Fig. 1

Actin polymerization at the membrane is mediated by Arp2/3 complex and CP. a Molecular components required to produce cytoskeletal vesicles: Actin, His-tagged VCA, Arp2/3 complex, CP and profilin were encapsulated inside of giant unilamellar vesicles (GUVs). They contained 10% Ni-NTA lipids to localize His tagged VCA to the membrane and 2.5% PEG2000 PE to avoid unspecific protein-membrane interactions. b, c Confocal images of the equatorial plane of the cytoskeletal vesicles are shown. The encapsulation of only 3 µM actin (red hot) resulted in pronounced bulk polymerization (b). The addition of 0.3 µM Arp2/3, 0.3 µM His-tagged VCA and 13.5 µM profilin to the solution prior to encapsulation induced localized actin polymerization at the membrane (c). d Pyrene fluorescence assays of actin polymerization in the dependence of actin-binding proteins (ABPs): The polymerization rate of 3 µM actin (squares) was increased upon addition of 0.3 µM VCA, 0.3 µM Arp2/3 complex and 13.5 µM profilin (circles). Further addition of 0.02 µM CP (diamonds) resulted in a distinct lag phase before polymerization was initiated. e The addition of 20 nM CP to 3 µM actin, 0.3 µM VCA, 0.3 µM Arp2/3 and 13.5 µM shifted actin polymerization to the membrane. Two different binding modes occurred. Confocal images of the equatorial plane of the vesicles show both modes: flat cortex (left side) and flat actin domains (right side). Scale bars are 20 µm