Additional file 2: Figure S2. of Automatic detection of diffusion modes within biological membranes using back-propagation neural network

- Comparison of the percentage of decision using the BPNN, Hidden Markov Modeling (HMM)-Bayes, Bayesian Information Criterion (BIC) or Support Vector Machines (SVM) algorithms. 200 simulated trajectories of 300 frames mimicking diffusion within plasma membranes, including one directed motion segment with velocity randomly ranging from 1 to 3 μm/s and one confinement segment with diameters ranging from 0.5 and 1.2 μm, were analyzed with BPPN, HMM-Bayes, BIC or SVM. Within a trajectory each 50 frames segment is always localized at the same position. The diffusion coefficient D is 0.25 μm2/s and the integration time 100 ms. A 30 nm localization noise Pn was added to the trajectory (see Material and Methods section). The percentage of decision based on BPNN corresponds to the number of positive decision for a specific motion mode detected for a given frame over 200 trajectories and normalized to 1 or-1 for confined (light grey) or directed (dark grey) trajectories, respectively. The HMM-Bayes and the BIC algorithms can only detect directed or confined segments within a trajectory, respectively. The tables at the bottom detail the performance of the 4 algorithms in terms of sensitivity and specificity for detecting confined and directed motion modes in the range of parameters tested in this study (D = 0.25 μm2/s, 1 μm/s < v < 3 μm/s, 0.5 μm < L < 1.2 μm). (PDF 400 kb

Expression and localization of M19 in muscle cells.

<p>(<b>A</b>) Coomassie-blue stained gel and Western blot analysis of M19 in extracts from C2C12 cells grown in proliferation medium (d0) or placed in differentiation-promoting conditions for 2 to 9 days (d2 to d9). M19 is detected by the rabbit polyclonal P70612 antibody. (<b>B</b>, <b>C</b>) C2C12 myoblasts were grown in proliferation medium or (<b>D</b>, <b>E</b>) were placed in differentiation medium for 6 days, and then were double-labeled with the specific P70612 antibody (<b>B</b>, <b>D; green</b>) and an anti-cytochrome c antibody (<b>C</b>, <b>E; red</b>). There is a co-localization between the 2 detected proteins in C2C12 myoblasts (<b>B</b>, <b>C, merge</b>) and myotubes (<b>D</b>, <b>E, merge</b>) as indicated by arrowheads. (<b>F</b>, <b>G</b>) Double-label indirect immunofluorescence of mouse <i>Tibialis anterior</i> sections showing M19 (<b>F; green</b>) and cytochrome c (<b>G; red</b>). (<b>H</b>) After C2C12 cell fractionation, proteins from the cytosolic and the mitochondria fractions were separated by SDS-PAGE. Tubulin, COX IV and M19 are detected by Western immunobloting. (<b>I</b>) Purified mitochondria are subjected to limited degradation using increasing concentration of trypsin, from 0 to 80 µg/ml. The mitochondria are then lysed in Laemmli buffer. Tom40 and M19 are detected by Western immunobloting. (<b>J</b>) Purified mitochondria are disrupted with freeze/thaw cycles, followed by Na₂CO₃ precipitation. After centrifugation, the membrane fraction (memb) and the matrix/intermembrane space fraction (matrix) are analyzed by Western immunobloting using a VDAC antibody and the P70612 antibody.</p

Reduced insulin secretion in M19-deficient INS-1 cells.

<p>(<b>A</b>) Northern blot analysis of the <i>M19</i> gene in human tissues. Pa: pancreas; Ki: kidney; Sk: skeletal muscle; Li: liver; Lu: lung; Pl: placenta; Br: brain; He: heart. Molecular markers are shown on the left. (<b>B</b>) Fluorescence microscopy of INS-1 cells double-labeled with the M19-specific P70612 antibody (M19, merge; green) and the MitoTracker dye (MitoTracker, merge; red). (<b>C</b>) Cell fractionation of INS-1 cells. Proteins of the total cell lysate (Lys), the cytosolic (Cyt) and the mitochondria (Mi) fractions were subjected to Western immunobloting. The cytosolic protein tubulin, the mitochondrial protein VDAC and M19 are detected. (<b>D</b>) INS-1 cells were transfected with a control pHYPER vector (sh control) or with the pHYPER vector encoding a M19-specific shRNA (sh M19). Western immunoblot analysis of the cell extracts shows expression levels of M19 and the control protein, tubulin. ATP production was determined in these cells (<b>E</b>), and insulin secretion was measured under basal glucose conditions (<b>F</b>). Results are the mean ± SEM of five (<b>E</b>), or four (<b>F</b>) independent experiments. (*) indicates statistical significance at p<0.05.</p

Identification of a mitochondrial targeting signal.

<p>(<b>A</b>) Prediction of the secondary structure of mouse M19 (<i>Mus musculus</i> NM026063) using 4 different algorithms: phyre, PSIPRED, SAM and jufo. The predicted α-helices are indicated by black lines along the amino-acid sequence. (<b>B</b>) Helical wheel presentation of the N-terminal α-helix of mouse M19, from amino acid 1 to 13. Hydrophobic residues are indicated in black circles while the positively charged amino acids are mentioned with a “+”. The first methionine (amino acid 1), at the top of the figure, is considered as a positively charged residue. (<b>C</b>, <b>D</b>) C2C12 myoblasts were transfected with the pQETriSystem vector encoding histidine-tagged M19 (<b>C</b>) or a histidine-tagged M19 mutant lacking amino acids 1 to 12 (<b>D</b>). Indirect immunofluorescence was performed using an anti-histidine antibody. (<b>E</b>–<b>J</b>) C2C12 myoblasts were transfected with the pEGFP-N1 vector encoding GFP alone (<b>E</b>, <b>F</b>), the pEGFP-N1 vector encoding the N-terminal M19 α-helix fused to the N-terminal end of GFP (<b>G</b>, <b>H</b>), and the PEGFP-C3 vector encoding the N-terminal M19 α-helix coupled to the C-terminal end of GFP (<b>I</b>, <b>J</b>). Fluorescence microscopy allows the direct detection of GFP constructs (<b>E</b>, <b>G</b>, <b>I; green</b>) and the indirect detection of cytochrome c using an anti-cytochrome c antibody (<b>F</b>, <b>H</b>, <b>J; red</b>).</p

M19 expression levels regulate mitochondrial ATP production in HeLa cells.

<p>(<b>A</b>, <b>B</b>) HeLa cells were transfected with a control siRNA (si control), a human M19-specific siRNA (si M19), a control siRNA associated with the pEGFP-N1 vector encoding mouse M19 (si control/M19GFP), or a human M19-specific siRNA associated with the pEGFP-N1 vector encoding mouse M19 (si M19/M19GFP). (<b>A</b>) Expression levels of the endogenous human M19 and the mouse GFP-coupled M19 are detected by Western immunobloting using the P70612 antibody. (<b>B</b>) ATP production is presented for these transfected-HeLa cells. Results are the mean ± SEM of five independent experiments. (***) indicates statistical significance at p<0.001, according to the unpaired Student's <i>t</i> test. (<b>C</b>) HeLa cells were transfected with a control siRNA (si control) or a human M19-specific siRNA (si M19). ATP production was then determined in untreated or in oligomycin-treated cells (oligo). Results are the mean ± SEM of four independent experiments. (**) indicates statistical significance at p<0.01, and (n.s.) means statistically non significant, according to the Tukey HSD test used after performing a one-way analysis of variance.</p

M19 expression levels regulate oxygen consumption and ATP production in C2C12 cells.

<p>(<b>A</b>–<b>C</b>) C2C12 myoblasts were transfected with a control pHYPER vector (sh control) or the pHYPER vector encoding a M19-specific shRNA (sh M19). Transfected cells were grown in proliferation medium for 2 days and were then lysed in Laemmli buffer. (<b>A</b>) Cell extracts were processed for immunoblot analysis with the P70612 antibody and an anti-tubulin antibody for loading control. (<b>B</b>) Oxygen consumption was evaluated for control cells (sh control) and for M19-specific shRNA cells (sh M19), in untreated cells or in cells treated with the ionophore CCCP. (<b>C</b>) ATP production was determined in control cells (sh control) and in M19-specific shRNA cells (sh M19). (<b>D</b>–<b>F</b>) C2C12 myoblasts were transfected with the empty pQETriSystem vector (pQE control) or the pQETriSystem vector encoding mouse M19 (pQE M19). (<b>D</b>) Cell extracts were resolved by SDS-PAGE. Endogenous M19 and exogenous histidine-tagged M19 were detected by Western immunobloting using the P70612 antibody. (<b>E</b>) Oxygen consumption was evaluated for control cells (pQE control) and for M19-overexpressing cells (pQE M19), in untreated or in CCCP-treated myoblasts. (<b>F</b>) ATP production was determined in control cells (pQE control) and in M19-overexpressing cells (pQE M19). Results are the mean ± SEM of two (<b>E</b>), three (<b>B</b>), or five (<b>C</b>, <b>F</b>) independent experiments. (*) and (**) indicate statistical significance at p<0.05 and p<0.01, respectively, according to the unpaired Student's <i>t</i> test.</p

Expression of late muscle differentiation markers is affected in differentiated M19-deficient C2C12 cells.

<p>(<b>A</b>, <b>B</b>) C2C12 myoblasts were transfected with a control siRNA (si control) or a M19-specific siRNA (si M19) and were then placed in differentiation medium for 7 days. Protein extracts from transfected cells grown in proliferation medium (d0) or in differentiation-promoting conditions for 3, 5 and 7 days (d3, d5, d7) were analyzed by Western immunobloting using the M19-specific P70612 antibody (<b>A</b>) and a MHCII antibody (<b>B</b>). Densitometry analysis of the detected bands is presented as the relative expression of M19 (<b>A</b>) and MHCII (<b>B</b>) normalized to tubulin. Results are the mean ± SEM of three independent experiments. (*) and (**) indicate statistical significance at p<0.05 and at p<0.01. In a similar experiment, C2C12 myoblasts were transfected with the M19-specific shRNA vector allowing the expression of the specific shRNA with GFP. Cells were placed in differentiation medium for 7 days. Fluorescence microscopy allows the direct visualization of GFP-labeled cells expressing the M19-specific shRNA (<b>C, merge; green</b>) and the detection of MHCII using an anti-MHCII antibody (<b>D, merge; red</b>). (<b>E</b>) Protein extracts from control shRNA-transfected C2C12 cells (sh control) and M19-specific shRNA-transfected C2C12 cells (sh M19) grown in differentiation-promoting conditions for 7 days were analyzed by Western immunoblotting. The expression of the late muscle differentiation markers α-actinin 2, troponin T, MHCI and MHCII is shown, as well as the control protein tubulin.</p