Fig. S6Tables S1 to S3 (as Excel files) Movie S1

Aged proteins can become hazardous to cellular function, by accumulating molecular damage. This implies that cells should preferentially rely on newly produced ones. We tested this hypothesis in cultured hippocampal neurons, focusing on synaptic transmission. We found that newly synthesized vesicle proteins were incorporated in the actively recycling pool of vesicles responsible for all neurotransmitter release during physiological activity. We observed this for the calcium sensor Synaptotagmin 1, for the neurotransmitter transporter VGAT, and for the fusion protein VAMP2 (Synaptobrevin 2). Metabolic labeling of proteins and visualization by secondary ion mass spectrometry enabled us to query the entire protein makeup of the actively recycling vesicles, which we found to be younger than that of non‐recycling vesicles. The young vesicle proteins remained in use for up to ~ 24 h, during which they participated in recycling a few hundred times. They were afterward reluctant to release and were degraded after an additional ~ 24–48 h. We suggest that the recycling pool of synaptic vesicles relies on newly synthesized proteins, while the inactive reserve pool contains older proteins.

mCLING is a novel membrane probe for the study of membrane trafficking with demonstrated value in both live and fixed cells across a wide range of biological systems.

Expansion microscopy is a recently introduced imaging technique that achieves super‐resolution through physically expanding the specimen by ~4×, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately fourfold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20–30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10× in each dimension, which corresponds to an expansion of the sample volume by more than 1,000‐fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25–30 nm on conventional epifluorescence microscopes. X10 provides multi‐color images similar or even superior to those produced with more challenging methods, such as STED, STORM, and iterative expansion microscopy (iExM). X10 is therefore the cheapest and easiest option for high‐quality super‐resolution imaging currently available. X10 should be usable in any laboratory, irrespective of the machinery owned or of the technical knowledge.

No abstract

Expansion microscopy is a recently introduced imaging technique that achieves super-resolution through physically expanding the specimen by ~4x, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately 4-fold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20-30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10x in each dimension, which corresponds to an expansion of the sample volume by more than 1000-fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25-30 nm on conventional epifluorescence microscopes. X10 provides multi-color images similar or even superior to those produced with more challenging methods, such as STED, STORM and iterative expansion microscopy (iExM), in both cell cultures and tissues.

Many proteins involved in synaptic transmission are well known, and their features, as their abundance or spatial distribution, have been analyzed in systematic studies. This has not been the case, however, for their mobility. To solve this, we analyzed the motion of 45 GFP‐tagged synaptic proteins expressed in cultured hippocampal neurons, using fluorescence recovery after photobleaching, particle tracking, and modeling. We compared synaptic vesicle proteins, endo‐ and exocytosis cofactors, cytoskeleton components, and trafficking proteins. We found that movement was influenced by the protein association with synaptic vesicles, especially for membrane proteins. Surprisingly, protein mobility also correlated significantly with parameters as the protein lifetimes, or the nucleotide composition of their mRNAs. We then analyzed protein movement thoroughly, taking into account the spatial characteristics of the system. This resulted in a first visualization of overall protein motion in the synapse, which should enable future modeling studies of synaptic physiology.

Highlights d Synaptic vesicle supply needs to be adapted to synaptic activity d Presynapses that recycle more SVs contain more newly synthesized proteins d Colchicine disrupts the correlation between synaptic activity and protein turnover d Chronic stimulation or depression also abolish this correlation