Cardiac leiomodin2 binds to the sides of actin filaments and regulates the ATPase activity of myosin.

Leiomodin proteins are vertebrate homologues of tropomodulin, having a role in the assembly and maintenance of muscle thin filaments. Leiomodin2 contains an N-terminal tropomodulin homolog fragment including tropomyosin-, and actin-binding sites, and a C-terminal Wiskott-Aldrich syndrome homology 2 actin-binding domain. The cardiac leiomodin2 isoform associates to the pointed end of actin filaments, where it supports the lengthening of thin filaments and competes with tropomodulin. It was recently found that cardiac leiomodin2 can localise also along the length of sarcomeric actin filaments. While the activities of leiomodin2 related to pointed end binding are relatively well described, the potential side binding activity and its functional consequences are less well understood. To better understand the biological functions of leiomodin2, in the present work we analysed the structural features and the activities of Rattus norvegicus cardiac leiomodin2 in actin dynamics by spectroscopic and high-speed sedimentation approaches. By monitoring the fluorescence parameters of leiomodin2 tryptophan residues we found that it possesses flexible, intrinsically disordered regions. Leiomodin2 accelerates the polymerisation of actin in an ionic strength dependent manner, which relies on its N-terminal regions. Importantly, we demonstrate that leiomodin2 binds to the sides of actin filaments and induces structural alterations in actin filaments. Upon its interaction with the filaments leiomodin2 decreases the actin-activated Mg2+-ATPase activity of skeletal muscle myosin. These observations suggest that through its binding to side of actin filaments and its effect on myosin activity leiomodin2 has more functions in muscle cells than it was indicated in previous studies

Egy rendszeres testmozgást biztosító geriátriai mozgásprogram szerepe a sikeres öregedésben

Bevezetés: A rendszeres fizikai aktivitásnak minden életkorban, de különösen az idős életszakaszban fontos szerepe van. Jelen retrospektív kutatásunk célja az volt, hogy megvizsgálja egy budapesti kerület által biztosított - heti egyszer, aerob gyakorlatokból, progresszíven nehezedő izomerősítő-, nyújtó-, és egyensúlyfejlesztő gyakorlatokból felépülő - többkomponensű mozgásprogram hatását otthonélő idősek önálló életvitelét leginkább meghatározó funkcionális képességeire (alsó végtagi izomerőre, járássebességre, statikus egyensúlyra). Módszer: A kutatásban két csoport teljesítményét hasonlítottuk össze: az aktív idősek csoportjának tagjai a tornát két évnél régebben végezték, az inaktív idősek a geriátriai tornát csak a felmérés után kezdték el. Az alsó végtag izomerejét az 5-felállás teszttel, a járássebességet a 4-méteres járásteszttel, a statikus egyensúlyt az egylábon állás teszttel mértük. Eredmények: Az a feltételezésünk beigazolódott, miszerint azoknál az időseknél, akik több éve rendszeresen részt vesznek a kutatásban vizsgált mozgásprogramon, az alsó végtagi izomzat szignifikánsan erősebb, mint az inaktív idősek izomereje (t(36)=2,602; p=0,013; Cohen'd=0,99). Tendenciaszintű szignifikanciát találtunk a két csoport között a járássebesség tekintetében (t(36)= 1,769; p=0,085; Cohen'd=0,66). Ugyanakkor nem tudtunk csoportközi különbséget kimutatni a statikus egyensúly tesztjeiben. Következtetés: A budai kerület által biztosított többkomponensű mozgásprogram ígéretes lehetőség lehet az idősek funkcionális mozgásképességének megőrzésére. Amennyiben nagyobb elemszámú, kontrollcsoportos további kutatások is bizonyítják ezt az eredményt, érdemes országszerte preventív eljárásként alkalmazni, hogy egyre több magyarországi idős ember érje el a legjobb állapotban az időskort

Functional consequences of F-actin binding by Lmod2.

<p>(A) The effect of Lmod2^FL on the Mg²⁺-ATPase activity of HMM measured with coupled assay. The Mg²⁺-ATPase activity of HMM (0.5 μM) in the absence and presence of actin and/or Lmod2^FL was measured under low salt conditions (10 mM KCl, 0.5 mM MgCl₂). (B) The Mg²⁺-ATPase activity of HMM (0.5 μM) in the presence of F-actin (1 μM) as the function of Lmod2^FL concentration. Data are presented as mean ± SD (n = 3).</p

Lmod2 influences pyrene emission in actin filaments.

<p>(A) Polymerisation kinetics of actin assembly (4 μM; 5% labelled) in the presence of Lmod2^FL show increased saturation of pyrene fluorescence emission in a concentration dependent manner. (B) Cterm does not affect the steady-state value of pyrene actin fluorescence. The actin concentration was 4 μM, containing 5% labelled actin. (C) Rapid kinetics measurements of the time dependent change in pyrene F-actin fluorescence. Stopped-flow measurements of the kinetics of pyrene fluorescence emission of prepolymerised F-actin (1 μM, 5% pyrene labelled) in the absence or presence of different concentrations of Lmod2^FL (1 μM, 6 μM and 12.5 μM, as indicated). Salt conditions: 100 mM KCl, 2 mM MgCl₂. Dashed lines show the fit using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186288#pone.0186288.e007" target="_blank">Eq 7</a>.</p

The effects of purified cardiac leiomodin2 on actin assembly dynamics.

<p>(A) Effects of cardiac Lmod2 and Cterm on the polymerisation kinetics of actin. The kinetics of actin assembly (4 μM, containing 5% pyrene labelled actin) in the absence and presence of different concentrations of Lmod2^FL or Cterm. Pyrene fluorescence was measured at excitation and emission wavelengths of 350 nm and 404 nm, respectively. Salt conditions: 100 mM KCl, 2 mM MgCl₂. (B) Cardiac Lmod2 influences actin polymerisation in a concentration dependent manner. Polymerisation rates were determined from the slope of the pyrene curves (shown on panel (A)) at 50% polymerisation and normalised by the rate measured for spontaneous actin assembly. The actin polymerisation rates are plotted as a function of Lmod2^FL (empty circles) and Cterm (filled circles) concentrations. (C) Ionic strength dependence of the effects of cardiac Lmod2 and Cterm on actin polymerisation. Pyrene-actin (4 μM, 5% labelled) was polymerised in the absence or presence of 100 nM Lmod2^FL (empty circles) or 4 μM Cterm (filled circles) under low (10 mM KCl, 0.5 mM MgCl₂), medium (50 mM KCl, 1 mM MgCl₂) and high (100 mM KCl, 2 mM MgCl₂) salt conditions. Normalised polymerisation rates were derived as described above and plotted as a function of ionic strength (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186288#pone.0186288.e004" target="_blank">Eq 4</a>). (D) The effect of cardiac Lmod2 on the critical concentration of actin assembly. Pyrene intensities as a function of actin concentration were measured in the absence (black circles) or presence of 100 nM Lmod2^FL (grey circles) or 4 μM Cterm (light grey circles) under high salt conditions (100 mM KCl, 2 mM MgCl₂). Actin (5% pyrene labelled) concentrations were 30; 50; 100; 300; 500; 700 nM and 1; 3; 5 μM. The critical concentrations were determined by using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186288#pone.0186288.e005" target="_blank">Eq 5</a> and were found to be 120 ± 83 nM in the absence of Lmod2^FL and 117 ± 68 nM and 132 ± 58 nM in the presence of Lmod2^FL and Cterm, respectively. Dashed lines in the corresponding colour show the fit to the data. (E) Skeletal tropomyosin (Tpm1.1/2.2) reduces the polymerisation activity of Lmod2. Pyrene-actin (4 μM, 5% labelled) was polymerised in the absence (empty squares) or presence (empty circles) of 1 μM Lmod2^FL or 4 μM Cterm (filled circles) under high salt conditions (100 mM KCl, 2 mM MgCl₂) in the presence of different concentrations of skeletal muscle tropomyosin. Data are presented as mean ± SD (n = 3).</p

The effect of leiomodin2 on the flexibility of actin filaments.

<p>(A) Emission spectra of IAEDANS (D, donor) in the absence and presence of IAF (A, acceptor), and in the absence and presence of Lmod2^FL. The actin filament concentration was 4 μM. Note the acceptor induced decrease in the donor emission due to FRET. (B) Temperature-dependent FRET measurements were performed on IAEDENS-IAF actin filaments (4 μM) in the absence and presence of different concentrations of Lmod2^FL. FRET efficiencies calculated from the emission spectra (representative spectra are shown on panel (A)) were normalised by FRET efficiency measured at the lowest temperature (relative f ‘, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186288#pone.0186288.e009" target="_blank">Eq 9</a>) and plotted as a function of temperature. Data obtained in the absence (filled circles) or presence (empty circles) of Lmod2^FL (5 μM) are shown. Data are presented as mean ± SD (n = 3).</p

Cardiac leiomodin2 binds to the sides of actin filaments.

<p>(A) In high-speed cosedimentation assays F-actin (4 μM) and Lmod2^FL (0–5 μM) was incubated under high salt conditions (100 mM KCl, 2 mM MgCl₂) and centrifuged at 258.000 g for 30 min at room temperature. The pellets were analysed by SDS-PAGE. Representative Coomassie stained SDS-PAGE gel of the pellets is shown. (B) Lmod2^FL (5 μM) does not sediment in the absence of actin. Leiomodin was centrifuged in the absence of F-actin (left two columns) or F-actin (5 μM) was pelleted in the presence of Lmod2^FL (right two columns). Pellets and supernatants are indicated on the figure. (C) Actin-Lmod2^FL ratios in pellet were calculated from SDS-PAGE analysis of under high salt (HS, 100 mM KCl, 2 mM MgCl₂) and medium salt (MS, 50 mM KCl, 1 mM MgCl₂) conditions, and plotted as the function of Lmod2^FL concentration. Dashed lines show the fit using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186288#pone.0186288.e006" target="_blank">Eq 6</a>. Data are presented as mean ± SD (n = 3).</p

Intrinsic structural properties of Lmod2.

<p>(A) Bioinformatics analysis of Lmod protein sequences predicts intrinsically unstructured protein regions. The disorder probability in <i>Homo sapiens</i> Lmod2 and <i>Rattus norvegicus</i> Lmod2 (Uniprot accession numbers are <a href="http://www.uniprot.org/uniprot/Q6P5Q4" target="_blank">Q6P5Q4</a> and <a href="http://www.uniprot.org/uniprot/A1A5Q0" target="_blank">A1A5Q0</a>, respectively). The analysis was performed by IUPred [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186288#pone.0186288.ref042" target="_blank">42</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186288#pone.0186288.ref043" target="_blank">43</a>]. The tryptophan residues of <i>Rattus norvegicus</i> Lmod2 are highlighted by red. (B) Structural features of the interactions of Tmod1 with actin. The complex between Tmod1 ABS1 and actin is shown (PDB ID: 4PKG). The position of W73 of <i>Rattus norvegicus</i> cardiac leiomodin2 is predicted by multiple sequence alignment and highlighted by red. (C) Structural features of the interactions of Lmod2 with actin. The complexes between Lmod2 ABS2/LRR or WH2 actin binding sites and actin monomers are shown (PDB ID: 4PKG). The position of W386 and W347 (red) of <i>Rattus norvegicus</i> cardiac leiomodin2 are predicted to localize in a flexible protein region (blue) by multiple sequence alignment (PDB ID: 4RWT). This flexible region between residues 339–388 is missing from the structure of 4RWT. (D) Tryptophan fluorescence spectra of Lmod2. Excitation (EX) and emission (EM) spectra of the intrinsic tryptophans of Lmod2^FL (1 μM, black line) and Cterm (4 μM, grey line) were obtained at emission wavelength of 354 nm and excitation wavelength of 282 nm.</p