EGF receptor lysosomal degradation is delayed in the cells stimulated with EGF-Quantum dot bioconjugate but earlier key events of endocytic degradative pathway are similar to that of native EGF

Abstract

Quantum dots (QDs) complexed to ligands recognizing surface receptors undergoing internalization are an attractive tool for live cell imaging of ligand-receptor complexes behavior and for specific tracking of the cells of interest. However, conjugation of quasi-multivalent large QD-particle to monovalent small growth factors like EGF that bound their tyrosine-kinase receptors may affect key endocytic events tightly bound to signaling. Here, by means of confocal microscopy we have addressed the key endocytic events of lysosomal degradative pathway stimulated by native EGF or EGF-QD bioconjugate. We have demonstrated that the decrease in endosome number, increase in mean endosome integrated density and the pattern of EEA1 co-localization with EGF-EGFR complexes at early stages of endocytosis were similar for the both native and QD-conjugated ligands. In both cases enlarged hollow endosomes appeared after wortmannin treatment. This indicates that early endosomal fusions and their maturation proceed similar for both ligands. EGF-QD and native EGF similarly accumulated in juxtanuclear region, and live cell imaging of endosome motion revealed the behavior described elsewhere for microtubule-facilitated motility. Finally, EGF-QD and the receptor were found in lysosomes. However, degradation of receptor part of QD-EGF-EGFR-complex was delayed compared to native EGF, but not inhibited, while QDs fluorescence was detected in lysosomes even after 24 hours. Importantly, in HeLa and A549 cells the both ligands behaved similarly. We conclude that during endocytosis EGF-QD behaves as a neutral marker for degradative pathway up to lysosomal stage and can also be used as a long-term cell marker.