Reducing cytochrome c enhances NLRP3 inflammasome activation while exogenous cytochrome c inhibits the caspase-1 activation in a reconstitution assay.

<p>(A) Immunoblots of cell supernatants and cell lysates from LPS primed THP-1 previously transfected with cytochrome c or control siRNAs. The indicated proteins were immunoblotted. (B) Quantification of the results from (A) and 2 other similar experiments. Results are shown as mean +/- SEM of the fold increase of the indicated protein as assessed by Image J. The difference between the control and cytochrome c siRNA treated cells were compared by Student <i>t</i> test using Prism software. ** indicates that p < 0.01. (C) Immunoblot to assess caspase-1 activation <i>in vitro</i> using a cell fraction enriched for mitochondria and another for cytosolic proteins. The mitochondrial fraction was prepared from HEK 293T cells that had been treated with or without ATP. The cytosolic fraction was prepared from LPS primed THP-1 cells. The two fractions were mixed and incubated with either BSA or cytochrome c. The levels of the indicate proteins in the various mixtures are shown. (D) Quantification of the results from (C) and 2 other similar experiments. Results are shown as mean +/- SEM. The intensity of the bands determined using Image J. The differences in processed caspase-1 (p20) levels in the reactions containing BSA or cytochrome c were compared by Student <i>t</i> test using Prism software. ** indicates that p < 0.01. The above experiments were respectively performed 3 times.</p

Increased cytosolic cytochrome c inhibits NLRP3, but not AIM2 inflammasome activation.

<p>(A) Immunoblots of cell lysates and supernatants for the expression of IL-1β, caspase-1, caspase-9, ASC, cytochrome c, and actin as indicated to assess the impact of cytochrome c on NLRP3 inflammasome activity. Cytochrome c or BSA was transduced into LPS-primed THP-1 cells for 3 hours, and during the last hour ATP was present in the cell culture. Cell lysates and supernatants were collected. Endogenous cytochrome c was visualized on longer exposure (not shown). (B) Quantification of the results from (A) and 2 other similar experiments. Results are shown as mean +/- SEM. The difference between the BSA and cytochrome c transduced cells were compared by Student <i>t</i> test using Prism software. ** indicates that p < 0.01. (C) Immunoblots of cell lysates for processed IL-1β and caspase-1 p20 to examine the impact of cytochrome c on AIM2 inflammasome activation. LPS primed (2 h) THP-1 cells were transduced with cytochrome c or BSA, and transfected with poly(dA-dT) for six hours. Supernatants were collected and analyzed. (D) Quantification of the results from (C) and 2 other similar experiments. Results are shown as mean +/- SEM. The difference between the BSA and cytochrome c transduced cells were compared by Student <i>t</i> test using Prism software. N.S. indicates non-significant. The above experiments were respectively performed 3 times.</p

Cytochrome c binds to the LRR repeats of NLRP3 and interferes with cardiolipin binding.

<p>(A) Immunoblots of cell lysates and either NLRP3 or sham immunoprecipitates prepared from LPS-primed THP-1 macrophage cells, stimulated with ATP or not, to detect an interaction between endogenous NLRP3 and cytochrome c. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J. (B) Immunoblots of cell lysates and either control or Myc immunoprecipitates to map the portion of NLRP3 important for the interaction with cytochrome c. Myc-tagged NLRP3 constructs were transfected into HEK 293T cells. The immunoprecipitates were washed, incubated with cytochrome c (50 ng), washed again, fractionated by SDS-PAGE, and immunoblotted. (C) Immunoblots of cell lysates and Myc-LRR domain immunoprecipitates incubated with cytochrome c, or not. A construct expressing a myc tagged NLRP3 LRR domain was transfected into HEK 293T cells. The Myc and control immunoprecipitates were incubated with cytochrome c (50 ng), washed, and immunoblotted. (D) Schematic of the constructs used in the above experiments (B & C). (E) Immunoblots of cell lysates and cardiolipin bead pull-downs to assess whether cytochrome c interferes with the interaction between cardiolipin and NLRP3. BSA (50 ng) or purified cytochrome c (50 ng) was added to lysates prepared from HEK 293T cells expressing NLRP3-Flag. Following a 30 minute incubation cardiolipin conjugated beads were added to the lysates for an additional 30 minutes. The caridolipin beads were washed; and the bound NLRP3 eluted in SDS-sample buffer, size fractionated by SDS PAGE, and quantitated by immunoblotting. The amount of NLRP3 in the cardiolipin pulldowns was normalized to the BSA control. The above experiments were respectively performed twice. (F) Cell lysates from cell treated with ATP, or not, were fractionated into cytosolic and mitochondrial fractions and the indicated proteins were immunoblotted. NLRP3 immunoprecipitates were prepared using the cytosolic fraction. An interaction between endogenous NLRP3 and cytochrome c was assess by immunoblotting. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J.</p

ATP treatment induces cytochrome c release.

<p>PMA differentiated THP-1 cells were primed with LPS 50 ng/ml overnight. One hour before harvesting the cells 5 mM ATP was added. Cell lysates were fractionated into a cytosolic and mitochondria enriched fractions. (A) Immunoblots of IL-1β and caspase-1 p20 present in the collected cell supernatants to verify NLRP3 inflammasome activation. (B) Immunoblots of cytosol and mitochondria enriched fractions from the above cells to assess cytochrome c release following NLRP3 inflammasome activation.</p

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 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 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