Figure 7.

Recombinant FH5 FH2 and FH5 FH1FH2 Nucleate Actin Polymerization from Monomeric Actin and Actin/Profilin Complexes.

(A) Time course of actin polymerization in the presence of FH5 FH1FH2 monitored by pyrene fluorescence. The signal is proportional to actin polymer concentration (Cooper et al., 1983). Different concentrations of FH5 FH1FH2 were added to 2 μM actin (10% pyrene labeled) before initiation of polymerization (from bottom to top: 0, 5, 7.5, 10, 12, 15, 20, 35, and 50 nM). a.u., arbitrary units.

(B) Time course of actin polymerization in the presence of FH5 FH2. Increasing amounts of FH5 FH2 were added to 2 μM actin (10% pyrene labeled) before initiation of polymerization (from bottom to top: 0, 10, 15, 20, 50, 80, 100, and 200 nM).

(C) Nucleation efficiency of FH5 FH2 and FH5 FH1FH2.

(D) and (E) Micrographs of actin filaments in the absence or presence of FH5 FH2. Actin alone (D); actin incubated with 100 nM FH5 FH2 (E). Bar = 5 μm.

(F) Mean length (±se) of actin filaments in the absence or presence of 100 nM formin.

(G) Time course of actin polymerization in the presence of AFH1 FH1FH2 and/or human profilin, monitored by pyrene fluorescence. From bottom to top: actin+profilin (black), actin alone (blue), actin+profilin+100 nM AFH1 FH1FH2 (red), and actin+100 nM AFH1 FH1FH2 (dark cyan).

(H) Time course of actin polymerization in the presence of FH5 FH1FH2 and/or human profilin, monitored by pyrene fluorescence. From bottom to top: actin+profilin (black), actin alone (pink), actin+profilin+10 nM FH5 FH1FH2 (red), actin+profilin+50 nM FH5 FH1FH2 (blue), actin+profilin+100 nM FH5 FH1FH2 (dark cyan), and actin+100 nM FH5 FH1FH2 (dark yellow).

(I) Time course of actin polymerization in the presence of FH5 FH1FH2 and lily profilin1 (Ll PRO1), monitored by pyrene fluorescence. From bottom to top: actin+LlPRO1 (black), actin+LlPRO1+10 nM FH5 FH1FH2 (red), actin+LlPRO1+20 nM FH5 FH1FH2 (blue), and actin+LlPRO1+50 nM FH5 FH1FH2 (dark cyan).