Fig. 8. L-plastin Ser5Glu has highly increased bundling activity when compared to non-phosphorylated wild type protein. (A) Co-sedimentation of WT L-plastin or S/E variant with F-actin bundles. Co-sedimentation of 12 µM G-actin with WT L-plastin or S/E variant as described in Fig. 7, with the exception that mixtures were centrifuged at low speed to sediment F-actin bundles. Coomassie-staining patterns of pellets (upper panels) and supernatants (lower panels) are shown. Lane 1, actin alone (input). The molar ratios of L-plastin to actin are indicated. (B) Quantification of F-actin bundle formation. Actin in the pellet (% of total) corresponding to bundled F-actin, was plotted as a function of increasing concentrations of WTL-plastin () or L-plastin Ser5Glu (). Each point is the mean of four experiments ± s.d. (C) Electron microscopy reveals increased bundle formation in the presence of S/E variant. Actin was copolymerised with L-plastin WT (left) or L-plastin S/E (right) at a molar ratio of 1:4 as described in A. Actin filaments were negatively stained with 1% uranyl acetate and analysed by transmission electron microscopy. Electron microscopy images are shown. (D) Effect of Ca²⁺ on bundling activities of L-plastin WT or S/E variant. Low-speed co-sedimentation of 12 µM G-actin with L-plastin WT () or S/E variant () was performed as described in A, at a 1:4 molar ratio, in the presence of various concentrations of Ca²⁺. pCa was varied by the addition of various volumes of 1 mM CaCl₂. pCa=8 (0.024 mM); pCa=7 (0.195 mM); pCa=6 (0.709 mM); pCa=5 (0.971 mM). Note that pCa=-log [Ca²⁺]. F-actin in the pellet (% of total) was plotted as a function of increasing concentrations of free Ca²⁺. Each point is the mean of three experiments ± s.d.