Cooperative function of synaptophysin and synapsin in the generation of\xa0synaptic vesicle-like clusters in non-neuronal cells

Daehun Park,Yumei Wu,Sunghoe Chang,Sang-Eun Lee,Pietro De Camilli,Seonyoung Jeong,Dragomir Milovanovic,Goeun Kim

doi:10.1038/s41467-020-20462-z

Abstract

Clusters of tightly packed synaptic vesicles (SVs) are a defining feature of nerve terminals. While SVs are mobile within the clusters, the clusters have no boundaries consistent with a liquid phase. We previously found that purified synapsin, a peripheral SV protein, can assemble into liquid condensates and trap liposomes into them. How this finding relates to the physiological formation of SV clusters in living cells remains unclear. Here, we report that synapsin alone, when expressed in fibroblasts, has a diffuse cytosolic distribution. However, when expressed together with synaptophysin, an integral SV membrane protein previously shown to be localized on small synaptic-like microvesicles when expressed in non-neuronal cells, is sufficient to organize such vesicles in clusters highly reminiscent of SV clusters and with liquid-like properties. This minimal reconstitution system can be a powerful model to gain mechanistic insight into the assembly of structures which are of fundamental importance in synaptic transmission.

Highlights

Clusters of tightly packed synaptic vesicles (SVs) are a defining feature of nerve terminals
It was recently suggested that principles of liquid-liquid phase separation (LLPS) may explain the formation and maintenance of these clusters[1]
We had previously shown that expression in fibroblastic CHO cells of synaptophysin (Syph), a major SV intrinsic membrane protein, results in its accumulation in synaptic-like microvesicles which, like bona fide SVs, are part of the recycling endocytic system, as they become labeled by endocytic tracers[13,14]

Summary

Introduction

Clusters of tightly packed synaptic vesicles (SVs) are a defining feature of nerve terminals. When expressed together with synaptophysin, an integral SV membrane protein previously shown to be localized on small synaptic-like microvesicles when expressed in non-neuronal cells, is sufficient to organize such vesicles in clusters highly reminiscent of SV clusters and with liquid-like properties. We had previously shown that expression in fibroblastic CHO cells of synaptophysin (Syph), a major SV intrinsic membrane protein, results in its accumulation in synaptic-like microvesicles which, like bona fide SVs, are part of the recycling endocytic system, as they become labeled by endocytic tracers[13,14] These vesicles are often arranged in very small clusters[13], but not in the larger tightly packed clusters typical of SVs in presynaptic nerve terminals[1]. Our results show that expression of these two neuronal proteins alone in fibroblasts is sufficient to generate clusters of small vesicles that are morphologically similar to those typical of synapses and that share some of the same properties

Methods

Results

Conclusion