Figure 6.

VLN1 Does Not Have Nucleation, Capping, or Severing Activity.

(A) VLN1 lacks nucleation activity. VLN1 or GS at the concentrations noted on the figure were incubated for 5 min with 2 μM actin (5% pyrene-labeled) before polymerization. Pyrene fluorescence was plotted versus time after addition of polymerization buffer.

(B) VLN1 lacks capping activity. Preformed F-actin (0.4 μM) seeds were incubated with different concentrations of VLN1 and GS, as noted on the figure. One micromolar G-actin saturated with 4 μM human profilin I was added to initiate actin elongation at the barbed end. The change in pyrene-actin fluorescence accompanying polymerization was plotted versus time after addition of G-actin.

(C) VLN1 lacks severing activity. Zea mays profilin (ZmPRO5; 2.5 μM) alone or ZmPRO5 together with VLN1 or GS were added to a F-actin solution prepared from 2.5 μM actin (40–50% pyrene-labeled). Depolymerization was recorded by reduction in pyrene fluorescence beginning from the addition of protein. All reactions are performed in the presence of 10 μM free Ca²⁺.