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. 2017 Aug 28;12(8):e0183760.
doi: 10.1371/journal.pone.0183760. eCollection 2017.

Histones Bundle F-actin Filaments and Affect Actin Structure

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Free PMC article

Histones Bundle F-actin Filaments and Affect Actin Structure

Edna Blotnick et al. PLoS One. .
Free PMC article

Abstract

Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III) and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Polymerization of CaATP-G-actin by histone mixture and H2A histone followed by increase in pyrene fluorescence.
(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.
Fig 2
Fig 2. 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.
(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 Fig 1. Sedimentation data were taken from experiments presented in Fig 2A and B. The presented data are mean and standard deviation of three independent experiments.
Fig 3
Fig 3. Histone mixture and H2A histone induced bundle formation of Mg-F-actin followed by low speed centrifugation and by light scattering.
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.
Fig 4
Fig 4. 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.
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.
Fig 5
Fig 5. 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.
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.
Fig 6
Fig 6. Effect of DNase1 on the light scattering and sedimentation of 4 μM MgF-actin bundled by histone mixture or H2A histone.
(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.
Fig 7
Fig 7. Effect of DNA on the bundling of F-actin by histone.
(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.
Fig 8
Fig 8. Effect of cofilin on the sedimentation of 4 μM MgF-actin bundled by histone mixture or H2A histone.
(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.
Fig 9
Fig 9. Effect of cofilin on the light scattering of histone mixture or H2A histone bundled MgF-actin.
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.
Fig 10
Fig 10. TIRF microscopy revealed a change in morphology of histone-induced F-actin bundles in the presence of cofilin.
In all experiments actin was polymerized by 1 mM MgCl2 containing in TIRF imaging buffer. (A), Actin (1μM) polymerized in TIRF imaging buffer for 15 minutes forms unbundled long filaments. (B), Actin (1μM) copolymerized with 1 μM cofilin in TIRF imaging buffer for 15 minutes. More short filaments were detected in the sample due to the cofilin-induced severing. (C-D), Actin (1μM) copolymerized with 1 μM (14 μg/ml) H2A histone in TIRF imaging buffer for 15 (C) and 30 minutes (D), respectively. Thin, cable-like F-actin bundles were detected. (E-F), Actin (1μM) copolymerized with 1 μM (14 μg/ml) H2A histone and 1 μM cofilin in TIRF imaging buffer for 15 (E) and 30 minutes (F). Star-like F-actin structures were detected under these conditions. Scale bars: 10 μm. Presented data are representative of three independently taken microscope images. TIRF imaging buffer: 10 mM Hepes, 1 mM MgCl2, 50 mM KCl, 0.2 mM EGTA (pH 7.4) supplemented with 50 mM DTT, 0.2 mM ATP, 0.05 mg/ml casein, 20 mM glucose, 0.25 mg/ml glucose oxidase, 50 μM catalase, 0.5% methyl cellulose. TIRF microscopy and Alexa 484 SE labeling were carried out as described in MATERIALS and METHODS.

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Grant support

This work was supported by Hebrew University of Jerusalem Science Fund (AM).
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