From this, the functional state of ErbB2 was determined as described in the following section. Statistical Method to Determine Functional State of ErbB2 In order to distinguish ErbB2 proteins forming homodimers from ones that were randomly grouped at closer vicinities, the label position data was statistically analyzed using the pair correlation function, with being 0.02 of the 200 nm in this case, and the factor of three originating from the Rose criterion (Rose, 1973). membrane ErbB2 in red. Cell marked with ? was lost during EM preparation process. (B) Corresponding low magnification scanning transmission electron micrograph of graphene covered breast cancer cells taken at the same area, Magnification = 1000. (C) High resolution scanning transmission electron (STEM) micrographs of region marked in a-b. QD-labels appear as white dots, = 100,000. (D) Pair correlation function of QD-labeled ErbB2 in flat regions without actin-GFP. Data_Sheet_1.PDF (2.8M) GUID:?0D6527ED-0B78-4093-AAFA-A14DD91207BA FIGURE S3: Correlative fluorescence microscopy and STEM of whole breast cancer cells after treatment with Cytochalasin D. (ACC) Cropped fluorescence micrographs of SKBR3 breast cancer Tipifarnib (Zarnestra) cells showing cellular actin-GFP in green, and QD-labeled membrane ErbB2 in red. Areas were both signals overlay appears yellow. Image A was taken at the baseline before treatment with Cytochalasin D (1 h, 2 M) and staining of QDs. Image B was acquired immediately after Cytochalasin D treatment and labeling of ErbB2 with QDs. Image C was generated of maximum intensity overlays of deconvoluted z-stacks from the same spot as in A and B. Cells that disappeared during Cytochalasin D Rabbit Polyclonal to NKX3.1 treatment and EM preparation process are marked with white asterisks in image A. (D) Corresponding low magnification scanning transmission electron micrograph of graphene covered breast cancer cells taken at the same area, = 1000. (E,F) High resolution scanning transmission electron micrographs of region marked in aCd. QD-labels appear as white dots in (F). E: = 30,000 , F: = 100,000. (G) Graph of pair correlation function of QD-labeled ErbB2 in peripheral cellular regions containing actin-GFP as displayed in (ACF). Note the peak at 20 nm which is similar to the one of actin-rich areas in untreated cells. Data_Sheet_1.PDF (2.8M) GUID:?0D6527ED-0B78-4093-AAFA-A14DD91207BA TABLE S1: Summary of scanning transmission electron microscopy (STEM) data used for label distribution analysis of STEM images acquired at actin-rich regions of Cytochalasin D (Cyt D) treated SKBR3 breast cancer cells that were transduced with actin-green fluorescent protein (GFP). Data_Sheet_1.PDF (2.8M) GUID:?0D6527ED-0B78-4093-AAFA-A14DD91207BA Data Availability StatementThe datasets generated for this study are available on request to the corresponding author. 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 Tipifarnib (Zarnestra) 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. = 63 pA, and an electron dose of 67 eC/?2 for a magnification of 100,000 (the pixel size was 1 nm with 2048 2048 image resolution). The camera length was set to 8 cm, leading to Tipifarnib (Zarnestra) a detector opening semi-angle Tipifarnib (Zarnestra) of 43 mrad for the angular dark field detector. This relates to the opening through which electrons pass the detector, although, the actual collection angle is larger. QD-Label Analysis For the detection of QD-labeled ErbB2 membrane proteins in the STEM micrographs, an automated analysis programmed in ImageJ (National Institute of Health, United States) was applied (Peckys et al., 2015). To avoid false counting of label positions, STEM images were preprocessed by checking for contaminating, electron-dense particles different from QDs that are of a large size. In case contaminating particles were present, STEM micrographs were.