A comparative analysis of the mobility of 45 proteins in the synaptic bouton.

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.

Comparative synaptosome imaging: a semi-quantitative method to obtain copy numbers for synaptic and neuronal proteins.

Protein copy numbers can be measured by biochemical methods ranging from quantitative Western Blotting to several mass spectrometry approaches. Such methods only provide average copy numbers, obtained over large cell numbers. However, copy number estimates for single cells or single organelles could be obtained by combining biochemical characterizations with an imaging approach. We performed this here for synaptic proteins, in a protocol that we termed comparative synaptosome imaging for semi-quantitative copy numbers (CosiQuant). In brief, in CosiQuant we immunostain in parallel biochemically-characterized synaptosomes, for which we have already determined the average protein copy numbers, and the samples of interest (such as neuronal cultures). We then derive the copy numbers in the samples of interest by comparing the immunofluorescence intensities. We measured the intensities not only in arbitrary fluorescence units, but also as numbers of antibodies per synaptosome, for a large number of targets. This implies that other groups can immediately apply CosiQuant for these targets, by simply estimating the number of antibodies per structure of interest. CosiQuant should therefore be a useful addition to the growing set of imaging techniques for synaptic neuroscience.