(A) In the absence of actin, our Mica supported model membrane revealed a phase transition at Tc ≈ 28°C, below which macroscopic Lo/Ld phase separation was observed. The membrane was stained with the Ld-marker DPPE-KK114 (magenta), and imaged by STED-microscopy with a lateral resolution of 70 nm. (B) The same membrane as in (A) but now in the presence of an actin network (green) bound to the membrane via a streptavidin linker. The Ld domains were strongly correlated to the actin network, as can be seen in the overlay, resulting in a meshwork like structure of Ld channels. This structure was stable even at temperatures above Tc. (C) Variation of the cumulant U1 of the images with temperature T in the absence of actin (red) and presence (blue). In the absence of actin, there is a transition toward a two-phase coexistence region at low temperature, as manifested by a pronounced drop of U1 toward unity. The transition occurs at Tc ≈ 28°C. In the presence of actin, no such transition is detected. (D) Typical domain size R as a function of T. In the absence of actin, there was pronounced domain coarsening below Tc. In the presence of actin, R was essentially temperature independent, and restricted to at most 90 nm. Note that R could not be measured for temperatures above T = 32°C as the contrast of the domains became too low. (E) Pearson correlation coefficient (PCC) between Ld domains and actin vs temperature. This coefficient measures the degree of correlation between Ld domains and the actin fibers. The correlation is largest at low temperature, but it persists at high temperature also, including the physiological temperature T = 37°C. Inset shows the graphical representation of the correlation between high intensities in the actin channel with high intensities in the lipid channel (ld phase). For another example of phase reorganization by actin binding see Figure 1—figure supplement 1. For actin meshwork binding to single component DOPC membranes see Figure 1—figure supplement 2.