Concentration dependent effects of IC261 on the cell cycle and apoptosis in CV-1 and AC1-M88 cells.

<p>CV-1 and AC1-M88 cells were cultivated for 12, 24 and 48 h with different concentrations of IC261 (0.2–3.2 µM) or DMSO (0.0 µM) and subsequently analyzed by FACS analysis with FACScan (Becton Dickinson) (as described in Material and Methods). IC261 induced a full G2/M arrest (arrow) at a cell type dependent concentration (CV1∶1.6 µM, AC1-M88∶0.8 µM). At half of this concentration IC261 induces an increase of the subG1 population (black triangle: CV-1∶0.8 µM, AC1-M88∶0.4 µM). Interestingly, at higher concentrations the amount of cells in subG1 population is smaller indicating less apoptosis (open triangle).</p

Subcellular association of CK1δ with membrane structures and COPI positive vesicles.

<p>NRK cells stably expressing the fusion protein TGN38-EGFP and untransfected NRK cells were either untreated (<b>A–C</b>), treated with BFA (10 µg/ml) (<b>D–F</b>) or IC261 (50 µM) (<b>G–I</b>) and prepared for analysis by immunofluorescence microscopy. The Golgi apparatus was labeled by using a specific antibody (MG160), COPI positive vesicles were labeled with a β-COP specific antibody, and CK1δ was labeled by using the specific antibody 128A.</p

Effect of IC261, nocodazole, taxol, taxol/IC261 on TGN morphology in NRK cells. Line 1–5:

<p>NRK cells stably expressing the fusion protein TGN38-EGFP were cultured in a flow-through chamber and observed by time-resolved fluorescence microscopy. At time point “0 min” cell were treated with DMSO (0.1%) <b>(line 1)</b>, 50 µM IC261 <b>(line 2)</b>, 5 µM nocodazole <b>(line 3)</b> or 10 µM taxol <b>(line 4)</b>. Here representative cells are shown for the stated time points (see video sequence, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100090#pone.0100090.s005" target="_blank">movie S1</a>). The solvent DMSO and the treatment with taxol showed no effect on the TGN structure. IC261 as well as nocodazole treatment fragmented the tubular membrane structure of the TGN into vesicles distributed throughout the cell. At time point “−10 min” cells were treated with 10 µM taxol and from time point “0 min” on with 10 µM taxol +50 µM IC261 <b>(line 5)</b>. Additional treatment with taxol could prevent the IC261 induced effects on the TGN.</p

Microtubule depolymerization by IC261 treatment is reversible.

<p>(<b>A</b>) CV-1 cells expressing EYFP-tubulin were treated at time point “0 min” with 3.2 µM IC261 and observed by time-resolved fluorescence microscopy (see video sequence, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100090#pone.0100090.s009" target="_blank">movie S5</a>). The spindle apparatus of the representative cell shown here was dissolved within 8 min. At time point “10 min” IC261 was removed by exchange of media. Within a few minutes spindle MTs were built up again (“15 min”) and 20 min after removal a morphologically unimpaired spindle apparatus had been developed (“30 min”). After 2 h the cell proceeded into anaphase and cytokinesis (“155 min”). (<b>B</b>) Densitometric analysis of grey values. For quantitative analysis the relative mean intensity of EYFP-tubulin fluorescence signal in a defined region of interest (ROI) around the spindle apparatus and in the cytoplasm was measured by the software CellR. Due to IC261 treatment at time point “0 min” (arrow up) the relative intensity immediately decreased due to MT depolymerization and subsequent removal of IC261 at time point “10 min” (arrow down) lead to a reconstruction of microtubules.</p

IP₃ 3-KA activity results in attenuation of ERK phosphorylation.

<p>IP₃ does not appear to be required for NGF driven neurite outgrowth. (A) Analysis of the effects of XeC blockade of the IP₃ receptor, on PC12 cells expressing high levels of IP₃ 3-KA and on wild type PC12 cells. XeC has no effect on control PC12 cells, but partially overcomes the inhibition of neurite outgrowth caused by IP₃ 3-KA expression. Data are presented as mean +/− SEM. **p<0.01, by ANOVA with post-hoc analysis. (B) Determination of ERK activation in control PC12 cells and IP₃ 3-KA expressing cells using a suboptimal dose of NGF, in the presence of either 40 µM C5 or vehicle. Lower blot represents ERK activation as detected by phospho-specific antibodies against ERK1 and ERK2. Upper blot represents total ERK, as detected using an antibody that detects both ERK1 and ERK2. All antibodies were visualised with an HRP conjugated secondary antibody. The blot shown is representative of three independent experiments. (C) Densitometric quantification of ERK activation, as depicted in (B). **p<0.01, Data are presented as mean +/− SEM. ***p<0.001, by ANOVA with post-hoc analysis. IP₃ 3-KA expression attenuates ERK activation, whilst C5 inhibition of IP₃ 3-kinase results in increased ERK activity.</p

IP₃ 3-KA co-localises with F-actin at the PC12 cell growth cone, and inhibits neurite outgrowth.

<p>(A) Growth cone of PC12 cell stably expressing high levels of IP₃ 3-KA-GFP, fixed ten days after culture in the presence of 100 ng/ml NGF, and labeled with phalloidin-594. IP₃ 3-KA-GFP co-localises with F-actin. Scale bar is 10 µm. (B) Quantitative analysis of the effects of IP₃ 3-KA-GFP expression on neurite outgrowth. Neurite outgrowth was quantified using the Cellomics Arrayscan Neurite Outgrowth algorithm to measure the percentage of cells with neurites, and average neurite length (described in full in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032386#s2" target="_blank">materials and methods</a>). IP₃ 3-KA expression results in an inhibition of neurite outgrowth. Data are presented as mean +/− SEM. ***p<0.001, by ANOVA with post-hoc analysis. (C) Arrayscan images of PC12 cells expressing varying levels of IP₃ 3-KA-GFP, fixed four days after culture in the presence of 100 ng/ml NGF, and labeled using Hoescht 33342 and the Cellomics Neurite Outgrowth HitKit to identify neuritis. Scale bar is 100 µm.</p

The effect of expression of Vpu on the lipid raft localisation of endogenous tetherin.

<p><b>A</b>. HeLa cells were subjected to extraction in ice-cold Triton X-100 (1%) prior to separation by sucrose density gradient centrifugation, then analysis of fractions from the gradients by immunoblot. Fractions 1-4 are considered as lipid raft fractions, fractions 5-12 as non-raft fractions. The top panel shows an immunoblot, using an antibody to tetherin, of fractions from untransfected HeLa cells and confirms that a significant proportion of endogenous tetherin is present in lipid rafts (i.e. in fractions 1-4). The second panel shows an immunoblot, using an antibody to tetherin, of fractions from HeLa cells that had been transfected to express Vpu-GFP and shows that the majority of endogenous tetherin is lost from lipid rafts (i.e. fractions 1-4) in the presence of Vpu-GFP: this experiment was performed in the absence of lysosomal enzyme inhibitors, the blot was exposed longer to ensure detection of tetherin. Panel 3 shows the distribution of Vpu-GFP across the sucrose gradient; it has a broad distribution across the gradient, indicating that it is present in a range of membrane environments, both raft and non-raft. Expression of another viroporin protein (the M2 protein of influenza virus) does not lead to a redistribution of tetherin from lipid rafts, as shown by the bottom two panels. Panel 4 shows an immunoblot, using an antibody to tetherin, of fractions from HeLa cells that had been transfected to express M2-GFP and shows that the distribution of endogenous tetherin is similar to that in non-transfected cells (top panel). The bottom panel shows the distribution of M2-GFP across the sucrose gradient. <b>B</b>. Graphical representation of the proportions of tetherin molecules in raft vs. non-raft fractions on control HeLa cell and HeLa cells expressing Vpu-GFP (as indicated, n=5).</p

Tetherin is in lipid rafts at the cell surface.

<p><b>A</b>. HeLa cells were labeled with a membrane impermeant biotin reagent at 4°C prior to either immediate lysis in ice cold Triton X-100, or incubation for 10 min at 37°C before lysis in ice cold Triton X-100. Lysates were then separated on sucrose density gradients and 1 ml fractions were taken. Fractions 1-4 were pooled as raft fractions, 5-8 and 9-12 as two separate non-raft fractions. Lysates were then incubated with streptavidin-agarose beads to separate biotinylated (cell surface in the case of immediate lysis after biotinylation at 4°C, or internalised in the case of the 10 min chase at 37°C, proteins) from non-biotinylated proteins. Total, plasma membrane, and internalised fractions were then subjected to immunoblot analysis using antibodies to endogenous tetherin, Transferrin receptor or Flotillin 2 (the latter two as controls) as indicated. The total population of tetherin molecules is ~ 35% raft localized (1-4 in Total), however the tetherin that is at the cell surface (i.e. the biotinylated tetherin at 0 min uptake at 37°C) is almost exclusively raft localized (1-4 in Plasma Membrane). After 10 min uptake at 37°C a significant proportion of biotinylated tetherin is present in non-raft fractions (Figure 4A Internalised). <b>B</b>. As in A, but using HeLa cells transfected to express Vpu-GFP.</p

Cartoon representation of the effect of Vpu on tetherin trafficking following internalisation from the cell surface.

<p>The left hand side of the cartoon (green) shows what happens to cell surface tetherin in the absence of Vpu. It is internalised and delivered to early endosomes. During this process it exits lipid rafts. The majority of tetherin molecules then end up being recycled to the cell surface, probably via one or more intermediate compartment (as indicated by the thick arrows) whilst some tetherin ends up in multivesicular bodies (MVBs)/late endosomes and is ultimately destined for lysosomal degradation. Thus the fate of internalised tetherin is finely balanced between recycling to the cell surface and lysosomal degradation. The right hand side of the cartoon (pink) shows what happens to cell surface tetherin in the presence of Vpu. Both are internalised and delivered to early endosomes (with tetherin once again exiting lipid rafts along the way) where they associate with one another. This association leads to a shift in the balance between recycling and lysosomal degradation for tetherin, with the majority of tetherin now destined for delivery to MVBs/late endosomes and then to lysosomes (as indicated by the thick black arrows). NB this cartoon illustrates only the effect of Vpu on the fate of internalised tetherin.</p

Lysosomal degradation of tetherin.

<p><b>A</b>.and <b>B</b>. Triple label fluorescence analysis (antibody detection of Vpu, tetherin and TGN46, and LysoTracker detection of lysosomes) of HeLa cells that had been incubated in the presence of lysosomal inhibitors for 24 hours prior to processing for immunofluorescence analysis. Cells expressing Vpu demonstrated significant accumulation of tetherin in lysosomes (<b>B</b>), but not in the TGN (<b>A</b>). Bar = 10µ. <b>C</b>. Quantification of co-localisation between LysoTracker-594 and tetherin, using both Pearson’s correlation coefficient and percentage pixel overlay. <b>D</b>. Immunoblot analysis, using an antibody to endogenous tetherin, of lysates from control HeLa cells, or HeLa cells expressing Vpu-GFP (Vpu) that had been incubated in the presence of lysosomal (24 hours) or proteosomal (12 hours) inhibitors (as indicated). The lower panels show immunoblots for tubulin as loading controls. <b>E</b>. Graphical representation of quantification of the data presented in <b>D</b>, P=0.0002, n = 3.</p