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Yet, we lack detailed mechanistic understanding of how lipid packing directly affects membrane-associated cellular processes. Here, we address this by focusing on caveolae, small \u03a9-shaped invaginations of the plasma membrane, which serve as key regulators of cellular lipid sorting and mechano-responses. In addition to caveolae coat proteins, the lipid membrane is a core component of caveolae that critically impacts their biogenesis, morphology, and stability. We show that the small compound Dyngo-4a adsorbs and inserts into the membrane, resulting in a dramatic dynamin-independent inhibition of caveola dynamics. Analysis of model membranes in combination with molecular dynamics simulations revealed that a substantial amount of Dyngo-4a was inserted and positioned at the level of cholesterol in the bilayer, affecting lipid order in a cholesterol-dependent manner. Dyngo-4a treatment resulted in decreased lipid packing of the plasma membrane. This prevented caveolae internalization and lateral diffusion without affecting their morphology, associated proteins, or the overall cell stiffness. Artificially increasing plasma membrane cholesterol levels was found to counteract the block in caveola dynamics caused by Dyngo-4a. Therefore, we propose that the outer leaflet lipid packing of cholesterol in the plasma membrane critically contributes to the confinement of caveolae to the plasma membrane.<\/jats:p>","DOI":"10.7554\/elife.108369","type":"journal-article","created":{"date-parts":[[2025,9,26]],"date-time":"2025-09-26T15:30:12Z","timestamp":1758900612000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.108369","source":"Crossref","is-referenced-by-count":0,"title":["Lipid packing contributes to the confinement of caveolae to the plasma membrane"],"prefix":"10.7554","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4224-8226","authenticated-orcid":true,"given":"Elin","family":"Larsson","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05kb8h459","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Medical and Translational Biology and Laboratory for Molecular Infection Medicine Sweden, Ume\u00e5 Centre for Microbial Research, SciLifeLab, Ume\u00e5 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