TAGLN2 stabilizes F-actin and competes with cofilin for binding to F-actin. (A) Actin polymerization was performed with pyrene-labeled 4 µM G-actin in APB containing 10 nM Arp2/3, 400 nM VCA, 4 µM TAGLN2, or Arp2/3 + VCA. The extent of polymerization is shown in normalized fluorescence units. (B) Pyrene-labeled G-actin was diluted to various concentrations in the presence or absence of an equimolar concentration of TAGLN2, and fluorescence was measured 4 h after adding APB to initiate polymerization. Inset graph indicates fluorescence intensities of 0–16μM pyrene-labeled G-actin mixed with same molar concentration of TAGLN2. (C) To visualize TAGLN2 and F-actin, G-actin was incubated for 1 h in APB in the presence or absence of TAGLN2 conjugated with Dylight 488. F-actin was stained with TRITC-phalloidin. (D) Depolymerization of actin filaments was performed in the presence of various concentrations of TAGLN2. 4 µM phalloidin was used as a positive control. Actin was polymerized in the presence of TAGLN2 or phalloidin for 1 h in APB and then diluted fivefold into G buffer to initiate depolymerization. The degree of depolymerization was monitored using a fluorometer. (E) Actin depolymerization was performed as in D in the presence or absence of 1 µM cofilin. T/C indicates the molar ratio of TAGLN2 (T) and cofilin (C) in the reaction. (top) Total change in pyrene intensities (ΔF) were calculated as described in Materials and methods. Results represent means ± SD of triplicate experiments. For confocal image analysis, pyrene-labeled G-actin was replaced with 488–G-actin. All data shown are representative of at least three independent experiments. (F) Prediction of the ribbon structure of a TAGLN2 homodimer using Chimera software. (left) Black arrows indicate the actin-binding motif (ABM). (right) TAGLN2 dimer and multimers are shown schematically. The TAGLN2 dimer is represented by triangular monomers with helices I and II, helices IV and V, and ABM at the three vertices represented by arcs. Bars, 5 μm.