Protein nanorotors control the size of lipid domains in phase-separated monolayers

Phase separation in lipid membranes leads to the formation of distinct lipid domains, which are influenced by kinetic factors and interfacial phenomena. While line tension has been considered a key determinant of domain size, studies suggest that kinetic effects play a significant role. We hypothesize that modifying in situ the surface pressure difference between coexisting lipid phases can regulate domain size. Specifically, the rotational activity of ATP synthase embedded in a specific phase may induce local changes in the lipid surface pressure, triggering the change in domain size.

To test this hypothesis, ATP synthase was incorporated into phase-separated lipid monolayers by leveraging its specific interaction with cardiolipin (CL). The ATP synthase assembly and its co-localization within CL-rich phases were characterized to assess the enzyme's role in domain modulation. The effect of rotational forces on phase dynamics was analyzed, with particular attention to the change in size of protein-enriched and protein-devoid lipid domains. The system was characterized using fluorescence video microscopy and quantitative analysis of domain contour fluctuations.

Upon ATP addition, protein-enriched domains increased in size, while protein-devoid domains contracted. The observed changes followed the 2D Young-Laplace equation, where the spinning motion of ATP synthase reduces the lateral pressure in the protein-enriched phase. The unbalanced surface pressure between phases drives the domain size modulation; which is sensitive to variations in the surface pressure difference between lipid phases as small as 10^-9N/m. These findings show that ATP synthase activity can dynamically regulate lipid phase separation by modifying interfacial properties and kinetic constraints.