Ingredients for a synaptic cocktail

Some weeks ago in Naukas.com I wrote (in Spanish) about the wonderful latest video released by the team responsible for the Inner Life of a Cell which deals with the intricacies of cell trafficking in the inside of a neuron. Today I want to discuss an article ¹ recently published in the journal Science and that expands on this topic.

To get an initial idea of what we’re dealing with I recommend to watch this video and take a look at the vesicle moving inside the neuron, those clathrin molecules coating and uncoating it in its process of evagination from the Golgi or the ER to whichever other intracellular location. A well studied process of cell trafficking has typically been the synaptic vesicle recycling after the neurotransmitters have been released to the synaptic cleft for synaptic transmission. It is a simple enough process -considering how complicated things can get inside a cell- which comprises only a few steps: First, those vesicles full of neurotransmitters dock into the release site of the plasma membrane of the synaptic boutons and after receiving the right signal fuse with the membrane and release their cargo (exocytosis). The vesicle particles are then recycled from the plasma membrane by endocytosis. All textbook knowlegde. One thing left to do, however, was to understand the quantitative organization of this particular pathway.

To do so, the team of Benjamin Wilhem, Sunit Mandad et al. first isolated and purified synaptosomes (synaptic boutons) from cells of adult rat cortex and cerebellum using a brain fractionation protocol and then separated the different cellular component in a density gradient which resulted in a quite heterogeneus sample that was analyzed with electronic microscopy. This way they determined that almost 60% of all organelles were vesicle loaded synaptosomes, this analysis also provided information on the size, surface and volume of synaptosomes, which are important parameters to get an understanding of protein concentration.

They tested the quality of their purification procedure by comparing their samples to others from unpurified brain with fluorescent microscopy. No protein changes could be observed due to the purification step.

Now to get started with the protein quantification they performed protein immunoblots for 62 synaptic proteins, and then to go from the amount of protein per microgram of synaptosomes to copy numbers per synaptosome, they determined the number of particles in the synaptosome prep by fluorescence microscopy (~17million) and the number of synaptosomes by electron microscopy and immunostaining of synaptic markers. By both means the percentage of synaptosomes is close to 60%, about 10 million synaptosomes per microgram.

By immunoblot they could also determine the mass of about 40.5% of proteins in the synaptosome, to extend the measurement they used mass spectrometry and together with the data provided by the immunoblots they could quantify protein weight in the sample up to 88.4% in the whole synaptosome preparation.

They found that even though the copy numbers of some proteins correlate well with the literature there are other factors that are either too abundant for the cell needs (exocytic SNAREs appeared in thousands even though only 1-3 would take part in vesicle fusion) or too scarce like the clathrin molecules which would allow for the enodcytosis of only 7% of all vesicles. To estimate the importance of protein localization since this could affect the local concentrations of different factors, they immunostained 62 proteins and used STED, a special type of fluorescent microscopy with very high spatial resolution, in their sample and 2 others for control: one from cultured hyppocampal neurons and another from a neuromuscular junction.

They found most proteins from the active zone were confined to this region whereas the rest were more or less distributed throughout the synaptic boutons, with a slight accumulation either in the active zone or the vesicle cluster but it doesn’t seem that localization would be a likely way of compensation for low copy number.

With all the data they created a three-dimensional model containing 60 proteins in a synaptic volume. This 3D model, which you can observe in this video is very interesting because it shows just how crowded the synaptic space is, which might impede to a certain extent organelle and protein diffusion. It could be, they propose, that the high copy numbers of vesicle exocytosis proteins are necessary to ensure fast neurotransmitter release. However, endocytosis of the recycled vesicles is much slower but the cell can afford it by having a high number of ready-to-go vesicles.

One last remark has to do with correlations in copy number among proteins involved in the same steps of vesicle recycling, which is peculiar since those proteins have different lifetimes and their precursors are also different. A possible explanation could lie in synaptic vesicle regulation: a mechanism by which vesicles bind and buffer proteins by means of cofactors. But it is yet to be determined how transmembrane proteins get regulated.

As a summary, this work is important because it not only gives us a measure of the composition of a cell structure as important as the synaptic bouton but it also puts it into a spatial perspective which helps us get at the function of its components and its relative importance -in numbers, at least-. Plus the 3d models are just incredible!