The Liquid Phase of a Protein Is Involved in Synaptic Vesicle Clustering

What's the science?

Synaptic vesicles carry neurotransmitters and thus are critical for neuron-to-neuron communication. Synaptic vesicles cluster together at the presynaptic membrane and yet are able to move within the neuron terminal prior to release. Why might this be? One protein called synapsin is among the most abundant proteins in presynaptic neuron terminals. It binds to synaptic vesicles and therefore might be involved in their release. Furthermore, recent research suggests that some proteins and molecules within the cytoplasm of cells (including neurons) may be able to organize themselves even in the absence of a membrane, via phase separation (e.g. like how oil and water separate). This week in Science, Milovanovic and colleagues test whether synapsin demonstrates properties of phase separation and whether it is involved in cluster forming or release of synaptic vesicles.

How did they do it?

The authors tagged synapsin with green fluorescent protein and incubated synapsin molecules with a salt containing liquid buffer (resembling typical physiological conditions) to test whether synapsin phase-separates (i.e. into two liquid phases). They also tested whether synapsin can recruit (into it’s phase, ie. liquid) other proteins within the presynaptic terminal. They then performed experiments to mimic the synaptic environment more closely (which is filled with many different molecules and organelles) by adding polyethelene glycol to the buffer as a crowding reagent. They tested how synapsin might interact with vesicles and be involved in their cluster forming by incubating small lipid vesicles with synapsin. Using green fluorescent protein tagging, fluorescence microscopy, and electron microscopy, they tested whether synapsin could recruit the lipid vesicles into its phase. They further tested whether calcium dependent phosphorylation of synapsin (which is known to occur in the synapse during neuron activity) resulted in a disassembly of the synapsin/lipid vesicle phase as this would indicate the involvement of synapsin in the vesicle dispersion that occurs in stimulated synapses.

What did they find?

Synapsin was confirmed to phase-separate under physiological conditions (i.e. similar to those occurring in neurons in the human brain) as was evident by droplets of synapsin forming within the liquid buffer solution. They found that many other proteins located in the presynaptic terminal with synaptic vesicles also underwent phase separation when incubated with synapsin. Therefore, they tested how these interacted with synapsin. Two proteins known to bind synapsin, intersectin and GRB2, were found to form droplets with synapsin in the liquid buffer. After adding a crowding reagent to mimic the environment of a synapse, droplets of synapsin (either alone or with its binding partners intersectin or GRB2) formed even more efficiently. After incubating synapsin with lipid vesicles, they found that the synapsin droplets sequestered lipid vesicles which resulted in the formation of droplets containing lipid vesicles. As a control experiment, they tested whether other liquid mediums containing proteins that phase separate (but do not bind lipid vesicles) could also sequester lipid vesicles, and found that they did not. Thus, synapsin specifically allows the clustering of lipid vesicles. Upon calcium dependent phosphorylation of synapsin, droplets of synapsin alone or synapsin + lipid vesicles dispersed, supporting the hypothesis that synapsin is important for vesicle sequestering and for their dispersion during stimulation.

What's the impact?

This is the first study to show that phase-separation of a protein, synapsin, within the presynaptic terminal plays an important role in vesicle clustering. More generally, it is the first demonstration that phase-separation of a protein can organize vesicles within a cell. We now know that synapsin, and importantly it’s liquid phase, is involved in sequestering and dispersing vesicles in the presynaptic terminal. Future studies should examine how molecules within neurons may organize themselves based on phase separation.

Milavanovic et al., A liquid phase of synapsin and lipid vesicles. Science (2018). Access the original scientific publication here.