Figure 5.

Villin Rapidly Bundles Actin Filaments But Does Not Affect the Rate of Actin Polymerization.

Conditions: 4 μM actin, 10 mM Tris-HCl, pH 7, 50 mM KCl, 2 mM MgCl₂, 1 mM EGTA, 0.2 mM ATP, 0.2 mM CaCl₂, 0.5 mM DTT, and 3 mM NaN₃ at 20°C.

(A) Time course of polymerization monitored by light scattering. Dashed line, no additions to actin; filled squares, addition of 0.2 μM villin; filled triangles, addition of 0.4 μM villin; filled circles, addition of 0.5 μM villin.

(B) Comparison of actin polymerization monitored by pyrene fluorescence and light scattering. Filled squares, polymerization of actin alone monitored by light scattering; filled circles, polymerization of actin in presence of 0.5 μM villin monitored by light scattering; open squares, polymerization of actin alone monitored by pyrene fluorescence; open circles, polymerization of actin in presence of 0.5 μM villin monitored by pyrene fluorescence.

(C) and (D) Micrograph of actin bundles in the presence of VLN1 in vitro. The ability of VLN1 to form actin bundles was visualized by fluorescence light microscope with rhodamine-phalloidin staining. Scale bar = 10 μm.

(C) Individual actin filaments formed in the absence of VLN1.

(D) Bundles of actin filaments in the presence of 500 nM VLN1.