Expression of Mfn2-GFP complements the phenotype of <i>mfn2</i> KO cells.

<p>Expression of Mfn2-GFP complements the phenotype of <i>mfn2</i> KO cells.</p

Fluorescence analysis of ER and mitochondria.

<p>WT (A) and <i>mfn2</i> KO cells (B) were co-transfected with a GFP-coupled ER marker (green) and an RFP-coupled mitochondrial marker (red). After fixation, confocal images were acquired and used to quantify Manders' colocalization coefficients (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046293#pone-0046293-t005" target="_blank">Table 5</a>). The arrowhead indicates a region of a <i>mfn2</i> KO cell where the ER appears dilated. Bar: 10 µm.</p

Complementation of mitofusin-2 KO cells.

<p>Mfn2 KO cells were transfected with a plasmid driving the expression of Mitofusin-2 fused to GFP. Cells expressing moderate levels of fluorescence were sorted, re-grown, fixed and processed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046293#pone-0046293-g001" target="_blank">Fig. 1</a>. For each individual cell profile, the frequency of ER-mitochondria tethering (expressed as % of the total mitochondrial perimeter) was determined, as well as the average width of ER cisternae. Mock-transfected <i>mfn2</i> KO cells (red circles) exhibited wider ER cisternae and more ER-mitochondria tethering than cells re-expressing Mitofusin-2 (green triangles). For each population of cells, the average and S.E.M. are indicated in black.</p

No significant alteration in the overall surface of mitochondria in <i>mfn2</i> KO cells.

<p>No significant alteration in the overall surface of mitochondria in <i>mfn2</i> KO cells.</p

Visualization of ER-mitochondria tethering by electron microscopy.

<p>Cells were fixed, embedded in Epon resin, sectioned and observed. (A) In WT MEFs expressing mitofusin-2, compartments continuous with ER cisternae were closely apposed to the mitochondrial outer membrane. Full arrowheads point to ribosomes attached to ER cisternae. Empty arrowheads designate the limits of the zone of intimate contact between ER and mitochondria. m: mitochondria. (B, C) Close ER-mitochondria apposition was also observed in <i>mfn2</i> KO cells. In these cells, ER cisternae often appeared swollen (asterisks), but regions of tethering were still very thin.</p

ER-mitochondria contacts are increased in <i>mfn2</i> KO cells.

<p>ER-mitochondria contacts are increased in <i>mfn2</i> KO cells.</p

ER cisternae are dilated in <i>mfn2</i> KO cells.

<p>ER cisternae are dilated in <i>mfn2</i> KO cells.</p

Manders' colocalization coefficients in WT and <i>mfn2</i> KO MEF cells.

<p>Manders' colocalization coefficients in WT and <i>mfn2</i> KO MEF cells.</p

PKD2 is not essential for folate chemotaxis.

<p>A) Cells were deposited at the surface of buffered agar plates 4 mm away to a source of folate (to the left), and allowed to migrate for 5 h. Phase-contrast pictures of one representative experiment after migration are shown. The drawn inner circle represents the position of the cells at time 0. Scale bar: 1 mm. B) Distance travelled by the front of cells between times 0 and 5 h. WT and <i>pkd2</i> KO cells moved towards folate with similar efficiency; n = 4. C) WT and <i>pkd2</i> KO cells submerged in phosphate buffer were allowed to move for 90 min towards a micropipette emitting folate. Phase-contrast pictures of one representative experiment are shown at times 0 and 90 min. D) In the experimental setup described in (C), persistence of cell movement was measured as the distance from the initial to the final cell position divided by the total travelled distance. No significant difference was observed between WT and <i>pkd2</i> KO cells; n = 4. E) The distance of each cell to the micropipette tip was measured at time 0 and 90 min to calculate the overall migration towards the source of folate (Δd). Negative values indicate that cells are moving towards the folate source. No significant difference was observed between WT and <i>pkd2</i> KO cells; n = 4.</p

<i>Dictyostelium</i> orthologs with a potential role in mechanosensing.

*<p>Similarity to the <i>Arabidopsis thaliana</i> ortholog (no human ortholog exists for this protein).</p>$<p>Considering only the VWA motif (see original paper for more information).</p