Colocalisation of PrP Sc with endosomal and lysosomal markers

In further localisation experiments of PrP^Sc in endosomal/lysososomal compartments ConA time series were carried out in parallel to determine the putative acceleration of PrP^Sc internalisation in endosomes and lysosomes although the internalisation of PrP^Sc with ConA could not be visualised directly (Figure 19 and 20). H6-22L cells were treated with ConA for 10, 30 and 100 min. After fixation, permeabilisation and denaturation with 6 M GdnHCl cells were stained with mAb 6H4 and with the early endosomal marker EEA1. Confocal images were analysed for colocalisation with PrP^Sc by applying the criterion of intracellular GdnHCl dependent PrP signals (see Figure 14). In almost every focal plane of a z-stack, one or more colocalisations were found, but the major part of PrP^Sc accumulated outside early endosomes (Figure 21). Colocalisation was not enhanced upon treatment with ConA.

Although H6-22L cells were not treated with PK it is unlikely that PrP^C contributed substantially to the PrP staining for the following reasons: First, the PrP^C signal was considerably weaker than the PrP^Sc signal. (Note that in Figure 21 the detector gain was set in such a way as to reduce the PrP^C signal almost completely.) Second, colocalisation only occurred at intracellular sites where PrP^C signals are known to be very weak (Figure 14).

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Figure 21: Colocalisation of PrP^Sc with early endosomal markers. H6-22L cells were incubated with unlabeled ConA (100 µg/ml) for 10, 30 and 100 min. The cells were fixed, permeabilised, denatured with 6 M GdnHCl and incubated with 6H4 and gαm A488 for PrP, and with EEA1 and dαg Cy3 for early endosome labelling.

Representative colocalisations are indicated by arrows. Note that colocalisation was rare and that ConA did not increase the number of colocalisation events between early endosomes and PrP. Z-stacks were performed with Laser Scanning Microscopy. One focal plane of a z-stack (0.08 µm x 0.08 µm x 0.38 µm) was shown in each row.

Scale bars = 10 µm.

Colocalisations were measured according to the signal intensities of PrP^Sc and EEA1. A scatter plot of Figure 21 (0 min ConA) was performed as an example for calculation of colocalisations (Figure 22). On the x-axis the intensity of the fluorophore Cy3 and on the y-axis the intensity of fluorophore A488 was measured. White pixels in the plot represented one Cy3 molecule of a certain intensity meeting one A488 molecule of another (orange arrow) or the same (green arrow) intensity. If PrP^Sc and EEA1 displayed the same signal intensity, they were interpreted as colocalisations (Figure 22, green arrow). Molecules of the same intensity are located on the diagonal through zero (in magenta). Pixels on this straight line represent a perfect colocalisation, with signals of same intensities (green arrow). In addition, it was also possible to interpet signals of different intensities (orange arrow) within a defined region (rectangle) as colocalisations. The data points within the rectangle were transferred to colocalisation points of the merged pictures (Figure 23, white dots in right column). Thus, not only colocalisations of perfect match were visible, indicated by arrows in Figure 23 but also a range of different intensities of fluorophores Cy3 and A488 were defined as colocalisations. In this case, more colocalisations between PrP^Sc and EEA1 could be found.

Nevertheless, a great part of PrP^Sc was not localised to early endosomes.

Figure 22: Scatter Plot of Figure 21 (0 min ConA) as an example for calculation of colocalisations with ImageJ.

On the x-axis the intensity of the fluorophore Cy3 and on the y-axis the intensity of fluorophore A488 were measured. White pixels in the plot represent one Cy3 molecule of a certain intensity meeting one A488 molecule of a higher (orange arrow) or the same (green arrow) intensity. Molecules of the same intensity are located on the diagonal through zero (in magenta). Pixels on this straight line represent a perfect colocalisation, with signals of same intensities (green arrow). The rectangle displays the plane which was used in Figure 23 for calculating all colocalisation points within this rectangle. The position of the rectangle was chosen to ensure that only strong signals were analysed. Obviously, also colocalisations of different intensities were measured.

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Figure 23: Merged pictures of Figure 21 were further analysed for colocalisation using ImageJ according the protocol explained above in Figure 22. White dots in “Colocalisation Points” image display all colocalisations found between PrP^Sc and EEA1. Arrows point to perfect colocalisations. Scale bar = 10 µm.

For localisation of PrP^Sc in late endosomes/lysosomes H6-22L cells were treated with ConA for 10, 30 and 100 min. After fixation, permeabilisation and denaturation with 6 M GdnHCl cells were stained with mAb 6H4 and the late endosomal/lysosomal marker Limp2. Confocal images were analysed for colocalisation with PrP^Sc by applying the criterion of intracellular GdnHCl-dependent PrP signals (see Figure 14). The result was similar to the localisation of PrP^Sc in early endosomes. Some colocalisations between PrP^Sc and Limp2 could be discovered in every focal plane of a z-stack but the majority of PrP^Sc was located outside of Limp2 positive compartments (Figure 24). Substantiating the finding from the analysis of early endosomal colocalisations (Figure 21) ConA did not enhance PrP^Sc endocytosis. As described above, the simultaneous detection of PrP^Sc and PrP^C was almost excluded. Only intracellular PrP^Sc signals were analysed and the detector gain was reduced to record only signals from PrP^Sc. Moreover, colocalisations were analysed in detail with a scatter plot to detect not only perfect colocalisations of same intensity between Cy3 and A488 but also colocalisations of different intensities within a special range (described in Figure 22). With this additional analysis more colocalisation events were found (Figure 25, white dots).

However, also in this case most of PrP^Sc was observed outside of Limp2 positive organelles.

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Figure 24: Colocalisation of PrP^Sc with the late endosomal/lysosomal marker Limp2. H6-22L cells were incubated with unlabeled ConA (100 µg/ml) for 10, 30 or 100 min. Cells were then fixed, permeabilised, denatured with 6 M GdnHCl and incubated with mAb 6H4 and gαm A488 for PrP, and with Limp2 and dαg Cy3 for late endosomes/lysosomes. Some of the rare colocalisations are indicated by arrows. Note that ConA did not increase the colocalisation events between late endosomes/lysosomes and PrP. Z-stacks were performed with Laser Scanning Microscopy. One slice of a z-stack (0.08 µm x 0.08 µm x 0.38 µm) was shown in each row.

Scale bar = 10 µm.

Figure 25: Merged pictures of Figure 23 were further analysed for colocalisation using ImageJ. White dots in

“Colocalisation Points” image display all colocalisations found between PrP^Sc and Limp2. Arrows point to perfect colocalisations. Scale bar = 10 µm.

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5.1.7 Visualisation of PrP^Sc in combination with endoplasmatic reticulum and Golgi