because the fluorescent label is pH sensitive and emits more strongly when encountering the extracellular space (pH ~7.4) than when in the vesicle (pH ~5.6). (B) Complete fusion predicts an expanding and dimming signal as the Incident Reflected Laser Video vesicle membrane protein diffuses into beam beam the plasma membrane [6]; kiss-and-run predicts a non-diffusing signal that Mirrors Current Biology increases in intensity as the EGFP tag was first exposed to pH 7.4 followed by a decrease as the vesicle reseals and reacidifies (see above); data support kiss-and-run. (C) Complete fusion predicts elimination of the negative vesicle image (‘ghost’) as the fluorescence 'fills in' the cytoplasm behind the lost vesicle, while kiss-and-run predicts that the ghost would remain in position. Upper data panel shows that the ghost remains in place (top data panel), conforming to kiss-and-run; lower data panel confirms that fusion occurs synchronously as contents escape (compare with (A)).

{

should be immediate. In this regard, the kinetics of resealing from the TIR microscopy experiments — less than 10 seconds — are entirely consistent with capacitance measurements in chromaffin cells which showed that, after minimal stimulation, rapid endocytosis of membrane occurs in seconds [8]. In the work of Artalejo et al. [8], more intense stimulation resulted in the suppression of this rapid endocytosis and the emergence of a much slower endocytosis, taking several minutes. Mechanistic analysis showed that the two processes are different: the rapid route is clathrin-independent, but the slower route requires clathrin. Interestingly, the two events involve different isoforms of dynamin; the neuronallyconcentrated dynamin-1 mediates rapid endocytosis, while the more generally distributed dynamin-2 mediates slow endocytosis. The lack of clathrin dependence in the fast process makes perfect sense if it represents the final step of transient fusion. The purpose of the clathrin cage is to deform the membrane, but as there is no collapse of the vesicle into the surface membrane in kiss-and-run, the requirement for a deformation step is eliminated.

Some may still argue that the cycling behaviour of dense-core vesicles differs in some fundamental way from small synaptic vesicles, and exo-endocytosis at fast synaptic terminals is mostly or entirely by a complete fusion-clathrin coated vesicle pathway, perhaps modified for speed [9]. For example, experiments in which synaptic transmission in the squid giant synapse was inhibited by injection of low affinity peptide antagonists of clathrin cage assembly were recently offered as support for this idea [10]. As yet, TIR microscopy, which might yield a definitive answer to this question, has not been applied to synaptic terminals labelled with protein markers. But capacitance analysis, while possible only at few synapses, supports the notion that endocytotic rates are plastic and vary widely with the degree of stimulation. Recent spectacular experiments at the Calyx of Held synapse [11] showed that low stimulation led to retrieval rates in the 100 milliseconds range, whereas tetanic pulses resulted in retrieval rates of tens of seconds. Genetic studies support the existence of alternative modes of vesicle cycling at excitatory synapses.

Current Biology R399

Elements of the elaborate machinery thought to mediate clathrin-based endocytosis have been genetically eliminated in flies, worms and mammals, and this was found to have little effect on synaptic transmission at physiological levels of stimulation. For example, ablation of the dynamin- and clathrin-binding protein amphiphysin, much touted as an essential factor in synaptic vesicle recycling [9], had no effect on synaptic transmission in Drosophila and only minimal effects in mice [12,13]. More tellingly, loss of endophilin in flies, which completely abrogates clathrin-dependent endocytosis, was found not to affect synaptic transmission at low rates [14], probably because of the persistence of a recycling pool of vesicles maintained by kiss-and-run [15,16]. While the endocytotic characteristics of transient vesicle fusion differentiate it from complete fusion scenarios, the mechanism of kiss-and-run exocytosis remains unclear. One obstacle is that the composition and operation of the putative fusion pore that mediates transmitter release is still obscure. Despite advances with model systems and several proposals as to the operation of this structure, its assembly and mode of action are still not understood. The fate of vesicular matrix proteins, such as the chromogranins, during kiss-and-run is also not known. It is predicted that such proteins would be retained in the granule lumen, and this should undoubtedly be amenable to investigation using TIR microscopy with GFP-chromogranin as the fluorescent reporter. Another perplexing issue is the fate of the SNARE complexes — believed to mediate membrane fusion events in the secretory pathway — in the kiss-and-run mechanism. The transition from a tight trans-SNARE complex to a cis-SNARE complex on the plasma membrane during the fusion reaction, followed by SNARE dissociation was thought to be an irreversible event, but if kiss-and-run is a reality then a vesicle that has already fused — made a connection to the outside of the cell via a fusion pore — can reverse the process and detach from the membrane. Might reversible association of v-SNAREs and t-SNAREs mediate secretion in the kiss-and-run mode, with retention of the v-SNARE by retrieved vesicles? The relationship between the scissioning action of dynamin GTPase and the closing of the fusion pore also requires further investigation. Dynamin-1, perhaps in collusion with as yet unspecified partner proteins, might mediate rapid vesicle fission [8]. Notwithstanding these molecular questions, analysis of the stimulation-dependent processes that control vesicle switching between kissand-run and full fusion modes is a fascinating area that may yield further insight into synaptic plasticity (see for example [17,18]).

References 1. Palfrey, H.C. and Artalejo, C.R. (1998). Vesicle recycling revisited: Rapid endocytosis may be the first step. Neuroscience 83, 969–989. 2. 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. 3. Ceccarelli, B., Hurlbut, W.P. and Mauro, A. (1973). Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J. Cell Biol. 57, 499–524.

4. Taraska, J.W., Perrais, D., Ohara-Imaizumi, M., Nagamatsu, S. and Almers, W. (2003). Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells. Proc. Natl. Acad. Sci. U.S.A. 100, 2070–2075. 5. Axelrod, D. (2001). Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764–774. 6. Schmoranzer, J., Goulian, M., Axelrod, D. and Simon, S.M. (2000). Imaging constitutive exocytosis with total internal reflection fluorescence microscopy. J. Cell Biol. 149, 23–32. 7. Holroyd, P., Lang, T., Wenzel, D., De Camilli, P. and Jahn, R. (2002). Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells. Proc. Natl. Acad. Sci. U.S.A. 99, 16806–16811. 8. Artalejo, C.R., Elhamdani, A. and Palfrey, H.C. (2002). Sustained stimulation shifts the mechanism of endocytosis from dynamin-1dependent rapid endocytosis to clathrin- and dynamin-2-mediated slow endocytosis in chromaffin cells. Proc. Natl. Acad. Sci. U.S.A. 99, 6358–6363. 9. De Camilli, P., Slepnev, V.I., Shupliakov, O. and Brodin, L. (2000). Synaptic vesicle endocytosis. In Synapses, W.M Cowan, T.C Sudhof and C.F. Stevens, eds. (Johns Hopkins, Baltimore) pp. 217–274. 10. Morgan, J.R., Prasad, K., Hao, W., Augustine, G.J. and Lafer, E.M. (2000). A conserved clathrin assembly motif essential for synaptic vesicle endocytosis. J. Neurosci. 20, 8667–8676. 11. Sun, J.Y., Wu, X.S. and Wu, L.G. (2002). Single and multiple vesicle fusion induce different rates of endocytosis at a central synapse. Nature 418, 89–92. 12. Zelhof, A.C., Bao, H., Hardy, R.W., Razzaq, A., Zhang, B. and Doe, C.Q. (2001). Drosophila amphiphysin is implicated in protein localization and membrane morphogenesis but not in synaptic vesicle endocytosis. Development 128, 5005–5015. 13. Di Paolo, G., Sankaranarayanan, S., Wenk, M.R., Daniell, L., Perucco, E., Caldarone, B.J., Flavell, R., Picciotto, M.R., Ryan, T.A., Cremona, O. and De Camilli, P. (2002). Decreased synaptic vesicle recycling efficiency and cognitive deficits in amphiphysin 1 knockout mice. Neuron 33, 789–804. 14. Verstreken, P., Kjaerulff, O., Lloyd, T.E., Atkinson, R., Zhou, Y., Meinertzhagen, I.A. and Bellen, H.J. (2002). Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release. Cell 109, 101–112. 15. Koenig, J. H.,and Ikeda, K. (1996). Synaptic vesicles have two distinct recycling pathways. J. Cell Biol. 135, 797-808. 16. Kjaerulff, O., Verstreken, P. and Bellen, H.J. (2002). Synaptic vesicle retrieval: still time for a kiss. Nat. Cell Biol. 4, E245–248. 17. Choi, S., Klingauf, J. And Tsien, R. W. (2000) Postfusional regulation of cleft glutamate concentration during LTP at ‘silent synapses’. Nat. Neurosci. 3, 330-336. 18. Zakharenko, S.S., Zablow, L. and Siegelbaum, S.A. (2002). Altered presynaptic vesicle release and cycling during mGluR-dependent LTD. Neuron 35, 1099–1110.