Functional recruitment of dynamin requires multimeric interactions for efficient endocytosis

Morgane Rosendale,Daniel Choquet,Léa Claverie,Thi Nhu Ngoc Van,Matthieu Sainlos,Stéphane Claverol,Dolors Grillo-Bosch,Isabel Gauthereau,David Perrais,Silvia Sposini

doi:10.1038/s41467-019-12434-9

Abstract

During clathrin mediated endocytosis (CME), the concerted action of dynamin and its interacting partners drives membrane scission. Essential interactions occur between the proline/arginine-rich domain of dynamin (dynPRD) and the Src-homology domain 3 (SH3) of various proteins including amphiphysins. Here we show that multiple SH3 domains must bind simultaneously to dynPRD through three adjacent motifs for dynamin’s efficient recruitment and function. First, we show that mutant dynamins modified in a single motif, including the central amphiphysin SH3 (amphSH3) binding motif, partially rescue CME in dynamin triple knock-out cells. However, mutating two motifs largely prevents that ability. Furthermore, we designed divalent dynPRD-derived peptides. These ligands bind multimers of amphSH3 with >100-fold higher affinity than monovalent ones in vitro. Accordingly, dialyzing living cells with these divalent peptides through a patch-clamp pipette blocks CME much more effectively than with monovalent ones. We conclude that dynamin drives vesicle scission via multivalent interactions in cells.

Highlights

During clathrin mediated endocytosis (CME), the concerted action of dynamin and its interacting partners drives membrane scission
We based our approach on rescuing CME in dynamin triple knock-out (TKO) mouse embryonic fibroblast (MEF) cells by re-expressing wild-type or mutant dynamin[2] tagged with GFP (Fig. 1a)
The data presented here support the idea that dynamin-Src-homology domain 3 (SH3) interactions rely on the presence of multiple interaction sites to achieve successful membrane scission

Summary

Introduction

During clathrin mediated endocytosis (CME), the concerted action of dynamin and its interacting partners drives membrane scission. Its low affinity for the SH3 domain of amphiphysin (amphSH3), 90 μM22, makes it a poor competitor of endogenous dynamin This suggests that the specific binding of a single SH3 to a given motif on dynPRD cannot solely account for the functional interaction of dynamin with its associated proteins and that avidity effects may be at play in vivo. We show that multiple neighbouring SH3 binding motifs in the dynPRD are necessary for CME rescue and for the timely recruitment of dynamin to nascent CCVs. Second, we develop divalent peptide-based amphSH3 ligands that are designed to mimic the native interactions and bind multimeric amphSH3 domains with high affinity. These results demonstrate the importance of multimeric interactions and reveal avidity effects in regulating the scission machinery

Methods

Results

Conclusion