Journal: eLife

Article Title: AMP-activated protein kinase fortifies epithelial tight junctions during energetic stress via its effector GIV/Girdin

doi: 10.7554/eLife.20795

Figure Lengend Snippet: ( A ) Subconfluent monolayers of MDCK cells grown on cover slips were fixed and stained for α-Tubulin (green), pS245-GIV (red) and DAPI (blue; nuclei) and analyzed by confocal microscopy. Image A shows an xy-plane maximum projection of a deconvolved stack of images, acquired at 20-nm z-intervals using a Olympus cellSens deconvolution (constrained iterative) and restoration system (Olympus FV3000) and a Apochromat 60XOSC2, NA 1.4 objective. The total thickness of the image stack is 4.8 mm. Two regions of interest (ROI), 1 and 2, are indicated with white boxes. ( B–D ) 3D reconstruction of each ROI shows the close proximity of pS245-GIV (red) to connecting microtubules (green) running along the cell-cell junctions (arrowheads). Little or no pS245-GIV staining (red pixels) is seen on microtubules at the 'free' cell border in panel D (arrows). ( E – H ) Maximum projected ROI‘s 1 and 2 are magnified in E and G , respectively. Individual Z-stacks of each ROI is shown in . Single Z-stacks of ROI‘s 1 and 2 are displayed in F and H , respectively. The insets in panels F and H show red, green and merged channels of respective figures. The white line indicates the pixels used for generating the RGB profile plots shown in I and J . RGB profiles show that pS245-GIV (red pixels) often co-localizes either completely with microtubule tracks (green pixels; I ) or lay between two microtubule tracks ( J ) at the cell-cell junctions. DOI: http://dx.doi.org/10.7554/eLife.20795.015

Article Snippet: Images were processed using the 3D deconvolution and 3D reconstructions tools of the Olympus cellSens (version Dimension Desktop 1.15) software.

Techniques: Staining, Confocal Microscopy