Proximal Protein Interaction Landscape of RAS Paralogs

International audienc

ADAM10 requirement for ExlA-dependent cadherin cleavage.

<p><b>A</b>. A549 cells were either treated with 0.1 μg/mL PMA or 5 μM ionomycin (Iono) for 30 min, or infected with CLJ1 (90 min), or left untreated/uninfected (NI). Cellular extracts were analysed by Western blot using E-cadherin and β-actin antibodies. FL, full-length; CTF, C-terminal fragment. The experiment was performed twice. <b>B</b>. A549 cells (left) or HUVECs (right) were pre-treated with DMSO, the general metalloprotease inhibitor GM6001 (10 μg/mL) or the specific ADAM10 inhibitor GI254023X (5 μM) and then incubated with CLJ1 or IHMA87 (90 min), or uninfected (NI). Cellular extracts were analysed as above. The experiment was performed twice for A549 and 3 times for HUVECs. <b>C</b>. A549 or ADAM10-deficient A549 (A549 <i>ADAM10</i>^<i>-/-</i>) cells were incubated with either CLJ1 or IHMA87. Cellular extracts were prepared at different time points post-infection as indicated and analysed by Western blot (left). The right panel shows the FACS analysis of ADAM10 surface expression of both cell lines, as well as the negative control. The experiment was performed 3 times. <b>D</b>. Similar experiment with HUVECs, either transfected with ADAM10 siRNA or untreated. The experiment was performed twice.</p

Intracellular calcium elevation generated by ExlA-secreting bacteria.

<p>Intracellular Ca²⁺ content was analysed by videomicroscopy using the permeant Fluo3-AM fluorescent probe. Plasma membrane rupture was evaluated using the non-permeant Draq7 fluorescent probe. A549 cells (<b>A,C,E,G,I,K</b>) or HUVECs (<b>B,D,F,H,J,L</b>) were either non-infected (<b>A,B</b>) or infected with PAO1F (<b>C,D</b>), CLJ1 (<b>E,F</b>), IHMA87 (<b>G,H</b>), IHMA87Δ<i>exlA</i> (<b>I,J</b>) or IHMA87Δ<i>exlA</i>::<i>exlBA</i> (<b>K,L</b>). The fluorescence intensities of five cells were analysed in each case; the Fluo3 intensities are represented by solid lines and the Draq7 intensities by dashed lines, using the same colour code for one cell. The data are representative of 4–7 experiments. Uninfected conditions were performed in each experiment.</p

Effects of S. marcescens ShlA on cadherin cleavage and calcium influx.

<p><b>A</b>. A549 cells (left) or HUVECs (right) were incubated with the <i>S</i>. <i>marcescens</i> ShlA-secreting strain Db11, or with the non-ShlA-secreting mutant 21C4. Cellular extracts were analysed for their E- or VE-cadherin contents. The experiment was performed twice for the left panel and once for the right panel. <b>B</b>. Similar analysis using A549 <i>ADAM10</i>^<i>-/-</i>. The experiment was performed once. <b>C</b>. Similar analysis using A549 cells, in presence/ absence of BAPTA-AM. <b>D-G</b>. Intracellular Ca²⁺ contents and plasma membrane permeability were measured using Fluo3-AM and Draq7 fluorescent probes, respectively. A549 cells (<b>D,F</b>) and HUVECs (<b>E,G</b>) were infected with Db11 (<b>D,E</b>) or 21C4 (<b>F,G</b>) and fluorescence was recorded on both channels by videomicroscopy. Five cells were analysed in each case; the Fluo3 intensities are represented by straight lines and the Draq7 intensities by dashed lines, using the same colour code for one cell. Data are representative of 8 and 5 independent experiments for A549 and HUVECs, respectively.</p

ExlA-dependent cleavage of E- and VE-cadherins.

<p><b>A</b>. A549 cells (left) or HUVECs (right) were incubated with various <i>P</i>. <i>aeruginosa</i> strains: CHA, PAO1F or CLJ1, or were mock-infected with LB (NI). Cell extracts were prepared at different times post-infection, as indicated and analysed by Western blot using E-cadherin (left) or VE-cadherin (right) antibodies. In both cases, the full-length (FL) and post-cleavage C-terminal (CTF) fragments are shown. β-actin was used as loading control. The experiment was performed 3 times for A549 and twice for HUVECs with similar results. <b>B</b>. A549 cells (left) and HUVECs (right) were incubated with IHMA87, IHMA87Δ<i>exlA</i> or IHMA87Δ<i>exlA/exlA</i> strains, and cellular extracts were analysed as above. The experiment was performed 3 times for A549 and once for HUVECs. <b>C</b>. Similar experiments with A549 cells (left) or HUVECs (right) incubated with <i>E</i>. <i>coli</i> containing the empty vector (<i>exlBA</i> -) or <i>E-coli</i> expressing ExlB-ExlA <i>(exlBA +)</i>. <b>D</b>. A549-E-cadherin-GFP cells were incubated with CLJ1, IHMA87, IHMA87Δ<i>exlA</i> or PAO1F bacteria. E-cadherin-GFP (green) as well as nuclei labelling by propidium iodide (red) were followed by confocal videomicroscopy. Times post-infection are shown as “h:min”. One z-position is represented. The experiment was performed twice, with 4–5 positions recorded each time. All films showed similar results. <b>E</b>. Mice (5 per condition) were infected by bacteria inhalation (2.5x10⁶), using CLJ1 strain, or were mock-infected with PBS (NI). Mice were euthanized at 18 h.p.i.; protein extracts were prepared from lungs and were analysed by Western blot using E- and VE-cadherin antibodies (left). Histograms (right) show the E-cadherin/ β-actin and VE-cadherin/ β-actin ratios of band intensities, represented as means + s.d. Significance was calculated using Mann-Whitney’s test, as variances were not equal. The experiment was repeated once, with similar results.</p

Mechanisms of ExlA/ShlA-induced cadherin cleavage.

<p>In uninfected cells, pro-ADAM10 is associated with calmodulin, preventing its maturation and export to the plasma membrane. Pore formation by ExlA or ShlA induces a massive Ca²⁺ influx in host cells. Intracellular Ca²⁺ interacts with the Ca²⁺-binding protein calmodulin, which detaches from pro-ADAM10, allowing its maturation to m-ADAM10. m-ADAM10 cleaves E- and VE-cadherin in epithelial and endothelial cells, respectively, provoking intercellular junction rupture.</p

ExlA necrotizing activity is preserved in ADAM10-deficient cells.

<p>Plasma membrane rupture was monitored by LDH release in the supernatant. A549 or A549<i>ADAM10</i>^<i>-/-</i> cells were incubated for 5 hours with IHMA87, IHMA87Δ<i>exlA</i> or IHMA87Δ<i>exlA/exlA</i> strains. The supernatants were the tested for LDH activity. The histograms show the mean ± s.d. of triplicates. The data are representative of 3 experiments.</p

Detection of known and novel ALK fusion transcripts in lung cancer patients using next-generation sequencing approaches

Abstract Rearrangements of the anaplastic lymphoma kinase (ALK) gene in non-small cell lung cancer (NSCLC) represent a novel molecular target in a small subset of tumors. Although ALK rearrangements are usually assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), molecular approaches have recently emerged as relevant alternatives in routine laboratories. Here, we evaluated the use of two different amplicon-based next-generation sequencing (NGS) methods (AmpliSeq and Archer®FusionPlex®) to detect ALK rearrangements, and compared these with IHC and FISH. A total of 1128 NSCLC specimens were screened using conventional analyses, and a subset of 37 (15 ALK-positive, and 22 ALK-negative) samples were selected for NGS assays. Although AmpliSeq correctly detected 25/37 (67.6%) samples, 1/37 (2.7%) and 11/37 (29.7%) specimens were discordant and uncertain, respectively, requiring further validation. In contrast, Archer®FusionPlex® accurately classified all samples and allowed the correct identification of one rare DCTN1-ALK fusion, one novel CLIP1-ALK fusion, and one novel GCC2-ALK transcript. Of particular interest, two out of three patients harboring these singular rearrangements were treated with and sensitive to crizotinib. These data show that Archer®FusionPlex® may provide an effective and accurate alternative to FISH testing for the detection of known and novel ALK rearrangements in clinical diagnostic settings