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

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-1sans-serifβ 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 γ-irradiation or alternatively neuregulin-1sans-serifβ 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.

Wrong place at the wrong time: how retrograde trafficking drives cancer metastasis through receptor mislocalization

“…Radiation treatment of cells is known to drive EGFR to the nucleus as part of the DNA-repair pathway mechanism, and studies have shown that treatment of irradiated cells with cetuximab will inhibit EGFR trafficking to the nucleus in both lung carcinoma and breast cancer cell lines [137] . Radiation treatment, along with neuregulin stimulation or trastuzumab will also promote retrograde trafficking of ErbB2-ErbB3 dimers to the nucleus, visible by super-resolution confocal microscopy as demonstrated by Pilarczyk et al [138] . Nuclear import might also be inhibited by targeting the importin-β1 molecule through treatment with small molecule inhibitors such as Karyostatin1A to disrupt importin interactions with the GTPase Ran [139] .…”

Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

“…In-house software (for detailed descriptions see [40,44,[78][79][80][81]) written in MATLAB was used to calculate signal point localizations from blinking events in raw image stacks. In brief, background signals were excluded, and the two-dimensional intensity profile of each fluorescent signal was subjected to Gaussian fitting through the whole image stack.…”

Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

“…It was shown that cells with different expression levels of EGF-receptors (e.g., ErbB2 in breast cancer or EGFRvIII in glioblastoma) show a different frequency of clustering, and differ in cluster size and molecular arrangements of the clustering receptor molecules [ 26 , 27 , 46 , 51 ]. Since the cluster formation is directly correlated to functional consequences of inter-receptor communication and signaling pathways [ 52 ], it is of high interest to analyze such clusters with respect to molecular treatment.…”

“…The results of localization microscopy measurements ( Figure 4 ) revealed a significant difference in the organization of GFP tags, and consequently, Smurf2. The over-expressed functional Smurf2, formed conformation clusters, as is typical for activated proteins (see for instance [ 52 ]) in a more equally distributed environment of single proteins. The over-expressed non-functional Smurf2-CG showed a much stronger formation of clusters in a random environment, which may be due to vesicle formation.…”