Phase-separated plasma membranes show elevated viscosity and reduced line tension of liquid-ordered domains

Membrane surface viscosity and line tension govern the behavior of lipid domains in plasma membranes and thereby their functions. These parameters have been quantified primarily in synthetic bilayers, although eukaryotic plasma membranes are much more complex. To enable direct quantification and comparison under quasi-identical conditions, we generated pore-spanning membranes (PSMs) and pore-spanning plasma membranes (PSPMs) on porous silicon chips with micrometer-sized pores. PSMs were formed by spreading giant unilamellar vesicles, whereas PSPMs were derived from giant plasma membrane vesicles, preserving the compositional heterogeneity of native membranes. The planar PS(P)M architecture allowed straightforward fluorescence imaging. Time-lapse recordings showed phase separation at room temperature into liquid-disordered (l_d) and liquid-ordered (l_o) domains. In the freestanding membrane regions of the PS(P)Ms, l_o domains remained mobile but exhibited confined diffusion. Using our previously established analysis of l_o-domain diffusion in two-dimensional confinement as a function of the domain radius, we extracted the membrane surface viscosity. Complementary analysis of two-dimensional shape undulations of l_o domains, applying capillary wave theory, yielded the domains' line tension. Comparison of synthetic PSMs and natural PSPMs revealed a threefold higher membrane surface viscosity and a threefold lower line tension for PSPMs. This combination of lower line tension and larger membrane viscosity may improve control over domain size and lifetime in plasma membranes, facilitating the formation of dynamic, stable signaling platforms.