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EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements

1
Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary
2
Max Planck Institute for Biophysical Chemistry, Laboratory of Cellular Dynamics, Am Fassberg 11, D-37077 Göttingen, Germany
3
Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands
4
MTA-DE Cell Biology and Signaling Research Group, Faculty of Medicine, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary
5
Faculty of Pharmacy, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(13), 3370; https://doi.org/10.3390/ijms20133370
Received: 15 June 2019 / Revised: 4 July 2019 / Accepted: 5 July 2019 / Published: 9 July 2019
(This article belongs to the Special Issue Imaging with Fluorescent Proteins)
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Abstract

To elucidate the molecular details of the activation-associated clustering of epidermal growth factor receptors (EGFRs), the time course of the mobility and aggregation states of eGFP tagged EGFR in the membranes of Chinese hamster ovary (CHO) cells was assessed by in situ mobility assays. Fluorescence correlation spectroscopy (FCS) was used to probe molecular movements of small ensembles of molecules over short distances and time scales, and to report on the state of aggregation. The diffusion of larger ensembles of molecules over longer distances (and time scales) was investigated by fluorescence recovery after photobleaching (FRAP). Autocorrelation functions could be best fitted by a two-component diffusion model corrected for triplet formation and blinking. The slow, 100–1000 ms component was attributed to membrane localized receptors moving with free Brownian diffusion, whereas the fast, ms component was assigned to cytosolic receptors or their fragments. Upon stimulation with 50 nM EGF, a significant decrease from 0.11 to 0.07 μm2/s in the diffusion coefficient of membrane-localized receptors was observed, followed by recovery to the original value in ~20 min. In contrast, the apparent brightness of diffusing species remained the same. Stripe FRAP experiments yielded a decrease in long-range molecular mobility directly after stimulation, evidenced by an increase in the recovery time of the slow component from 13 to 21.9 s. Our observations are best explained by the transient attachment of ligand-bound EGFRs to immobile or slowly moving structures such as the cytoskeleton or large, previously photobleached receptor aggregates. View Full-Text
Keywords: Fluorescence correlation spectroscopy; FCS; fluorescence recovery after photobleaching; FRAP; epidermal growth factor receptor; translational diffusion; EGFR–eGFP fusion protein Fluorescence correlation spectroscopy; FCS; fluorescence recovery after photobleaching; FRAP; epidermal growth factor receptor; translational diffusion; EGFR–eGFP fusion protein
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Vámosi, G.; Friedländer-Brock, E.; Ibrahim, S.M.; Brock, R.; Szöllősi, J.; Vereb, G. EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements. Int. J. Mol. Sci. 2019, 20, 3370.

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