Figure 4

A nuclear indentation detected at the interface between the invadopodia core and the juxtaposed nucleus.

(A) A375 cells were transfected with LifeAct-GFP (green) and mCherry-vinculin (red) and plated on gelatin-coated coverslips for 2 h. Fluorescence microscopy of the double-labeled cell is shown in the left panel. The invadopod marked as #1 was further examined by FIB-SEM (A′). A FIB-SEM image revealed an indentation in the nucleus, coinciding with the interface between the dorsal tip of the invadopod core actin bundle and the nuclear membrane (red arrow). (B) 3D reconstruction of the FIB-SEM view of the whole cell presented in (A). Red arrows mark three nuclear indentations. (C) TEM image of an invadopod (side view), showing the core actin bundle (dense area, devoid of organelles) (Arrow 1), the nuclear indentation and the invasion into the gelatin layer (Arrow 2). (D) A375 cells, cultured for 2 h on a gelatin-Alexa 350-coated glass- bottomed dish. The images (from left to right): (a) actin, marker for invadopodia core. Note the invadopodia, indicated by the arrow; (b) Lamin A/C imaged by TIRF microscopy. The dark region indicated by the arrow corresponds to the invadopodia-associated indentation in the nucleus; (c) Lamin A/C imaged by epi-fluorescence in a higher focal plan, showing an intact nuclear lamina; (d) Fluorescently-tagged gelatin. The dark spot indicated by the arrow corresponds to the area degraded by the invadopod; (e) Overlay of actin (red), Lamin A/C TIRF (green) and Lamin A/C epi-fluorescence (blue). (E) TIRF imaging of the nuclear indentation, using variable TIRF angles and producing evanescence layers of increasing thickness enabling the imaging of the entire indentation. From left to right, three TIRF angles (Z = 1–3) show different focal planes of the indentation. Top-down view of three-dimensional reconstruction of the indentation (yellow), invadopodia-associated F-actin bundle, inside the indentations, is shown in white. The reconstruction is for illustration only and was performed by setting an artificial Z pixel size (in the order of the X-Y pixel size). Triple overlay of actin (red), Lamin A/C TIRF (green) and Lamin A/C epi-fluorescence (blue).