Figure 7.

FH1FH2 Preferentially Binds MTs and Crosslinks AFs and MTs in Vitro. (A) Time course of actin polymerization in the presence of FH1FH2 and MTs monitored by pyrene fluorescence. Different concentrations of taxol-stabilized MTs were incubated with 200 nM FH1FH2 and 2 μM actin (10% pyrene labeled) before initiation of polymerization. a.u., arbitrary units. (B) Time course of actin polymerization in the presence of FH1FH2 and tubulin monomers monitored by pyrene fluorescence. Different concentrations of tubulin monomers were incubated with 200 nM FH1FH2 and 2 μM actin (10% pyrene labeled) before initiation of polymerization. a.u., arbitrary units. (C) Preformed AFs labeled with Alexa-488 phalloidin without FH1FH2. (D) AFs exhibited considerable bundles 30 min after addition of FH1FH2. (E) and (F) Addition of preformed taxol-stabilized rhodamine-conjugated MTs to the reaction in (D) resulted in the formation of MT bundles and AF bundles decrease within 30 min (E) and 4 h (F). (G) Taxol-stabilized rhodamine-conjugated MTs without FH1FH2. (H) MTs exhibited considerable bundles 30 min after addition of FH1FH2. (I) and (J) MTs remained bundled and AFs were randomly scattered throughout the solution 30 min (I) and 4 h (J) after the addition of preformed AFs to the reaction in (H). (K) AFs and MTs were scattered randomly throughout the suspension without FH1FH2. (L) MT bundles formed at 30 min after the addition of low concentration FH1FH2 (500 nM) to the reaction in (K), but AF bundles were not formed. (M) and (N) Excess FH1FH2 proteins (2 μM) induced bundling of both MTs and AFs at 30 min (M) and crosslinking bundles of the MTs and AFs at 4 h (N) after addition. Bar in (L) = 10 μm.