Resistance to Inhibitors of Cholinesterase (Ric)-8A and Gα_i 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_i, Gα_q, and Gα_12/13, 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_i 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_i and LGN, where these proteins functioned to regulate Vps34 phosphatidylinositol 3-kinase activity.</p></div

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_i 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

Inhibition of Ric-8A or Gα_i 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_i3 wt-YFP, or Gα_i3 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 inhibition increases the length of the intercellular bridge, delays abscission time and promotes multinucleation.

<p>(<b>A</b>) Immunoblot of Ric-8A and Gα_i3 protein expressions after treatment of HeLa cells with siRNA control or siRNA directed at Ric-8A or at Gα_i 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