Biological imaging: the diffraction barrier broken.
Nature. 2006 Apr 13;440(7086):879-80.
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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.
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.