Functional recruitment of dynamin requires multimeric interactions for efficient endocytosis

During clathrin mediated endocytosis (CME), membrane scission is achieved by the concerted action of dynamin and its interacting partners. The dynamin-mimicking peptide D15 inhibits endocytosis in living cells by disrupting the interaction between the proline/arginine-rich domain of dynamin (dynPRD) and the Src-homology domain 3 of amphiphysin (amphSH3). However, the low affinity of D15 for amphSH3 raises a question: how could a weak dynamin-amphiphysin interaction result in the precise spatio-temporal recruitment of dynamin? Here, we propose that oligomerisation increases the avidity of the interaction by bringing multiple binding sites in close proximity. We found that divalent dynPRD-derived peptides bind more strongly than monovalent ones to multimers of amphSH3. Consistently, divalent peptides block endocytosis more effectively than monovalent ones in living cells. Moreover, while a mutant dynamin modified in the D15 motif alone partially rescues endocytosis in dynamin triple knock-out cells, mutations in flanking SH3-binding sites prevent that rescue. Finally, the frequency of endocytic events decreases with hypomorphic rescue mutants but the kinetics of dynamin recruitment is unaffected. This suggests that PRD-SH3 interactions act upstream of dynamin accumulation at the neck of nascent vesicles. We conclude that dynamin drives vesicle scission via multivalent interactions in vivo.