biostudies-literatureHancock LPBiotechnology and Biological Sciences Research Council759https://www.ebi.ac.uk/biostudies/studies/S-EPMC12081870biostudies-literatureUnknown8(1)Eukaryotic actin filaments bind factors that regulate their assembly and disassembly creating a self-organising system, the actin cytoskeleton. Despite extensive knowledge of signals that modulate actin organisation, significant gaps remain in our understanding of spatiotemporal regulation of de novo filament initiation. Yeast Las17/WASP is essential for actin polymerisation initiation supporting membrane invagination in Saccharomyces cerevisiae endocytosis and therefore its tight regulation is critical. The adaptor protein Sla1 inhibits Las17 but mechanisms underpinning Las17 activation remain elusive. Here we show that Las17 binding of tandem Sla1 SH3 domains is >100-fold stronger than single domains. Furthermore, SH3 domains directly compete with G-actin for binding in the Las17 polyproCommunications biologyCompetitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation.PMC12081870BB/N007581/1BB/R000727/1BB/M011151/1Rowe MLPalmer JSSmaczynska-de Rooij IIHancock LPWilliamson MPAyscough KRAllwood EGHodder AJfalseCompetitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation.Eukaryotic actin filaments bind factors that regulate their assembly and disassembly creating a self-organising system, the actin cytoskeleton. Despite extensive knowledge of signals that modulate actin organisation, significant gaps remain in our understanding of spatiotemporal regulation of de novo filament initiation. Yeast Las17/WASP is essential for actin polymerisation initiation supporting membrane invagination in Saccharomyces cerevisiae endocytosis and therefore its tight regulation is critical. The adaptor protein Sla1 inhibits Las17 but mechanisms underpinning Las17 activation remain elusive. Here we show that Las17 binding of tandem Sla1 SH3 domains is >100-fold stronger than single domains. Furthermore, SH3 domains directly compete with G-actin for binding in the Las17 polypro2025-01-01T00:00:00Z2025 May2026-04-08T19:51:56.93Z2026-04-08T14:31:18.951ZS-EPMC120818704037477610.1038/s42003-025-08188-4