Figure 2
From: Mechanical interplay between invadopodia and the nucleus in cultured cancer cells
Three-dimensional imaging of invadopodia, using correlative light-FIB-SEM microcopy.
(A) A375 cells co-expressing LifeAct-GFP (green) and mCherry vinculin (red) were plated on gelatin gel-coated gridded coverslips and cultured for 2 h (see Materials and Methods). Left panel: Invadopodia as imaged by fluorescence microscopy, using a 100x/1.3 oil objective. Invadopodia #1–3 were then identified in the FIB-SEM and imaged using the “slice and view” mode (10 nm-thick slices; total thickness of the imaged area: 10 μm). Right panel: Two slices viewed by FIB-SEM. Invadopodia that correspond to the fluorescence image are denoted by the numbers 1 and 2 and by red arrows. NM: nuclear membrane; PM: plasma membrane. (B) Reconstruction of the “top-down view” obtained by the FIB-SEM and showing the slice closest to the matrix (invadopodia marked 1, 2 and 3). (B′) The same FIB-SEM top-down view is shown, overlaid with actin (red) and vinculin (green). (B″) The same FIB-SEM top-down view, overlaid with vinculin (red), showing the adhesion ring surrounding the invadopod core. (B′″) Magnification of invadopod #3′ (as in “B”, shown here in inverted contrast), superimposed on a schematic representation of vinculin (red ring) and F-actin (green) immunofluorescence.
