Nanodomains in the secretory pathway investigated by super-resolution microscopy
Final Report Abstract
The mechanisms behind many cellular pathways are well understood, and their molecular players are becoming increasingly clear. However, the quantitative organization of cellular pathways is still unavailable. It is unclear how many molecules are involved in a given pathway, and whether their numbers correlate in any fashion. We have addressed this issue here, by quantifying the organization of synaptic vesicle recycling, a pathway that has long served as a model for the general mechanisms of cellular trafficking. We used quantitative Western Blotting to measure the absolute copy numbers of 62 proteins totaling more than 40% of the synaptic protein weight, and we estimated their locations by super-resolution fluorescence imaging. We used mass spectrometry to estimate the abundance of a further ~1100 proteins. Together with electron microscopy measurements of synaptic morphology, these data allowed us to generate a model of the synapse showing both protein numbers and structures. We found that protein copy numbers varied over more than three orders of magnitude, from around 150 for endosomal fusion proteins to 20,000 for exocytotic proteins. The copy numbers of functionally related proteins correlated closely, for all of the known steps of synaptic vesicle recycling. This finding points to a very tight, hitherto unknown, mechanism of regulation for cellular pathways. The proteins that make up the different organelles of the synapse seem to be stably organized, in domains that maintain their structure over time. One of the main factors supporting the stable arrangement of proteins is cholesterol. Protein clustering in the plasma membrane is eliminated by removing cholesterol. Just as in the plasma membrane, proteins are clustered in the membranes of early endosomes, and cholesterol depletion causes their dispersion. On the other hand, mitochondrial membranes, which contain 40-fold lower levels of cholesterol than the plasma membrane, are dominated by a homogeneous protein distribution. Loading them with cholesterol induced the formation of clusters. This suggests that cholesterol is a major membrane-organizing molecule, able to act even in membranes where it is not normally present at high levels. We conclude that organelles in the trafficking pathways are patterned, and that their cholesterol contents determines the patterning phenotype.
Publications
- 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
(See online at https://doi.org/10.1126/science.1252884) - Correlated optical and isotopic nanoscopy. Nat Commun. 2014, 5:3664
Saka SK, Vogts A, Kröhnert K, Hillion F, Rizzoli SO, Wessels JT
(See online at https://doi.org/10.1038/ncomms4664) - 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
(See online at https://doi.org/10.1038/ncomms5509)
