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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 <jats:italic>Saccharomyces cerevisiae<\/jats:italic> 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 &gt;100-fold stronger than single domains. Furthermore, SH3 domains directly compete with G-actin for binding in the Las17 polyproline region, thus rationalising how SH3 interactions can affect actin polymerisation despite their distance from C-terminal actin-binding and Arp2\/3-interacting VCA domains. Our data and proposed model also highlight the likely importance of multiple weak interactions that together ensure spatial and temporal regulation of endocytosis.<\/jats:p>","DOI":"10.1038\/s42003-025-08188-4","type":"journal-article","created":{"date-parts":[[2025,5,15]],"date-time":"2025-05-15T22:21:54Z","timestamp":1747347714000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Competitive binding of actin and SH3 domains at proline-rich regions of Las17\/WASP regulates actin polymerisation"],"prefix":"10.1038","volume":"8","author":[{"given":"Lewis P.","family":"Hancock","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John S.","family":"Palmer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ellen G.","family":"Allwood","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Iwona I.","family":"Smaczynska-de Rooij","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony J.","family":"Hodder","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9237-1540","authenticated-orcid":false,"given":"Michelle L.","family":"Rowe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5572-1903","authenticated-orcid":false,"given":"Mike P.","family":"Williamson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3716-4490","authenticated-orcid":false,"given":"Kathryn R.","family":"Ayscough","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,5,15]]},"reference":[{"key":"8188_CR1","doi-asserted-by":"publisher","first-page":"28","DOI":"10.1016\/j.ceb.2008.12.001","volume":"21","author":"MA Chesarone","year":"2009","unstructured":"Chesarone, M. 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