Endosomal sorting of readily releasable synaptic vesicles

TLDR

Neurotransmitter release relies on synaptic vesicle fusion with the plasma membrane, followed by endocytosis, and a long‑standing hypothesis posits that endosomes mediate a slow recycling pathway that sorts vesicle components and generates exocytosis‑competent vesicles. The authors examined endosomal function in hippocampal neurons, synaptosomes, and PC12 cells using stimulated emission depletion microscopy, photooxidation electron microscopy, and conventional microscopy assays. They discovered that endosomal sorting is a rapid pathway that recycles the readily releasable pool, with vesicle composition shifting after endocytosis to enrich plasma‑membrane proteins and forming clustered protein domains that segregate from plasma‑membrane components to facilitate vesicle budding.

Abstract

Neurotransmitter release is achieved through the fusion of synaptic vesicles with the neuronal plasma membrane (exocytosis). Vesicles are then retrieved from the plasma membrane (endocytosis). It was hypothesized more than 3 decades ago that endosomes participate in vesicle recycling, constituting a slow endocytosis pathway required especially after prolonged stimulation. This recycling model predicts that newly endocytosed vesicles fuse with an endosome, which sorts (organizes) the molecules and buds exocytosis-competent vesicles. We analyzed here the endosome function using hippocampal neurons, isolated nerve terminals (synaptosomes), and PC12 cells by stimulated emission depletion microscopy, photooxidation EM, and several conventional microscopy assays. Surprisingly, we found that endosomal sorting is a rapid pathway, which appeared to be involved in the recycling of the initial vesicles to be released on stimulation, the readily releasable pool. In agreement with the endosomal model, the vesicle composition changed after endocytosis, with the newly formed vesicles being enriched in plasma membrane proteins. Vesicle proteins were organized in clusters both in the plasma membrane (on exocytosis) and in the endosome. In the latter compartment, they segregated from plasma membrane components in a process that is likely important for sorting/budding of newly developed vesicles from the endosome.