mGluR1 conserved protein isoforms with <i>In silico</i> analysis of <i>GRM1</i> missense substitution.

<p>(A) Partial nucleotide sequence alignment of human <i>GRM1</i> and its homologues in the animal kingdom (CLUSTAL2.1). The position of the missense mutation in family 9000105 is indicated by an arrow. (B) Partial amino acid sequence alignment of the human mGluR1 protein and its homologues in the animal kingdom. (C) mGluR1 protein isoforms. Domain LBD/ATD = ligand binding domain/amino terminal domain, C = cysteinerich domain in red domain, TMD = transmembrane domain, CTD = C-terminal domain, H = Homer 1-binding motif.^<b>42</b> (D) Ribbon presentation of the mGluR1 protein. The protein is colored grey, the side chain of the mutated residue, p.Leu454Phe, is colored magenta and shown as small balls. (E) Close-up of the p.Leu454Phe mutation. The protein is colored grey, the side chains of both the wild-type and the mutant residue are shown and colored green and red, respectively. The p.Leu454Phe position is indicated.</p

Braining imaging study.

<p>Brain imaging of the patient (IV: 4.1) with Congenital Cerebellar Ataxia; the images in T1 and T2 show cerebellar atrophy and severe increased number of cerebellar cisterns.</p

Clinical features of affected individuals from family 9000114 with a new mutation in the <i>TRMT1</i> gene and from family M300 previously reported (Najmabadi et al., 2011).

<p>Clinical features of affected individuals from family 9000114 with a new mutation in the <i>TRMT1</i> gene and from family M300 previously reported (Najmabadi et al., 2011).</p

The novel <i>TRMT1</i> gene alteration and its complete segregation in 9000114 family.

<p>(A) Scheme of the <i>TRMT1</i> gene structure; arrows indicated the mutation identified in this study (c.1332_1333delGT) and the one published by Najmabadi et al. (see text). (B) Sequence chromatograms showing the complete segregation of the homozygous c.1332-1333delGT in 900114 family in the patients (5.2, 5.4 and 5.5), the healthy sibling (5.6) and mother (4.2) are heterozygous for deletion.</p

The candidate mutation in the <i>GRM1</i> gene and its segregation within the family.

<p>(A) Scheme of the <i>GRM1</i> gene structure; arrows indicated the mutation identified in this study (c.1360C>T) and those published by Guerguelcheva et al. (B). Sequence chromatograms showing the complete segregation of the missense mutation c.1360C>T in family 9000105 in the patients.</p

Functional consequences of the absence of BOD1 in patient-derived fibroblasts.

<p>(A) Flow cytometric analysis of cell cycle profile in WT and <i>BOD1</i>^-/- primary fibroblasts electroporated with control or <i>BOD1</i> siRNA. Error bars represent standard deviation. (B) Immunoblotting of BOD1 and tubulin from cell lysates simultaneously electroporated with samples analysed in (A). (C) Representative DIC timelapse imaging of primary fibroblast cells undergoing mitosis. Nuclear Envelope Breakdown (NEB) and Anaphase Onset (AO) are indicated. (D) Cumulative timing of NEB to AO timing in Primary Fibroblast cell lines. Error bars represent standard deviation. P<0.001 for <i>BOD1</i>^-/- cells to each WT sample. Insufficient data collected for <i>BOD1</i>^-/- cells to determine statistical significance. (E) Immunofluorescence localization of PP2A-B56 in WT and <i>BOD1</i>^-/- Primary fibroblasts. DAPI (blue), centromeres (detected with ACA) (green), anti-PP2A-B56α (red). (F) Mean B56α levels at kinetochores of WT and <i>BOD1</i>^-/- Primary fibroblasts (P<0.001). Error bars represent SEM. (G) Immunofluorescence localization of PLK1 in WT and <i>BOD1</i>^-/- Primary fibroblasts. DAPI (white), anti-PLK1 (green), ACA (blue). (H-J) Mean PLK1 levels at kinetochores and centrosomes of WT and <i>BOD1</i>^-/- Primary fibroblasts, respectively (P<0.001 in each instance). Error bars represent SEM. (K) Immunoblotting of PLK1, BOD1 and tubulin in asynchronous WT and <i>BOD1</i>^-/- primary fibroblasts. (L) Immunoblotting of PLK1, BOD1 and tubulin in asynchronous and Monastrol arrested WT and <i>BOD1</i>^-/- Primary fibroblasts. (M) Immunofluorescence localization of Bod1 in WT Primary Fibroblasts. DAPI (white), ACA (blue), anti-Plk1 (red), anti-Bod1 (green). Scale = 5 μm. Inset shows a single bioriented kinetochore pair. (N) Immunoblotting of PP2A-B56δ, PLK1 and tubulin in WT primary fibroblast electroporated with indicated combinations of CTR, B56-pool or <i>BOD1</i> siRNA. Rescue of WT primary fibroblasts after siRNA depletion of Bod1 with plasmids expressing GFP fused to either siRNA resistant WT Bod1 or Bod1^T95E. (O) Mitotic profile of WT primary fibroblasts and <i>BOD1</i>^-/- fibroblasts 1 hr after release from RO 3306 into the indicated concentrations of BI 2536. Results show average of three independent experiments. A minimum of 100 mitotic cells counted per condition per experiment. Error bars represent SEM.</p

Identification of the homozygous c.2503A>G in <i>MUSK</i>, hereditary transmission and localization of the mutation in a three-dimensional model of the intracellular part of MuSK.

<p>(<b>A</b>) Sequence chromatograms from a normal individual (control), affected proband IV: 1 and his parents (parents) are shown. (<b>B</b>) Pedigree of the Iranian family. The proband is indicated by an arrow. The expected nucleotide change c.2503A>G transmitted in this consanguineous Iranian family is indicated below the symbols when determined. (<b>C</b>) 3D model of the cytoplasmic kinase domain dimer of human MuSK built by homology modeling based on the structure of rat MuSK (PDB code: 1LUF) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053826#pone.0053826-Till1" target="_blank">[11]</a> superimposed on the structure of the Insulin-like Growth factor 1 (PDB code 3D94), as proposed recently <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053826#pone.0053826-Bergamin1" target="_blank">[34]</a>. The model is shown in cartoon representation; also represented in sticks are the catalytic residues (Tyrosines 751, 755 and 756, Aspartate 725 and Arginine 729) and residues whose mutations are associated with myasthenic syndromes A727/M605, V790 and M835). N-terminus, C-terminus and a loop not resolved in the structure are also indicated.</p

Electron microscopy in control and patient.

<p>(<b>A</b>) Control endplate Patient’s endplates: (<b>B</b>) reinnervated endplate shows decreased and enlarged post-synaptic folds, a vanished basal lamina, and absence of active zones. (<b>C</b>) Ectopic NMJ is characterized by the total absence of subneural folds indicating that they are at an early stage of synaptogenesis. (<b>D</b>) An axon with a smooth endoplasmic reticulum (SER) network, a sign of impaired axonal flow. Zooms show aggregation of unidentified proteins on SER networks. Scale bars represent 500 nm for the four low-magnification prints and 3 µm for both zooms.</p

Immunocytochemistry of MuSK on cryostat sections.

<p>In the control, nAChR and MuSK are strongly expressed and perfectly colocalized. In the patient, nAChR expression is reduced whereas MuSK expression remains normal and extends slightly in the perisynaptic area. The scale bar represents 10 µm and applies to the six prints.</p

Expression of BOD1 and PLK1 in human tissues.

<p>(A) BOD1-specific quantitative RT-PCR experiments were carried out in triplicates, using RNA from the indicated tissues. All splice variants (indicated by the respective exon combinations) were investigated. Error bars represent the SEM. (B) Expression levels i.e. reads per kilobase of transcript per million reads mapped (RPKM), corresponding to BOD1 (NM_138369.2) and PLK1 (NM_005030.5) obtained by RNA-Sequencing of commercially available RNA-samples from different brain tissues, induced pluripotent stem cells (IPSC) and human embryonic stem cells (hES).</p