The presequence of β-F1-ATPase precursor targets gfp to mitochondria.

<p>The expression of gfp in C9 (<b>A,B)</b> and BHK (<b>C)</b> cells was assessed by fluorescence (<b>A</b>), immunofluorescence (<b>B</b>) and immunoelectron (<b>C</b>) microscopy. <b>A,</b> Illustrates by phase contrast (upper panel) and immunofluorescence (lower panel) the same low magnification field of C9-pβGFP3′β cells. <b>B,</b> C9-pβGFP3′β cells analyzed by immunofluorescence microscopy using anti-β-F1-ATPase antibody at 63× magnification. Upper panel, green gfp fluorescence; middle panel, red β-F1-ATPase immunostaining; lower panel, yellow merged image. <b>C,</b> Specific immunogold labeling (10 nm gold) of BHK mitochondria (m). Note the lack of gold labeling of other structures in the cytoplasm or in the nucleus (n) of the cell. <b>D</b>, Western blots of C9 (lane 1) and BHK cells (lanes 2–4) transfected with the pβGFP3′β construct. Fractionated proteins from the cellular extracts were probed with anti-gfp and anti-tubulin, the later as loading control. The migration of the pβ-gfp chimera is also indicated. In lane 3, BHK cells have been previously treated with FCCP (4 µM) plus oligomycin (2 µM) for 1 hour. Note the accumulation of pβ-gfp. In lane 4, BHK cells treated as in lane 3 were washed for 40 minutes before fractionation. Note the processing of pβ-gfp to mature gfp.</p

Changes in mitochondrial constituents during the cell cycle.

<p>Synchronized C9 cells (<b>A–C</b>) were recovered at the indicated time intervals and the relative cellular content of mtDNA (<b>A</b>) and proteins (<b>B–C</b>) determined. No significant changes in the cellular abundance of the mitochondrial 12S gene are observed. However, the cellular content of β-F1-ATPase, Hsp60 and cyclin B1 showed a significant increase at 4 h after release from the metabolic arrest. Representative experiments are shown (<b>A–C</b>). * and **, P<0.05 and <0.005 when compared with 0–2 h. <b>D</b>, The fluorescence intensity of the NAO probe was used to determine changes on mitochondrial mass during the cell cycle of non-synchronized C9 cells. The results shown are the means±SEM of three experiments. *, P<0.05 when compared with cells in G0/G1.</p

Morphological changes in the cellular mitochondrial network during mitosis.

<p>Synchronized mitochondria-tagged C9-pβGFP3′β cells were analyzed by immunofluorescence microscopy at four hours after release from the metabolic block. Typical morphologies of cells in interphase (a,f), prophase (b), metaphase (c,g), anaphase (d) and telophase (e,h) are shown at 60× magnification. The red fluorescence identifies the cytoskeletal proteins α-tubulin (a–e) and β-actin (f–h). The green fluorescence identifies mitochondria. The blue fluorescence reveals the stained nuclear DNA with the To-Pro probe.</p

Asynchronous accumulation of mitochondrial proteins and development <i>ΔΨ</i>m during the cell cycle.

<p><b>A</b>, Determination of the expression level of β-F1-ATPase (β-F1), Hsp60 and cytochrome c oxidase subunits I (COXI) and IV (COXIV) in C9 cells in the different phases of cell cycle by flow cytometry. Open bars, G0/G1; grey bars, S and black bars, G2/M. The results shown are the means±SEM of four experiments. * and ^#, P< 0.05 when compared with cells in G0/G1 and S, respectively. <b>B</b>, C9 cells were sorted by flow cytometry according to their DNA content and the protein extracts fractionated and blotted with the indicated antibodies. The two bands shown under each condition are from two independent experiments. The results shown in the graph are the means±SEM of three experiments. *, P<0.05 when compared with cells in S phase. <b>C</b>, Synchronized C9 cells were recovered at the indicated time intervals and the relative cellular content of β-F1-ATPase mRNA determined by Northern-blot analysis. The mitochondrial encoded ATPase 6–8 mRNA and 12S rRNA and the nuclear encoded 18S rRNA were also determined. The results shown are the means±SEM of four experiments. <b>D</b>, FACS determination of the mitochondrial membrane potential (<i>ΔΨ</i>m) in C9 cells in the different phases of the cell cycle. The results shown are the means±SEM of three experiments. *, P<0.05 when compared with cells in G0/G1.</p

Detailed morphological parameters for WT and Gdap1^-/- mitochondria in mouse motorneuron primary culture.

<p>Mitochondrial shape descriptors were measured in 20 WT and 30 <i>Gdap1</i>^-/- motorneurons. Student’s t test was performed for normal distributed parameters (number of mitochondria, circularity, roundness and aspect ratio) and Mann-Whitney U test for those that were non-normal distributed (surface area, Feret´s diameter and perimeter). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005115#pgen.1005115.g006" target="_blank">Fig 6</a> for a visual representation.</p><p>*p<0.05,</p><p>**p<0.01.</p><p>Detailed morphological parameters for WT and Gdap1^-/- mitochondria in mouse motorneuron primary culture.</p

SOCE alteration in <i>Gdap1</i>^<i>-/-</i> embryonic motor neurons <b>(A)</b> Fura-2 [Ca²⁺] signals of embryonic MNs from WT (black) and <i>Gdap1</i>^<i>-/-</i> (grey) mice.

<p>After Ca²⁺ release from cell stores with 5 μM thapsigargin (TG) treatment during 7 min in Ca²⁺ free medium, SOCE was activated by adding 2 mM of CaCl₂. Traces were used to obtain <b>(B)</b> maximum Ca²⁺ peak during SOCE and <b>(C)</b> SOCE Ca²⁺ influx (slope). <b>(D)</b> Fura-2 recordings of 5 μM ionomycin elicited [Ca²⁺]_cyt peak in Ca²⁺-free medium. <b>(E)</b> Maximum [Ca²⁺]_cyt peak obtained in Ca²⁺-free medium represents total amount of cytoplasmic Ca²⁺ after cell stores Ca²⁺ release. Traces were obtained averaging at least 100 cells from each genotype. Error bars represent S.E.M. (***p<0.001, Student’s <i>t</i> test).</p

Generation of <i>Gdap1</i>^<i>-/-</i> mice.

<p><b>(A)</b> Schematic representation of <i>Gdap1</i>^<i>-/-</i> targeting strategy. Diagram is not to scale. Hatched rectangles represent <i>Gdap1</i> exons 1 to 6, solid line represents mouse chromosome 1. FRT sites are represented by double triangles and <i>lox</i>P sites are right-faced triangles. <b>(B)</b> GDAP1 protein expression was assessed by immunoblotting of selected tissue homogenates prepared from 2 months-old wild-type (WT), <i>Gdap1</i>^<i>+/-</i> (+/-) and <i>Gdap1</i>^-/- (-/-) mice.</p

Diagnostic performance of metabolic biomarkers.

<p>ROC curves were plotted to describe performance characteristics of the indicated metabolites in the 57 subject cohort. The Area under the curve (AUC), 95% range of the interval of confidence (IC) and P values are indicated.</p

Postranscriptional modification of the tubulin cytoskeleton in primary sensory and motor neuron cultures.

<p><b>(A)</b> DRG sensory neurons and <b>(B)</b> embryonic MNs were double-stained for acetylated α-tubulin (acetylated α-tub, green) and β-III tubulin (β-III tub, red). As indicated by respective histograms there is a significant reduction of acetylated α-tubulin in both MN and sensory neurites in <i>Gdap1</i>^<i>-/-</i> mice. Graph represents means and S.E.M of 3 independent culture preparation per genotype. Student’s <i>t</i> test ***p<0.001.</p

Behavioural testing and electrophysiological measurements on <i>Gdap1</i>^<i>-/-</i> mice.

<p><b>(A)</b> Upper panel shows photographs of 3 months-old mice suspended by its tail. WT mice show a characteristic response trying to escape by splaying its hind limbs away from the trunk of its body. In contrast, hind limbs of <i>Gdap1</i>^-/- mice are held tonically against its trunk in an abnormal dystonic posture. Lower panels display a low body position and a dragging tail present in <i>Gdap1</i>^<i>-/-</i> mice as compared to age-matched WT mice. <b>(B)</b> Motor coordination was assessed by rotarod test, (n = 10 for each genotype and at each age group). <b>(C)</b> Representative hind limb walking patterns of 5 months-old WT and <i>Gdap1</i>^-/- mice where the stride length (SL) and stride angle (SA) have been depicted. Footprints revealed that <i>Gdap1</i>^<i>-/-</i> mice walk with an abnormal gait. The scheme of a hindpaw footprint indicating measured parameters (PL: plantar length; TS: toe spreading) has been included. <b>(D)</b> Quantification of various parameters obtained from the gait analysis of WT (black columns) and <i>Gdap1</i>^<i>-/-</i> (grey columns) animals at 5 and 12 months of age. Upper graphs show stride length (left) and stride angle (right). Lower graphs show the quantitative analysis of the hindpaw footprint parameters toe spreading (left) and plantar length (right). Analysis was conducted on 10 clearly visible footprints at 5 animals per genotype. Determination of sciatic nerve compound muscle action potential (CMAP) amplitudes at both distal and proximal <b>(E)</b> as well as motor nerve conduction velocities (MNCV) <b>(F)</b> measured in WT and <i>Gdap1</i>^-/- mice at 2 and 5 months of age (n = 4). Error bars indicate standard error of the mean (S.E.M.). <i>p</i> values were calculated using Student's <i>t</i> test,*p<0.05, **p<0.001, ***p<0.0001.</p