VLN2 Can Bundle and Cap MFs in Vitro.
(A) Purification of recombinant VLN2 protein in E. coli. Two micrograms of purified VLN2-6×His tag fusion protein was loaded and stained by Coomassie blue.
(B) VLN2 binds to actin filaments. A high-speed cosedimentation assay was used to determine the binding of VLN2 to filamentous actin. Three micromolars of preassembled F-actin was incubated with 0.5 μM VLN2 for 30 min at room temperature and sedimented at 100,000g for 1 h at 4°C. The proteins in the supernatant (S) and pellet (P) were resolved by SDS-PAGE.
(C) Determination of the equilibrium dissociation constant (K d) for VLN2 binding to actin filaments. The K d value calculated for VLN2 in this representative experiment is 0.87 μM.
(D) VLN2 bundles actin filaments. A low-speed cosedimentation assay was used to determine the bundling activity of VLN2 on MFs. Three micromolars of preassembled F-actin was incubated with 0.5 μM VLN2 for 30 min at room temperature and sedimented at 13,600g for 1 h at 4°C. The proteins in the supernatant and pellet were resolved by SDS-PAGE.
(E) and (F) Micrographs showing the actin filament status in the absence ([E]; filamentous actin) or presence ([F]; actin bundles) of VLN2. Bars = 50 μm.
(G) VLN2 inhibits seeded actin elongation. VLN2 inhibited the addition of the profilin/actin complex onto the barbed end of actin filaments in a dose-dependent manner.
(H) The initial rates of elongation were plotted for the representative experiments to determine the capping activity of VLN2. In this representative experiment, the K d value for the binding of VLN2 to the barbed ends of MFs is 41.8 nM.
(I) VLN2 stabilizes actin filaments from dilution-mediated depolymerization in the presence of 10 nM Ca2+.
