Stoichiomeric biology and life cycle of the SNAREs
Zusammenfassung der Projektergebnisse
We have originally asked what the life cycle and the organization of SNARE molecules is in neuronal membranes. To understand this in detail, we first analyzed the general distribution of proteins in membranes. Most proteins have uneven distributions in the plasma membrane. This may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. The latter hypothesis has been difficult to test in the past. We introduced several approaches based on click chemistry, through which we studied the distribution of membrane proteins in living cells, as well as in membrane sheets. We found that the plasma membrane proteins form multiprotein assemblies that are long lived (minutes), and in which protein diffusion is restricted. The formation of the assemblies is dependent on cholesterol. They are separated and anchored by the actin cytoskeleton. Specific proteins are preferentially located in different regions of the assemblies, from their cores to their edges. SNAREs such as SNAP25 and syntaxin 1 accumulate at an off-center position in these assemblies, while the structurally similar, but functionally different SNAP23 and syntaxin 13 do not. We concluded that the assemblies constitute a basic mesoscale feature of the membrane, which affects the patterning of most membrane proteins, including SNAREs, and possibly also their activity. We have determined the cluster organization of the SNAREs in synapses, and found them to be quite different from each other: synaptobrevin 2 (VAMP2) was present on synaptic vesicles, syntaxin 1 formed clusters in the vicinity of the active zone, and SNAP25 formed randomly distributed clusters. Their copy numbers were all at ~20-26,000 per synaptic bouton. We have also determined the lifetime of these proteins, which averaged to ~10 days in living mice. Finally, we found that the SNARE SNAP25 is transported from the cell body to the synapse via free diffusion. Moreover, in synapses syntaxin 1 and SNAP25 are differently treated during membrane trafficking within the synapse. SNAP25, but not syntaxin 1, seems to be picked up by recycling vesicles. Synaptobrevin, which could, in principle, be lost from synaptic vesicles during recycling, does not appear to do so. In a technological sense, we have established the tools to investigate the turnover of SNARE-containing membranes, combining fluorescence microscopy with isotopic mass spectrometry imaging. The results so far indicate that organelles containing SNAP25 turn over slowly, while those that contain syntaxin 1 do not. However, this observation, performed on cell cultures, needs to be verified in the future in neurons, and eventually in living organisms.
Projektbezogene Publikationen (Auswahl)
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Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science. 2014, 344: 1023-1028
Wilhelm BG, Mandad S, Truckenbrodt S, Kröhnert K, Schäfer C, Rammner B, Koo SJ, Claßen GA, Krauss M, Haucke V, Urlaub H, Rizzoli SO
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Correlated optical and isotopic nanoscopy. Nat Commun. 2014, 5:3664
Saka SK, Vogts A, Kröhnert K, Hillion F, Rizzoli SO, Wessels JT
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Multi-protein assemblies underlie the mesoscale organization of the plasma membrane. Nat Commun. 2014, 5:4509
Saka SK, Honigmann A, Eggeling C, Hell SW, Lang T, Rizzoli SO
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Secondary ion mass spectrometry of genetically-encoded targets. Angewandte Chemie International Edition. 2015, 51: 13221-13224
Vreja IC, Kabatas S, Saka SK, Kröhnert K, Höschen C, Opazo F, Diederichsen U, Rizzoli SO
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Synaptic vesicle pools: classical and emerging roles. Book chapter in: Presynaptic Terminals. Editor: Sumiko Mochida. Springer Japan, 2015
Truckenbrodt S, Rizzoli SO
