Dual Role of BAR Domain-containing Proteins in Regulating Vesicle Release Catalyzed by the GTPase, Dynamin-2

TLDR

Dynamin-2 (Dyn2) is ubiquitously expressed and catalyzes membrane fission during clathrin‑mediated endocytosis in nonneuronal cells. The study hypothesizes that endocytic accessory proteins generate the curvature needed to scaffold Dyn2 assembly and trigger fission. The authors examined BAR domain‑containing proteins that bind Dyn2’s PRD via SH3 domains, assessing their ability to generate membrane curvature and synergize with Dyn2ΔPRD or full‑length Dyn2 to release vesicles. Amphiphysin and endophilin, but not SNX9 or ENTH, generate tubules and synergize with Dyn2ΔPRD to release vesicles; SH3‑PRD interactions inhibit scaffold assembly, and only full‑length amphiphysin synergizes with full‑length Dyn2, demonstrating that curvature‑generating potency and SH3‑mediated regulation coordinate membrane curvature, dynamin assembly, and fission in clathrin‑mediated endocytosis.

Abstract

Dynamin-2 (Dyn2) is ubiquitously expressed and catalyzes membrane fission during clathrin-mediated endocytosis in nonneuronal cells. We have previously shown that Dyn2 inefficiently generates membrane curvature and only mediates fission of highly curved membranes. This led to the hypothesis that other endocytic accessory proteins (EAPs) generate curvature needed to sculpt a sufficiently narrow neck to trigger Dyn2 assembly and fission. Candidates for this activity are EAPs that bind to the dynamin proline/arginine-rich domain (PRD) through their SH3 (src homology-3) domains and also encode curvature-generating BAR (Bin/Amphiphysin/Rvs) domains. We show that at low concentrations, amphiphysin and endophilin, but not SNX9 or the curvature-generating epsin N-terminal homology (ENTH) domain, are able to generate tubules from planar membrane templates and to synergize with Dyn2ΔPRD to catalyze vesicle release. Unexpectedly, SH3-PRD interactions were inhibitory and reciprocally regulate scaffold assembly. Of the three proteins studied, only full-length amphiphysin functions synergistically with full-length Dyn2 to catalyze vesicle release. The differential activity of these proteins correlates with the relative potency of their positive, curvature-generating activity, and the negative regulatory effects mediated by SH3 domain interactions. Our findings reveal opportunities for the spatio-temporal coordination of membrane curvature generation, dynamin assembly, and fission during clathrin-mediated endocytosis.