Fig. 2

Distinct domain morphologies appear at the membrane dependent on CP concentration. a-c The equatorial plane of the vesicle, a confocal z-projection of the lower vesicle’s hemisphere, the front and side view of digitally 3D reconstituted membrane localized actin domains are shown for the three different shape deformations: a Spatially confined actin protrusions emerged at the membrane, which pushed the membrane forward and such induced a positive bending of the membrane. b Thick and laterally expanded actin patches grew at the membrane. The membrane sided surface was concave and imitated negative curvatures within the membrane. c Thin and laterally expanded actin domains grew along the membrane and did not induce any shape deformations of the membrane. d The observed actin shapes depended on CP concentration. Shown are exemplary confocal micrographs of Ni-NTA containing vesicles at different CP concentrations: actin protrusions (40 nM CP), concave actin domains (60 nM CP), concave actin cortices (120 nM CP) and undeformed cortices (180 nM CP). Top panels show the equatorial plane and bottom panels a z-stack projection of the lower hemisphere of the vesicles. The white-dashed line indicates the vesicle’s membrane. e, f Domain formation of actin was not induced by VCA or Arp2/3 complex clustering. e A total concentration of 0.3 µM encapsulated VCA bound homogeneously to the Ni-NTA membrane in the absence of actin and any ABPs. A 25% of VCA was labelled with Atto 647N. f A total concentration of 0.3 µM of Arp2/3 bound uniformly to the Ni-NTA vesicles in the presence of 0.3 µM unlabelled VCA. A 35% of the Arp2/3 was labelled with Atto 647N. g VCA clustering induced by actin polymerization is shown. The full system containing 3 µM actin, 0.3 µM VCA, 0.3 µM Arp2/3 complex, 60 nM CP and 13.5 µM profilin was encapsulated inside of Ni-NTA vesicles. A 25% of VCA was labelled with Atto 647N and 10% of actin was labelled with Atto 488. All scale bars are 20 µm