Fig 1.
Labeling approaches for focal adhesions and HER2 used in this study.
Focal adhesion complexes are formed once integrins (orange) bind to the extracellular matrix (cyan). Focal adhesion complex proteins in the cytoplasm (inside, upper part) connect the focal adhesion to the actin cytoskeleton (purple). Human epidermal growth factor receptor 2 (HER2) (pale green, indicated as a receptor dimer) is located in the membrane near focal adhesions and may interact with members thereof. For extracellular labeling of HER2 (outside, lower part), a biotin (small red) conjugated anti-HER2 affibody was used. For intracellular labeling of HER2 (inside, upper part), an anti-HER2 biotinylated antibody directed against the intracellular domain of HER2 was used. The primary biotinylated label was linked to a streptavidin (blue)-coated quantum dot nanoparticle (strept-QD, magenta). Note that for the affibody, one biotin binding site is available, whereas the antibody carries multiple biotins. Expression of green fluorescent protein (GFP) or enhanced green fluorescent protein (eGFP) (green) fused talin or vinculin was used to mark focal adhesion spots.
Fig 2.
Co-labeling of focal adhesion markers vinculin or talin with HER2.
Direct interference contrast (DIC) and wide field fluorescence images of SKBR3 cells seeded on glass-bottom dishes. The cells were either transfected with eGFP-vinculin (A and B) or transduced with talin- GFP (C) to label focal adhesion spots (GFP signal, magenta arrow heads). HER2 was labeled by biotinylated anti-HER2 affibody coupled to strept-QD (HER2-Aff-QD). (A) The focus was adjusted to the basal side of the cells at the interface with the substrate. Images were acquired using a 63x objective. (B) Similar images as in (A), but with the focus adjusted to apical side of the cell. (C) Images of SKBR3 cells with labeled HER2 and transfected with talin-GFP. Images were acquired using a 40x objective. Colors in merged images: yellow for GFP and cyan for HER2-Aff-QD. Scale bars: 20 μm and 10 μm for the insets.
Table 1.
Information is provided about the labeling method, the microscopy method, the total number of analyzed images, the number of analyzed cells, and the amount of focal adhesion marker expressing cells. Figs 2–4 show representative images for each experiment (see also related S1–S3 Figs).
Fig 3.
Analysis of intracellular HER2 labeling and co-labeling with talin confirms HER2 expression at the cell substrate interface.
(A and B) Total internal reflection fluorescence (TIRF) microscopy of SKBR3 cells transduced with talin-GFP on glass-bottom dishes analyzed with a 100x oil TIRF optimized objective. The intracellular domain of HER2 was labeled with strept-QD via biotinylated anti-HER2 antibody coupled to strept-QD (HER2-QD). Outlined regions indicate HER2-low expression areas. (C) Control experiment using the same protocol as A and B, but without anti-HER2 antibody. (D) Box plot graph displaying the ratios of talin-GFP to HER2-QD corrected integrated fluorescence intensities (CFI) measured at focal adhesions (left) and HER2-enriched regions (right). (E) Box plot graph displaying the CFI for HER2-QD measured at focal adhesions or at HER2-enriched regions. (D, E) **** for p < 0.001, unpaired, two-tailed t-test, n = 339 for talin spots and n = 95 for HER2-enriched regions, obtained from 16 different cells expressing variable amounts of talin. Box plot graphs show min to max representations with indicated median value with each point representing one measurement. Colors in merged images: yellow for GFP and cyan for strept-QD. Scale bars: 20 μm and 5 μm for insets.
Fig 4.
Correlative light- and electron microscopy (CLEM) of intracellularly HER2-QD, and talin-GFP at focal adhesions.
(A-E) Example image of a SKBR3 cell grown on a silicon nitride window of a silicon microchip suitable for correlative light- and electron microscopy. Shown are DIC (A), fluorescence images for talin-GFP (B), HER2-QD (C), the fluorescent channel overlay image (D), and an 800x low magnification (low mag.) bright field scanning transmission electron microscopy (STEM) image (E) of the same cell. HER2 low regions are outlined in blue. The rectangle outlined in red indicates the magnified region shown in (F) acquired with dark field STEM and overlaid with the corresponding magnified merge fluorescent image. (G) displays a zoom of the highlighted region in blue in (F). A further magnified region is shown in the inlet (white rectangle). Note that the talin-positive region shows a lower abundance of HER2-QDs (yellow stripe). (H) Box plot graph displaying the ratios of talin-GFP to HER-QD CFIs measured at focal adhesions (left) and HER2-enriched regions (right). (I) Box plot graph displaying the CFI for HER2-QD measured at focal adhesions or at HER2-enriched regions. **** for p < 0.001, unpaired, two-tailed t-test, n = 192 for talin spots and n = 48 for HER2-enriched regions. (J) Box plot graph displaying the HER2-QD density measured from the STEM image for focal adhesion versus non-focal adhesion regions. ** for p < 0.01 unpaired, two-tailed t-test, n = 14 for both talin spots and non-talin spots. Graphs display min to max representations with indicated median value. Each point represents one CFI measurement (H and I) or the particle density of one analyzed image (J). Colors in merged images: yellow for GFP and cyan for HER2-QD. Scale bars: 10 μm in (A), 2 μm (F), 0.5 μm and 0.2μm for (G).