localised volume of 0.4. Intriguingly, this value seen at 60 min was retained after 8 hrs incubation at 39uC. With SVLP being internalised into similar structures as DQ- Ova, suggested similar routes of uptake and processing. The relatively slow rate of the process suggested a role for macro- pinocytosis; caveolar uptake was unlikely due to the aforemen- tioned absence of SVLP co-localisation with caveolin. According- ly, the association of the SVLP with other intracellular structures involved in different antigen processing routes was investigated. (ii) MHCII and Microtubules. As in Fig. 5A, de-quenched SVLP was associated with EEA-1+ early endosomes by 2 hrs at 39uC (Fig. 5D, red on green signal). In contrast, it was rare to visualise de-quenched SVLP associated with MHCII-rich com- partments (Fig. 5D, blue on green signal). This may have reflected an association with MHCII-positive structures typifying more advanced structures in antigen processing, wherein the SVLP had been degraded beyond detectability. Alternatively, a slow rate of processing may have led SVLP into MHCII+ compartments at levels too low to be detected. DC which had endocytosed SVLP were also stained for MHCII and microtubules. Fig. 6A shows an apposition of MHCII+ structures (blue) with microtubules (red), but this was distinct from the SVLP-containing vesicles (green). The majority of the SVLP- containing vesicles were observed appositional to the microtubules (Fig. 6A). It was considered that those SVLP-containing vesicles not associated with microtubules may have been in an earlier stage of internalisation. Certainly, when the SVLP were detectably polarised (as observed with lipid raft association using CTB – see Fig. 3), there was a tight apposition with the microtubules nearer the leading edge of the cell (Fig. 6A, right-hand image). Further analyses looked at the relative positioning of the SVLP signal in the DC. During the initial 20 min after shifting to 39uC, SVLP were often found internalised at the leading edge of the cell (see Fig. 3 and 6A). Such internalisation yielded a polarised positioning (Fig. 6B), distinctive from the less peripheral and more perinuclear location of the MHC+ structures (Fig. 6B). At later time points, such as the 2h shown in Fig. 6C, the SVLP-containing structures, including those associated with EEA-1+ structures, were only rarely associating with MHCII+ structures. By employing quenched SVLP, so that the signal would only appear upon enzymatic digestion, association of the SVLP signal with MHCII+ structures remained rare, in contrast to association with EEA-1+ structures (Fig. 6C; see also Fig. 5D). When the occasional interaction with MHC II+ structures could be noted (Fig. 6C white arrow), the signal was in the more peripheral ‘‘EEA-1+ region’’, which may explain the more common association with the EEA-1+ structures compared with MHCII. (iii) Lysosomes and Endoplasmic Reticulum. Although the results indicate that processing of the SVLP was occurring, albeit at a slow rate, attempts to show association of the SVLP with more degradative lysotracker+ organelles was unsuccessful (data not shown). However, DC are reported to express low lysosomal proteolytic activity [9,24,25]. As an alternative to loss of de- tectability during SVLP processing, the SVLP may have been diverted prior to entering lysotracker+ organelles. An alternative processing route to maturing endosomes and lysosomes is the transfer into the ER. However, no SVLP signal could be found associated with the ER using ER-tracker or the chaperones calnexin and calreticulin (data not shown). Association of SVLP with markers of different endocytic pathways (i) Clathrin. Considering that DC possess different endocytic routes, a role for clathrin-dependent or -independent endocytosis DC Endocytosis of SVLP was analysed. Labelling of the DC with antibody against clathrin at different times after the shift to 39uC revealed that the SVLP were mostly associated with structures free of clathrin (data not shown). Only at early time points such as 10 min was there evidence of co-localisation (Fig. 7A). Again, the majority of SVLP- structures were not clathrin+, and no such association was observed after this 10 min time point (data not shown). (ii) Transferrin. Alexa-labelled transferrin was used as a marker for clathrin-mediated endocytosis, as well as an aid for identifying sorting endosomal structures; following the shift from ice to 39uC, unbound SVLP were removed, but presence of the transferrin was maintained for the different time points of incubation. The earliest time point of 5 seconds (plus the time for washing) after the shift to 39uC showed no association of transferrin and SVLP (data not shown). From 5 min, both transferrin and the SVLP were becoming less associated with the cell periphery, but there were still no signs of co-localisation (Fig. 7B, top row). Between 10 and 60 min, the transferrin continued to be translocated from the periphery (replaced presumably by newly endocytosed transferrin at the periphery), but the SVLP tended to remain more peripheral than the transferrin (Fig. 7B, 2nd and 3rd rows). It was during this period that SVLP associating with transferrin-containing vesicles was noted, also more peripheral (Fig. 7B, arrows). By the 2 hrs time point, the SVLP-containing structures were showing evidence that they were now translocating from the cell surface in larger numbers, but were distinct from the transferrin-bearing structures (Fig. 7B, 4th row), Interestingly, a number of particularly perinuclear transferrin+ structures were seen, but the SVLP remained to the peripheral side of these (Fig. 7B, 4th row). (iii) Dextran. The evidence pointing to the clathrin-in- dependent endocytic process of macropinocytosis was interesting for SVLP considering that many viral pathogens employ this route to infect host cells including DC [26]. Accordingly, high molecular weight dextran was employed as a marker for macropinocytosis [27], due to its accumulation in macropinosomes; the high molecular weight of the dextran prevents its translocation from the vesicular structures. Cells were given high molecular weight dextran 30 min at 39uC prior to adding the SVLP, allowing the dextran to accumulate, and ensure that there would not be confusion with any dextran still entering the cells. DC were then washed and pulsed with SVLP on ice for 20 min, unbound SVLP washed away, and the cells shifted to 39uC for different time points. By 20 min of incubation at 39uC most of the SVLP+ vesicles were associating with dextran+ vesicles (Fig. 8A, yellow dots). Interestingly, the SVLP+ vesicles still at the leading edge of the cells were distinct from the dextran-containing vesicles (Fig. 8A, white arrow). It was also observed in 3D-analyses that some dextran+ vesicles were seen in apposition with SVLP-vesicles (Fig. 8A, right-hand image, yellow arrows). Nevertheless, algorith- mic analysis of co-localised voxels demonstrated a high confidence in co-localisation (Fig. 8B), and this was time-dependent. SVLP internalisation by immature and mature DC Further analysis of macropinocytosis used the characteristic of mature DC having reduced activity compared with immature DC [28]. The uptake of SVLP by mature and immature DC was therefore compared, using DC matured with interferon- a (1000 U/ml) plus lipopolysaccharide (10 mg/ml) for 24 hrs: the immature DC were left untreated. For macropinocytosis, the uptake of high molecular weight dextran (500 KD) and ovalbumin were used as markers. Mature DC did not endocytose high molecular weight dextran, unlike immature DC, confirming the relative states of maturation (Fig. 8C). Ovalbumin uptake was PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e43248