FIGURE 2.

Shift in T_misc due to membrane-associated actin networks. (A) Illustration of domain stabilization by actin networks. T_ma and T_m denote T_misc for the vesicles with and without actin, respectively. T_R and T_H indicate room temperature and high temperature, respectively. (B) T_misc increases for GUVs associated with an actin network (red squares) compared to those without (black squares). T_m and T_ma were determined to be 33.4°C ± 0.5°C and 40.6°C ± 0.3°C by fitting the data points to a sigmoidal curve (black and red line). When actin was polymerized at T_H, T_misc of individual vesicles (n = 3) was found to be 40.3°C ± 0.8°C (blue square) by lowering temperature. (C) Dependence of T_misc shift on model system composition. A substantial increase in T_misc was not observed when N-WASP or TMR-PIP₂ were omitted from the model system (bars 4 and 5), indicating that PIP₂-N-WASP linked through actin filaments is responsible for the observed increase in T_misc. When the actin network was removed from membrane by incubation with Proteinase K or when TMR-PIP₂ was replaced by DOPS, a substantial increase in T_misc was not observed (bars 9 and 10). However, when TMR-PIP₂ was replaced with unlabeled-PIP₂, a significant increase in T_misc was measured (bars 2 and 3). Phase separation of membrane was determined by lis-DPPE fluorescence for bars 2, 3, and 10, and fluo-DOPE for all other experiments. T_misc was determined as in B.