Quantum dots implementation as a label for analysis of early stages of EGF receptor endocytosis: a comparative study on cultured cells
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Anna V. Salova1, Tatiana N. Belyaeva1, Ekaterina A. Leontieva1, Maria V. Zlobina1,4, Marianna V. Kharchenko1, Elena S. Kornilova1,2,3
1Institute of Cytology of the Russian Academy of Sciences, St. Petersburg, Russia
2Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia
3St. Petersburg State University, St. Petersburg, Russia
4Central European Institute of Technology, Brno, Czech Republic
Elena S. Kornilova, e-mail: [email protected]
Keywords: EGF receptor, quantum dots, endocytosis, EEA1, HRS
Received: July 28, 2015 Accepted: November 25, 2015 Published: December 22, 2015
EGF complexed to fluorescent photostable quantum dots by biotin-streptavidin system (bEGF-savQD) is attractive for both the basic research and therapeutic application such as targeted drug delivery in EGF-receptor (EGFR) expressing cancers. However, compared to native EGF, the large size of QD and its quasi-multivalency can have unpredictable effects on EGFR endocytosis changing the internalization portal and/or endosomal processing tightly bound to EGF signaling. We have found that bEGF-savQDs enter HeLa cells via the temperature-dependent clathrin-mediated EGF-receptor-specific pathway characteristic for native EGF. We also found that EGF-to-QD concentration ratios used for the complex preparation and the level of EGF receptor expression affect the number and integral densities of the formed endosomes. So, at EGF-to-QD ratio from 4:1 to 12:1 (at nanomolar bEGF concentrations) on average 100 bright endosomes per HeLa cell were formed 15 min after the complex addition, while 1:1 ratio resulted in formation of very few dim endosomes. However, in A431 cells overexpressing EGFR 1:1 ratio was effective. Using dynamin inhibition and Na-acidic washout we showed that bEGF-savQDs bind surface receptors and enter clathrin-coated pits slower than the same ligands without QD. Yet, the bEGF-savQD demonstrated similar to native EGF and bEGF-savCy3 co-localization dynamics with tethering protein EEA1 and HRS, the key component of sorting ESCRT0 complex. In conclusion, our comparative study reveals that in respect to entrapment into coated pits, endosomal recruitment, endosome fusions, and the initial steps of endosomal maturation, bEGF-savQD behaves like native EGF and QD implementation does not affect these important events.
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