The “kiss-and-run” theory of synaptic vesicle recycling posits that vesicles grace the synaptic membrane sufficiently to expel their neurotransmitters, but not enough to risk full-blown fusion. Kiss-and-run makes a lot of sense, because it predicts that after flirting with the membrane, the vesicles maintain their independence prior to endocytosis—components are not lost to diffusion through the cell membrane and do not need to be resorted and reassembled. In effect, the vesicle acts like a drop of oil on water. Whether or not this type of courtship actually goes on in neurons has been a bit of a sticking point, but in today’s Nature, researchers from Germany report that they have captured the vesicles in the act.

The major obstacle to proving the kiss-and-run theory has been visually resolving synaptic vesicles. Light microscopes cannot distinguish objects that are closer than one half of the wavelength of light being used because diffraction blurs the elements into a single fuzzy image. So though the best confocal microscopes can discriminate objects as close as 200 nm, they are not capable of resolving single synaptic vesicles, which are only about 40-50 nm across. But now, microscopist Stefan Hell and neurobiologist Reinhard Jahn, both at the Max Planck Institute for Biophysical Chemistry in Gottingen, Germany, have teamed up to overcome that diffraction problem and track recycling vesicles in stimulated neurons.

Joint first authors Katrin Willig and Silvio Rizzoli and colleagues used a technique called stimulated emission depletion (STED) to break the diffraction barrier and improve resolution manyfold. Pioneered by Hell, STED uses a deceptively simple trick to improve the resolution of fluorescence microscopy. Superimposed on the regular excitation beam is a donut-shaped beam of light that de-excites fluorophores by driving electrons back toward the ground state from their excited states. In effect, STED quenches fluorescence and acts as an aperture to narrow fluorescence emission to a focal spot about 60 nm in diameter, effecting resolution to 45 nm.

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A Microscope in Good Stead
STED microscopy improves resolution to about 45 nm. Compared to confocal microscopy (left), STED (right) can resolve numerous spots of synaptotagmin, a major synaptic vesicle protein, within synaptic boutons in primary hippocampal neurons (see insets). [Image courtesy of Reinhard Jahn and Stefan Hell, Max Planck Institute for Biophysical Chemistry, Gottingen, Germany.]

The authors used STED to study the behavior of synaptotagmin, a major synaptic vesicle protein, in hippocampal primary neurons. They used the microscope to visualize both internal and cell surface protein. They labeled the latter by adding fluorescent synaptotagmin antibodies to cultures on ice. Internal protein was labeled once the cultures were warmed to 37 degrees centigrade in the presence of calcium—this stimulated endocytosis of synaptotagmin-bound antibodies.

The authors found that both pools of protein appeared as discrete dots rather than diffuse patches, suggesting that even when fused to the cell membrane, synaptotagmin remains in small clusters. This in itself lends support to the kiss-and-run theory, but to capture it in action, the researchers examined how synaptotagmin behaves when synaptic vesicle recycling is ramped up. When they stimulated the neurons with potassium chloride, total internal and cell surface staining was dramatically increased. However, the pattern remained unchanged—synaptotagmin on the cell surface still appeared as discrete dots, demonstrating that at least this one vesicle component does not diffuse through the cell membrane once synaptic vesicles dock there.

“Their study provides some of the most compelling evidence to date that at least some membrane constituents remain grouped together after vesicles fuse with the plasma membrane (rather than diffusing freely within the membrane like a drop of water on water), which is consistent with the kiss-and-run theory,” writes Garth Simpson, Purdue University, West Lafayette, Indiana, in an accompanying Nature News & Views. Should the same be seen for other vesicle components, then support for the kiss-and-run theory should receive a huge boost.

In the meantime, it is worth noting that there is no theoretical limit to the resolution attainable by STED microscopy. By narrowing the aperture of the STED beam, the resolution can be improved further, potentially allowing measurements of single molecules. In fact, Hell’s group has recently achieved a focal spot of only 16 nm across (see Westphal and Hell, 2005).—Tom Fagan.

References:
Willig KI, Rizzoli SO, Westphal V, Jahn R, Hell SW. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature. April 13, 2006;440:935-939. Abstract

Simpson GJ. The diffraction barrier broken. Nature. April 13, 2006;440:879-880. Abstract

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  1. The paper by Willig et al., is a technical tour de force. In this study, the authors use stimulated emission depletion (STED) microscopy to visualize fluorescent spots 10-fold smaller than the light diffraction limit. Breaking the diffraction limit in light microscopy opens up several new avenues in addressing biological questions. Using this technique, one can directly visualize the organization of molecular signaling complexes, membrane domains, and cellular organelles with nanometer resolution using the entire arsenal of tools available to light microscopy. This study also provides the first direct answer to a fundamental question in neurobiology, the fate of a single synaptic vesicle after fusion with the plasma membrane and release of its neurotransmitter cargo.

    What happens to a synaptic vesicle after it fuses with the plasma membrane? This question is not as esoteric as it sounds. Synapses desperately depend on retrieval and reuse of synaptic vesicles to maintain neurotransmission during bursts of stimuli. The rapidity of this vesicle retrieval impacts the reliability of neurotransmission during activity. Synapses, therefore, fine-tune this process to adjust to varying levels of activity. The Willig et al. study shows that synaptic vesicles retain their protein composition or “identity” following fusion. This finding suggests that synaptic vesicles are not reassembled after each fusion and thus can be retrieved swiftly with high fidelity without losing their competence for subsequent rounds of fusion. The next questions are whether this process is regulated and whether complex brain disorders involve deficits in synaptic vesicle retrieval that ultimately result in neurotransmission failures.

  2. The extremely small size of nerve terminals is one of the major handicaps to studying synaptic function in the brain. That problem gets even more challenging when studying the life cycle of synaptic vesicles, tiny membrane-bound organelles (~40 nm in diameter) that store and release neurotransmitters. In molecular terms, synaptic vesicles are probably the best described organelle in the cell. Until last week, unitary synaptic vesicles were invisible to conventional confocal microscopy that, limited by the diffraction barrier, only resolves structures larger than ~200 nm. Now, the groups of Reinhard Jahn and Stefan Hell have made an enormous step toward visualizing nerve terminals in cultured neurons with a novel microscopy technique that overcomes the diffraction limits. The so-called stimulation emission depleted (STED) microscopy has inaugurated its career in biology with a brilliant work resolving spots of synaptotagmin molecules that, strikingly, present a size coincident with the size of single synaptic vesicles.

    Synaptotagmin, the essential Ca2+ sensor for fast exocytosis, is an integral membrane protein of the synaptic vesicle. Using STED, the authors have followed the fate of synaptotagmin molecules at different stages of the synaptic vesicle cycle. A classical view of the synaptic vesicle cycle would predict that, upon exocytosis, the full collapse of the vesicle with the plasma membrane would lead to the spread and diffusion of synaptic vesicle proteins. Interestingly, what Willig, Rizzoli, and collaborators have observed is exactly the opposite: synaptotagmin molecules always kept together, forming individual clusters of rather uniform size. Most likely, those “synaptotagmin quanta” correspond to the synaptotagmin content of single synaptic vesicles, indicating the existence of strong mechanisms to maintain the functional and structural integrity of unitary vesicles through the vesicle cycle. It would not be surprising that perturbation of those mechanisms could lead to nerve terminal dysfunctions or neurodegeneration disorders. Certainly, the observations by Willig et al. are consistent with a model of exo- and endocytosis where neurotransmitter release occurs through the reversible opening of a transient fusion pore (“kiss-and-run”). Nevertheless, many laboratories would like to look through the STED microscope to ask, for example: Where is clathrin? Undoubtedly, the STED microscope promises to clarify those and other difficult questions in contemporary biology.

References

Paper Citations

  1. . Nanoscale resolution in the focal plane of an optical microscope. Phys Rev Lett. 2005 Apr 15;94(14):143903. PubMed.
  2. . STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature. 2006 Apr 13;440(7086):935-9. PubMed.
  3. . Biological imaging: the diffraction barrier broken. Nature. 2006 Apr 13;440(7086):879-80. PubMed.

Further Reading

Papers

  1. . STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature. 2006 Apr 13;440(7086):935-9. PubMed.
  2. . Biological imaging: the diffraction barrier broken. Nature. 2006 Apr 13;440(7086):879-80. PubMed.

Primary Papers

  1. . STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature. 2006 Apr 13;440(7086):935-9. PubMed.
  2. . Biological imaging: the diffraction barrier broken. Nature. 2006 Apr 13;440(7086):879-80. PubMed.