SVLP association with clathrin and transferrin.

<p>(A) DC were incubated with 2.5 µg/ml SVLP (green) on ice, shifted to 39°C for different times, fixed/permeabilised, and labelled using antibody targeting clathrin (red). Acquisition was by confocal microscopy; high-resolution stacks were prepared using IMARIS, including algorithmic co-localisation analysis for co-localised voxels. Arrows show co-localisation on the merged image. Scale bars: 20 µm (top-left), 5 µm (zooms). (B) DC were incubated with 2.5 µg/ml SVLP (green) and 10 µg/ml transferrin-546 (red) for 20 min on ice, washed and shifted to 39°C with addition of fresh transferrin-546 for different times at 39°C. Acquisition was by confocal microscopy; high-resolution stacks were prepared using IMARIS. Arrows show co-localisation on the merged image. Scale bars: 30 µm (left-panel) and 5 µm (zooms).</p

Synthetic Virus-Like Particles Target Dendritic Cell Lipid Rafts for Rapid Endocytosis Primarily but Not Exclusively by Macropinocytosis

<div><p>DC employ several endocytic routes for processing antigens, driving forward adaptive immunity. Recent advances in synthetic biology have created small (20–30 nm) virus-like particles based on lipopeptides containing a virus-derived coiled coil sequence coupled to synthetic B- and T-cell epitope mimetics. These self-assembling SVLP efficiently induce adaptive immunity without requirement for adjuvant. We hypothesized that the characteristics of DC interaction with SVLP would elaborate on the roles of cell membrane and intracellular compartments in the handling of a virus-like entity known for its efficacy as a vaccine. DC rapidly bind SVLP within min, co-localised with CTB and CD9, but not caveolin-1. In contrast, internalisation is a relatively slow process, delivering SVLP into the cell periphery where they are maintained for a number of hrs in association with microtubules. Although there is early association with clathrin, this is no longer seen after 10 min. Association with EEA-1⁺ early endosomes is also early, but proteolytic processing appears slow, the SVLP-vesicles remaining peripheral. Association with transferrin occurs rarely, and only in the periphery, possibly signifying translocation of some SVLP for delivery to B-lymphocytes. Most SVLP co-localise with high molecular weight dextran. Uptake of both is impaired with mature DC, but there remains a residual uptake of SVLP. These results imply that DC use multiple endocytic routes for SVLP uptake, dominated by caveolin-independent, lipid raft-mediated macropinocytosis. With most SVLP-containing vesicles being retained in the periphery, not always interacting with early endosomes, this relates to slow proteolytic degradation and antigen retention by DC. The present characterization allows for a definition of how DC handle virus-like particles showing efficacious immunogenicity, elements valuable for novel vaccine design in the future.</p> </div

SVLP associating with early endosomes and CD9.

<p>(A) Pre-chilled DC were incubated with SVLP and CTB as in Fig. 3, or with antibody against CD-9 for 30 min on ice. Samples were then washed and shifted to 39°C for 2 hrs, followed by confocal microscopy analysis. High-resolution stacks prepared using IMARIS are shown for each label, arrows indicating co-localisation. Scale bars: 20 µm (left image), 5 µm (zooms). (B) DC were treated with 20 mM MBCD for 20 min at 39°C, followed by washing to remove the MBCD and pre-chilling for 30 min on ice. DC were then pulsed with 2.5 µg/ml SVLP for 20 min on ice (“0” time point) and then washed with pre-chilled medium, prior to receiving fresh warm (39°C) medium containing 2 mM MBCD and shifting back to 39°C. DC were treated or not with PK to define internalisation at the time points shown. Analyses were by flow cytometry; all differences at the 39°C time points between 0 MBCD and 20 mM MBCD treatment are significant, as are the differences between “No PK” and “PK-treated” for the 20 mM MBCD samples (p≤0.001). (C) DC were incubated with 2.5 µg/ml SVLP at 39°C for 10 min, then fixed/permeabilised and co-labelled with anti-EEA-1 antibody prior to confocal microscopy analysis. Scale bar: 3 µm. (D) A 3-dimensional analysis of the circle shown in (C); the right image shows a further analysis of the circle in the left image, analysed with the aid of Spots module in IMARIS to define centre points for SVLP (green) and EEA-1 (red) labelling. Scale bars: 3 µm (blend), 1 µm (zoom; spot module to enhance visualisation, filtered on mean intensity).</p

SVLP association with lipid rafts.

<p>(A) Pre-chilled DC were treated with 2.5 µg/ml SVLP (green) and 5 µg/ml CTB (red), followed by 30 min incubation on ice and washing. Samples were fixed and analysed by confocal microscopy. Arrows indicate SVLP and CTB localisation. The white square is the zoomed area in the lower panel; the lower panel shows a different section to that in the upper panel, to demonstrate that areas with apparently no co-localisation of the SVLP with CTB could overlay areas showing co-localisation. Scale bars: 50 µm (upper panel) and 5 µm (lower panel). (B) DC treated as (A) were given warm medium and shifted to 39°C for 10 min. Arrowheads indicate SVLP and CTB co-localisation on polarised membranes at intercellular contacts; arrows indicate internalisation at polarisation sites. Scale bars: 10 µm (upper panel) and 5 µm (lower panel). (C) Pre-chilled DC incubated with 2.5 µg/ml SVLP (green) on ice for 20 min followed by washing and shifting to 39°C for 10 min. The image in the lower panel is a zoom of the white square in the upper panel. Arrows show SVLP associated with lamellipodia. Scale bars: 30 µm (left) and 10 µm (right). (D) DC were pulsed with 2.5 µg/ml SVLP and 5 µg/ml CTB for 30 min on ice, then shifted to 39°C for 30 min. High-resolution stacks, 3D images and co-localisation analysis, using IMARIS, demonstrate co-localisation of SVLP with CTB; this included algorithmic analysis to identify co-localised voxels. Scale bar: 8 µm. (E) Algorithmic analysis of the results from (D) performed using IMARIS.</p

SVLP binding to DC.

<p>(A) Graphic representation of SVLP. (B) DC were incubated with different concentrations of SVLP in medium without serum on ice for 20 min. Samples were washed and analysed by flow cytometry. (C) Pre-chilled DC were incubated with 2.5 µg/ml for SVLP for 5 or 30 seconds, then washed and fixed (10min, room temperature, 4% w/v p-formaldehyde) prior to analysing by confocal microscopy. Scale bars: 5 µm. (D) DC were incubated with high molecular weight dextran-546 (500 kDa), Ovalbumin-488, transferrin-546 (artificially coloured blue to aid visualisation) or PBS (mock) for 1 min at 39°C followed by analysis using confocal microscopy. Scale bars: 5 µm. (E) Pre-chilled DC were incubated with Ovalbumin-488 or PBS for 10 or 60 min on ice, followed by washing and analysis by flow cytometry. (F) Receptor-mediated binding of SVLP. DC were treated with pronase for 25 min at 39°C. Cells were then pre-cooled on ice for 30 min and washed 5 times. SVLP (1 µg/ml), or antibody against MHCI, CD172a or CD14, were added for 20 min on ice (antibody binding was then detected with Alexa₄₈₈-conjugated anti mouse immunoglobulin F(ab')₂). The cells were also treated with 3 mM NaN₃ to impair recycling of CD172a or CD14. Cells were analysed by flow cytometry. For the % SVLP positive cells, difference was significant between cells treated with pronase at “0.5 mg/ml” and “1 mg/ml” (p = 0.004), and also between “no pronase” and “0.5 mg/ml pronase” (p = 0.014). Results (B, E–F) are means of three samples ± s.d.</p

SVLP association with high molecular weight dextran and its uptake by mature or immature DC.

<p>(A) DC were incubated with 50 µg/ml high molecular weight dextran for 30 min at 39°C, followed by “on ice” for 30 min. Cells were then treated with 2.5 µg/ml SVLP and incubated for 30 min, then shifted to 39°C for different times. Acquisition was by confocal microscopy; high-resolution stacks were prepared using IMARIS. White arrows: SVLP near the leading edge. Yellow arrows: Dextran⁺ vesicles in apposition with SVLP-vesicles. Scale bars: 50 µm (top-left), 3 µm (zooms). (B) Algorithms of SVLP co-localisation with dextran. (C) Four-day old DC were treated or not with interferon-α (1000 U/ml) and lipopolysaccharide (10 µg/ml) for 24 hrs to mature. High molecular weight dextran (50 µg/ml) or ovalbumin (10 µg/ml) was added, and incubated for 5, 30 or 60 min at 39°C. The number of positive cells was measured by flow cytometry; each bar represents means of three values and standard deviation. (D) Internalisation of SVLP (1 µg/ml) by the immature and mature DC used in (C). SVLP were added for 30 min on ice, then shifted to 39°C for different times, when the cultures were chilled and treated or not for 30 min with PK on ice. The number of positive cells was measured by flow cytometry; each bar represents means of three values and standard deviation.</p

SVLP association with DC microtubules, leading edge and MHCII.

<p>(A) Pre-chilled DC were incubated with 2.5 µg/ml SVLP for 20 min on ice, followed by shifting to 39°C for 10 min. Samples were then fixed, permeabilised and labelled with antibody targeting α-tubulin (red) and MHCII (blue). Acquisition was by confocal microscopy; high-resolution stacks were prepared and analysed using Filament Tracer and Spots modules in IMARIS. Visualisation was enhanced using filament tracer (microtubules) and spot (SVLP, MHCII) modules. Scale bars: 5 µm (left), 2 µm (right). (B) Pre-chilled DC were incubated with 2.5 µg/ml SVLP for 30 min on ice, followed by washing and shifting to 39°C for 20 min. Cells were then fixed, permeabilised and labelled with antibody targeting MHCII (blue). Acquisition was by confocal microscopy; high-resolution stacks were prepared using IMARIS. Scale bar: 5 µm. (C) The same cultures were used as in (B), but incubated for 2 hours. Cells were then fixed, permeabilised and labelled with antibody targeting EEA-1 (red) or MHCII (blue). The arrows indicate sites of potential SVLP and MHCII association. Acquisition was by confocal microscopy; high-resolution stacks were prepared using IMARIS. The arrow shows a vesicle in which SVLP and MHCII were present. Scale bar: 5 µm.</p

SVLP associating with early endosomes, MHCII and DQ-Ova.

<p>(A) Application of quenched SVLP signal to identify endosomal enzyme activity leading to de-quenching of the signal. DC were incubated with 2.5 µg/ml quenched SVLP (green) for different incubation times at 39°C. Samples were fixed, permeabilised and labelled with antibody targeting EEA-1 (red). Acquisition of the images was by confocal microscopy, followed by analysis and merging using IMARIS. Scale bars: 30 µm. (B) Cells were treated as for (A), but also with DQ- Ovalbumin. Acquisition of the images was by confocal microscopy, followed by analysis and merging using IMARIS. Scale bar: 20 µm. (C) Algorithmic co-localisation analysis of SVLP and DQ-Ova performed using IMARIS. (D) Cells were treated as for (A), but stained also for MHCII molecules (blue). Acquisition of the images was by confocal microscopy; high-resolution stacks were prepared using IMARIS. Scale bars: 20 µm (top left), 5 µm (zooms).</p

Internalisation of SVLP by DC.

<p>(A) DC incubated with 2.5 µg/ml SVLP (green) for 30 min at 39°C. High resolution stacks (1024×1024 voxels) were prepared, and 3D-imaging preformed using IMARIS. Scale bar: 5 µm. (B) SVLP (1 µg/ml) were added to pre-chilled DC (SVLP) or not (Mock) for 30 min on ice; pre-chilled DC were treated with PK before (DC + PK then SVLP) or after (DC + SVLP then PK) incubation with SVLP on ice. PK was added for 30 min on ice, then 10% serum added to “neutralise” PK activity. Cells were analysed by flow cytometry. The % Alexa₄₈₈ positive cells were significantly different between SVLP (cells not treated with PK; “SVLP (positive control)”) and “DC + PK then SVLP” (p = 0.006). (C) SVLP (1 µg/ml) were first pre-treated with PK as in (B). The treated SVLP were added to DC on ice for 30 min. Cells were washed and analysed by flow cytometry. (D) Pre-chilled DC were pulsed with 1 µg/ml SVLP for 20 min on ice and then shifted to 39°C for different time points, followed by treating or not with PK for 30 min on ice, and analysed by flow cytometry. (E) DC were incubated with different concentrations of SVLP for 24, 48 or 72 hrs at 39°C. Following washing, the cells were treated with PI and analysed by flow cytometry. The “0” on the x-axis indicates the cell control untreated with SVLP. (F) DC were incubated with 1 µg/ml SVLP for 20 min on ice, then different incubation time at 39°C, followed by flow cytometry analysis. Results are means of three samples ± s.d.</p

Multiple poliovirus-induced organelles suggested by comparison of spatiotemporal dynamics of membranous structures and phosphoinositides

<div><p>At the culmination of poliovirus (PV) multiplication, membranes are observed that contain phosphatidylinositol-4-phosphate (PI4P) and appear as vesicular clusters in cross section. Induction and remodeling of PI4P and membranes prior to or concurrent with genome replication has not been well studied. Here, we exploit two PV mutants, termed EG and GG, which exhibit aberrant proteolytic processing of the P3 precursor that substantially delays the onset of genome replication and/or impairs virus assembly, to illuminate the pathway of formation of PV-induced membranous structures. For WT PV, changes to the PI4P pool were observed as early as 30 min post-infection. PI4P remodeling occurred even in the presence of guanidine hydrochloride, a replication inhibitor, and was accompanied by formation of membrane tubules throughout the cytoplasm. Vesicular clusters appeared in the perinuclear region of the cell at 3 h post-infection, a time too slow for these structures to be responsible for genome replication. Delays in the onset of genome replication observed for EG and GG PVs were similar to the delays in virus-induced remodeling of PI4P pools, consistent with PI4P serving as a marker of the genome-replication organelle. GG PV was unable to convert virus-induced tubules into vesicular clusters, perhaps explaining the nearly 5-log reduction in infectious virus produced by this mutant. Our results are consistent with PV inducing temporally distinct membranous structures (organelles) for genome replication (tubules) and virus assembly (vesicular clusters). We suggest that the pace of formation, spatiotemporal dynamics, and the efficiency of the replication-to-assembly-organelle conversion may be set by both the rate of P3 polyprotein processing and the capacity for P3 processing to yield 3AB and/or 3CD proteins.</p></div