Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin.

Indra Navina Dahmke,Patrick Trampert,Florian Weinberg,Zahra Mostajeran,Niels De Jonge,Franziska Lautenschläger

doi:10.3389/fcell.2020.00521

Abstract

Epidermal growth factor receptor 2 (ErbB2) is found overexpressed in several cancers, such as gastric, and breast cancer, and is, therefore, an important therapeutic target. ErbB2 plays a central role in cancer cell invasiveness, and is associated with cytoskeletal reorganization. In order to study the spatial correlation of single ErbB2 proteins and actin filaments, we applied correlative fluorescence microscopy (FM), and scanning transmission electron microscopy (STEM) to image specifically labeled SKBR3 breast cancer cells. The breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and labeled with quantum dot (QD) nanoparticles attached to specific anti-ErbB2 Affibodies. FM was performed to identify cellular regions with spatially correlated actin and ErbB2 expression. For STEM of the intact plasma membrane of whole cells, the cells were fixed and covered with graphene. Spatial distribution patterns of ErbB2 in the actin rich ruffled membrane regions were examined, and compared to adjacent actin-low regions of the same cell, revealing an association of putative signaling active ErbB2 homodimers with actin-rich regions. ErbB2 homodimers were found absent from actin-low membrane regions, as well as after treatment of cells with Cytochalasin D, which breaks up larger actin filaments. In both latter data sets, a significant inter-label distance of 36 nm was identified, possibly indicating an indirect attachment to helical actin filaments via the formation of heterodimers of ErbB2 with epidermal growth factor receptor (EGFR). The possible attachment to actin filaments was further explored by identifying linear QD-chains in actin-rich regions, which also showed an inter-label distance of 36 nm.

Highlights

The membrane protein ErbB2 named HER2 is a member of the receptor tyrosine kinase family of epidermal growth factor receptors together with the epidermal growth factor receptor (EGFR), named ErbB1, ErbB3, and ErbB4
A correlative microscopy method was established to analyze the spatial correlation of the functional state of ErbB2 with the abundance of actin in whole cancer cells (Figure 1)
SKBR3 breast cancer cells grown on silicon microchips were first transduced with an actin-green fluorescent protein (GFP) baculovirus construct, and cultivated for 40 h

Summary

Introduction

The membrane protein ErbB2 named HER2 is a member of the receptor tyrosine kinase family of epidermal growth factor receptors together with the epidermal growth factor receptor (EGFR), named ErbB1, ErbB3, and ErbB4. ErbB2 plays a key role in cancer cell growth, and metastasis and, as such, resembles an important target for cancer treatment ErbB2 can homodimerize with another ErbB2 protein, or heterodimerize with other tyrosine kinases leading to the activation of the respective cell signaling pathways (Citri and Yarden, 2006). Overexpression of ErbB2 affects the actin cytoskeleton, and plays a role in the metastatic progression in cancer cells (Brix et al, 2014; Day et al, 2017), which resembles the major cause of mortality (Dillekas et al, 2019). HER2 homodimers were found to preferentially reside in membrane ruffles (Peckys et al, 2015)

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