Scientific Understanding of Consciousness
Synaptic Architecture 3D Model
(Science 30 May 2014: Vol. 344 no. 6187 pp. 1023-1028)
Science 30 May 2014: Vol. 344 no. 6187 pp. 1023-1028
Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins
Benjamin G. Wilhelm, et.al.
1Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, European Neuroscience Institute, Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Göttingen, Germany.
2International Max Planck Research School Neurosciences, 37077 Göttingen, Germany.
3Bioanalytical Mass Spectrometry Group, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
4Bioanalytics, Department of Clinical Chemistry, University Medical Center Göttingen, 37075 Göttingen, Germany.
5International Max Planck Research School Molecular Biology, 37077 Göttingen, Germany.
6Leibniz Institut für Molekulare Pharmakologie, Department of Molecular Pharmacology and Cell Biology, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
Synaptic vesicle recycling has long served as a model for the general mechanisms of cellular trafficking. We used an integrative approach, combining quantitative immunoblotting and mass spectrometry to determine protein numbers; electron microscopy to measure organelle numbers, sizes, and positions; and super-resolution fluorescence microscopy to localize the proteins. Using these data, we generated a three-dimensional model of an “average” synapse, displaying 300,000 proteins in atomic detail. The copy numbers of proteins involved in the same step of synaptic vesicle recycling correlated closely. In contrast, copy numbers varied over more than three orders of magnitude between steps, from about 150 copies for the endosomal fusion proteins to more than 20,000 for the exocytotic ones.
The quantitative organization of cellular pathways is not well understood. One well-researched membrane trafficking pathway, synaptic vesicle recycling, occupies its own compartment, the synaptic bouton, and can therefore be studied in isolation. It is a relatively simple pathway, comprising only a few steps. First, neurotransmitter-filled synaptic vesicles dock to the release site (active zone), are primed for release, and then fuse with the plasma membrane (exocytosis). The vesicle molecules are later sorted and retrieved from the plasma membrane (endocytosis). An additional sorting step in an early endosome may take place before the vesicle refills with neurotransmitter.
To quantify the organization of synaptic vesicle recycling, we first purified synaptic boutons (synaptosomes) from the cellular layers of the cortex and cerebellum of adult rats, using a modified version of a classical brain fractionation protocol. The different cellular components were separated by Ficoll density gradients, resulting in a heterogeneous sample, which we first analyzed by electron microscopy. About 58.5% of all organelles were resealed, vesicle-loaded synaptosomes. Most of the remaining organelles, such as mitochondria (~20%) and myelin (8%), contained few proteins relevant to synaptic vesicle recycling and thus did not bias synaptic protein quantification. The electron microscopy analysis of the synaptosomes also provided their spatial parameters (size, surface, and volume), which are critical in understanding protein concentrations.
The 3D model suggests that the synaptic space is rather crowded, especially inside the vesicle cluster and at the active zone. This probably places constraints on both organelle and protein diffusion. The high copy numbers of exocytosis-related proteins may have evolved as a mechanism to cope with these constraints, to ensure the high speed of neurotransmitter release. In contrast, endocytosis can take place for many tens of seconds after exocytosis. This allows endocytosis to proceed with proportionally lower numbers of cofactor proteins. In principle, the synaptic boutons could increase the speed of endocytosis by accumulating larger amounts of endocytotic proteins. This, however, would result in an even greater congestion of the synaptic space, which presumably might perturb synaptic function. A simpler solution for the problem of balancing rapid release with slow vesicle retrieval appears to have been to maintain a large enough reservoir of vesicles.
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