peerj_json_files/PeerJ_Json_132.json

{
    "v1_Abstract": "Tropomyosins are actin-binding regulatory proteins, which overlap end-to-end along the filament. High resolution structures of the overlap regions were determined for muscle and non-muscle tropomyosins, however conformations of the junction regions in complex with actin are unknown. In this work, orientation of the overlap on actin alone and on actin-myosin complex was evaluated by measuring FRET distances between a donor (AEDANS) attached to tropomyosin and an acceptor (DABMI) bound to actin\u2019s Cys374. Donor was attached to the Cys residue introduced by site-directed mutagenesis near the C-terminal half of the overlap. The recombinant alpha-tropomyosin isoforms used in this study \u2013 skeletal muscle skTM, nonmuscle TM2 and TM5a, and chimeric TM1b9a had various amino acid sequences of the Nand C-termini involved in the end-to-end overlap. The donor-acceptor distances calculated for each isoform varied between 36.4 and 48.1 . Rigor binding of myosin S1 increased the \u01fa \u01fa apparent FRET distances of skTM and TM2, but decreased the distances separating TM5a and TM1b9a from actin. The results show that isoform-specific sequences of the end-to-end overlaps determine orientations and dynamics of tropomyosin isoforms on actin. This can be important for specificity of tropomyosin in the regulation of actin filament diverse functions.",
    "v1_col_introduction": "introduction  : Tropomyosins are, a family of two-chain coiled coil proteins, and are regarded as actin\n\u201cgate keepers\u201d, which control access of numerous actin-binding proteins to actin filaments (Gunning et al. 2008). Tropomyosin (TM) binds cooperatively to actin and due to end-to-end overlap between adjacent molecules it forms long chains along both sides of the filament. Binding of TM to actin involves weak, but specific electrostatic interactions between periodic actin-binding sites on TM\u2019s coiled-coil and residues exposed on actin subunits (Barua et al. 2011; Li et al. 2011). In this closed state, TM inhibits activation of actomyosin ATPase at low myosin concentrations (Lehrer & Morris 1982). Strongly bound myosin heads (S1) cooperatively shift the filament into the open state, which is associated with an azimuthal shift of TM away from the position occupied in the closed state (Lehman & Craig 2008). The S1induced shift from the closed to the open state is a universal mechanism of actin filament activation executed in the presence of muscle and non-muscle TM isoforms.\nTM isoforms are generated by several genes (four in vertebrates), selection of\nalternative promoters, and alternative splicing of the transcripts. In \u03b1-tropomyosins, the\nproducts of the TPM1 gene, the N- and C-terminal regions are encoded respectively by two (1a and 1b) and four (9a-d) alternative exons. Selection of the alternative promoter gives rise to high molecular weight (HMW) and low molecular weight (LMW) isoforms of TM. HMW tropomyosins bind along seven actin subunits, whereas LMW isoforms along six actin subunits.The main structural difference between these two TM types is the N-terminal sequence, which is encoded by exon 1a or 1b respectively in HMW and LMW isoforms, respectively (Lees-Miller & Helfman 1991).\n6\n7\n8\n9\n10\n11\n12\n13\n14\n15\n16\n17\n18\n19\n20\n21\n22\n23\n24\n25\n26\n27\nPeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013)\nR ev ie w in g M an\nus cr ip t\nStructures of tropomyosin intermolecular junctions were studied with the use of model\npeptides imitating sequences of the end-to-end overlaps. Peptides with sequences of skeletal TM encoded by exons 1a and 9a (Greenfield et al. 2006; Murakami et al. 2008), non-muscle TM encoded by exons 1b and 9d (Greenfield et al. 2009), and smooth muscle TM encoded by exons 1a and 9d (Frye et al. 2010) were analyzed to obtain NMR and X-ray structures. Although the structures differ in the number of amino acids forming the overlap and in\nspecific interactions between amino acid chains, the three complexes are similar \u2013- the two \u03b1-\nhelical chains of the C-terminus spread apart and interlock with the N-terminal coiled coils. The structures revealed, however, a slightly different tilt of the axis of the C- and N-terminal coiled coil (Frye et al. 2010).\nFor understanding of the molecular mechanisms controlling numerous functions of\nactin, high- resolution structures of F-actin in complex with different TM isoforms are required. Models of actin complexes with skeletal and cardiac muscle TMs in different activation states are available (Barua et al. 2013; Barua et al. 2011; Barua et al. 2012; Behrmann et al. 2012; Li et al. 2011; Miki et al. 2012). The models show periodic interactions between conserved residues repeting along TM and charged or hydrophobic residues exposed on actin (Barua et al. 2011; Li et al. 2011). When bound to actin alone, the N-terminal residues Lys6 and Gln9, located with the overlap region, make important interactions with Asp25 on actin. On the other hand, the C-terminal half of the overlap does not contribute directly to actin binding. This region could may be important for regulation of myosin interactions with the filament, however but there is no experimental data supporting this hypothesis.\nIn our earlier work, we used steady-state F\u00f6rster resonance energy transfer (FRET) to\ndetermine apparent distances between donors specifically attached to the N-terminal regions of different TM isoforms. Our data suggested that, in closed and myosin-induced open states the N-\n28\n29\n30\n31\n32\n33\n34\n35\n36\n37\n38\n39\n40\n41\n42\n43\n44\n45\n46\n47\n48\n49\n50\n51\nPeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013)\nR ev ie w in g M an\nus cr ip t\nterminal segments of tropomyosin isoforms are differently oriented on F-actin (Sliwinska et al. 2011). However, the results did not provide any insight into the orientationposition of C-terminal segments of the studied isoforms. We do not know whether the regions located in the close vicinity to the of end-to-end junctions are stiff or rather flexible. Various flexibilities within this region of TM might be an important determinant of differences between TM isoforms in regulation of actin-myosin interactions.\nThe aim of this study was to analyze the orientation positions of the C-terminal regions,\nadjacent to the end-to-end overlap, in four tropomyosin isoforms. Steady-state FRET between a donor attached to the Cys residue introduced into the C-terminal segment of each isoform and acceptor bound to actin\u2019s penultimate Cys374 was used. The data shows that orientation localization of the C-terminal region in relation to actin\u2019s C-terminus is unique for each type of the studied isoform. Specific conformational changes associated with activation of the filament by strongly bound myosin heads suggest that depending on the sequence, the end-to-end overlap regions have different flexibilities.",
    "v2_Abstract": "Tropomyosins are actin regulatory proteins, which overlap end-to-end along the filament. High resolution structures of the overlap regions were determined for muscle and non-muscle tropomyosins, however conformations of the junction regions in complex with actin are unknown. In this work, orientation of the overlap on actin alone and on actin-myosin complex was evaluated by measuring FRET distances between a donor (AEDANS) and an acceptor (DABMI) bound to actin\u2019s Cys374. Donor was attached to the Cys residue introduced by sitedirected mutagenesis near the C-terminal half of the overlap. The recombinant alphatropomyosin isoforms used in this study \u2013 skeletal muscle skTM, non-muscle TM2 and TM5a, and chimeric TM1b9a had various amino acid sequences of the Nand C-termini involved in the end-to-end overlap. The donor-acceptor distances calculated for each isoform varied between 36.4 and 48.1 . Rigor binding of myosin S1 increased the apparent FRET \u01fa \u01fa distances of skTM and TM2, but decreased the distances separating TM5a and TM1b9a from actin. The results show that isoform-specific sequences of the end-to-end overlaps determine orientations and dynamics of tropomyosin isoforms on actin. This can be important for specificity of tropomyosin in the regulation of actin filament diverse functions.",
    "v2_col_introduction": "introduction  : Tropomyosins, a family of two-chain coiled coil proteins, are regarded as actin \u201cgate\nkeepers\u201d, which control access of numerous actin-binding proteins to actin filaments (Gunning et al. 2008). Tropomyosin (TM) binds cooperatively to actin and due to end-to-end overlap between adjacent molecules it forms long chains along both sides of the filament. Binding of TM to actin involves weak, but specific electrostatic interactions between periodic actin-binding sites on TM\u2019s coiled-coil and residues exposed on actin subunits (Barua et al. 2011; Li et al. 2011). In this closed state TM inhibits activation of actomyosin ATPase at low myosin concentrations (Lehrer & Morris 1982). Strongly bound myosin heads cooperatively shift the filament into open state, which is associated with an azimuthal shift of TM away from the position occupied in closed state (Lehman & Craig 2008). The S1-induced shift from closed to open state is a universal mechanism of actin filament activation executed in the presence of muscle and non-muscle TM isoforms.\nTM isoforms are generated by several genes (four in vertebrates), selection of alternative\npromoter, and alternative splicing of the transcripts. In \u03b1-tropomyosins, products of TPM1 gene,\nthe N- and C-terminal regions are encoded respectively by two (1a and 1b) and four (9a-d) alternative exons. Selection of alternative promoter gives rise to high molecular weight (HMW) and low molecular weight (LMW) isoforms of TM. HMW tropomyosins bind along seven, wereas LMW isoforms along six actin subunits.The main structural difference between these two TM types is the N-terminal sequence, which is encoded by exon 1a or 1b respectively in HMW and LMW isoforms (Lees-Miller & Helfman 1991).\nStructures of tropomyosin intermolecular junctions were studied with the use of model\npeptides imitating sequences of the end-to-end overlaps. Peptides with sequences of skeletal TM encoded by exons 1a and 9a (Greenfield et al. 2006; Murakami et al. 2008), non-muscle TM encoded by exons 1b and 9d (Greenfield et al. 2009), and smooth muscle TM encoded by exons 1a and 9d (Frye et al. 2010) were analyzed to obtain NMR and X-ray structures. Although the structures differ in the number of amino acids forming the overlap and in specific interactions\nbetween amino acid chains, the three complexes are similar - the two \u03b1-helical chains of the\nC-terminus spread apart and interlock with the N-terminal coiled coil. The structures revealed, however, a slightly different tilt of the axis of the C- and N-terminal coiled coil (Frye et al. 2010).\n8\n9 10 11 12 13 14 15 16 17 18 19 20 21\n22 23 24 25 26 27 28 29 30 31 32 33 34\n35 36 37\nPeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013)\nR ev ie w in g M an\nus cr ip t\nFor understanding the molecular mechanisms controlling numerous functions of actin,\nhigh resolution structures of F-actin in complex with different TM isoforms are required. Models of actin complexes with skeletal and cardiac muscle TMs in different activation states are available (Barua et al. 2013; Barua et al. 2011; Barua et al. 2012; Behrmann et al. 2012; Li et al. 2011; Miki et al. 2012). The models show periodic interactions between conserved residues repeting along TM and charged or hydrophobic residues exposed on actin (Barua et al. 2011; Li et al. 2011). When bound to actin alone, the N-terminal residues Lys6 and Gln9, located with the overlap region, make important interaction with Asp25 on actin. On the other hand, the C-terminal half of the overlap does not contribute directly to actin binding. This region could be important for regulation of myosin interctions with the filament, however there is no experimental data supporting this hypothesis.\nIn our earlier work we used steady-state F\u00f6rster resonance energy transfer (FRET) to\ndetermine apparent distances between donor specifically attached to N-terminal regions of different TM isoforms. Our data suggested that in closed and myosin-induced open states the N-terminal segments of tropomyosin isoforms are differently oriented on F-actin (Sliwinska et al. 2011). However, the results did not provide insight into the orientation of C-terminal segments of the studied isoforms. We do not know whether the regions located in the close vicinity of end-to-end junctions are stiff or rather flexible. Various flexibilities within this region of TM might be an important determinant of differences between TM isoforms in regulation of actin-myosin interactions.\nThe aim of this study was to analyze orientation of C-terminal regions, adjacent to the\nend-to-end overlap, in four tropomyosin isoforms. Steady-state FRET between donor attached to Cys residue introduced in C-terminal segment of each isoform and acceptor bound to actin\u2019s penultimate Cys374 was used. The data shows that orientation of the C-terminal region in relation to actin\u2019s C-terminus is unique for each type of the studied isoform. Specific conformational changes associated with activation of the filament by strongly bound myosin heads suggest that depending on the sequence, the end-to-end overlap regions have different flexibilities.",
    "v1_text": "materials and methods : Chicken skeletal muscle \u03b1-Aactin, chicken skeletal myosin subfragment 1 (S1) and recombinant rat \u03b1-tropomyosin isoforms were used. TM2, TM5a and TM1b9a were obtained as described in (SSliwinska et al. (2011). Recombinant skTM was modified by insertion of AlaSer at the N-terminus to compensate for low actin affinity of recombinant skTM due to the lack of N-terminal acetylation obtained as described in (Robaszkiewicz et al. (2012). The Department of Biochemistry and Cell Biology is authorized by the Minister of the 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t Environment (Poland) for laboratory use of genetically modified organisms (permit nr GMO: 01-112/112). PCR-based oligonucleotide-directed mutagenesis (Stratagene) was used to create an attachment site for a fluorescent probe at the C-terminal region of TM. First, cDNA encoding all TM isoforms used in this study was mutated to replace the single Cys codon for Ser. The procedure was described in (Sliwinska et al. (2011). Thean cDNA from the first stage of mutagenesis was used to create Ala269Cys-skTM, Ser269Cys-TM2, Ala232Cys-TM1b9a and Ser232Cys-TM5a mutants. The oligonucleotides used at this stage were the following: 9a Ala269(232)Cys: 5\u2019-ctgaagtacaagtgcatcagcgaggagctggaccacg-3\u2019 9d Ser269(232)Cys: 5\u2019-gccaaagaagaaaacctttgcatgcaccagatgctggac-3\u2019 All primers were synthesized and HPLC purified by the Laboratory of DNA Sequencing and Oligonucleotide Synthesis, Institute of Biochemistry and Biophysics, Polish Academy of Science (Warsaw, Poland). Tropomyosin mutants were labeled with AEDANS and actin was labeled with DABMI according to the procedure described before previously (Sliwinska et al. 2011). Tropomyosin affinity for actin was measured in a co-sedimentation assay. Increasing amounts of tropomyosin (final concentrations varied between 0 and 10 \u00b5M) were mixed with actin (5 \u00b5M) in F-buffer: 2 mM HEPES, pH 7.6, 40 mM NaCl, 5 mM MgCl2 at 22 \u00b0C and ultracentrifuged. Protein content in supernatants and pellets was analyzed electrophoretically on SDS-PAGE as described in (Skorzewski et al. 2009). The activity of actomyosin ATPase was measured in F-buffer. Myosin S1 concentration was at 0.8 \u00b5M, F-actin was 9.2 \u00b5M, and tropomyosin was 1.2 \u00b5M. The reaction was started by 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t addition of MgATP to 5 mM and stopped after 10 min. with 3.3% SDS and 30 mM EDTA. The amount of liberated phosphate was measured colorimetrically according to the method described in (White (1982). Fluorescence anisotropy, FRET experiments, calculation of F\u00f6rster critical distance (R0), and donor-acceptor distance (R) were conducted according to the methods described in (Sliwinska et al. (2011). results : Rationale for the labeling -site selection and characterization of the labeled tropomyosin isoforms End-to-end overlap sequences present in the studied TM isoforms were formed by two N-terminal and two C-terminal variants of alternative sequences of rat \u03b1TM. A Sschematic illustration of the overlap regions are is shown in Fig. 1. In skeletal muscle \u03b1TM isoform (skTM), non-muscle isoforms (TM2, TM5a), and in chimeric TM1b9a, an isoform an isoform whichthat has no natural counterpart, N-termini encoded by exons 1a or 1b formed complexes with C-termini encoded by exons 9a or 9d. In order to create a specific reactive site for attachment of a fluorescent probe to the C-terminal regions of tropomyosin isoforms, residue 269 in HMW isoforms or its counterpart in LMW isoforms (residue 232) were was changed for to Cys (Table. 1). This residue was selected for the following reasons: (a) it is located at the outskirts of the end-to-end junction (Frye et al. 2010; Greenfield et al. 2006; Greenfield et al. 2009), ); (b) it is not involved in any interactions within the studied end-to-end complexes (Frye et al. 2010),; and (c) it is located on the outer surface of the two-chain coiled-coil 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t tropomyosin (position c of the coiled-coil heptapeptide repeat), which allows for free motions of the attached label, (d) it is part of the flexible region, where coiled coil structure turns into parallel helices (Frye et al. 2010; Greenfield et al. 2003). Thus we expected that the attached label was sensitive to conformational differences within the end-to-end overlap. Replacement of Cys for Ser in the central region of TM did not affect the basic functions such as actin binding and regulation of actomyosin ATPase activity (Sliwinska et al. 2011). In order to check whether these functions were conserved in TMs with Cys mutations in the C-terminal region, we measured actin binding regulatory functions of all studied AEDANS-labeled TM isoforms. Binding constants (Kapp) obtained in the co-sedimentation assay show that all TM mutants bound to actin with high affinity (Table 2), consistent with previous work (Moraczewska et al., 1999). The mutations also did not change significantly change the interactions between actin and myosin heads. In the presence of wild -type and mutant TMs the activities of actin-myosin S1 ATPase were similar (Table 2). To check whether all isoforms of AEDANS-labeled TM bound to DABMI-labeled actin stoichiometrically, TMs were titrated with increasing concentrations of actin. The titration curves shown in Fig. 2 indicate that the fluorescence was maximally quenched at the ratio of TM to F-actin, which that was close to stoichiometric. For skTM and TM2 the maximal quenching was reached at 1 TM to 6-7 actins and in the case of TM5a and TM1b9a at 1 TM to 4-5 actins. The results confirm high actin affinity of TM isoforms labeled in the Cterminal segment. Additionally, the titration curves show that in case of the isoforms with the 9d-encoded C-terminus (TM2 and TM5a), the maximal quenching of AEDANS was reached at a lower TM/actin molar ratio than in the case of their 9a-encoded counterparts (skTM and TM1b9a). Because As fluorescence quenching was due to FRET, the observed differences suggest that the C-terminal sequence encoded by exon 9d facilitates interactions of the Cterminal regions of TM2 and TM5a with actin. 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t Distances separating the donor attached to the C-terminal region of tropomyosin from the acceptor bound to actin Random orientation of the labels is important for FRET distance measurements (Lakowicz 1999). To ensure that the probe was flexibly attached to Cys269/232 in all studied TM isoforms, we measured AEDANS fluorescence anisotropies. As shown in Table 3, the obtained anisotropies were low, and therefore we concluded that the label bound to free TM as well as to TM in complex with actin and with myosin S1 was randomly oriented. Thus, the orientation of the donor did not limit the FRET distance measurements. Quantum yield of the donor (QD) and spectral overlap between emission of the donor and absorption of the acceptor (J) affect donor-acceptor critical distance (R0) (Lakowicz 1999). Because the local environment surrounding AEDANS bound to Cys269/232 could influence both parameters, QD and J were determined for each labeled TM mutant in the presence of unlabeled actin. The results show that the label attached to the C-terminal segment of the studied TM isoforms was exposed to different environments, which caused variations in QD, small shifts in J and, in consequence, differences in R0 (Table 4). The FRET efficiency (E) was calculated from the fluorescence intensity of AEDANS- labeled TM in the absence and in the presence of acceptor (TM saturated with DABMI-Factin). The fluorescence of the donor was corrected for the increase caused by binding of unlabeled actin (3-7% depending on TM isoform). The efficiencies obtained for the studied TM isoforms were used for calculations of the apparent distances (R) separating donor and acceptor. All calculated FRET parameters are collected in Table 4. The results suggest isoform-specific localization of the overlap region. The differences between the distances obtained for HMW isoforms (skTM, TM2) were small, which indicates similar position of the 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t donor in relation to the acceptor. In LMW isoforms (TM5a and TM1b9a) the donor-acceptor distances were larger. It is worth noting that the distance obtained for TM1b9a was far from R0, thus the sensitivity of FRET measurements was limited and the result can only be regarded as a rough estimate. Changes in tropomyosins\u2019 C-termini as an effect of myosin S1 binding to actin Strong binding of myosin heads (S1) to the filament increases affinity of TM to actin and induces an azimuthal shift of TM chains. This changes the TM interactions with actin and activates the filament allowing for actin-myosin cross-bridge cycling (Lehman & Craig 2008; Moraczewska 2002). Saturation of the filament with myosin S1 caused a significant increase of the fluorescence of AEDANS bound to C-terminal segments of all studied TM isoforms. The average increase was about 14% and 18% for skTM and the non-muscle isoforms, respectively. Since the fluorescence intensity of the probe attached to the N-terminal segment increased by about 2-9% (Sliwinska et al. 2011), it appeared that the fluorophore bound to the C-terminal segment of TM was more sensitive to myosin binding. The change of fluorescence observed in this work suggested that the C-terminal region of TM either directly interacted with myosin or significantly changed conformation upon myosin binding to actin. To explore the later possibility, changes in FRET distances in the presence of myosin were analyzed. Strongly bound S1 shifted the C-terminal regions of all TM isoforms, however but the direction of the shift observed for HMW and LMW isoforms was different. As compared to the TM-actin complex, in the presence of S1 the energy transfer efficiency between AEDANS attached to skTM or TM2 and DABMI-actin decreased. In contrast, when the energy donor was attached to TM5a or TM1b9a, an increase in transfer efficiency was observed (Table 5). Binding of unlabeled actin-S1 did not shift the maximum of the fluorescence spectrum, thus 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t the spectral overlap (J) was unchanged. However, the quantum yield of AEDANS-TM bound to unlabeled actin-S1 increased (Table 5), which. This called for recalculation of the critical distances (see equation 5 in (Sliwinska et al. (2011)). Based on the new values of R0 and transfer efficiencies (E) obtained for AEDANS-TMs saturated with DABMI-actin-S1, donoracceptor distances were calculated (Table 5). The degree of the maximal shift induced by S1 binding to actin (\u0394RS1) was calculated as the difference between the donor-acceptor distance in the absence and in the presence of S1. The data indicates that the C-terminal region of the HMW isoforms was shifted away from the donor, whereas in LMW it was shifted closer to the donor. The S1-induced activation of the filament is a very cooperative process, which. It means that the final change in TM orientation is achieved at S1 concentrations much far below the concentrations required for actin saturation (Eaton 1976; Moraczewska 2002). Fig. 3 shows the effects of increasing S1 concentrations on energy transfer between donor-labeled TM and acceptor-labeled actin. The experimental data was normalized and fit to the Hill equation. The ratios of strongly bound myosin heads to actin required for the half-maximal change in the energy transfer (S1/actin molar ratio) are collected shown in Table 5. The numbers in parentheses show the S1/TM molar ratio, obtained by multiplying S1/actin by the number of actin subunits bound by one TM molecule. The data shows that the cooperativity was very high for each of the isoforms. The differences depended on the type of end-toend overlap. acknowledgements : We thank William Lehman and Edward Li for sharing with us the coordinates of the actin-TM model. The authors thank Katarzyna Robaszkiewicz and Ireneusz Moraczewski for their help with preparation of Figures 1 and 4. discussion : The position tropomyosin assumes on the filament controls interactions of the filament with many actin-binding proteins, thus it is an important determinant of actin filament functions. The present work is a continuation of our previous studies on the structural diversity among 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t tropomyosin isoforms determined by sequences of the end-to-end junctions (Sliwinska et al. 2011). Here In this study, orientations positions of the C-terminal segments of four tropomyosin isoforms relative to the actin C-terminal region werewas analyzed with the use of steady-state FRET. Distances were measured between a donor specifically attached to the C-terminal segment of TM and an acceptor bound to actin\u2019s penultimate Cys374, which is located in the outer domain of the filament. The TM-actin distances obtained by steady-state FRET do not reflect the single donor- acceptor distance, because donor bound to a specific site on TM transfers energy to several acceptors attached to actin subunits. However, as discussed in our earlier work (Sliwinska et al. 2011), the apparent distances calculated from steady-state FRET data were comparable to the distances obtained by modeling of lifetime fluorescence data, which estimated transfer efficiency from single donor to the closest single acceptor (Bacchiocchi et al. 2004). According to the NMR as well as the crystal structures of muscle and non-muscle TM model peptides, the overlap region is flexible. When forming a complex with the N-terminus, the C-terminal helices open up and interlock with the N-terminal coiled-coil crystal structures of Cterminal segments of muscle TMs, the C-terminal helices open upon formation of the end-to-end complex (Greenfield et al. 2006; Greenfield et al., 2009; Frye et al. 2010). Although the high resolution structure of tropomyosin overlap bound to actin is not known, most probably this mode of tropomyosin end-to-end interaction along the actin filament is maintained, as it fits well into atomic models of F-actin-TM (Barua et al., 2011; Li et al., 2011). The results obtained in this work suggest that, when bound to actin, the overlap complex remains flexiblerevealed that the orientation of C-terminal segments of the studied TM isoforms was not determined by exon 9- encoded sequence, but rather by the sequence of the end-to-end overlap complex. The donoracceptor distance obtained for the isoforms with the same C-terminal sequence encoded by exon 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t 9a (skTM and TM1b9a) or 9d (TM2 and TM5a) differed by about 6-10 \u00c5. This suggests shows that interdigitation of the C-terminal sequences with different types of N-termini changes conformation of the C-terminal helices with by various degrees. The position of each C-terminal segment of the studied TM isoforms was not determined by the exon 9-encoded sequence, but rather by the sequence of the end-to-end overlap complex. Interestingly, in the case of TM1b9a, which shares the same C-terminal sequence with skTM (encoded by exon 9a), the donor was separated from the acceptor actin by a much larger distance than in the three other isoforms indicating . This shows that the non-muscle 1bencoded N-terminus strongly distorts the structure of the striated muscle-specific 9a-encoded C-terminus., which is specific for skeletal muscle TM and forms complex with the 1a-encoded N-terminus. In the earlier study, we usedthree out of the four TMs used in this work were studied: TM2, TM5a , and TM1b9a. The TMs were labeled with AEDANS in the N-terminal segment (residues 23 or 28) to measure the distance separating the donor located near the N-terminal half of the end-to-end junction and the acceptor bound to actin\u2019s (Cys374) (Sliwinska et al. 2011). In TM2, the FRET distances obtained for donors attached to the N-terminal and the C-terminal regions were 40.2 and 36.4 \u00c5, respectively. In the case of TM5a, the respective distances were 39.3 and 42.7 \u00c5. Taking into account the length of the donor and acceptor probes (about 10 \u00c5) and their random distribution, the differences of about 3.5 \u00c5 between both distances in the two isoforms were small. Thus, when bound to actin, TM2 and TM5a seem to be slightly bent within the end-to-end junction. In contrast, in TM1b9a the FRET distances measured from the N- and Cterminus were 34.8 and 48.1 \u00c5 respectively. The 13.3 \u00c5 difference suggests that the overlap of this isoform is bent or even broken. The cartoon shown in Fig. 4 compares the FRET distances obtained in this and the previous work. To localize the ends on the surface of actin monomer, we positioned the N-terminus of TM2, encoded by exon 1a, over a path of actin amino acid residues 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t D25, K326 and K328, which, according to the models of TM-actin, directly interact with TM\u2019s K6, E16, and D20 (Barua et al. 2013; Barua et al. 2011; Li et al. 2011). Since the C-terminal residues of skeletal TM do not seem to contribute to the TM-actin interface (Barua et al. 2013; Barua et al. 2011; Li et al. 2011), this region was positioned arbitrarily, to illustrate the apparent distance separating Cys269 from actin. Atomic models of actin in complex with LMW TMs are not available, thus our FRET distances were used to position the ends of TM5a and TM1b9a relative to the position of TM2. Differences in FRET distances measured from C- and N-termini of TM2 and TM5a were 3.8 \u00c5 and 3.4 \u00c5, respectively, therefore it appears that in complex with actin the N- and C-terminal regions of these two isoforms were slightly bent. The difference between donor-acceptor distances measured from C- and N-termini of TM1b9a was about 13.3 \u00c5, which suggested that the end-to-end junction of this isoform was either strongly bent or even broken. Significant curvature of the end-to-end overlap was also observed in X-ray and NMR structures of muscle and non-muscle TMs (Frye et al. 2010; Greenfield et al. 2009; Murakami et al. 2008). Together the results suggest that curvature of the overlap region is an inherent attribute of all TM isoforms. As suggested before, bending is important to adopt the helical structure of the actin filament (Holmes & Lehman 2008). Our recent studies have shown that dDuring the activation of the filament by strongly boundbinding of myosin heads (open state), the N-terminal segments of TM isoforms awere differently shifted from the positions they occupiedy on the filament in the absence of strongly bound myosin heads (Sliwinska et al. 2011). In the presentthis work work we have shownobserved that the donor-acceptor distances measured for the C-terminal segments of HMW isoforms (skTM and TM2) increased the C-terminal segments of the isoforms are shifted by S1 not only by different distances but also in the opposite directions. upon binding of myosin heads to actin, whereas in LMW isoforms (TM5a and TM1b9a) the distances decreased. This shows that the extent of the S1-induced shift of the C-terminus is determined 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t by the type of the N-terminal sequence. However, the direction of the shift is not certain. If FRET measured a single donor-acceptor distance between AEDANS bound to TM and DABMI attached to Cys374 of only one actin subunit, shortening the distance would mean shifting the C-terminal segment towards actin\u2019s subdomain 1, where myosin-binding sites are located. This, however would be in discrepancy with the earlier observations that LMW isoforms are better activators of actomyosin ATPase than HMW isoforms (Skorzewski et al., 2009). However, the single TM-bound donor in the TM-actin complex is surrounded by multiple acceptors attached to actin subunits, which contribute to the energy transfer with various efficiencies (Bacchiocchi et al. 2004). According to 3D reconstructions of the filaments\u2019 electron micrographs, binding of myosin to actin shifts TM azimuthally towards the inner domain of actin filament (Lehman et al. 1994; Lehman et al. 2000; Xu et al. 1999). Such a shift might increase FRET transfer efficiency between donor and acceptors attached to actin subunits, which belong to the second chain of the long-pitch actin helix. To verify this possibility, we used atomic models of actin-TM (Li et al., 2011) and actin-TM-S1 (Behrman et al., 2012) and analyzed changes in distances between Cys190 in the central region of TM and the five closest Cys374 residues. The cysteines were located in three actin subunits, which directly bound TM along the filament (A-1, A0 and A+1), and in two subunits across the filament (A-2 and A+2). The analysis showed that in the absence of S1 Cys374 in A0, A-1 and A-2 were the closest to Cys190. In the presence of S1 Cys190 was shifted away from Cys374 in A0 and A-1, whereas it was moved towards Cys374 in A+2 and A-2. Thus, in the S1-induced open state the acceptors bound to actin subunits across the filament significantly contributed to FRET efficiency. Even though the ends are missing in the actin-TM-S1 model, and in the actin-TM model the overlap is not resolved, we assume that these considereations also hold true for the end-to-end junction. Binding of S1 diminished the FRET distances of LMW 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t isoforms, and therefore it appears that, in these isoforms, the end-to-end overlap was more strongly shifted towards actin subunits across the filament than in HMW TM isoforms. According to the actin-TM-S1 model (Behrman et al., 2012), binding of myosin heads to actin shifts TM by as much as 23 \u00c5. The S1-induced shift which we observed in this and the previous work was much shorter, which could be explained by the multi-acceptor system discussed above as well as by local differences in TM isoforms bending. C-termini of HMW and LMW isoforms were shifted respectively further or closer to actin\u2019s outer domain. However, at this stage of investigation it is difficult to locate the junction region of the isoforms in the open state. The reconstructions of the filament electronmicrographs consistently show that upon binding of myosin, TM chains shift away from actin\u2019s outer domain towards inner domain (Lehman et al. 1994; Lehman et al. 2000; Xu et al. 1999). Thus, shortening the donor-acceptor distance, observed by steady-state FRET, might indicate that the actual shift is not in the direction of outer domain but towards acceptors attached to the second chain of actin subunits, which become significant contributors to the transfer. Our data has also shown very high cooperativity of the S1-induced activation of the filament. Depending on the isoform, the maximal shift was completed when about 1-2 myosin heads per one TM molecule was bound. It is worth noting that the C-terminal segment of TM1b9a showed similar cooperativity as the C-termini of the other isoforms. However, it was much larger than the cooperativity of the N-terminal segment of TM1b9a, which required about 0.4 S1/actin for half maximal saturation of the changes in FRET distance (Sliwinska et al. 2011). This result supports our conclusion that both ends of this chimeric TM are not compatible with each other. conclusions : The FRET data gives us an insight into the dynamic changes in the positions of various end-to-end junctions. The isoform-specific sequences determine dDifferences in FRET distances 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t measured between C-termini of tropomyosin isoforms and actin in two (closed and open) filament activation states of the filament are determined by isoform-specific sequences. Based on the results of this and the earlier work, we conclude that the end-to-end junction of tropomyosin isoforms are dassume differently oriented positions on actin filament. and tThe degree of TM shift in response to the filament activation by myosin is individually determined by the sequences of both ends for each isoform. The results agree very well with the observation based on crystal structure of the TM end-to-end overlap that the intermolecular junction is flexible (Greenfield et al. 2009; Frye et al. 2010). Because TM isoforms are functionally specific, the present data give a structural explanation of this specificity and helps us to understand the steric and cooperative mechanisms of the thin filament regulation. quenching of aedans-tm fluorescence by dabmi-actin. : sk TM (closed circles, solid line), TM2 (open circles, long dash), TM5a (open squares, short dash) and TM1b9a (closed squares, dotted line) at 0.6 \u00b5M were titrated with DABMI-actin. The data was normalized using equation: (I - I min ) / (I max -I min ), where I max is the minimal intensity of the fluorescence of AEDANS-TM alone and I min is the minimal fluorescence intensity obtained in the presence of DABMI-actin. The lines were generated by fitting the experimental points to ligandbinding equation in Sigma Plot. Conditions: 2 mM HEPES, pH 7.6, 40 mM NaCl, 5 mM MgCl 2 at 22 \u00b0C. Excitation and emission wavelengths were 340 nm and 495 nm, respectively. The points were averaged from 3 to 5 independent experiments. PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t Figure 3 Myosin S1-induced changes in fluorescence of AEDANS-TM bound to DABMI-actin. (A) HMW TM isoforms: skTM (closed circles, solid line), TM2 (open circles, long dash line) (B) LMW TM isoforms: TM5a (open squares, short dash line), TM1b9a (closed squares, dotted line). AEDANS-TM isoforms at 0.6 \u00b5M were bound to 4.8 \u00b5M DABMI-actin and titrated with myosin S1. The data obtained for HMW isoforms was normalized by using the equation (I-I A )/(I S1 -I A ). For LMW isoforms the equation was: (I-I S1 )/(I A -I S1 ), where I was the fluorescence intensity at given titration point; I A was the intensity of TM-F-actin complex in the absence of S1; I S1 was the fluorescence intensity of AEDANS-TM at maximal S1 concentration. The points were averaged from 3 to 4 independent experiments. The lines were obtained by fitting the experimental data to the Hill equation. Conditions as described under Table 3. PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t Table 1(on next page) Localization of cysteine mutation sites in C-termini encoded by exons 9a and 9d The upper row shows positions of the amino acid residues in the coiled-coil heptapeptide repeat. Ala or Ser residues changed into Cys to create AEDANS attachment sites are in red. PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t f g a b c d e f g a b c d e f g a b c d e f g a b c d 9 a D E L Y A Q K L K Y K A/ I S E E L D H A L N D M T S I 9 d E K V A H A K E E N L S/ C M H Q M L D Q T L L E L N N M 1 2 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t Table 2(on next page) Functional properties of wild type and mutant tropomyosin isoforms labeled with aedans : Conditions: 2 mM HEPES, pH 7.6, 40 mM NaCl, 5 mM MgCl 2 at 22 \u00b0C. The numbers are average values \u00b1 S.E. taken from 2 to 9 independent experiments. PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t TM isoform Kapp (x 107 M-1) Actin-myosin S1 ATPase activity (nmol Pi/mg S1/min.) skTM 1.63 \u00b1 0.57 103.0 \u00b1 8.0 skTM/A269C 1.80 \u00b1 0.26 103.8 \u00b1 7.0 TM2 2.25 \u00b1 0.76 91.7 \u00b1 13.0 TM2/S269C 2.04 \u00b1 0.45 112.0 \u00b1 10.0 TM5a 1.42 \u00b1 0.36 169.0 \u00b1 16.0 TM5a/S232C 1.49 \u00b1 0.58 183.8 \u00b1 20.0 TM1b9a 1.27 \u00b1 0.40 108.2 \u00b1 9.0 TM1b9a/A232C 1.24 \u00b1 0.44 125.5 \u00b1 9.0 1 2 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t Table 3(on next page) Anisotropy of AEDANS bound to C-terminal cysteine residues of TM isoforms Conditions: 0.6 \u00b5M AEDANS-labeled TM alone and with 4.8 \u00b5M actin \u00b1 5 \u00b5M S1 in 2 mM HEPES, pH 7.6, 40 mM NaCl, 5 mM MgCl 2 at 22 \u00b0C. Excitation and emission wavelength were 340 nm and 495 nm, respectively. Average values \u00b1 S.E. were taken from 3 to 8 independent measurements. PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t TM isoform TM alone TM-actin TM-actin-S1 skTM/A269C 0.073 \u00b1 0.008 0.086 \u00b1 0.008 0.123 \u00b1 0.012 TM2/S269C 0.072 \u00b1 0.002 0.106 \u00b1 0.005 0.137 \u00b1 0.004 TM5a/S232C 0.066 \u00b1 0.003 0.096 \u00b1 0.004 0.124 \u00b1 0.004 TM1b9a/A232C 0.095 \u00b1 0.008 0.104 \u00b1 0.003 0.127 \u00b1 0.009 1 2 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t Table 4(on next page) Spectral parameters of FRET between AEDANS-labeled tropomyosins and DABMIlabeled actin Conditions as given under Table 3. Average values \u00b1 S.E. were taken from 10 to 14 independent experiments. PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t TM isoform QD J (x1014 nm4 M-1 cm-1) R0 (\u00c5) E R (\u00c5) skTM/A269C 0.14 6.838 34.7 0.40 \u00b1 0.01 37.3 \u00b1 0.21 TM2/S269C 0.08 6.924 32.1 0.32 \u00b1 0.01 36.4 \u00b1 0.21 TM5a/S232C 0.20 7.012 37.1 0.32 \u00b1 0.03 42.7 \u00b1 1.20 TM1b9a/A232 C 0.12 7.087 34.3 0.13 \u00b1 0.02 48.1 \u00b1 1.45 1 2 3 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t Table 5(on next page) S1-induced changes in FRET between AEDANS-labeled TMs and DABMI-labeled actin Conditions as given under Table 3. Average values \u00b1 S.E. were taken from 6 to 9 independent experiments. S1/actin molar ratio is the ratio of strongly bound myosin heads required for half-maximal change in FRET. In parentheses, S1/TM molar ratio obtained by multiplying S1/actin by the number of actin subunits bound by one molecule of each TM isoform. PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t TM isoform QD R0 E R (\u00c5) \u0394RS1 (\u00c5) S1/actin molar ratio skTM/A269C 0.15 35.0 0.28 \u00b1 0.01 41.0 \u00b1 0.1 3.7 0.15 \u00b1 0.02 (1.05) TM2/S269C 0.09 32.5 0.26 \u00b1 0.02 38.8 \u00b1 0.2 2.4 0.12 \u00b1 0.01 (0.84) TM5a/S232C 0.22 37.6 0.44 \u00b1 0.02 38.9 \u00b1 0.4 -3.8 0.08 \u00b1 0.02 (0.48) TM1b9a/A232C 0.14 35.0 0.24 \u00b1 0.03 42.6 \u00b1 1.2 - 5.5 0.14 \u00b1 0.03 (0.84) 1 2 PeerJ reviewing PDF | (v2013:07:647:1:0:NEW 26 Sep 2013) R ev ie w in g M an us cr ip t",
    "v2_text": "materials and methods : Actin, myosin S1 and recombinant rat tropomyosin isoforms TM2, TM5a and TM1b9a were obtained as described in (Sliwinska et al. 2011). Recombinant skTM was obtained as in 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t (Robaszkiewicz et al. 2012). The Department of Biochemistry and Cell Biology is authorized by Minister of the Environment (Poland) for laboratory use of genetically modified organisms (permit nr GMO: 01-112/112). PCR-based oligonucleotide-directed mutagenesis (Stratagene) was used to create an attachment site for a fluorescent probe at the C-terminal region of TM. First, cDNA encoding all TM isoforms used in this study was mutated to replace the single Cys codon for Ser. The procedure was described in (Sliwinska et al. 2011). Than cDNA from the first stage of mutagenesis was used to create Ala269Cys-skTM, Ser269Cys-TM2, Ala232Cys-TM1b9a and Ser232Cys-TM5a mutants. The oligonucleotides used at this stage were the following: 9a Ala269(232)Cys: 5\u2019-ctgaagtacaagtgcatcagcgaggagctggaccacg-3\u2019 9d Ser269(232)Cys: 5\u2019-gccaaagaagaaaacctttgcatgcaccagatgctggac-3\u2019 All primers were synthesized and HPLC purified by Laboratory of DNA Sequencing and Oligonucleotide Synthesis, Institute of Biochemistry and Biophysics, Polish Academy of Science (Warsaw, Poland). Tropomyosin mutants were labeled with AEDANS and actin was labeled with DABMI according to the procedure described before (Sliwinska et al. 2011). Tropomyosin affinity for actin was measured in co-sedimentation assay. Increasing amounts of tropomyosin (final concentrations varied between 0 and 10 \u00b5M) were mixed with actin (5 \u00b5M) in F-buffer: 2 mM HEPES, pH 7.6, 40 mM NaCl, 5 mM MgCl2 at 22 \u00b0C and ultracentrifuged. Protein content in supernatants and pellets was analyzed electrophoretically on SDS-PAGE as described in (Skorzewski et al. 2009). The activity of actomyosin ATPase was measured in F-buffer. Myosin S1 concentration was at 0.8 \u00b5M, F-actin was 9.2 \u00b5M and tropomyosin was 1.2 \u00b5M. The reaction was started by addition of MgATP to 5 mM and stopped after 10 min. with 3.3% SDS and 30 mM EDTA. The amount of liberated phosphate was measured colorimetricaly according to the method described in (White 1982). Fluorescence anisotropy, FRET experiments, calculation of F\u00f6rster critical distance (R0) and donor-acceptor distance (R) were conducted according to the methods described in (Sliwinska et al. 2011). Results 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t Rationale for the labeling site selection and characterization of the labeled tropomyosin isoforms End-to-end overlap sequences present in the studied TM isoforms were formed by two N-terminal and two C-terminal variants of alternative sequences of rat \u03b1TM. Schematic illustration of the overlap regions are shown in Fig. 1. In skeletal muscle isoform (skTM), non-muscle isoforms (TM2, TM5a), and in chimeric TM1b9a, an isoform which has no natural counterpart, N-termini encoded by exons 1a or 1b formed complexes with C-termini encoded by exons 9a or 9d. In order to create a specific reactive site for attachment of fluorescent probe to the C-terminal regions of tropomyosin isoforms, residue 269 in HMW isoforms or its counterpart in LMW isoforms (residue 232) were changed for Cys (Table. 1). This residue was selected for the following reasons: (a) it is located at the outskirts of the end-to-end junction (Frye et al. 2010; Greenfield et al. 2006; Greenfield et al. 2009), (b) it is not involved in any interactions within the studied end-to-end complexes (Frye et al. 2010), (c) it is located on the outer surface of the two-chain coiled-coil tropomyosin (position c of the coiled-coil heptapeptide repeat), which allows for free motions of the attached label, (d) it is part of the flexible region, where coiled coil structure turns into parallel helices (Frye et al. 2010; Greenfield et al. 2003). Thus we expected that the attached label was sensitive to conformational differences within the end-to-end overlap. Replacement of Cys for Ser in the central region of TM did not affect the basic functions such as actin binding and regulation of actomyosin ATPase activity (Sliwinska et al. 2011). In order to check whether these functions were conserved in TMs with Cys mutations in the C-terminal region, we measured actin binding regulatory functions of all studied AEDANS-labeled TM isoforms. Binding constants (Kapp) obtained in co-sedimentation assay show that all TM mutants bound to actin with high affinity (Table 2). The mutations also did not change significantly interactions between actin and myosin heads. In the presence of wild type and mutant TMs the activities of actin-myosin S1 ATPase were similar (Table 2). To check whether all isoforms of AEDANS-labeled TM bound to DABMI-labeled actin stoichiometrically, TMs were titrated with increasing concentrations of actin. The titration curves shown in Fig. 2 indicate that the fluorescence was maximally quenched at the ratio of TM to F-actin, which was close to stoichiometric. For skTM and TM2 the maximal quenching was reached at 1 TM to 6-7 actins and in the case of TM5a and TM1b9a at 1 TM to 4-5 actins. The results confirm high actin affinity of TM isoforms labeled in C-terminal segment. Additionally, 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t the titration curves show that in case of the isoforms with 9d-encoded C-terminus (TM2 and TM5a), the maximal quenching of AEDANS was reached at lower TM/actin molar ratio than in case of their 9a-encoded counterparts (skTM and TM1b9a). Because fluorescence quenching was due to FRET, the observed differences suggest that the C-terminal sequence encoded by exon 9d facilitates interactions of the C-terminal regions of TM2 and TM5a with actin. Distances separating donor attached to C-terminal region of tropomyosin from acceptor bound to actin Random orientation of the labels is important for FRET distance measurements (Lakowicz 1999). To ensure that the probe was flexibly attached to Cys269/232 in all studied TM isoforms, we measured AEDANS fluorescence anisotropies. As shown in Table 3, the obtained anisotropies were low, therefore we concluded that the label bound to free TM as well as to TM in complex with actin and with myosin S1 was randomly oriented. Thus, the orientation of the donor did not limit the FRET distance measurements. Quantum yield of the donor (QD) and spectral overlap between emission of the donor and absorption of the acceptor (J) affect donor-acceptor critical distance (R0) (Lakowicz 1999). Because local environment surrounding AEDANS bound to Cys269/232 could influence both parameters, QD and J were determined for each labeled TM mutant in the presence of unlabeled actin. The results show that the label attached to C-terminal segment of the studied TM isoforms was exposed to different environments, which caused variations in QD, small shifts in J and, in consequence, differences in R0 (Table 4). The FRET efficiency (E) was calculated from the fluorescence intensity of AEDANS-labeled TM in the absence and in the presence of acceptor (TM saturated with DABMI-F-actin). The fluorescence of the donor was corrected for the increase caused by binding of unlabeled actin (3-7% depending on TM isoform). The efficiencies obtained for the studied TM isoforms were used for calculations of the apparent distances (R) separating donor and acceptor. All calculated FRET parameters are collected in Table 4. The results suggest isoform-specific localization of the overlap region. The differences between the distances obtained for HMW isoforms (skTM, TM2) were small, which indicates similar position of the donor in relation to the acceptor. In LMW isoforms (TM5a and TM1b9a) the donor-acceptor distances were larger. It is worth noting that the distance obtained for TM1b9a was far from R0, 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t thus the sensitivity of FRET measurements was limited and the result can only be regarded as a rough estimate. Changes in tropomyosins\u2019 C-termini as an effect of myosin S1 binding to actin Strong binding of myosin heads (S1) to the filament induces an azimuthal shift of TM chains. This changes TM interactions with actin and activates the filament allowing for actin-myosin cross-bridge cycling (Lehman & Craig 2008; Moraczewska 2002). Saturation of the filament with myosin S1 caused a significant increase of the fluorescence of AEDANS bound to C-terminal segments of all studied TM isoforms. The average increase was about 14% and 18% for skTM and the non-muscle isoforms, respectively. Since the fluorescence intensity of the probe attached to the N-terminal segment increased by about 2-9% (Sliwinska et al. 2011), it appeared that the fluorophore bound to the C-terminal segment of TM was more sensitive to myosin binding. The change of fluorescence observed in this work suggested that the C-terminal region of TM either directly interacted with myosin or significantly changed conformation upon myosin binding to actin. To explore the later possibility, changes in FRET distances in the presence of myosin were analyzed. Strongly bound S1 shifted the C-terminal regions of all TM isoforms, however the direction of the shift observed for HMW and LMW isoforms was different. As compared to TM-actin complex, in the presence of S1 the energy transfer efficiency between AEDANS attached to skTM or TM2 and DABMI-actin decreased. In contrast, when energy donor was attached to TM5a or TM1b9a, an increase in transfer efficiency was observed (Table 5). Binding of unlabeled actin-S1 did not shift the maximum of fluorescence spectrum, thus the spectral overlap (J) was unchanged. However, the quantum yield of AEDANS-TM bound to unlabeled actin-S1 increased (Table 5). This called for recalculation of the critical distances (see equation 5 in (Sliwinska et al. 2011)). Based on the new values of R0 and transfer efficiencies (E) obtained for AEDANS-TMs saturated with DABMI-actin-S1, donor-acceptor distances were calculated (Table 5). The degree of the maximal shift induced by S1 binding to actin (\u0394RS1) was calculated as the difference between the donor-acceptor distance the absence and in the presence of S1. The data indicates that the C-terminal region of the HMW isoforms was shifted away from the donor, whereas in LMW it was shifted closer to the donor. 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t The S1-induced activation of the filament is a very cooperative process. It means that the final change in TM orientation is achieved at S1 concentrations much below the concentrations required for actin saturation (Eaton 1976; Moraczewska 2002). Fig. 3 shows the effects of increasing S1 concentrations on energy transfer between donor-labeled TM and acceptor-labeled actin. The experimental data was normalized and fit to Hill equation. The ratios of strongly bound myosin heads to actin required for half-maximal change in the energy transfer (S1/actin molar ratio) are collected in Table 5. The numbers in parentheses show S1/TM molar ratio, obtained by multiplying S1/actin by the number of actin subunits bound by one TM molecule. The data shows that the cooperativity was very high for each of the isoforms. The differences depended on the type of end-to-end overlap. acknowledgements : The authors thank Katarzyna Robaszkiewicz and Ireneusz Moraczewski for their help with preparation of Figures 1 and 4. References 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t Bacchiocchi C, Graceffa P, and Lehrer SS. 2004. Myosin-induced movement of alphaalpha, alphabeta, and betabeta smooth muscle tropomyosin on actin observed by multisite FRET. Biophysical Journal 86:2295-2307. Barua B, Fagnant PM, Winkelmann DA, Trybus KM, and Hitchcock-Degregori SE. 2013. A periodic pattern of evolutionarily-conserved basic and acidic residues constitutes the binding interface of actin- tropomyosin. Journal of Biological Chemistry. Barua B, Pamula MC, and Hitchcock-DeGregori SE. 2011. Evolutionarily conserved surface residues constitute actin binding sites of tropomyosin. Proceedings of the National Academy of Sciences of the United States of America 108:10150-10155. Barua B, Winkelmann DA, White HD, and Hitchcock-DeGregori SE. 2012. Regulation of actin-myosin interaction by conserved periodic sites of tropomyosin. Proceedings of the National Academy of Sciences of the United States of America 109:18425-18430. Behrmann E, Muller M, Penczek PA, Mannherz HG, Manstein DJ, and Raunser S. 2012. Structure of the rigor actin-tropomyosin-myosin complex. Cell 150:327-338. Eaton BL. 1976. Tropomyosin binding to F-actin induced by myosin heads. Science 192:1337-1339. Frye J, Klenchin VA, and Rayment I. 2010. Structure of the tropomyosin overlap complex from chicken smooth muscle: insight into the diversity of N-terminal recognition. Biochemistry 49:4908-4920. Greenfield NJ, Huang YJ, Swapna GV, Bhattacharya A, Rapp B, Singh A, Montelione GT, and Hitchcock-DeGregori SE. 2006. Solution NMR structure of the junction between tropomyosin molecules: implications for actin binding and regulation. Journal of Molecular Biology 364:80-96. Greenfield NJ, Kotlyanskaya L, and Hitchcock-DeGregori SE. 2009. Structure of the N terminus of a nonmuscle alpha-tropomyosin in complex with the C terminus: implications for actin binding. Biochemistry 48:1272-1283. Greenfield NJ, Swapna GV, Huang Y, Palm T, Graboski S, Montelione GT, and Hitchcock-DeGregori SE. 2003. The structure of the carboxyl terminus of striated alpha-tropomyosin in solution reveals an unusual parallel arrangement of interacting alpha-helices. Biochemistry 42:614-619. Gunning P, O'Neill G, and Hardeman E. 2008. Tropomyosin-based regulation of the actin cytoskeleton in time and space. Physiological Reviews 88:1-35. 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t Holmes KC, and Lehman W. 2008. Gestalt-binding of tropomyosin to actin filaments. Journal of Muscle Research and Cell Motility 29:213-219. Lakowicz J. 1999. Principles of Fluorescence Spectroscopy. New York, Boston, Dordrecht, London, Moscow: Kluwer Academic/Plenum Publishers. Lees-Miller JP, and Helfman DM. 1991. The molecular basis for tropomyosin isoform diversity. Bioessays 13:429-437. Lehman W, and Craig R. 2008. Tropomyosin and the steric mechanism of muscle regulation. Advances in Experimental Medicine and Biology 644:95-109. Lehman W, Craig R, and Vibert P. 1994. Ca(2+)-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. Nature 368:65-67. Lehman W, Hatch V, Korman V, Rosol M, Thomas L, Maytum R, Geeves MA, Van Eyk JE, Tobacman LS, and Craig R. 2000. Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments. Journal of Molecular Biology 302:593-606. Lehrer SS, and Morris EP. 1982. Dual effects of tropomyosin and troponin-tropomyosin on actomyosin subfragment 1 ATPase. Journal of Biological Chemistry 257:8073-8080. Li XE, Tobacman LS, Mun JY, Craig R, Fischer S, and Lehman W. 2011. Tropomyosin position on F-actin revealed by EM reconstruction and computational chemistry. Biophysical Journal 100:1005-1013. Miki M, Makimura S, Sugahara Y, Yamada R, Bunya M, Saitoh T, and Tobita H. 2012. A three-dimensional FRET analysis to construct an atomic model of the actin-tropomyosin-troponin core domain complex on a muscle thin filament. Journal of Molecular Biology 420:40-55. Moraczewska J. 2002. Structural determinants of cooperativity in acto-myosin interactions. Acta Biochimica Polonica 49:805-812. Murakami K, Stewart M, Nozawa K, Tomii K, Kudou N, Igarashi N, Shirakihara Y, Wakatsuki S, Yasunaga T, and Wakabayashi T. 2008. Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T. Proceedings of the National Academy of Sciences of the United States of America 105:7200-7205. Robaszkiewicz K, Dudek E, Kasprzak AA, and Moraczewska J. 2012. Functional effects of congenital myopathy-related mutations in gamma-tropomyosin gene. Biochimica et Biophysica Acta 1822:1562-1569. 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t Skorzewski R, Sliwinska M, Borys D, Sobieszek A, and Moraczewska J. 2009. Effect of actin C-terminal modification on tropomyosin isoforms binding and thin filament regulation. Biochimica et Biophysica Acta 1794:237-243. Sliwinska M, Zukowska M, Borys D, and Moraczewska J. 2011. Different positions of tropomyosin isoforms on actin filament are determined by specific sequences of end-to-end overlaps. Cytoskeleton (Hoboken) 68:300-312. White HD. 1982. Special instrumentation and techniques for kinetic studies of contractile systems. Methods in Enzymology 85:698-708. Xu C, Craig R, Tobacman L, Horowitz R, and Lehman W. 1999. Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy. Biophysical Journal 77:985-992. 342 343 344 345 346 347 348 349 350 351 352 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t Table 1(on next page) Localization of cysteine mutation sites in C-termini encoded by exons 9a and 9d The upper row shows positions of the amino acid residues in the coiled-coil heptapeptide repeat. Ala or Ser residues changed into Cys to create AEDANS attachment sites are in red. Amino acids, which form coiled coil structure are in black. Amino acids within the flexible region, where coiled coil structure turns into parallel helices are in green, the unstructured C- terminal residues are in blue. PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t f g a b c d e f g a b c d e f g a b c d e f g a b c d 9a D E L Y A Q K L K Y K A/ I S E E L D H A L N D M T S I 9d E K V A H A K E E N L S/C M H Q M L D Q T L L E L N N M 1 2 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t Table 2(on next page) Functional properties of wild type and mutant tropomyosin isoforms labeled with discussion : The position tropomyosin assumes on the filament controls interactions of the filament with many actin-binding proteins, thus it is an important determinant of actin filament functions. The present work is a continuation of our previous studies on the structural diversity among tropomyosin isoforms determined by sequences of end-to-end junction (Sliwinska et al. 2011). Here orientations of the C-terminal segments of four tropomyosin isoforms relative to actin C-terminal region was analyzed with the use of steady-state FRET. Distances were measured between donor specifically attached to C-terminal segment of TM and acceptor bound to actin\u2019s penultimate Cys374, which is located in the outer domain of the filament. The TM-actin distances obtained by steady-state FRET do not reflect the single donor-acceptor distance, because donor bound to a specific site on TM transfers energy to several acceptors attached to actin subunits. However, as discussed in our earlier work (Sliwinska et al. 2011), the apparent distances calculated from steady-state FRET data were comparable to the distances obtained by modeling of lifetime fluorescence data, which estimated transfer efficiency from single donor to the closest single acceptor (Bacchiocchi et al. 2004). According to the crystal structures of C-terminal segments of muscle TMs, the C-terminal helices open upon formation of the end-to-end complex (Frye et al. 2010). The results obtained in this work revealed that the orientation of C-terminal segments of the studied TM isoforms was not determined by exon 9-encoded sequence, but rather by the sequence of the end-to-end overlap complex. This suggests that interdigitation of C-terminal sequences with different types of N-termini changes conformation of the C-terminal helices with various degrees. Interestingly, TM1b9a, which shares the same C-terminal sequence with skTM (encoded by exon 9a), was 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t separated from actin by a much larger distance than the three other isoforms. This shows that 1b-encoded N-terminus strongly distorts the structure of the 9a-encoded C-terminus, which is specific for skeletal muscle TM and forms complex with the 1a-encoded N-terminus. In the earlier study we used TM2, TM5a and TM1b9a labeled with AEDANS in N-terminal segment (residues 23 or 28) to measure distance separating donor located near the N-terminal half of the end-to-end junction and acceptor bound to actin (Cys374) (Sliwinska et al. 2011). The cartoon shown in Fig. 4 compares the FRET distances obtained in this and the previous work. To localize the ends on the surface of actin monomer, we positioned the N-terminus of TM2, encoded by exon 1a, over a path of actin amino acid residues D25, K326 and K328, which, according to the models of TM-actin, directly interact with TM\u2019s K6, E16, and D20 (Barua et al. 2013; Barua et al. 2011; Li et al. 2011). Since the C-terminal residues of skeletal TM do not seem to contribute to the TM-actin interface (Barua et al. 2013; Barua et al. 2011; Li et al. 2011), this region was positioned arbitrarily, to illustrate the apparent distance separating Cys269 from actin. Atomic models of actin in complex with LMW TMs are not available, thus our FRET distances were used to position the ends of TM5a and TM1b9a relative to the position of TM2. Differences in FRET distances measured from C- and N-termini of TM2 and TM5a were 3.8 \u00c5 and 3.4 \u00c5, respectively, therefore it appears that in complex with actin the N- and C-terminal regions of these two isoforms were slightly bent. The difference between donor-acceptor distances measured from C- and N-termini of TM1b9a was about 13.3 \u00c5, which suggested that the end-to-end junction of this isoform was either strongly bent or even broken. Significant curvature of the end-to-end overlap was also observed in X-ray and NMR structures of muscle and non-muscle TMs (Frye et al. 2010; Greenfield et al. 2009; Murakami et al. 2008). Together the results suggest that curvature of overlap region is an inherent attribute of all TM isoforms. As suggested before, bending is important to adopt the helical structure of the actin filament (Holmes & Lehman 2008). Our recent studies have shown that during the activation of the filament by strong binding of myosin (open state), N-terminal segments of TM isoforms are differently shifted from the positions they occupy on the filament in the absence of strongly bound myosin heads (Sliwinska et al. 2011). In this work we have shown that the C-terminal segments of the isoforms are shifted by S1 not only by different distances but also in the opposite directions. C-termini of HMW and LMW isoforms were shifted respectively further or closer to actin\u2019s outer domain. However, at this stage of investigation it is difficult to locate the junction region of the isoforms in the open state. The reconstructions of the filament electronmicrographs consistently show that upon 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t binding of myosin, TM chains shift away from actin\u2019s outer domain towards inner domain (Lehman et al. 1994; Lehman et al. 2000; Xu et al. 1999). Thus, shortening the donor-acceptor distance, observed by steady-state FRET, might indicate that the actual shift is not in the direction of outer domain but towards acceptors attached to the second chain of actin subunits, which become significant contributors to the transfer. Our data has also shown very high cooperativity of the S1-induced activation of the filament. Depending on the isoform, the maximal shift was completed when about 1-2 myosin heads per one TM molecule was bound. It is worth noting that the C-terminal segment of TM1b9a showed similar cooperativity as the C-termini of the other isoforms. However, it was much larger than the cooperativity of the N-terminal segment of TM1b9a, which required about 0.4 S1/actin for half maximal saturation of the changes in FRET distance (Sliwinska et al. 2011). This result supports our conclusion that both ends of this chimeric TM are not compatible. conclusions : The isoform-specific sequences determine differences in FRET distances measured between C-termini of tropomyosin isoforms and actin in two (closed and open) filament activation states. Based on the results of this and earlier work we conclude that tropomyosin isoforms are differently oriented on actin filament and the degree of TM shift in response to the filament activation by myosin is individual for each isoform. The results agree very well with the observation based on crystal structure of TM end-to-end overlap that the intermolecular junction is flexible (Frye et al. 2010). Because TM isoforms are functionally specific, the present data give structural explanation of this specificity and help to understand steric and cooperative mechanisms of the thin filament regulation. aedans : Conditions: 2 mM HEPES, pH 7.6, 40 mM NaCl, 5 mM MgCl 2 at 22 \u00b0C. The numbers are average values \u00b1 S.E. taken from 2 to 9 independent experiments. PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t TM isoform Kapp (x 107 M-1) Actin-myosin S1 ATPase activity (nmol Pi/mg S1/min.) skTM 1.63 \u00b1 0.57 103.0 \u00b1 8.0 skTM/A269C 1.80 \u00b1 0.26 103.8 \u00b1 7.0 TM2 2.25 \u00b1 0.76 91.7 \u00b1 13.0 TM2/S269C 2.04 \u00b1 0.45 112.0 \u00b1 10.0 TM5a 1.42 \u00b1 0.36 169.0 \u00b1 16.0 TM5a/S232C 1.49 \u00b1 0.58 183.8 \u00b1 20.0 TM1b9a 1.27 \u00b1 0.40 108.2 \u00b1 9.0 TM1b9a/A232C 1.24 \u00b1 0.44 125.5 \u00b1 9.0 1 2 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t Table 3(on next page) Anisotropy of AEDANS bound to C-terminal cysteine residues of TM isoforms Conditions: 0.6 \u00b5M AEDANS-labeled TM alone and with 4.8 \u00b5M actin \u00b1 5 \u00b5M S1 in 2 mM HEPES, pH 7.6, 40 mM NaCl, 5 mM MgCl 2 at 22 \u00b0C. Excitation and emission wavelength were 340 nm and 495 nm, respectively. Average values \u00b1 S.E. were taken from 3 to 8 independent measurements. PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t TM isoform TM alone TM-actin TM-actin-S1 skTM/A269C 0.073 \u00b1 0.008 0.086 \u00b1 0.008 0.123 \u00b1 0.012 TM2/S269C 0.072 \u00b1 0.002 0.106 \u00b1 0.005 0.137 \u00b1 0.004 TM5a/S232C 0.066 \u00b1 0.003 0.096 \u00b1 0.004 0.124 \u00b1 0.004 TM1b9a/A232C 0.095 \u00b1 0.008 0.104 \u00b1 0.003 0.127 \u00b1 0.009 1 2 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t Table 4(on next page) Spectral parameters of FRET between AEDANS-labeled tropomyosins and DABMIlabeled actin Conditions as given under Table 3. Average values \u00b1 S.E. were taken from 10 to 14 independent experiments. PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t TM isoform QD J (x1014 nm4 M-1 cm-1) R0 (\u00c5) E R (\u00c5) skTM/A269C 0.14 6.838 34.7 0.40 \u00b1 0.01 37.3 \u00b1 0.21 TM2/S269C 0.08 6.924 32.1 0.32 \u00b1 0.01 36.4 \u00b1 0.21 TM5a/S232C 0.20 7.012 37.1 0.32 \u00b1 0.03 42.7 \u00b1 1.20 TM1b9a/A232 C 0.12 7.087 34.3 0.13 \u00b1 0.02 48.1 \u00b1 1.45 1 2 3 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t Table 5(on next page) S1-induced changes in FRET between AEDANS-labeled TMs and DABMI-labeled actin Conditions as given under Table 3. Average values \u00b1 S.E. were taken from 6 to 9 independent experiments. S1/actin molar ratio is the ratio of strongly bound myosin heads required for half-maximal change in FRET. In parentheses, S1/TM molar ratio obtained by multiplying S1/actin by the number of actin subunits bound by one molecule of each TM isoform. PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t TM isoform QD R0 E R (\u00c5) \u0394RS1 (\u00c5) S1/actin molar ratio skTM/A269C 0.15 35.0 0.28 \u00b1 0.01 41.0 \u00b1 0.1 3.7 0.15 \u00b1 0.02 (1.05) TM2/S269C 0.09 32.5 0.26 \u00b1 0.02 38.8 \u00b1 0.2 2.4 0.12 \u00b1 0.01 (0.84) TM5a/S232C 0.22 37.6 0.44 \u00b1 0.02 38.9 \u00b1 0.4 -3.8 0.08 \u00b1 0.02 (0.48) TM1b9a/A232C 0.14 35.0 0.24 \u00b1 0.03 42.6 \u00b1 1.2 - 5.5 0.14 \u00b1 0.03 (0.84) 1 2 PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t Figure 1 Schematic illustration of the types of end-to-end junctions present in TM isoforms used in this work. C-terminal sequences encoded by exon 9a (green) or 9d (red) overlapped with N-terminal sequences encoded by exons 1a (orange) or 1b (blue). The residues in C-terminal segments, which were mutated to cysteines are shown. PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t Figure 2 quenching of aedans-tm fluorescence by dabmi-actin. : sk TM (closed circles, solid line), TM2 (open circles, long dash), TM5a (open squares, short dash) and TM1b9a (closed squares, dotted line) at 0.6 \u00b5M were titrated with DABMI-actin. The data was normalized using equation: (I - I min ) / (I max -I min ), where I max is the minimal intensity of the fluorescence of AEDANS-TM alone and I min is the minimal fluorescence intensity obtained in the presence of DABMI-actin. The lines were generated by fitting the experimental points to ligandbinding equation in Sigma Plot. Conditions: 2 mM HEPES, pH 7.6, 40 mM NaCl, 5 mM MgCl 2 at 22 \u00b0C. Excitation and emission wavelengths were 340 nm and 495 nm, respectively. The points were averaged from 3 to 5 independent experiments. PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t Figure 3 Myosin S1-induced changes in fluorescence of AEDANS-TM bound to DABMI-actin. (A) HMW TM isoforms: skTM (closed circles, solid line), TM2 (open circles, long dash line) (B) LMW TM isoforms: TM5a (open squares, short dash line), TM1b9a (closed squares, dotted line). AEDANS-TM isoforms at 0.6 \u00b5M were bound to 4.8 \u00b5M DABMI-actin and titrated with myosin S1. The data obtained for HMW isoforms was normalized by using the equation (I-I A )/(I S1 -I A ). For LMW isoforms the equation was: (I-I S1 )/(I A -I S1 ), where I was the fluorescence intensity at given titration point; I A was the intensity of TM-F-actin complex in the absence of S1; I S1 was the fluorescence intensity of AEDANS-TM at maximal S1 concentration. The points were averaged from 3 to 4 independent experiments. The lines were obtained by fitting the experimental data to the Hill equation. Conditions as described under Table 3. PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t Figure 4 Positions of the overlap regions of TM isoforms relative to actin monomer. Two chains of TM coiled coil (black and grey lines) are located on actin monomer (blue shadow). Positions of actin amino acid residues D25, K326 and K328 are marked with yellow dot clusters. Donors attached to N- and C-terminal regions of TM are marked with red dots. Approximate position of the acceptor is marked by green circle. The numbers show FRET distances in \u00c5 between donors attached to C-terminal (purple) and N-terminal (navy) regions of TM isoforms and acceptor bound to actin. PeerJ reviewing PDF | (v2013:07:647:0:0:NEW 19 Aug 2013) R ev ie w in g M an us cr ip t",
    "url": "https://peerj.com/articles/182/reviews/",
    "review_1": "John Hutchinson \u00b7 Sep 29, 2013 \u00b7 Academic Editor\nACCEPT\nI am totally satisfied with the revisions, which I have checked- the authors are to be commended for a particularly fastidious and patient revision of the paper! This is a rigorous magnum opus that will be a boon to palaeontology. It is wonderful that PeerJ is publishing it.",
    "review_2": "John Hutchinson \u00b7 Jun 2, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nThe authors are to be commended on a rigorous, substantive body of work in this manuscript; it is certain to be well cited in future literature. The reviewers have all made constructive suggestions for changes and uniformly agree that it should be published, with these minor revisions. Please include a point-by-point Response to Reviewers with your revised MS. In particular, I agree that a wider consideration of heterochrony as a concept and in non-dinosaurs would improve the paper, as would illustration of \"ontogenetic changes in the external morphology of the cranial bones, even if such changes are hypothetical\".",
    "review_3": "Reviewer 1 \u00b7 Jun 2, 2013\nBasic reporting\nThis paper is generally well-written and accurate. I think that the sections of the anatomical descriptions dealing with ontogenetic change could be more consistently delineated from the remainder, however. Almost every element should have some differences \u2013 see, e.g., multiple papers on turtle ontogeny by G.S. Bever, alligator ontogeny by Vickaryous and Hall (2008, J. Morph.), Bhullar et al. (2012, Nature) on bird and dinosaur ontogeny and heterochrony, several papers my Maxwell on birds, the supplement to Bhullar (2012, JEZ B), Maisano (2002, JMorph), and several papers by Tarazona, all of which have extensive descriptions of bone-by-bone ontogenetic change in reptiles: turtles, crocodylians, birds, and squamates, respectively.\nOn this note, a table of ontogenetic changes in Parasaurolophus element-by-element and as compared to other hadrosaurs would be useful.\nThere is one persistent grammatical mistake \u2013 the use of a hyphen following adverbs describing adjectives, e.g., 727 \u201cpoorly-preserved,\u201d 908 \u201csimilarly-interpreted\u201d, twice in 1131, etc. throughout the manuscript.\nExperimental design\nThe discussion of heterochrony needs some citations \u2013 at the least, Gould (1977) and Alberch et al. (1979). Additionally, it would be interesting to know whether overall skull shape change (not just in the crest) is decoupled from size between Parasaurolophus and Corythosaurus. This would allow a more nuanced approach to potential heterochrony in these forms.\nRegarding the nasal cavity work, the endocasts are interesting and impressive. A few words about which parts of the cartilaginous nasal capsule the various chambers relate to would be welcome. Regarding the brain endocast, Gans (1980, JHerp), Bever (2005, Palaeo electronica), and Bhullar et al. (2012, Nature) among others addressed ontogenetic changes in reptile brains and braincases and will provide comparative insight to the changes described here. For instance, the (de)flexure and rotation of the brain during ontogeny is well-known in archosaurs.\nIn the section on the axial skeleton, a little more attention to potential ontogenetic changes would have been welcome, despite the poor preservation. In the appendicular section, a bit of comparison to changes in birds and crocodylians would be enlightening as to the generality of the transformations described in, for example, the pelvis.\nValidity of the findings\nAnatomical descriptions appear accurate and thorough. Histological descriptions are excellent. I would have liked to see a formal phylogenetic analysis at least attempted to place this animal as Parasaurolophus.\nAdditional comments\nThis paper is thorough and interesting and certainly of interest to a number of audiences. It is, in its current form, a little myopic in its focus on the dinosaurian literature and could benefit from broader comparisons. Certainly the papers mentioned above on ontogeny and heterochrony in various reptiles (including dinosaurs!) should be considered. Erickson et al. (2012, Science) should probably be cited in the section on jaw mechanics. The discussion of heterochrony should reference foundational works on the subject and perhaps try to couch the potential heterochrony in hadrosaurs in the formal structure set out therein.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Ontogeny in the tube-crested dinosaur Parasaurolophus (Hadrosauridae) and heterochrony in hadrosaurids (v0.1)\". PeerJ https://doi.org/10.7287/peerj.182v0.1/reviews/1",
    "review_4": "David Weishampel \u00b7 May 29, 2013\nBasic reporting\nSee below\nExperimental design\nSee below\nValidity of the findings\nSee below\nAdditional comments\nNote from staff: The original comments from this reviewer did not make it into the system correctly. Therefore Dr Weishampel supplied comments direct to staff as per the text below:\n\n>my review was very simple: The Farke et al. MS is spot- on, well written, and complete as far as the bony descriptions and the synthetic discussions go. The only thing I would ask them to spend some more time on is the issue of the S-Loop of the crest and its phylogenetic distribution - parasaurolophins don't have one (that is, they've highly modified the vestibule to make their entire crest) and corythosaurins have an S-Loop. That's about it. In lieu of an official review, I hope that this will suffice.\nCite this review as\nWeishampel D (2013) Peer Review #2 of \"Ontogeny in the tube-crested dinosaur Parasaurolophus (Hadrosauridae) and heterochrony in hadrosaurids (v0.1)\". PeerJ https://doi.org/10.7287/peerj.182v0.1/reviews/2",
    "pdf_1": "https://peerj.com/articles/182v0.2/submission",
    "pdf_2": "https://peerj.com/articles/182v0.1/submission",
    "review_5": "Reviewer 3 \u00b7 May 21, 2013\nBasic reporting\nThis paper provides a thorough documentation and discussion of an important and unique specimen of lambeosaurine hadrosaurid. It is very informative, containing new insights into the anatomy and life history of one of the most iconic, yet still enigmatic taxa. I have only a few remarks; otherwise, I recommend this paper for publication, with minor revisions.\nExperimental design\nNo comments.\nValidity of the findings\nTitle and line 3 of the abstract: \u201cHadrosaurs\u201d is too imprecise, unless you mean Hadrosauria (sensu Wagner and Lehman 2009), which I don\u2019t think you do. Thus, I advise to better use \u201chadrosaurid(s)\u201d.\n\nPage 3, line 49: Usage of \u201ccorythosaurins\u201d should be replaced for \u201clambeosaurins\u201d. As recommended by Sullivan et al. (2011): \u201cAccording to to Article 37.1 of the International Code of Zoological Nomenclature (1999): When a family-group taxon is subdivided, the\nsubordinate name that contains the taxon that contains the type genus of the superior taxon is denoted by the same name the nominotypical name. Thus, given that \u201cCorythosaurini\u201d is a subdivision (tribe) of Lambeosaurinae coordinate with Parasaurolophini, the proper name of the tribe should be Lambeosaurini, not \u201cCorythosaurini.\u201d (Sullivan et al. 2001:406).\n\nPage 8, lines 259 and 260. The terms \u201claterotemporal\u201d and \u201cdorsotemporal\u201d are not how these opening are typically referred too and they seem like an odd choice. I\u2019d recommend using the more common terms \u201cinfratemporal\u201d and \u201csupratemporal\u201d fenestrae.\n\nPage 9, line 281, 284: \u201cnaris\u201d. It would be better to further specify that it is the \u201cexternal naris\u201d or \u201cbony naris\u201d.\n\nFigure 6 and the premaxilla-nasal articulation: on Fig. 6 the nasal bifurcates ventrally to receive a finger-shaped process of the premaxilla. This configuration is similar to that of juvenile Corythosaurus (Evans et al. 2005:Fig. 18.1). It is not clear to me how could the authors derived with such detail this articular morphology from RAM 14000? As seen in Fig. 5, such articulation is nowhere to be seen (unpreserved?).\n\nPage 34, line 1379 and following lines: Regarding the interesting discussion of the extent of the nasal and premaxilla in Parasaurolophus, I think the paper would be even more informative than already is if it included a figure showing ontogenetic changes in the external morphology of the cranial bones, even if such changes are hypothetical at this juncture, much like they did with the nasal passages in Fig. 11.\n\nReferences\n\n\nEvans D. C., Forster, C. A., and Reisz R. R. 2005. The type specimen of Tetragonosaurus erectofrons (Ornithischia: Hadrosauridae) and the identification of juvenile lambeosaurines. In: Currie PJ, Koppelhus EB, eds. Dinosaur provincial park: a\nspectacular ecosystem revealed. Bloomington: Indiana University Press, 349\u2013366.\n\nWagner, J. R., and Lehman, T. M. 2009. An enigmatic lambeosaurine hadrosaur (Reptilia: Dinosauria) from the Upper Shale Member of the Campanian Aguja Formation of Trans-Pecos Texas. Journal of Vertebrate Paleontology 29:605-611.\n\nSullivan, R. M., Jasinski, S. E., Guenter, M., and Lucas, S. G. 2011. The first lambeosaurine (Dinosauria, Hadrosauridae, Lambeosaurinae) from the Upper Cretaceus Ojo Alamo Formation (Naashoibito Member), San Juan Basin, New Mexico. New Mexico Museum of Natural History and Science 53:405-417.\nAdditional comments\nTitle and line 3 of the abstract: \u201cHadrosaurs\u201d is too imprecise, unless you mean Hadrosauria (sensu Wagner and Lehman 2009), which I don\u2019t think you do. Thus, I advise to better use \u201chadrosaurid(s)\u201d.\n\nPage 3, line 49: Usage of \u201ccorythosaurins\u201d should be replaced for \u201clambeosaurins\u201d. As recommended by Sullivan et al. (2011): \u201cAccording to to Article 37.1 of the International Code of Zoological Nomenclature (1999): When a family-group taxon is subdivided, the\nsubordinate name that contains the taxon that contains the type genus of the superior taxon is denoted by the same name the nominotypical name. Thus, given that \u201cCorythosaurini\u201d is a subdivision (tribe) of Lambeosaurinae coordinate with Parasaurolophini, the proper name of the tribe should be Lambeosaurini, not \u201cCorythosaurini.\u201d (Sullivan et al. 2001:406).\n\nPage 8, lines 259 and 260. The terms \u201claterotemporal\u201d and \u201cdorsotemporal\u201d are not how these opening are typically referred too and they seem like an odd choice. I\u2019d recommend using the more common terms \u201cinfratemporal\u201d and \u201csupratemporal\u201d fenestrae.\n\nPage 9, line 281, 284: \u201cnaris\u201d. It would be better to further specify that it is the \u201cexternal naris\u201d or \u201cbony naris\u201d.\n\nFigure 6 and the premaxilla-nasal articulation: on Fig. 6 the nasal bifurcates ventrally to receive a finger-shaped process of the premaxilla. This configuration is similar to that of juvenile Corythosaurus (Evans et al. 2005:Fig. 18.1). It is not clear to me how could the authors derived with such detail this articular morphology from RAM 14000? As seen in Fig. 5, such articulation is nowhere to be seen (unpreserved?).\n\nPage 34, line 1379 and following lines: Regarding the interesting discussion of the extent of the nasal and premaxilla in Parasaurolophus, I think the paper would be even more informative than already is if it included a figure showing ontogenetic changes in the external morphology of the cranial bones, even if such changes are hypothetical at this juncture, much like they did with the nasal passages in Fig. 11.\n\nReferences\n\n\nEvans D. C., Forster, C. A., and Reisz R. R. 2005. The type specimen of Tetragonosaurus erectofrons (Ornithischia: Hadrosauridae) and the identification of juvenile lambeosaurines. In: Currie PJ, Koppelhus EB, eds. Dinosaur provincial park: a\nspectacular ecosystem revealed. Bloomington: Indiana University Press, 349\u2013366.\n\nWagner, J. R., and Lehman, T. M. 2009. An enigmatic lambeosaurine hadrosaur (Reptilia: Dinosauria) from the Upper Shale Member of the Campanian Aguja Formation of Trans-Pecos Texas. Journal of Vertebrate Paleontology 29:605-611.\n\nSullivan, R. M., Jasinski, S. E., Guenter, M., and Lucas, S. G. 2011. The first lambeosaurine (Dinosauria, Hadrosauridae, Lambeosaurinae) from the Upper Cretaceus Ojo Alamo Formation (Naashoibito Member), San Juan Basin, New Mexico. New Mexico Museum of Natural History and Science 53:405-417.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #3 of \"Ontogeny in the tube-crested dinosaur Parasaurolophus (Hadrosauridae) and heterochrony in hadrosaurids (v0.1)\". PeerJ https://doi.org/10.7287/peerj.182v0.1/reviews/3",
    "all_reviews": "Review 1: John Hutchinson \u00b7 Sep 29, 2013 \u00b7 Academic Editor\nACCEPT\nI am totally satisfied with the revisions, which I have checked- the authors are to be commended for a particularly fastidious and patient revision of the paper! This is a rigorous magnum opus that will be a boon to palaeontology. It is wonderful that PeerJ is publishing it.\nReview 2: John Hutchinson \u00b7 Jun 2, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nThe authors are to be commended on a rigorous, substantive body of work in this manuscript; it is certain to be well cited in future literature. The reviewers have all made constructive suggestions for changes and uniformly agree that it should be published, with these minor revisions. Please include a point-by-point Response to Reviewers with your revised MS. In particular, I agree that a wider consideration of heterochrony as a concept and in non-dinosaurs would improve the paper, as would illustration of \"ontogenetic changes in the external morphology of the cranial bones, even if such changes are hypothetical\".\nReview 3: Reviewer 1 \u00b7 Jun 2, 2013\nBasic reporting\nThis paper is generally well-written and accurate. I think that the sections of the anatomical descriptions dealing with ontogenetic change could be more consistently delineated from the remainder, however. Almost every element should have some differences \u2013 see, e.g., multiple papers on turtle ontogeny by G.S. Bever, alligator ontogeny by Vickaryous and Hall (2008, J. Morph.), Bhullar et al. (2012, Nature) on bird and dinosaur ontogeny and heterochrony, several papers my Maxwell on birds, the supplement to Bhullar (2012, JEZ B), Maisano (2002, JMorph), and several papers by Tarazona, all of which have extensive descriptions of bone-by-bone ontogenetic change in reptiles: turtles, crocodylians, birds, and squamates, respectively.\nOn this note, a table of ontogenetic changes in Parasaurolophus element-by-element and as compared to other hadrosaurs would be useful.\nThere is one persistent grammatical mistake \u2013 the use of a hyphen following adverbs describing adjectives, e.g., 727 \u201cpoorly-preserved,\u201d 908 \u201csimilarly-interpreted\u201d, twice in 1131, etc. throughout the manuscript.\nExperimental design\nThe discussion of heterochrony needs some citations \u2013 at the least, Gould (1977) and Alberch et al. (1979). Additionally, it would be interesting to know whether overall skull shape change (not just in the crest) is decoupled from size between Parasaurolophus and Corythosaurus. This would allow a more nuanced approach to potential heterochrony in these forms.\nRegarding the nasal cavity work, the endocasts are interesting and impressive. A few words about which parts of the cartilaginous nasal capsule the various chambers relate to would be welcome. Regarding the brain endocast, Gans (1980, JHerp), Bever (2005, Palaeo electronica), and Bhullar et al. (2012, Nature) among others addressed ontogenetic changes in reptile brains and braincases and will provide comparative insight to the changes described here. For instance, the (de)flexure and rotation of the brain during ontogeny is well-known in archosaurs.\nIn the section on the axial skeleton, a little more attention to potential ontogenetic changes would have been welcome, despite the poor preservation. In the appendicular section, a bit of comparison to changes in birds and crocodylians would be enlightening as to the generality of the transformations described in, for example, the pelvis.\nValidity of the findings\nAnatomical descriptions appear accurate and thorough. Histological descriptions are excellent. I would have liked to see a formal phylogenetic analysis at least attempted to place this animal as Parasaurolophus.\nAdditional comments\nThis paper is thorough and interesting and certainly of interest to a number of audiences. It is, in its current form, a little myopic in its focus on the dinosaurian literature and could benefit from broader comparisons. Certainly the papers mentioned above on ontogeny and heterochrony in various reptiles (including dinosaurs!) should be considered. Erickson et al. (2012, Science) should probably be cited in the section on jaw mechanics. The discussion of heterochrony should reference foundational works on the subject and perhaps try to couch the potential heterochrony in hadrosaurs in the formal structure set out therein.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Ontogeny in the tube-crested dinosaur Parasaurolophus (Hadrosauridae) and heterochrony in hadrosaurids (v0.1)\". PeerJ https://doi.org/10.7287/peerj.182v0.1/reviews/1\nReview 4: David Weishampel \u00b7 May 29, 2013\nBasic reporting\nSee below\nExperimental design\nSee below\nValidity of the findings\nSee below\nAdditional comments\nNote from staff: The original comments from this reviewer did not make it into the system correctly. Therefore Dr Weishampel supplied comments direct to staff as per the text below:\n\n>my review was very simple: The Farke et al. MS is spot- on, well written, and complete as far as the bony descriptions and the synthetic discussions go. The only thing I would ask them to spend some more time on is the issue of the S-Loop of the crest and its phylogenetic distribution - parasaurolophins don't have one (that is, they've highly modified the vestibule to make their entire crest) and corythosaurins have an S-Loop. That's about it. In lieu of an official review, I hope that this will suffice.\nCite this review as\nWeishampel D (2013) Peer Review #2 of \"Ontogeny in the tube-crested dinosaur Parasaurolophus (Hadrosauridae) and heterochrony in hadrosaurids (v0.1)\". PeerJ https://doi.org/10.7287/peerj.182v0.1/reviews/2\nReview 5: Reviewer 3 \u00b7 May 21, 2013\nBasic reporting\nThis paper provides a thorough documentation and discussion of an important and unique specimen of lambeosaurine hadrosaurid. It is very informative, containing new insights into the anatomy and life history of one of the most iconic, yet still enigmatic taxa. I have only a few remarks; otherwise, I recommend this paper for publication, with minor revisions.\nExperimental design\nNo comments.\nValidity of the findings\nTitle and line 3 of the abstract: \u201cHadrosaurs\u201d is too imprecise, unless you mean Hadrosauria (sensu Wagner and Lehman 2009), which I don\u2019t think you do. Thus, I advise to better use \u201chadrosaurid(s)\u201d.\n\nPage 3, line 49: Usage of \u201ccorythosaurins\u201d should be replaced for \u201clambeosaurins\u201d. As recommended by Sullivan et al. (2011): \u201cAccording to to Article 37.1 of the International Code of Zoological Nomenclature (1999): When a family-group taxon is subdivided, the\nsubordinate name that contains the taxon that contains the type genus of the superior taxon is denoted by the same name the nominotypical name. Thus, given that \u201cCorythosaurini\u201d is a subdivision (tribe) of Lambeosaurinae coordinate with Parasaurolophini, the proper name of the tribe should be Lambeosaurini, not \u201cCorythosaurini.\u201d (Sullivan et al. 2001:406).\n\nPage 8, lines 259 and 260. The terms \u201claterotemporal\u201d and \u201cdorsotemporal\u201d are not how these opening are typically referred too and they seem like an odd choice. I\u2019d recommend using the more common terms \u201cinfratemporal\u201d and \u201csupratemporal\u201d fenestrae.\n\nPage 9, line 281, 284: \u201cnaris\u201d. It would be better to further specify that it is the \u201cexternal naris\u201d or \u201cbony naris\u201d.\n\nFigure 6 and the premaxilla-nasal articulation: on Fig. 6 the nasal bifurcates ventrally to receive a finger-shaped process of the premaxilla. This configuration is similar to that of juvenile Corythosaurus (Evans et al. 2005:Fig. 18.1). It is not clear to me how could the authors derived with such detail this articular morphology from RAM 14000? As seen in Fig. 5, such articulation is nowhere to be seen (unpreserved?).\n\nPage 34, line 1379 and following lines: Regarding the interesting discussion of the extent of the nasal and premaxilla in Parasaurolophus, I think the paper would be even more informative than already is if it included a figure showing ontogenetic changes in the external morphology of the cranial bones, even if such changes are hypothetical at this juncture, much like they did with the nasal passages in Fig. 11.\n\nReferences\n\n\nEvans D. C., Forster, C. A., and Reisz R. R. 2005. The type specimen of Tetragonosaurus erectofrons (Ornithischia: Hadrosauridae) and the identification of juvenile lambeosaurines. In: Currie PJ, Koppelhus EB, eds. Dinosaur provincial park: a\nspectacular ecosystem revealed. Bloomington: Indiana University Press, 349\u2013366.\n\nWagner, J. R., and Lehman, T. M. 2009. An enigmatic lambeosaurine hadrosaur (Reptilia: Dinosauria) from the Upper Shale Member of the Campanian Aguja Formation of Trans-Pecos Texas. Journal of Vertebrate Paleontology 29:605-611.\n\nSullivan, R. M., Jasinski, S. E., Guenter, M., and Lucas, S. G. 2011. The first lambeosaurine (Dinosauria, Hadrosauridae, Lambeosaurinae) from the Upper Cretaceus Ojo Alamo Formation (Naashoibito Member), San Juan Basin, New Mexico. New Mexico Museum of Natural History and Science 53:405-417.\nAdditional comments\nTitle and line 3 of the abstract: \u201cHadrosaurs\u201d is too imprecise, unless you mean Hadrosauria (sensu Wagner and Lehman 2009), which I don\u2019t think you do. Thus, I advise to better use \u201chadrosaurid(s)\u201d.\n\nPage 3, line 49: Usage of \u201ccorythosaurins\u201d should be replaced for \u201clambeosaurins\u201d. As recommended by Sullivan et al. (2011): \u201cAccording to to Article 37.1 of the International Code of Zoological Nomenclature (1999): When a family-group taxon is subdivided, the\nsubordinate name that contains the taxon that contains the type genus of the superior taxon is denoted by the same name the nominotypical name. Thus, given that \u201cCorythosaurini\u201d is a subdivision (tribe) of Lambeosaurinae coordinate with Parasaurolophini, the proper name of the tribe should be Lambeosaurini, not \u201cCorythosaurini.\u201d (Sullivan et al. 2001:406).\n\nPage 8, lines 259 and 260. The terms \u201claterotemporal\u201d and \u201cdorsotemporal\u201d are not how these opening are typically referred too and they seem like an odd choice. I\u2019d recommend using the more common terms \u201cinfratemporal\u201d and \u201csupratemporal\u201d fenestrae.\n\nPage 9, line 281, 284: \u201cnaris\u201d. It would be better to further specify that it is the \u201cexternal naris\u201d or \u201cbony naris\u201d.\n\nFigure 6 and the premaxilla-nasal articulation: on Fig. 6 the nasal bifurcates ventrally to receive a finger-shaped process of the premaxilla. This configuration is similar to that of juvenile Corythosaurus (Evans et al. 2005:Fig. 18.1). It is not clear to me how could the authors derived with such detail this articular morphology from RAM 14000? As seen in Fig. 5, such articulation is nowhere to be seen (unpreserved?).\n\nPage 34, line 1379 and following lines: Regarding the interesting discussion of the extent of the nasal and premaxilla in Parasaurolophus, I think the paper would be even more informative than already is if it included a figure showing ontogenetic changes in the external morphology of the cranial bones, even if such changes are hypothetical at this juncture, much like they did with the nasal passages in Fig. 11.\n\nReferences\n\n\nEvans D. C., Forster, C. A., and Reisz R. R. 2005. The type specimen of Tetragonosaurus erectofrons (Ornithischia: Hadrosauridae) and the identification of juvenile lambeosaurines. In: Currie PJ, Koppelhus EB, eds. Dinosaur provincial park: a\nspectacular ecosystem revealed. Bloomington: Indiana University Press, 349\u2013366.\n\nWagner, J. R., and Lehman, T. M. 2009. An enigmatic lambeosaurine hadrosaur (Reptilia: Dinosauria) from the Upper Shale Member of the Campanian Aguja Formation of Trans-Pecos Texas. Journal of Vertebrate Paleontology 29:605-611.\n\nSullivan, R. M., Jasinski, S. E., Guenter, M., and Lucas, S. G. 2011. The first lambeosaurine (Dinosauria, Hadrosauridae, Lambeosaurinae) from the Upper Cretaceus Ojo Alamo Formation (Naashoibito Member), San Juan Basin, New Mexico. New Mexico Museum of Natural History and Science 53:405-417.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #3 of \"Ontogeny in the tube-crested dinosaur Parasaurolophus (Hadrosauridae) and heterochrony in hadrosaurids (v0.1)\". PeerJ https://doi.org/10.7287/peerj.182v0.1/reviews/3\nReview 6: \n",
    "LLM_extracted_review": "['Review 1: John Hutchinson \u00b7 Sep 29, 2013 \u00b7 Academic Editor  \\nI am totally satisfied with the revisions, which I have checked- the authors are to be commended for a particularly fastidious and patient revision of the paper! This is a rigorous magnum opus that will be a boon to palaeontology. It is wonderful that PeerJ is publishing it.\\n\\nReview 2: John Hutchinson \u00b7 Jun 2, 2013 \u00b7 Academic Editor  \\nThe authors are to be commended on a rigorous, substantive body of work in this manuscript; it is certain to be well cited in future literature. The reviewers have all made constructive suggestions for changes and uniformly agree that it should be published, with these minor revisions. Please include a point-by-point Response to Reviewers with your revised MS. In particular, I agree that a wider consideration of heterochrony as a concept and in non-dinosaurs would improve the paper, as would illustration of \"ontogenetic changes in the external morphology of the cranial bones, even if such changes are hypothetical\".\\n\\nReview 3: Reviewer 1 \u00b7 Jun 2, 2013  \\nThis paper is generally well-written and accurate. I think that the sections of the anatomical descriptions dealing with ontogenetic change could be more consistently delineated from the remainder, however. Almost every element should have some differences \u2013 see, e.g., multiple papers on turtle ontogeny by G.S. Bever, alligator ontogeny by Vickaryous and Hall (2008, J. Morph.), Bhullar et al. (2012, Nature) on bird and dinosaur ontogeny and heterochrony, several papers my Maxwell on birds, the supplement to Bhullar (2012, JEZ B), Maisano (2002, JMorph), and several papers by Tarazona, all of which have extensive descriptions of bone-by-bone ontogenetic change in reptiles: turtles, crocodylians, birds, and squamates, respectively.  \\nOn this note, a table of ontogenetic changes in Parasaurolophus element-by-element and as compared to other hadrosaurs would be useful.  \\nThere is one persistent grammatical mistake \u2013 the use of a hyphen following adverbs describing adjectives, e.g., 727 \u201cpoorly-preserved,\u201d 908 \u201csimilarly-interpreted\u201d, twice in 1131, etc. throughout the manuscript.  \\nThe discussion of heterochrony needs some citations \u2013 at the least, Gould (1977) and Alberch et al. (1979). Additionally, it would be interesting to know whether overall skull shape change (not just in the crest) is decoupled from size between Parasaurolophus and Corythosaurus. This would allow a more nuanced approach to potential heterochrony in these forms.  \\nRegarding the nasal cavity work, the endocasts are interesting and impressive. A few words about which parts of the cartilaginous nasal capsule the various chambers relate to would be welcome. Regarding the brain endocast, Gans (1980, JHerp), Bever (2005, Palaeo electronica), and Bhullar et al. (2012, Nature) among others addressed ontogenetic changes in reptile brains and braincases and will provide comparative insight to the changes described here. For instance, the (de)flexure and rotation of the brain during ontogeny is well-known in archosaurs.  \\nIn the section on the axial skeleton, a little more attention to potential ontogenetic changes would have been welcome, despite the poor preservation. In the appendicular section, a bit of comparison to changes in birds and crocodylians would be enlightening as to the generality of the transformations described in, for example, the pelvis.  \\nAnatomical descriptions appear accurate and thorough. Histological descriptions are excellent. I would have liked to see a formal phylogenetic analysis at least attempted to place this animal as Parasaurolophus.  \\nThis paper is thorough and interesting and certainly of interest to a number of audiences. It is, in its current form, a little myopic in its focus on the dinosaurian literature and could benefit from broader comparisons. Certainly the papers mentioned above on ontogeny and heterochrony in various reptiles (including dinosaurs!) should be considered. Erickson et al. (2012, Science) should probably be cited in the section on jaw mechanics. The discussion of heterochrony should reference foundational works on the subject and perhaps try to couch the potential heterochrony in hadrosaurs in the formal structure set out therein.\\n\\nReview 4: David Weishampel \u00b7 May 29, 2013  \\nThe Farke et al. MS is spot-on, well written, and complete as far as the bony descriptions and the synthetic discussions go. The only thing I would ask them to spend some more time on is the issue of the S-Loop of the crest and its phylogenetic distribution - parasaurolophins don\\'t have one (that is, they\\'ve highly modified the vestibule to make their entire crest) and corythosaurins have an S-Loop. That\\'s about it.\\n\\nReview 5: Reviewer 3 \u00b7 May 21, 2013  \\nThis paper provides a thorough documentation and discussion of an important and unique specimen of lambeosaurine hadrosaurid. It is very informative, containing new insights into the anatomy and life history of one of the most iconic, yet still enigmatic taxa. I have only a few remarks; otherwise, I recommend this paper for publication, with minor revisions.  \\nTitle and line 3 of the abstract: \u201cHadrosaurs\u201d is too imprecise, unless you mean Hadrosauria (sensu Wagner and Lehman 2009), which I don\u2019t think you do. Thus, I advise to better use \u201chadrosaurid(s)\u201d.  \\nPage 3, line 49: Usage of \u201ccorythosaurins\u201d should be replaced for \u201clambeosaurins\u201d. As recommended by Sullivan et al. (2011): \u201cAccording to to Article 37.1 of the International Code of Zoological Nomenclature (1999): When a family-group taxon is subdivided, the subordinate name that contains the taxon that contains the type genus of the superior taxon is denoted by the same name the nominotypical name. Thus, given that \u201cCorythosaurini\u201d is a subdivision (tribe) of Lambeosaurinae coordinate with Parasaurolophini, the proper name of the tribe should be Lambeosaurini, not \u201cCorythosaurini.\u201d (Sullivan et al. 2001:406).  \\nPage 8, lines 259 and 260. The terms \u201claterotemporal\u201d and \u201cdorsotemporal\u201d are not how these openings are typically referred to and they seem like an odd choice. I\u2019d recommend using the more common terms \u201cinfratemporal\u201d and \u201csupratemporal\u201d fenestrae.  \\nPage 9, line 281, 284: \u201cnaris\u201d. It would be better to further specify that it is the \u201cexternal naris\u201d or \u201cbony naris\u201d.  \\nFigure 6 and the premaxilla-nasal articulation: on Fig. 6 the nasal bifurcates ventrally to receive a finger-shaped process of the premaxilla. This configuration is similar to that of juvenile Corythosaurus (Evans et al. 2005:Fig. 18.1). It is not clear to me how could the authors derive with such detail this articular morphology from RAM 14000? As seen in Fig. 5, such articulation is nowhere to be seen (unpreserved?).  \\nPage 34, line 1379 and following lines: Regarding the interesting discussion of the extent of the nasal and premaxilla in Parasaurolophus, I think the paper would be even more informative than already is if it included a figure showing ontogenetic changes in the external morphology of the cranial bones, even if such changes are hypothetical at this juncture, much like they did with the nasal passages in Fig. 11.']"
}