Compartmentalization of soluble endocytic proteins in synaptic vesicle clusters by phase separation

Tomofumi Yoshida; Koh-Ichiro Takenaka; Hirokazu Sakamoto; Yusuke Kojima; Takumi Sakano; Koyo Shibayama; Koki Nakamura; Kyoko Hanawa-Suetsugu; Yasunori Mori; Yusuke Hirabayashi; Kenzo Hirose; Shigeo Takamori

doi:10.1016/j.isci.2023.106826

Compartmentalization of soluble endocytic proteins in synaptic vesicle clusters by phase separation

iScience. 2023 May 6;26(6):106826. doi: 10.1016/j.isci.2023.106826. eCollection 2023 Jun 16.

Affiliations

¹ Laboratory of Neural Membrane Biology, Graduate School of Brain Science, Doshisha University, Kyoto 610-0394, Japan.
² Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
³ Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo 113-0033, Japan.
⁴ International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo, Tokyo 113-0033, Japan.

Abstract

Synaptic vesicle (SV) clusters, which reportedly result from synapsin's capacity to undergo liquid-liquid phase separation (LLPS), constitute the structural basis for neurotransmission. Although these clusters contain various endocytic accessory proteins, how endocytic proteins accumulate in SV clusters remains unknown. Here, we report that endophilin A1 (EndoA1), the endocytic scaffold protein, undergoes LLPS under physiologically relevant concentrations at presynaptic terminals. On heterologous expression, EndoA1 facilitates the formation of synapsin condensates and accumulates in SV-like vesicle clusters via synapsin. Moreover, EndoA1 condensates recruit endocytic proteins such as dynamin 1, amphiphysin, and intersectin 1, none of which are recruited in vesicle clusters by synapsin. In cultured neurons, like synapsin, EndoA1 is compartmentalized in SV clusters through LLPS, exhibiting activity-dependent dispersion/reassembly cycles. Thus, beyond its essential function in SV endocytosis, EndoA1 serves an additional structural function by undergoing LLPS, thereby accumulating various endocytic proteins in dynamic SV clusters in concert with synapsin.

Keywords: Biological sciences; Cell biology; Molecular biology.