Figure 4: ExoY association with F-actin can interfere with the regulation of filament dynamics by eukaryotic cytoskeletal proteins.
(a) MBP-ExoYK81M mediated acceleration of G-actin-ATP-Mg self-assembly rate and its inhibition by profilin. Four micromolar G-actin-Mg-ATP (3% pyrenyl labelled) was polymerized in the absence (continuous lines) or presence (dashed lines) of 12 μM profilin at the indicated concentrations (μM) of MBP-ExoYK81M. MBP by itself (4 μM, brown) did not stimulate polymerization. (b) ExoYK81M binding to filaments inhibits their spontaneous disassembly kinetics from free barbed- or pointed-ends. Disassembly from free barbed-ends (continuous lines): F-actin (2 μM, 50% pyrenyl) at steady state was diluted to 50 nM in the presence of 0 to 250 nM ExoYK81M. Disassembly from pointed-ends (dotted lines): filaments with their barbed-ends capped by Gelsolin (10 μM actin, 33.3 nM Gelsolin) at steady state were diluted to 300 nM in the presence of 0 to 250 nM ExoYK81M. (c) Binding affinity of MBP-fused ExoY and ExoYK81M for F-actin (MA-L) measured in high-speed cosedimentation assays. Left panels: Representative Coomassie blue stained SDS–PAGE (10%) gel images from the supernatant (S) and pellet (P) fractions using 3 μM of F-actin-ADP-BeF3− mixed with increasing amount of MBP-ExoY (0 to 17.9 μM). Without F-actin (lanes 1 and 2) no more than 7% of MBP-ExoY was found in the pellet. Right panel: The increasing amounts of MBP-ExoY/ExoYK81M bound to filaments were measured by densitometry, normalized and fitted to derive the equilibrium dissociation constant (Kd) of the proteins for F-actin-ADP-BeF3−. Error bars are s.e. (n≥3). (d) ExoYK81M inhibits the acceleration of filament formation induced by VCA-activated Arp2/3 complex. Three micromolar G-actin-Mg-ATP (3% pyrenyl) was polymerized with 7.5 μM profilin, 0.2 μM NWASP-VCA, in the absence (black) or presence of 35 nM Arp2/3 and 0 to 600 nM ExoYK81M. (e) ExoY/ExoYK81M inhibits the acceleration of F-actin-ADP disassembly promoted by ADF. F-actin-ADP (9 μM, 50% pyrenyl) at steady state was diluted to 4 μM and preincubated for 2 min with 0 to 630 nM ExoY (w.t., continuous lines) or ExoYK81M (dotted lines) before depolymerization assays without ADF (0 nM ExoY/ExoYK81M, black) or with 4 μM ADF (grey, cyan or blue curves).
