发现表皮生长因子受体2(ErbB2)在几种癌症中过度表达,比如胃,和乳腺癌,而且是,因此,一个重要的治疗靶点。ErbB2在癌细胞侵袭中起着核心作用,并与细胞骨架重组有关。为了研究单个ErbB2蛋白与肌动蛋白丝的空间相关性,我们应用相关荧光显微镜(FM),和扫描透射电子显微镜(STEM)成像特别标记的SKBR3乳腺癌细胞。乳腺癌细胞在微芯片上生长,转化表达肌动蛋白-绿色荧光蛋白(GFP)融合蛋白,并标记有量子点(QD)纳米颗粒,该纳米颗粒附着在特定的抗ErbB2抗体上。进行FM以鉴定具有空间相关的肌动蛋白和ErbB2表达的细胞区域。对于完整细胞的完整质膜的STEM,细胞被固定并用石墨烯覆盖。检查了富含肌动蛋白的皱褶膜区域中ErbB2的空间分布模式,并与同一细胞的相邻肌动蛋白低区域进行比较,揭示了推定的信号传导活性ErbB2同源二聚体与富含肌动蛋白的区域的关联。发现肌动蛋白低膜区不存在ErbB2同源二聚体,以及用细胞松弛素D处理细胞后,会破坏更大的肌动蛋白丝。在后面的两个数据集中,识别出36nm的显著标签间距离,可能表明通过与表皮生长因子受体(EGFR)形成ErbB2的异二聚体间接连接到螺旋肌动蛋白丝。通过识别富含肌动蛋白的区域中的线性QD链,进一步探索了对肌动蛋白丝的可能附着。这也显示了36nm的标记间距离。
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.