Localisation Microscopy of Breast Epithelial ErbB-2 Receptors and Gap Junctions: Trafficking after γ-Irradiation, Neuregulin-1β, and Trastuzumab Application.

Götz Pilarczyk,Ines Nesnidal,Manuel Gunkel,Margund Bach,Michael Hausmann,Felix Bestvater

doi:10.3390/ijms18020362

Abstract

In cancer, vulnerable breast epithelium malignance tendency correlates with number and activation of ErbB receptor tyrosine kinases. In the presented work, we observe ErbB receptors activated by irradiation-induced DNA injury or neuregulin- application, or alternatively, attenuated by a therapeutic antibody using high resolution fluorescence localization microscopy. The gap junction turnover coinciding with ErbB receptor activation and co-transport is simultaneously recorded. DNA injury caused by 4 Gray of 6 MeV photon -irradiation or alternatively neuregulin- application mobilized ErbB receptors in a nucleograde fashion—a process attenuated by trastuzumab antibody application. This was accompanied by increased receptor density, indicating packing into transport units. Factors mobilizing ErbB receptors also mobilized plasma membrane resident gap junction channels. The time course of ErbB receptor activation and gap junction mobilization recapitulates the time course of non-homologous end-joining DNA repair. We explain our findings under terms of DNA injury-induced membrane receptor tyrosine kinase activation and retrograde trafficking. In addition, we interpret the phenomenon of retrograde co-trafficking of gap junction connexons stimulated by ErbB receptor activation.

Highlights

The presented matter focuses on ErbB receptor protein molecules, either as isolated residues in the cell plasma membrane or in the form of dimers and oligomeres spanning the plasma membrane or being packed into intracellular carriers
An experimental approach combining a controlled stimulation of ErbB receptor and gap junction physiological activity with a close observation of the mentioned proteins by microscopy will greatly benefit from addressing the discrete appearance of the proteins as individual molecules, and mentioning the protein ensembles as dense and continuous protein populations at several intracellular locations [79]
Such a combination of two different microscopy approaches accounts for the discrete and the continuous nature of ErbB receptor and gap junction residues

Summary

Introduction

The presented matter focuses on ErbB receptor protein molecules, either as isolated residues in the cell plasma membrane or in the form of dimers and oligomeres spanning the plasma membrane or being packed into intracellular carriers. An increased copy number or transcription of the ErbB gene (Her2/neu) results in an abnormally high receptor density in malignant breast tissue [2] and in bad patient prognosis [3]. The ErbB-2/ErbB-3 dimer is the main combination responsible for receptor activity [4]. This activation is accompanied by an increase in the density of exclusively activated ErbB-2 receptors, further by the inclusion into membrane-coated transport vesicles and by the nucleograde transport across the cytosol. To visualize structural alterations during receptor mobilization, the nanometer regime for receptor aggregates and the μm scale for cytosolic transport have to be observed

Methods

Results

Conclusion