Functional recruitment of dynamin requires multimeric interactions for efficient endocytosis

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Clathrin-mediated endocytosis (CME) is a vital cellular process for membrane trafficking.
• Dynamin, a GTPase, plays a critical role in the final membrane scission step of CME.
• Dynamin interacts with various proteins, notably amphiphysins, via its proline/arginine-rich domain (dynPRD) and their SH3 domains.

Purpose of the Study:

• To investigate the necessity of multiple SH3 domain interactions with dynamin for efficient CME.
• To determine the role of simultaneous binding of SH3 domains to dynPRD motifs in dynamin function.
• To explore the potential of multivalent interactions in enhancing dynamin's role in membrane scission.

Main Methods:

• Utilized mutant dynamins with modifications in dynPRD motifs in dynamin triple knock-out cells.
• Designed and synthesized divalent dynPRD-derived peptides.
• Employed patch-clamp pipettes for delivering peptides to living cells.
• Performed in vitro binding assays to assess ligand affinity.

Main Results:

• Mutations in a single dynPRD motif only partially rescued CME, while mutations in two motifs largely abolished rescue.
• Divalent dynPRD-derived peptides exhibited over 100-fold higher affinity for amphiphysins SH3 domain multimers compared to monovalent peptides in vitro.
• Cellular delivery of divalent peptides via patch-clamp pipettes significantly blocked CME more effectively than monovalent peptides.

Conclusions:

• Simultaneous binding of multiple SH3 domains to adjacent motifs on dynPRD is essential for efficient dynamin recruitment and function in CME.
• Multivalent interactions significantly enhance the efficacy of dynamin in driving membrane scission during endocytosis.
• Dynamin utilizes multivalent interactions to drive vesicle scission within cells.