Polycation induced actin bundles

Abstract

Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4Å) MTS-1 (1,1-methanedyl bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder.

Figure 1. Polymerization and bundle formation of 2 µM pyrene-labeled MgATP-G-actin upon addition of 2 µM lysozyme at low ionic strength

Polymerization and bundling was monitored by following pyrene fluorescence and light scattering changes, respectively, as described in Materials and Methods. Curve 1, bundling; curve 2, polymerization. Addition of 2 µM lysozyme is shown by an arrow.

Figure 2. Bundle formation of Mg-F-actin by polycations and dissolution of Mg-F-actin bundles by 100 mM NaCl

Bundling was monitored by light scattering changes. (a), Bundling of 2 µM MgF-actin by 4 µM lysozyme (1, pink), 0.5 mM spermine (2, dark blue) and 4 µM polylysine (3, green); unbundling by 100 mM NaCl. Addition of polycations and NaCl is shown by arrows. Light scattering change was followed in a PTI fluorometer. (b), Bundling of 2 µM MgF-actin by 4 µM polylysine. Green curve, fluorometer tracing; black curve, fitting to a two phase association. Light scattering was measured in a stopped flow fluorometer. (c), 100 mM NaCl dissolves bundles of 2 µM MgF-actin formed by 4 µM polylysine. Triangles, fluorometer readings; curve, fitting to a single exponential decay. Light scattering was measured in a stopped flow apparatus as described in Materials and Methods.

Figure 2. Bundle formation of Mg-F-actin by polycations and dissolution of Mg-F-actin bundles by 100 mM NaCl

Bundling was monitored by light scattering changes. (a), Bundling of 2 µM MgF-actin by 4 µM lysozyme (1, pink), 0.5 mM spermine (2, dark blue) and 4 µM polylysine (3, green); unbundling by 100 mM NaCl. Addition of polycations and NaCl is shown by arrows. Light scattering change was followed in a PTI fluorometer. (b), Bundling of 2 µM MgF-actin by 4 µM polylysine. Green curve, fluorometer tracing; black curve, fitting to a two phase association. Light scattering was measured in a stopped flow fluorometer. (c), 100 mM NaCl dissolves bundles of 2 µM MgF-actin formed by 4 µM polylysine. Triangles, fluorometer readings; curve, fitting to a single exponential decay. Light scattering was measured in a stopped flow apparatus as described in Materials and Methods.

Figure 2. Bundle formation of Mg-F-actin by polycations and dissolution of Mg-F-actin bundles by 100 mM NaCl

Bundling was monitored by light scattering changes. (a), Bundling of 2 µM MgF-actin by 4 µM lysozyme (1, pink), 0.5 mM spermine (2, dark blue) and 4 µM polylysine (3, green); unbundling by 100 mM NaCl. Addition of polycations and NaCl is shown by arrows. Light scattering change was followed in a PTI fluorometer. (b), Bundling of 2 µM MgF-actin by 4 µM polylysine. Green curve, fluorometer tracing; black curve, fitting to a two phase association. Light scattering was measured in a stopped flow fluorometer. (c), 100 mM NaCl dissolves bundles of 2 µM MgF-actin formed by 4 µM polylysine. Triangles, fluorometer readings; curve, fitting to a single exponential decay. Light scattering was measured in a stopped flow apparatus as described in Materials and Methods.

Figure 3. Bundling of Mg-F-actin by polycations in the presence and absence of 100 mM NaCl

Actin bundling was monitored by low speed sedimentation as described in Materials and Methods. Error bars correspond to mean errors from three independent experiments. (a), Effects of polycation concentration and ionic strength on the bundling. Diamonds, lysozyme; squares, polylysine; full circles, spermine. Bundling in the presence and absence of 100 mM NaCl is shown in pink and dark blue, respectively. (b) Effect of F-actin concentration on its bundling. Solid line, polylysine; dashed line, lysozyme. Polycation concentration is shown by symbols: diamonds, 10 µM; squares 25 µM; triangles, 50 µM polycation.

Figure 3. Bundling of Mg-F-actin by polycations in the presence and absence of 100 mM NaCl

Actin bundling was monitored by low speed sedimentation as described in Materials and Methods. Error bars correspond to mean errors from three independent experiments. (a), Effects of polycation concentration and ionic strength on the bundling. Diamonds, lysozyme; squares, polylysine; full circles, spermine. Bundling in the presence and absence of 100 mM NaCl is shown in pink and dark blue, respectively. (b) Effect of F-actin concentration on its bundling. Solid line, polylysine; dashed line, lysozyme. Polycation concentration is shown by symbols: diamonds, 10 µM; squares 25 µM; triangles, 50 µM polycation.

Figure 4. Dissolution of polycation induced actin bundles by cofilin

(a), 8 µM cofilin was added to 8 µM Mg-F-actin bundled by 16–100 µM lysozyme or polylysine in the presence of 100 mM NaCl. Bundling was monitored by low speed sedimentation as described in Materials and Methods. Diamonds, polylysine without cofilin; squares, polylysine and cofilin; triangles, lysozyme without cofilin; full circles, lysozyme and cofilin. (b) Effect of 8 µM cofilin on the light scattering of 8 µM Mg-F-actin bundled by 50 µM lysozyme (black trace) and the effect of 50 µM lysozyme on the scattering of 8 µM Mg-F-actin in the presence of 8 µM cofilin (half tone trace). Lysozyme and cofilin addition, block and simple arrows, respectively. (c) Effect of cofilin on the bundling of 4 µM ANP-Mg-F-actin (squares) and cross-linked ANP-Mg-F-actin (diamonds). Bundling was monitored by low speed sedimentation as described in Materials and Methods. Error bars correspond to mean errors from three independent experiments.

Figure 4. Dissolution of polycation induced actin bundles by cofilin

(a), 8 µM cofilin was added to 8 µM Mg-F-actin bundled by 16–100 µM lysozyme or polylysine in the presence of 100 mM NaCl. Bundling was monitored by low speed sedimentation as described in Materials and Methods. Diamonds, polylysine without cofilin; squares, polylysine and cofilin; triangles, lysozyme without cofilin; full circles, lysozyme and cofilin. (b) Effect of 8 µM cofilin on the light scattering of 8 µM Mg-F-actin bundled by 50 µM lysozyme (black trace) and the effect of 50 µM lysozyme on the scattering of 8 µM Mg-F-actin in the presence of 8 µM cofilin (half tone trace). Lysozyme and cofilin addition, block and simple arrows, respectively. (c) Effect of cofilin on the bundling of 4 µM ANP-Mg-F-actin (squares) and cross-linked ANP-Mg-F-actin (diamonds). Bundling was monitored by low speed sedimentation as described in Materials and Methods. Error bars correspond to mean errors from three independent experiments.

Figure 4. Dissolution of polycation induced actin bundles by cofilin

(a), 8 µM cofilin was added to 8 µM Mg-F-actin bundled by 16–100 µM lysozyme or polylysine in the presence of 100 mM NaCl. Bundling was monitored by low speed sedimentation as described in Materials and Methods. Diamonds, polylysine without cofilin; squares, polylysine and cofilin; triangles, lysozyme without cofilin; full circles, lysozyme and cofilin. (b) Effect of 8 µM cofilin on the light scattering of 8 µM Mg-F-actin bundled by 50 µM lysozyme (black trace) and the effect of 50 µM lysozyme on the scattering of 8 µM Mg-F-actin in the presence of 8 µM cofilin (half tone trace). Lysozyme and cofilin addition, block and simple arrows, respectively. (c) Effect of cofilin on the bundling of 4 µM ANP-Mg-F-actin (squares) and cross-linked ANP-Mg-F-actin (diamonds). Bundling was monitored by low speed sedimentation as described in Materials and Methods. Error bars correspond to mean errors from three independent experiments.

Figure 5. Effect of heparin on the dissolution of the bundles of 6 µM Mg-F-actin by 6 µM cofilin

Mg-F-actin was bundled by 50 µM lysozyme in the presence of 100 mM NaCl. Unbundling was followed by low speed sedimentation as described in Materials and Methods. Diamonds, cofilin absent; squares, cofilin present. Error bars correspond to mean errors from three independent experiments.

Figure 6. Detection of antiparallel interfilament dimers in Mg-F-actin bundles by MTS-1 cross-linking

4µM Mg-F-actin - bundled by polycations - treated with 3 µM MTS-1 and analyzed by SDS-PAGE as described in Materials and Methods. Squares, polylysine; diamonds, lysozyme; full circles, spermine. Error bars correspond to mean errors from three independent experiments.

Figure 7. Dissolving MTS-1 cross-linked and uncross-linked Mg-F-actin bundles by DTT and NaCl

4µM Mg-F-actin was bundled by 6 µM lysozyme at low ionic strength and cross-linked with 3 µM MTS-1 as described in Materials and Methods. NaCl (100mM) and DTT (1 mM) were added and the samples were centrifuged at 16K for 8 min. The supernatants were analyzed for the actin and lysozyme content by SDS-PAGE. Additions to 4µM Mg-F-actin and 6 µM lysozyme: (A), none; (B), 100 mM NaCl; (C), 100 mM NaCl and 1 mM DTT; (D), 3 µM MTS-1; (E), 100 mM NaCl and 3 µM MTS-1; (F), 3 µM MTS-1, 100 mM NaCl and 1 mM DTT and. Amount of actin in bundles as percentage of total actin, empty bars; amount of lysozyme in bundles as percentage of total lysozyme, full bars. Error bars correspond to mean errors from three independent experiments.