Fig. 4. ABBA binds ATP-G-actin with a high affinity through its C-terminal WH2 domain but does not bundle actin filaments. (A-D) A change in fluorescence of NBD-labelled ATP- and ADP-G-actin was measured over a range of concentrations of ABBA_274-715. Symbols represent mean data from three experiments and solid lines indicate fitted binding curves with a 1:1 stoichiometry. ABBA interacts with ATP-actin monomers with four times higher affinity than ADP-actin monomers. ABBA-mutWH2 did not display detectable binding to G-actin. (E) Low-speed cosedimentation analysis measuring actin-filament-bundling activity of ABBA IM domain (ABBA-IMD). In the presence of -actinin, majority of actin filaments were in the pellet fraction `P', demonstrating the actin-filament-bundling or crosslinking activity of the protein. By contrast, under identical conditions, ABBA IMD did not induce detectable actin-filament bundling or crosslinking, and the majority of actin was in the supernatant `S'. (F) Quantification of bundling activities of ABBA-IMD and -actinin from two independent experiments. Data are means ± s.e.m. (G) A three-dimensional confocal microscopy analysis from C6-R cells expressing GFP-tagged ABBA-IMD revealed that ABBA-IMD does not localise to peripheral actin bundles (white arrowhead), but instead localises to the plasma membrane surrounding the protruding bundles (black arrowhead). F-actin is red and ABBA-IMD green. Scale bars: 10 µm (left) and 2 µm (magnified region on right).