Abstract
Membrane nanodomains based on phase-segregation of lipids have emerged as a key organizing principle of the plasma membrane. They have been shown to play important roles in signal transduction and membrane trafficking. We have developed lipid-like probes carrying multivalent nitrilotriacetic acid (tris-NTA) head groups for selective targeting of His-tagged proteins into liquid ordered or liquid disordered lipid phases. The stable, non-covalent interaction of His-tagged proteins to the tris-NTA moiety can be employed not only for efficient specific tethering of spectroscopic probes, but also for versatile manipulation of membrane nanodomains. In giant unilamellar vesicles strong partitioning of tris-NTA lipids into different lipid phases was observed. For a saturated tris-NTA lipid, at least 10-fold preference for the liquid ordered phase was found. In contrast, an unsaturated NTA lipid shows a comparable preference for the liquid disordered phase. Simular partitioning of the tris-NTA lipids was observed in solid-supported membranes on mica. Partitioning into submicroscopic membrane domains formed in solid-supported membranes was confirmed by superresolution imaging techniques (FPALM, STED). Single molecule tracking of His-tagged proteins tethered to solid-supported phase-separating membranes revealed clear differences in the diffusion behavior of the different NTA-lipids. By using vesicles as a carrier, multivalent NTA lipids were efficiently incorporated into the plasma membrane of live cells. After formation of giant plasma membrane vesicles (GPMV), efficient partitioning of the lipid probes into the respective membrane phases was confirmed. We have employed these probes for exploring lipid diffusion, morphology and spatiotemporal dynamics of membrane nanodomains in vitro and live cells by single molecule tracking and STED FCS.
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