Larsson, E.; Kabedev, A.; Pace, H.; Lindwall, J.; Bano, F.; Parton, R. G.; Bergstrom, C. A. S.; Parmryd, I.; Bally, M.; Lundmark, R.

Lipid packing is a fundamental characteristic of bilayer membranes. It affects all membrane-associated processes ranging from curvature generation to membrane fission. Yet, we lack detailed, mechanistic understanding of how lipid packing directly affects these processes in cellular membranes. Here, we address this by focusing on caveolae, small {Omega}-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 both the biogenesis, morphology and stability of such membrane invaginations. We show that the small compound Dyngo-4a inserts into the membrane, resulting in a dramatic dynamin-independent inhibition of caveola scission. 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. Dyngo-4a-treatment resulted in decreased lipid packing in the outer leaflet of the plasma membrane preventing scission without affecting caveola morphology, caveolae- associated proteins, or the overall membrane stiffness. Artificially increasing plasma membrane cholesterol levels was found to counteract the block in caveola scission caused by Dyngo-4a-mediated lipid packing frustration. Therefore, we propose that the packing of cholesterol in the outer leaflet of the plasma membrane critically controls the confinement of caveolae to the plasma membrane.