4.4 Localization of the three WS-TDI derivatives in
Figure 4.13: Endocytic pathways, adapted from Alberts et al.182 The early and late endosomes are highlighted with a green dotted rectangle.
In order to address the identity of the vesicles stained by the new dyes, co-localization assays were performed using HuH-7 cells that express either Rab5 fused to the Green Fluorescent Protein (GFP) or Rab9 fused to GFP (constructed in our group by Nadia Ruthardt). Each of those cell lines were marked with the three WS-TDI derivatives. The dye concentration was in each case 5×10−6 mol/L (see experimental section for details).
GFP was excited with a 532 nm laser line whereas the WS-TDI derivatives were excited with a 633nm laser line. The fluorescence emission spectra of the WS-TDIs and GFP are well separated, which allows imaging the WS-TDI derivatives and the Rab GTPases simultaneously on two separated channels, allowing to address any co-localization be-tween the endosomes labelled by the dye and Rab5 labelled early endosomes or Rab9 labelled late endosomes.
The HuH-7 cells marked with the WS-TDI derivatives were imaged with a confocal mi-croscope using simultaneously two laser lines of 532 nm and 633 nm. The time evolution of the fluorophores in the endocytic pathway was observed for HuH-7/Rab5-GFP and HuH-7/Rab9-GFP at three different time points: (1) After 40 min of incubation time with the fluorophore, (2) 30 min after washing of the remaining dye and (3) 16 h 30 min after washing, which corresponds to a very long time in living cell experiments.
Figure 4.14 shows overlays of the two channels where the red color corresponds to the fluorescent signal of the WS-TDI derivatives and the green color to the emission of the GFP Rab GTPases. Direct co-localization of the vesicle containing both Rab GTPases and a WS-TDI derivative will appear in yellow.
Figure 4.14: Fluorescence images of Human Hapatoma (HuH-7) living cells with Rab5 or Rab9 GTPases fused to the the Green Fluorescent Protein (GFP), and stained with WS-TDI or Alexa647 / dextran. A 532 nm laser was used for excitation of GFP-GTPases, which appear as green spots, and a 633 nm laser was used for excitation of WS-TDI (red spots). The endosomes containing both a Rab GTPase and WS-TDI appear as yellow dots. (a) and (b) HuH-7 cells/Rab5 and HuH-7 cells/Rab9 stained with WS-TDI, 40 min after addition of the dye. (c) and (d) 7 cells/Rab5 and HuH-7 cells/Rab9 stained with WS-TDI, 30 min after removing of the excess of dye. (e) and (f) HuH-HuH-7 cells/Rab9 stained with Alexa647 / dextran and WS-TDI respectively, 5 h 30 min after washing of the excess of dye.
Figure 4.14a shows a fluorescence image of HuH-7/Rab5-GFP cells after 40 min incuba-tion in a soluincuba-tion of WS-TDI. A first observaincuba-tion is that part of the vesicles appear in yellow, indicating that they contain both Rab5-GFP and WS-TDI. A close inspection of the remaining endosomes reveals that the green dots, i.e. Rab5-GFP containing endo-somes, exhibit also a small fluorescent signal in the red detection channel. This means that they also contain WS-TDI, however at lower concentrations. The red dots do not show any signal in the green detection channel i.e. here WS-TDI is not encapsulated in Rab5 containing early endosomes, but in another type of endosomes. One can thus conclude that WS-TDI is able to stain early endosomes according to the overlay of red and green dots. However, there is a second population which doesn’t co-localize with Rab5-GFP.
The identity of the second population of WS-TDI can be addressed investigating HuH-7 cells/Rab9-GFP stained with WS-TDI (at the same time point, see Figure 4.14b).
Here some endosomes appear in yellow, which means that WS-TDI is contained in late endosomes (identified by Rab9-GFP). Moreover, endosomes containing Rab9-GFP but not WS-TDI can be observed (green dots), as well as endosomes containing WS-TDI without Rab9-GFP (red dots). This shows that after 40 min of incubation a part of the Rab9-GFP containing endosomes are already labelled with WS-TDI.
Moreover, the transition to the late endosomes is not completed after 40 min since some Rab9-GFP vesicles do not show the presence of WS-TDI i.e. they appear as green dots.
The dye uptake into endosomes can be investigated by washing out the cells with fresh medium in order to deplete dye molecules from the medium. Figure 4.14c and 4.14d show overlayed fluorescence images 30 min after washing out the dye of HuH-7/Rab5-GFP cells and HuH-7/Rab9-HuH-7/Rab5-GFP cells respectively. Nearly no co-localization between the Rab5-GFP and WS-TDI containing endosomes can be observed in the image shown in Figure 4.14c. In contrast, Only yellow dots are visible in Figure 4.14d which i.e. WS-TDI is contained in the totality of the observable late endosomes. This demonstrates that WS-TDI is incorporated in the early endosomes which mature into late endosomes as long as the dye uptake process is possible, but that this process stops as soon as no dye is available anymore in the medium outside the cells. Moreover, after a certain time (already 30 min after washing), all the late endocytic system is labelled with WS-TDI.
The robustness of the labelling of the late endosomes with WS-TDI on longer timescale can be checked by monitoring the fluorescence of HuH-7/Rab9-GFP cells on a much longer timescale. Figure 4.14e which was recorded 16 h 30 min after washing. All the visible endosomes appear as bright yellow dots. This confirms that the late endosome system is still strongly labelled with WS-TDI even after longer time.
This ability of WS-TDI to label the late endocytic systems can be compared with those of a classical fluid phase tracer such as Alexa647/dextran. Figure 4.14e shows a fluorescence image of HuH-7/Rab9-GFP cells stained with Alexa647/dextran (which can be excited at 633 nm like WS-TDI) similarly to the previous experiment, and 16 h 30 after washing of the excess of fluorophore. This image is very similar to Figure 4.14f that is the totality of the late endosomes are labelled withAlexa647/dextran and appear as yellow spots.
This confirms again that WS-TDI can be used like endosomal uptake markers since it follows the endocytic pathway.
Finally, all the measurements described above were performed with TDI and WS-TDI dodecyl as well, with strictly identical results. To conclude, these data shed light on the pathway followed by the three water-soluble terrylene dyes after uptake in living cells. The fluorophores act as endosomal uptake markers: the dye molecules are first incorporated (within several minutes) in the early endosomes of the cells, then they are progressively transferred to the late endocytic system where the fluorescence signal is stably observed for several hours.