Effect of 0–400 mM NaCl on the sedimentation of 63 μg/ml histone mixture or 4 μM (56 μg/ml) H2A histone bundled 4 μM MgF-actin as measured by low speed centrifugation and light scattering.

<p>Sedimentation: (A), Bundling by histone mixture and H2A histone. Samples were centrifuged at 20800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: actin lanes, representatives of three independent experiments, from the SDS-PAGE of supernatants after low speed centrifugation. Light scattering: (B), 4x100 mM NaCl was added to histone mixture bundled 4 μM MgF-actin, (C), 3x100 mM NaCl was added to H2A histone bundled 4 μM MgF-actin and the light scattering was measured. Asterisks* represent 100 mM NaCl addition. Light scattering change was followed as described in MATERIALS and METHODS. Presented data are representative of three independent experiments.</p

Histone mixture and H2A histone induced bundle formation of Mg-F-actin followed by low speed centrifugation and by light scattering.

<p>5.25–84 μg/ml histone mixture (A), or 1–4 μM (14–56 μg/ml) H2A histone (B), were added to 4 μM MgF-actin in pH7.4 F-buffer and centrifuged at low speed. Samples were centrifuged at 20,800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: actin lanes, representatives of three independent experiments, from SDS-PAGE of low speed centrifugation supernatants. (C) 5.25–42 μg/ml histone mixture or (D) 1–4 μM (14–56 μg/ml) H2A histone were added to 4 μM MgF-actin in pH7.4 F-buffer and the light scattering change was followed as described in MATERIALS and METHODS. Presented data are representative of three independent experiments. All measurements were done at pH7.4 in F-buffer.</p

Polymerization of CaATP-G-actin by histone mixture and H2A histone followed by increase in pyrene fluorescence.

<p>(A) 5.25–126 μg/ml histone mixture, (B) 0.5–6 μM (7–84 μg/ml) H2A histone, or 2 mM MgCl₂ were added to pyrene labeled (10% labeling ratio) 4 μM CaATP-G-actin in pH 7.4 CaATP-G-buffer. (C), 21 μg/ml histone mixture was added to pyrene labeled (10% labeling ratio) 4 μM CaATP-G-actin in pH 6.5, 7.4 and 8.2 CaATP-G-buffer or 2 mM MgCl₂ was added in pH 6.5 CaATP-G-buffer. Fluorescence measurements were carried out as given in MATERIALS and METHODS. Presented data are representative of three independent experiments.</p

Effect of DNA on the bundling of F-actin by histone.

<p>(A), Effect of 25–400 μg/ml DNA on the sedimentation of 4 μM MgF-actin bundled by 42 μg/ml histone mixture or 3 μM (42 μg/ml) H2A histone. (B), Effect of 0–200 μg/ml DNA digested by Staphylococcus aureus micrococcal DNase on the sedimentation of 4 μM MgF-actin bundled by 42 μg/ml histone mixture or by 3 μM (42 μg/ml) H2A histone. 4 μg/ml DNA was digested by 20 μg/ml micrococcal DNase at 37°C for 30 min. Samples were centrifuged at 20800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: actin lanes, representatives of three independent experiments, from the SDS-PAGE of low speed centrifugation supernatants.</p

Effect of DNase1 on the light scattering and sedimentation of 4 μM MgF-actin bundled by histone mixture or H2A histone.

<p>(A) Effect of 9 μM DNase1 on the light scattering of 4 μM MgF-actin bundled by 63 μg/ml histone mixture or 3 μM (42 μg/ml) H2A histone. Light scattering change was followed as described in MATERIALS and METHODS. Presented data are representative of three independent experiments. (B). Effect of 2–15 μM DNase1 on the sedimentation of 4 μM MgF-actin bundled by 63 μg/ml histone mixture or 3 μM (42 μg/ml) H2A histone. The difference between the amount of actin sedimented following DNase1 treatment of histone mixture and H2A histone bundled actin is highly significant. Samples were centrifuged at 20800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: actin lanes, representatives of three independent experiments, from SDS-PAGE of low speed centrifugation supernatants.</p

Effect of cofilin on the sedimentation of 4 μM MgF-actin bundled by histone mixture or H2A histone.

<p>(A), 2–8 μM cofilin added to 4 μM F-actin bundled by 10.5 and 63 μg/ml histone mixture or (B) by 1 μM (14 μg/ml) and 3 μM (42 μg/ml) H2A histone. (C), 42 μg/ml histone mixture or 4 μM (56 μg/ml) H2A histone and 2.5 or 5 μM cofilin were added simultaneously to 4 μM F-actin. Samples were centrifuged at 20800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: lanes of SDS-PAGE gels, representatives of three independent experiments, obtained from SDS-PAGE of low speed centrifugation supernatants.</p

Effect of 3 μM (42 μg/ml) H2A histone and 6 μM cofilin on the viscosity of 4 μM MgF-actin measured by Viscous Aqua fluorescence viscosity probe.

<p>Viscous Aqua in original Ursa BioScience vial was dissolved in 50 μl methanol then diluted 50 times in actin buffer and added to actin containing solutions in 1 to 50 ratio in pH 7.4 buffer. The fluorescence of the mixtures was measured as described in MATERIALS and METHODS. The fluorescence values (in artificial units, A.U.) of the samples at 492 nm emission maximum minus the fluorescence of the buffer are given in the figure. The data obtained were compared by statistical analysis and the significance of the differences was indicated. * = p<0.05, ** = p<0.01 *** = p<0.005. The fluorescence emission increases with the increasing viscosity of the samples. The presented data are mean and standard deviation of at least three independent experiments.</p

Polymerization of CaATP-G-actin by histone mixture and H2A histone followed by high speed centrifugation was compared with the plateaus of the pyrene fluorescent measurements.

<p>(A) 5.25–63 μg/ml histone mixture, or (B) 0.5–4 μM (7–56 μg/ml) H2A histone was added to 4 μM CaATP-G-actin in pH7.4 CaATP-G-buffer. Samples were centrifuged at 129,151xg for 2h, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. Fig 2A, inset: SDS-PAGE, left, actin and histone mixture before centrifugation; right, molecular weight marker. Fig 2B, inset: actin lanes from the SDS-PAGE of supernatants after high speed centrifugation. All SDS-PAGE gels are representatives of three independent experiments. Actin sedimentation values were compared with plateaus of pyrene fluorescence upon addition of histone mixture (C) or H2A histone (D). Pyrene fluorescence values were taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183760#pone.0183760.g001" target="_blank">Fig 1</a>. Sedimentation data were taken from experiments presented in Fig 2A and B. The presented data are mean and standard deviation of three independent experiments.</p

Effect of cofilin on the light scattering of histone mixture or H2A histone bundled MgF-actin.

<p>4x 2 μM cofilin was added to 4 μM MgF-actin bundled by 63 μg/ml histone mixture (A), or by 4 μM (56 μg/ml) H2A histone (B). Stars indicate addition of 2 μM cofilin. Light scattering change was followed as described in MATERIALS and METHODS. Presented data are representative of three independent experiments.</p

LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

<div><p>Actin exists as a monomer (G-actin) which can be polymerized to filaments) F-actin) that under the influence of actin-binding proteins and polycations bundle and contribute to the formation of the cytoskeleton. Bundled actin from lysed cells increases the viscosity of sputum in lungs of cystic fibrosis patients. The human host defense peptide LL-37 was previously shown to induce actin bundling and was thus hypothesized to contribute to the pathogenicity of this disease. In this work, interactions between actin and the cationic LL-37 were studied by optical, proteolytic and surface plasmon resonance methods and compared to those obtained with scrambled LL-37 and with the cationic protein lysozyme. We show that LL-37 binds strongly to CaATP-G-actin while scrambled LL-37 does not. While LL-37, at superstoichiometric LL-37/actin concentrations polymerizes MgATP-G-actin, at lower non-polymerizing concentrations LL-37 inhibits actin polymerization by MgCl₂ or NaCl. LL-37 bundles Mg-F-actin filaments both at low and physiological ionic strength when in equimolar or higher concentrations than those of actin. The LL-37 induced bundles are significantly less sensitive to increase in ionic strength than those induced by scrambled LL-37 and lysozyme. LL-37 in concentrations lower than those needed for actin polymerization or bundling, accelerates cleavage of both monomer and polymer actin by subtilisin. Our results indicate that the LL-37-actin interaction is partially electrostatic and partially hydrophobic and that a specific actin binding sequence in the peptide is responsible for the hydrophobic interaction. LL-37-induced bundles, which may contribute to the accumulation of sputum in cystic fibrosis, are dissociated very efficiently by DNase-1 and also by cofilin.</p> </div