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 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

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 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

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

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

C-Abl expression in developing microfilariae decreases over the course of maturation.

<p>C-Abl expression was measured as mean fluorescence intensity in the internal structures of adult worms. (<i>A</i>, <i>C</i>) The intensity of fluorescence between the reproductive apparatus (<i>A</i>, uterus, ovaries, early morula, pretzel; <i>C</i>, vas deferens, spermatids), muscle, glycogen, and intestine is significantly more in the structures treated with anti-c-Abl antibody compared with isotype control. (<i>A</i>) Adult females show over the course of embryonic development a decrease in c-Abl expression from the early morula to the pretzel stage (p = 0.0035). Higher c-Abl expression is also seen in muscle compared with glycogen in females (<i>A</i>, p = 0.0187) as well as males (<i>C</i>, p = 0.0155). (<i>B</i>, <i>D</i>) Fluorescence intensity generated by the nuclear stain DAPI, is essentially identical between the c-Abl and isotype control conditions. Three sections per condition were analyzed, and the experiment was repeated 3 times; means ± SEM.</p

Defining the target and the effect of imatinib on the filarial c-Abl homologue

<div><p>Background</p><p>Previously we demonstrated the micro- and macrofilaricidal properties of imatinib <i>in vitro</i>. Here we use electron and multiphoton microscopy to define the target of imatinib in the adult and microfilarial stages of <i>Brugia malayi</i> and assess the effects of pharmacologically relevant levels of imatinib on the adult parasites.</p><p>Methods</p><p>After fixation of adult <i>B</i>. <i>malayi</i> males and females, sections were stained with polyclonal rabbit anti-c-Abl antibody (or isotype control) and imaged with multiphoton fluorescent microscopy. Microfilariae were fixed and labeled with rabbit anti-c-Abl IgG primary antibody followed by anti-rabbit gold conjugated secondary antibody and imaged using transmission electron microscopy (TEM; immunoEM). In addition, adult <i>B</i>. <i>malayi</i> males and females were exposed to 0 or 10μM of imatinib for 7 days following which they were prepared for transmission electron microscopy (TEM) to assess the drug’s effect on filarial ultrastructure.</p><p>Results</p><p>Fluorescent localization of anti-c-Abl antibody demonstrated widespread uptake in the adult filariae, but the most intense signal was seen in the reproductive organs, muscle, and intestine of both male and female worms. Fluorescence was significantly more intense in the early microfilarial stage (i.e. early morula) compared with later development stages (i.e. pretzel). Anti-c-Abl antibody in the microfilariae localized to the nuclei. Based on TEM assessment following imatinib exposure, imatinib appeared to be detrimental to embryogenesis in the adult female <i>B</i>. <i>malayi</i>.</p><p>Conclusions</p><p>At pharmacologically achievable concentrations of imatinib, embryogenesis is impaired and possibly halted in adult filariae. Imatinib is likely a slow microfilaricide due to interference in intra-nuclear processes, which are slowly detrimental to the parasite and not immediately lethal, and thus may be used to lower the levels of <i>L</i>. <i>loa</i> microfilariae before they are treated within the context of conventional mass drug administration.</p></div

Nerve and intestine unchanged in adult female <i>B</i>. <i>malayi</i> treated for 7 days with 10 μM imatinib compared with untreated cultured control worms.

<p>B) Nerve chord of treated adult female (9,300X) without a significant difference as compared to controls (A). D) No change in the intestine morphology (6,800X) compared with untreated controls (C). n–Nucleus, bi–Basal infoldings, mu–Muscle, b–Basement membrane, g–Glycogen, nc–Nerve Cord, mi–Mitochondria, mv–Microvilli, tj–Tight Junction, lv–Lipid Vacuole, iw–Intestinal Wall.</p