Analyse der Clathrin vermittelten Endocytose von Chemokin Rezeptoren
Zusammenfassung der Projektergebnisse
The aim of this research project was to use the chemokine receptors CXCR4 and CCR5 for a detailed analysis of clathrin mediated endocytosis using high resolution live cell microscopy, electron microscopy, biochemistry and RNAi inhibition studies. The idea was to investigate whether functionally distinct populations of clathrin coated structures exist. Another question was to see whether receptors induced to internalize through different activation mechanisms (CXCR4) would use distinct populations of endocytic clathrin coated structures and probably face different intracellular sorting. A third aim was to show how chemokine receptors are recruited to and internalized through clathrin coated vesicles. Research conducted on the internalization of CXCR4 proved that the receptor gets internalized via a clathrin mediated pathway regardless of the stimulus triggering internalization. Further research on the trafficking and sorting of the receptor revealed that there is no difference between agonist or phorbol ester induced internalization. In any case the receptor is targeted for lysosomal degradation with similar kinetics. I did not observe indications for functionally distinct clathrin coated structures for the internalization of CXCR4. I then refocused my research on the internalization of CCR5 via clathrin mediated endocytosis using TIRF (Total Internal Reflection Fluorescence) microscopy. Upon Rantes (agonist) treatment the labeled receptor could be seen to move rapidly into large and stable clathrin coated assemblies. In our model system CHO CCR5 cells the chemokine receptor seemed to be almost exclusively using the route via long lived extended clathrin coated structures. The endocytosis via single clathrin coated pits arising at the plasma membrane next to the cargo as reported for other receptors (for example Fotin et al. 2004) seemed to play a neglectable role and was hardly observed. Immuno electron microscopical data from untreated cells in our lab had frequently shown the chemokine receptor CCR5 aligned to the actin cytoskeleton like pearls on a string. The receptor also features a putative PDZ-ligand interaction motif at the C-terminus. This led to the assumption that CCR5 might be tethered to the actin cytoskeleton. To prove this idea experiments for high speed TIRF microscopy of single receptor molecules at a video rate of 30 frames per second were planned. Experiments conducted with JSPS funding in the laboratory of Prof. Kusumi at the university of Kyoto could show that untreated CCR5 displays a similar movement at the plasma membrane as the transferrin receptor with cargo. Only a small proportion of the receptor (9%) was found to be immobile, while the majority was moving rapidly along the surface of the plasma membrane (Average mean square displacement 0,15 µm²/200 ms). Upon Rantes treatment the number of immobile chemokine receptors rapidly increased from 9% to 30% of total. This happens presumably as the receptor gets trapped in clathrin coated structures. This also results in a lower mean average mean square displacement of the receptor. A CCR5 mutant with an obliterated PDZ-ligand interaction motif showed a very similar behaviour and the same percentage in increase in immobile receptors. This means that an interaction of CCR5 with the actin cytoskeleton can be largely ruled out. Additionally the method of single molecule tracking TIRF microscopy has shown to be a powerful tool to study clathrin mediated endocytosis, which seems to have many advantages to the more conventional TIRF technology used in many publications in the field.
