ECAM shows a change in gel mobility and overall structure upon activation.

<p>(A) Native PAGE showing the migration profile of ECAM. Lanes 1) native molecule; 2) methylamine-treated; 3) after reaction with chymotrypsin; 4) native molecule; 5) after reaction with porcine pancreatic elastase (B) Small-angle X-ray scattering (SAXS) results for native, methylamine-activated, elastase- and chymotrypsin-reacted ECAM. The radially averaged scattered X-ray intensity was plotted as a function of the momentum transfer <i>s</i>. Scattering patterns for ECAM in native form (black), after reaction with methylamine (green), elastase (blue) and chymotrypsin (red) were recorded in different concentrations (from 0.5 to 8 mg/mL) but only the curves relating to the highest concentration are shown. Inset, detail of differences in distinct side maxima. (C) Distance distributions <i>p</i>(r) of native, methylamine-reacted, elastase, and chymotrypsin of ECAM. All curves were normalized. Inset, detail of maxima of <i>p</i>(r) functions.</p

Electron microscopy reveals that ECAM is an elongated, flexible molecule.

<p>(A) Comparison between raw images, which were low-path filtered at 25 Š(lines 1, 3) and re-projections of the obtained 3D reconstructions (lines 2, 4). (B) Isosurface representations of the 3D reconstruction obtained for the activated form of ECAM (gray) and comparison with the 15 Šfiltered structure of C3b (PDB 2I07, blue). For the isosurface representations, an averaged mass density of 0.84 Da/ų and a molecular weight value of 190 kDa were used. Black arrows represent top (1) and bottom (2) views, while the red arrows point to the regions of potentially greatest flexibility.</p

The structures of both native and activated forms of ECAM resemble those of C3 and C3b.

<p>Surface representations of ECAM in native (A) and methylamine-activated (B) states based on SAXS measurements. Structures of C3 (PDB 2A73) and C3b (PDB 2I07, in blue) were modeled manually into the ECAM <i>ab initio</i> SAXS models. Note that the difference in position of the C3/C3b TED (thioester-containing) domain can be well accounted for in the SAXS envelopes of both native (A) and methylamine-activated (B) forms of ECAM. (C) The X-ray structure of C3b (PDB 2I07) was manually fitted into the 3D EM envelope of the activated ECAM. The size of the macromolecule as well as the MG ring and the TED domain are in comparable positions. The unoccupied density, shown with red arrows, indicates the different potential position of the C-terminus domain and indicates high flexibility in this region. The lack of sequence similarity between the C-terminal domain of ECAM and C3b (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035384#pone.0035384.s006" target="_blank">Fig. S6</a>) could also account for the differences observed.</p

<i>Ab initio</i> models of ECAM generated by SAXS.

<p>Each model results from averaging 10 individual models calculated by the program GASBOR using: (A) native ECAM, (B) methylamine-treated, (C) chymotrypsin-treated, and (D) elastase-treated ECAM. Note the appearance of a central cavity in all of the treated forms of the molecule. GASBOR was used in “user" mode, following default options, except for the total number of residues, which corresponded to total ECAM (1653 residues). The envelopes are based on the <i>p</i>(r) funtions shown on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035384#pone-0035384-g003" target="_blank">Fig. 3</a>, and the GNOM files generated were used as input for GASBOR. The models are drawn to scale.</p

Structural parameters calculated from the models generated from SAXS measurements.

<p>Structural parameters calculated from the models generated from SAXS measurements.</p

Schematic representations of human α-macroglobulin (α₂M), C3 convertase (C3), and <i>E. coli</i> α-macroglobulin (ECAM).

<p>Domain assignments for α₂M and C3 were based on their respective crystal structures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035384#pone.0035384-Marrero1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035384#pone.0035384-Janssen1" target="_blank">[17]</a>. Assignments for ECAM were performed with the JPRED server, supported by the analysis performed by Doan and Gettins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035384#pone.0035384-Doan1" target="_blank">[27]</a>. Note the similarity in domain predictions, including MG and TED domains. The CLEQ sequence, a signature of the thioester bond, is present in all proteins. For simplicity, only a limited number of the domains identified or predicted for the different molecules are depicted, and only one monomer of α₂M is shown. The C-terminus of ECAM displays low sequence similarity to that of C3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035384#pone.0035384.s006" target="_blank">Fig. S6</a>).</p

TrkC-KF is translocated to the nucleus by importins.

<p>(A) Schematic representation of TrkC-KF, generated after TrkC cleavage by caspase in D495 and D641, in absence of NT-3. A second fragment, TrkC 642–825, is also generated but has no apoptotic activity. (B) Expression of TrkC-FL, TrkC-KF, TrkC 642–825, and Neo-IC fused to GFP in N2A murine neuroblastoma cells. Nuclei are stained with DAPI. (C) Proximity ligation assay (DuoLink) using a pan-importins antibody and an anti-GFP antibody on untransfected SHEP cells or those transfected with TrkC-KF-GFP or TrkC-642-825-GFP: The protein–protein interactions are visualized by red fluorescent spots (Cy3). (D) Quantification of the proximity ligation assay presented in (C): Data represent mean ± SEM (at least 2 independent fields). *<i>p</i> < 0.05. <i>t</i> test compared with control (untransfected). (E) SHEP cells transfected with either TrkC-KF or Neo- IC-ΔNES and treated or not with 10 μM of pan-importin inhibitor Ivermectin were fractionated into cytoplasmic (Cytoplasm, marker: GAPDH) and nuclear (Nucleus, marker: histone) fractions. (F) Localization of the 2 putative NLSs in TrkC-KF sequence: Mutations of these NLSs used in experiment (G) are indicated in red. TrkC-KF-ΔNLS1/2 is mutated on both NLS1 and NLS2. (G) SHEP cells transfected with the NLS-mutated constructs of TrkC-KF were fractionated into cytoplasmic (Cytoplasm, marker: GAPDH) and nuclear (Nucleus, marker: histone) fractions. (H) Cell death quantification by trypan blue exclusion after transfection of SHEP cells with TrkC-KF, TrkC-KF-ΔNLS1/2, or Bax. Data represent mean ± SEM (<i>n</i> = 3). *<i>p</i> < 0.05. <i>t</i> test. (I) Quantification of luciferase activity in SHEP cells transfected with DCC-IC, TrkC-KF, or TrkC-492-825 constructs fused to the Gal4 DBD, and the gene coding for luciferase under the UAS-Gal4 control. Data represent mean ± SEM. *<i>p</i> < 0.05. <i>t</i> test compared to control (Gal4-DBD). Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002912#pbio.2002912.s001" target="_blank">S1 Data</a>. Bax, B cell lymphoma 2–associated X; DBD, DNA-binding domain; DCC-IC, deleted in colorectal cancer intracellular domain; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; N2A, Neuro2a; Neo-IC, intracellular fragment of Neogenin; Neo-IC-ΔNES, Neo-IC deleted for its nuclear export sequence; NES, nuclear export sequence; NLS, nuclear localization sequence; ns, nonsignificant; NT-3, neurotrophin-3; TrkC, tropomyosin receptor kinsase C; TrkC-FL, full-length TrkC; TrkC-KF, TrkC killer-fragment; UAS, upstream activating sequence; WB, western blot.</p

TrkC-KF and Hey1 cooperate to inhibit MDM2 transcription by direct binding on its promoter.

<p>(A) MDM2 mRNA expression was assessed by RT-QPCR on SHEP cells transfected with the indicated constructs (TrkC-DM). Data represent mean ± SEM (<i>n</i> = 3) relative to HPRT mRNA expression (housekeeping gene) and indexed to control. **<i>p</i> < 0.01. Two-sided Mann-Whitney test compared to control. Some cells were treated with z-vad, a general caspase inhibitor preventing TrkC cleavage. (B) MDM2 mRNA expression was assessed by RT-QPCR on SHEP cells transfected with the indicated constructs and with an siRNA control or an siRNA targeting Hey1. Data represent mean ± SEM (<i>n</i> = 3) relative to HPRT mRNA expression (housekeeping gene) and indexed to control. *<i>p</i> < 0.05. Two-sided Mann-Whitney test. (C) MDM2 protein level was assessed by WB in SHEP cells transfected with the indicated constructs. Actin was used as a loading control. (D) Schematic representation of <i>MDM2</i> promoter. Exons are indicated as gray boxes. An E-box contained in the regulated promoter of <i>MDM2</i> is indicated as a black box. The couples of primers a, b, and c used in the ChIP experiments described in figures (E) and (F) are also indicated. (E,F) ChIP assays using chromatin isolated from SHEP cells transfected with a control plasmid or TrkC-KF-Flag (E) or transfected with TrkC-KF-Flag or Hey1 (F). Proteins were immunoprecipitated with an anti-Flag antibody for TrkC-KF and an anti-Hey1 antibody for Hey1, as indicated. DNA sequences from <i>MDM2</i> promoter were amplified by RT-QPCR using couples of primers a (upstream control), b (binding site), and c (downstream control). Data represent mean ± SEM (<i>n</i> = 3) indexed to isotypic IgG value. *<i>p</i> < 0.05 <i>t</i> test. (G,H) ChIP assays using chromatin isolated from SHEP cells, transfected with TrkC-KF-Flag, and an siRNA control (G) or an siRNA targeting Hey1 (H). Proteins were immunoprecipitated with a control antibody (white bars), an anti-Flag antibody for TrkC-KF (gray bars), and an anti-Hey1 antibody for endogenous Hey1 (black bars). DNA sequences from <i>MDM2</i> promoter were amplified by RT-QPCR using couples of primers a (upstream control), b (binding site), and c (downstream control), indicated on (D). Data represent mean values ± SEM (<i>n</i> = 3) indexed to isotypic IgG value. (I) Sequences of <i>MDM2</i> promoter WT E-box and mutated E-box used in (J), (K), and (L). (J,K,L) DNA pull down assay using 80 bp double-stranded oligonucleotides (probe) corresponding to TrkC-KF and Hey1 binding site on <i>MDM2</i> promoter: WT probe (WT) contains the WT sequence of E-box; mut contains the mutated E-box indicated in (I). Biotinylated probes were incubated with nuclear lysates from SHEP cells transfected with TrkC-KF-GFP, Hey1-Flag, or both. Hey1 was revealed by an anti-Flag WB (J), TrkC-KF was revealed by an anti-GFP WB (K) and (L). “Input” corresponds to protein expression in whole nuclear lysate before incubation with the probes. (L) Biotinylated probes were incubated with nuclear lysate from SHEP cells transfected with an siRNA control or an siRNA targeting endogenous Hey1. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002912#pbio.2002912.s001" target="_blank">S1 Data</a>. ChIP, chromatin immunoprecipitation; E-box, enhancer box; GFP, green fluorescent protein; HPRT, hypoxanthine phosphoribosyltransferase; IgG, immunoglobulin G; MDM2, mouse double minute 2 homolog; mut, mutated probe; Ptc, Patched; RT-QPCR, quantitative reverse transciption PCR; siRNA, small interfering RNA; TrkC, tropomyosin receptor kinase C; TrkC-DM, TrkC-D495N/D641N; TrkC-FL, full-length TrkC; TrkC-KF, TrkC killer-fragment; WB, western blot; WT, wild-type.</p

TrkC-KF associates to the transcription factor Hey1 in the nucleus, and both bind to the chromatin.

<p>(A,B) Expression of TrkC-KF-GFP and Hey1-RFP in N2A cells indicates a partial colocalization in the nucleus, as shown by confocal analysis (A) and by the associated Pearson’s coefficient (B). As controls, TrkC, TrkC-642-825, and Neo-IC (fused to GFP) were also transfected. Data represent mean ± SEM (2 independent experiments). *<i>p</i> < 0.05. <i>t</i> test compared to control (TrkC-GFP). (C) Proximity ligation assay (DuoLink) using an anti-Hey1 antibody (recognizing endogenous Hey1) and an anti-GFP antibody on SHEP cells transfected with TrkC-KF-GFP, TrkC-642-825-GFP, GFP, or TrkC-KF-GFP and an siRNA against Hey1: The protein–protein interactions are visualized by red fluorescent spots (Cy3). (D) Quantification of the proximity ligation assay presented in (C): Data represent mean ± SEM (4 independent fields). **<i>p</i> < 0.01. <i>t</i> test compared with TrkC-KF. (E) Immunoprecipitation of TrkC-KF-GFP and TrkC-642-825-GFP using an anti-GFP antibody in HEK293T transfected cells. Hey1 and HeyL are revealed by an anti-Flag WB. (F) HEK293T cells transfected with TrkC-KF-GFP and Hey1-Flag constructs were fractionated into cytoplasmic, nuclear/DNA-unbound, and nuclear/DNA-bound fractions. Actin and Histone H3 are used as loading controls. “Input” corresponds to construct expression in whole cell lysates. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002912#pbio.2002912.s001" target="_blank">S1 Data</a>. GFP, green fluorescent protein; HEK293T, human embryonic kidney 293 T; IP, immunoprecipitation; N2A, Neruo2a; Neo-IC, intracellular fragment of Neogenin; RFP, red fluorescent protein; siRNA, small interfering RNA; TrkC, tropomyosin receptor kinase C; TrkC-FL, full-length TrkC; TrkC-KF, TrkC killer-fragment; WB, western blot.</p

Hey1 is essential for the cell death mediated by TrkC.

<p>(A) Quantification of cell death by trypan blue exclusion in N2A cells transfected with the indicated constructs (Ptc) and an siRNA control or an siRNA targeting Hey1. Expression of each construct was assessed by WB (lower panel). Data represent mean ± SEM (<i>n</i> = 5). **<i>p</i> < 0.01. <i>t</i> test. (B) Caspase-3 activity assay on SHEP cells transfected with siRNA control, siRNA NT-3, and siRNA Hey1. Data represent mean ± SEM (<i>n</i> = 3) indexed to control. **<i>p</i> < 0.01. <i>t</i> test. (C) Hey1 mRNA expression assessed by RT-QPCR on SHEP clones isolated from SHEP cells transiently transfected with a CAS9-only-expressing vector (Control clones 1 and 2) or a CAS9- and Hey1-targeting gRNA-expressing vector (Hey1-KO clones 1 and 2). Data represent mean ± SEM (<i>n</i> = 3) relative to HPRT mRNA expression (housekeeping gene) and indexed to control. Parental SHEP are used as control. (D) Immunofluorescence staining using an anticleaved caspase-3 antibody (Cy3) performed on parental, control, and Hey1-KO SHEP cells and clones after transfection with siRNA control or siRNA NT-3. A representative picture is shown for each condition. Nuclei are stained with DAPI. (E) Quantification of the cleaved caspase-3 staining shown in (D) as a percentage of total cell number measured by DAPI staining. Data represent mean ± SEM (<i>n</i> = 3 independent fields). (F) Cleaved-PARP protein level was assessed by WB in parental, control, and Hey1-KO SHEP cells and clones after transfection with siRNA control or siRNA NT-3. GAPDH is used as a loading control. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002912#pbio.2002912.s001" target="_blank">S1 Data</a>. CAS9, clustered regularly interspaced short palindromic repeat–associated protein 9; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; gRNA, guide RNA; HPRT, hypoxanthine phosphoribosyltransferase; KO, knock-out; N2A, Neuro-2a; NT-3, neurotrophin-3; PARP, poly [ADP-ribose] polymerase; Ptc, Patched; RT-QPCR, quantitative real-time PCR; siRNA, small interfering RNA; TrkC, tropomyosin receptor kinase; TrkC-FL, full-length TrkC; TrkC-KF, TrkC killer-fragment; WB, western blot.</p