25 research outputs found
Resistance to Inhibitors of Cholinesterase (Ric)-8A and Gα<sub>i</sub> Contribute to Cytokinesis Abscission by Controlling Vacuolar Protein-Sorting (Vps)34 Activity
<div><p>Resistance to inhibitors of cholinesterase (Ric)-8A is a guanine nucleotide exchange factor for Gα<sub>i</sub>, Gα<sub>q</sub>, and Gα<sub>12/13</sub>, which is implicated in cell signaling and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes. Ric-8A, Gα<sub>i</sub> subunits, and their regulators are localized at the midbody prior to abscission and linked to the final stages of cell division. Here, we identify a molecular mechanism by which Ric-8A affects cytokinesis and abscission by controlling Vps34 activity. We showed that Ric-8A protein expression is post-transcriptionally controlled during the cell cycle reaching its maximum levels at mitosis. A FRET biosensor created to measure conformational changes in Ric-8A by FLIM (Fluorescence Lifetime Imaging Microscopy) revealed that Ric-8A was in a close-state during mitosis and particularly so at cytokinesis. Lowering Ric-8A expression delayed the abscission time of dividing cells, which correlated with increased intercellular bridge length and multinucleation. During cytokinesis, Ric-8A co-localized with Vps34 at the midbody along with Gα<sub>i</sub> and LGN, where these proteins functioned to regulate Vps34 phosphatidylinositol 3-kinase activity.</p></div
Ric-8A inhibition increases the length of the intercellular bridge, delays abscission time and promotes multinucleation.
<p>(<b>A</b>) Immunoblot of Ric-8A and Gα<sub>i3</sub> protein expressions after treatment of HeLa cells with siRNA control or siRNA directed at Ric-8A or at Gα<sub>i</sub> subunit 1–3. (<b>B</b>) Panels show a representative deconvolution of z-stacks and 3D reconstruction of siRNA control (top panel) or Ric-8A siRNA (bottom panel) treated HeLa cells. Scale bar is 5 µm. Magnification of midbody region and Ric-8A localization is shown in the white squares. (<b>C</b>) Intercellular bridge length quantified from 3 independent experiments (*, p<0.05) by immunostaining with an anti-α-tubulin antibody. (<b>D</b>) Distribution and the average abscission time of HeLa cells transiently transfected with siRNA-Ric-8A or siRNA-control and the photo-activable GFP plasmid. Results from 12 cells were acquired in 4 independent experiments (*, p<0.05). (<b>E</b>) Histogram represents the percentage of total of cells with a mitotic phenotype (prophase to cytokinesis) and the number of cells showing an intercellular bridge. At least 500 cells were analyzed in 3 independent experiments, (n.s. stands for non-significant). (<b>F–G</b>) HeLa cells transiently transfected with siRNA-Ric-8A or siRNA-control were analyzed for their phospho histone H3-ser10 levels either by western blotting technique (<b>F</b>) or by flow cytometry technique (<b>G</b>) focusing on G2-M positive cells. (<b>H</b>) The percentage of multinucleated HeLa cells among cells transiently transfected with siRNA Control, siRNA Ric-8A, siRNA RGS14, siRNA GRK2 or siRNA LGN was assessed by flow cytometry 48 h after second siRNA transfection, (*, p<0.05, n = 3). The percentage in white indicates the amount of reduction in mRNA content for the targeted gene.</p
Ric-8A conformational change occurs during mitosis.
<p>(<b>A</b>) HeLa cells were transiently transfected with GFP-Ric-8A-mCherry. Focusing on metaphase cells using transmission light imaging, FRET by FLIM measurements were done on live cells. The panel shows a representative lifetime imaging and an electronic magnification of the interchromosome area (square 1) containing the midbody is displayed (<b>B</b>) Results of the average lifetime in whole cells at different stages of the cell cycle. Each point represents a single cell. (<b>C</b>) Results of the average lifetime in the different sub-cellular compartments at different stages of the cell cycle. The data were acquired from 4 independent transfections and from 6–15 cells for each bar.</p
Inhibition of Ric-8A or Gα<sub>i</sub> activity decreases the production of PtdIns(3)P in a Vps34 activity dependent manner.
<p>(<b>A</b>) Vps34, GFP and actin immunoblots of cell lysates immunoprecipitate from HeLa cells transiently transfected with vectors expressing YFP, Gα<sub>i3</sub> wt-YFP, or Gα<sub>i3</sub> QL-YFP. Experiments were repeated twice with similar results. (<b>B</b>) 3D reconstruction of confocal images of HeLa cells expressing GFP-Ric-8A immunostained for endogenous Vps34. Scale bar is 5 µm. The right panels show an electronic magnification of midbody area (white square). (<b>C–D</b>) Co-immunoprecipitation of endogenous Vps34 and endogenous Ric-8A in HeLa cells. The experiment was repeated 3 times with similar results. (<b>E</b>) Asynchronous HeLa cells lysates treated with PTX (200 ng/mL, 3 h) or with its vehicle were immunoprecipitated using anti-Vps34 antibody and immunoblotted for Ric-8A or Vps34. (<b>F</b>) Quantification of PtdIns(3)P production after purification of endogenous Vps34 kinase in asynchronous or G2/M enriched HeLa cells treated with PTX (200 ng/mL, 3 h) or siRNA control versus siRNA Ric-8A. Quantification was performed from 4 independent experiments (*, p<0.05).</p
Ric-8A is phosphorylated on S501 during G2/M phase.
<p>Asynchronous or nocodazole G2/M arrested HeLa cells were treated with cyclin dependent kinase inhibitors (Roscovitine, 3 h, 1 µM or Olomoucine, 3 h, 1 µM) prior to be assessed as indicated below. (<b>A</b>) A representative Ric-8A and actin immunoblots of anti- phospho-p190 antibody immunoprecipitates prepared from HeLa cells (<b>B</b>) Graph represents the percentage of phosphorylated Ric-8A as assessed in A. Results are expressed as a percentage of the nocodazole treated condition from 4 independent experiments (*, p<0.05). (<b>D</b>) Lysates from nocodazole G2/M enriched GFP, GFP-Ric-8A wt, GFP-Ric-8A S88A, GFP-Ric-8A S155A or GFP-Ric-8A S501A expressing HeLa cells were immunoprecipitated using anti-phospho-p190 antibody, resolved on a Tris-glycine gel, and immunoblotted for Ric-8A.</p
Ric-8A protein expression is increased during G2/M phase.
<p>(<b>A</b>) HeLa cells were blocked at the G1/S boundary by thymidine block and then released for the indicated times. Asynchronous or G2/M arrested cells (nocodazole 1 µM, 16 h) were used as controls. Ric-8A expression was detected by immunoblot analysis. Synchronization efficiency was verified by CyclinB1 immunoblotting. (<b>B</b>) Ric-8A and actin immunoblots of lysates prepared from HeLa cells enriched in G1, S or G2/M phase cells by cell sorting according to their DNA content. Numbers are the fold increase in Ric-8A expression normalized to actin expression using the level found in G1 phase as a baseline. (<b>C</b>) Quantification of Ric-8A and LGN mRNA expression detected by quantitative RT-PCR in nocodazole G2-M enriched HeLa cells. mRNA expression was normalized to β-actin mRNA expression, (*, p<0.05, n = 3).</p
Inhibition of Ric-8A or Gα<sub>i</sub> activity decreases the recruitment of PtdIns(3)P sensor.
<p>(<b>A</b>) Snapshots from live cell imaging of GFP-2XFYVE accumulation at the cytokinesis stage. HeLa cells were transiently transfected with GFP-2XFYVE and α-tubulin-RFP (vehicle versus PTX, 2 left panels); GFP-2XFYVE and DsRed-shRNA control or shRNA Ric-8A (middle two panels); or GFP-2XFYVE, α-tubulin-RFP and siRNA control or siRNA LGN (right two panels). Scale bar is 5 µm. Full length movies are available in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086680#pone.0086680.s002" target="_blank">movies S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086680#pone.0086680.s003" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086680#pone.0086680.s004" target="_blank">S3</a>). (<b>B</b>) The number of GFP-2XFYVE vesicles was quantified from at least 12 cells in 3 independent experiments and represented on a whisker graph (quartile) where+represents the mean for each condition (*, p<0.05).</p
DHA inhibits the translocation of NF-κB to the nucleus.
<p>FFAR4-GFP expressing LPS primed non-differentiated THP-1 cells were stimulated with ATP or nigericin. DHA (100 µM) was added or not at the priming step. PFA fixed cells were permeablized and then stained with p65 antibody and analysis was performed using an ImageStream instrument. Shown are the (A) Imaging results and (B) Flow cytometry results. (C) ImageStream analysis of nuclear p65 NF-κB in non-differentiated THP-1 cells expressing FFAR1-GFP or FFAR4-GFP and exposed to LPS, or not, in the presence of DHA (50 µM), or not. (D) Confocal microscopy of BMDMs LPS primed and treated with nigericin in the presence, or absence of DHA, to examine the status of NF-κB translocation by p65 immunostaining. Shown are representative individual images. Scale bar is 20 µM. Whisker plot shows the amount of nuclear p65 immunofluorescence in the nuclei of BMDMs treated as indicated. Data are representative of three independent experiments. **P<0.001, n.s-not significant.</p
DHA regulates inflammasome activity by promoting autophagy.
<p>(A) Confocal microscopy of BMDMs from GFP-LC3 mice primed with LPS and treated with nigericin. DHA (50 µM) was added at the priming step. Scale bar is 5 µM. (B) Number of cells with >5 GFP-LC3 puncta per cell were determined for each treatment group. Data are from 3 independent experiments. (C) ELISA to measure IL-1β in supernatants conditioned by BMDMs prepared from <i>Atg7</i><sup>flox/flox</sup> or <i>Atg7</i><sup>flox/flox </sup><i>Vav1-Cre</i> mice. The cells were primed with LPS and treated with ATP. DHA was added at the priming step. Results are from 3 independent experiments. (D) Western blot analysis of supernatants and cell lysates from BMDMs prepared from <i>Atg7</i><sup>flox/flox</sup> or <i>Atg7</i><sup>flox/flox </sup><i>Vav1-Cre</i> mice. Cells were LPS primed and treated with ATP. DHA was added at the time of priming. The numbers indicate the relative expression of IL-1β in the cell supernatants. **P<0.001, ***P<0.0002, ****P<0.0001. (E) DHA and ATP triggered changes in intracellular Ca<sup>+2</sup> in mouse BMDMs. The cells were exposed to the indicated concentrations of DHA and/or ATP. Results from one experiment performed in triplicate. Representative of three experiments performed.</p
DHA elicits a calcium flux and its suppression of inflammasome activity partially depends upon β-arrestin2 not β-arrestin1.
<p>(A) DHA elicited changes in intracellular Ca<sup>2+</sup> in BMDMs treated with pertussis toxin (200 ng/ml), or not, for 2 hours prior to the assay. (B) BRET analysis using HeLa cells expressing a BRET donor (RLuc-β-arrestin1 or β-arrestin2-RLuc) and increasing amounts of a BRET acceptor (FFAR4-GFP or FFAR1-GFP) stimulated with LPS, or not, prior to addition of DHA. The BRET ratios were calculated and the change in BRET determined. (C) Confocal microscopy of HeLa cells expressing FFAR4-GFP and β-arrestin-2 mCherry following exposure to DHA. Red arrow indicates β-arrestin-2 recruitment and the white arrows intracellular co-localization of β-arrestin-2 and FFAR4. Scale bar is 10 µM. (D) Intracellular calcium response to DHA of BMDMs prepared from wild type and β-arrestin knock-out mice. (E) ELISA to measure IL-1β in cell supernatants conditioned by LPS primed and ATP treated BMDMs from WT, <i>arrb1</i><sup>−/−</sup>, and <i>arrb2</i><sup>−/−</sup> mice. DHA (100 M) was added at the priming step. Results are from 3 independent experiments. * P<0.05, ****P<0.0001.</p