1 Color WIF-B.jpg (193632 bytes)

1 Morphology.jpg (273409 bytes)

1 Blue-Clip Art.jpg (107658 bytes)

WIF-B cells are highly-polarized. Triple-immunofluorescence labeling of apical (blue), basolateral (red) and Golgi (green) proteins in fully polarized WIF-B cells.  Fixed and permeabilized cells were incubated with anti-HA4, anti-HA321 and anti-albumin antibodies of three different species (mouse, rabbit and guinea pig, respectively) followed by appropriate fluorescently-labeled secondary antibodies.  The fluorescence image of each label was recorded on a separate channel of a confocal laser scanning microscope though three ~1 um optical sections starting ~6 um from the bottom.  The images were merged into one image and pseudocolored.  The apical protein, HA/4cell-CAM105/ ectoATPase, is localized to the membrane lining the dilated spaces, whereas the HA321/BEN protein is found along the basolateral surface and albumin is concentrated in the Golgi.  The nuclear-Golgi-apical axis is clearly evident in these cells.   Phase contrast morphology of living WIF-B cells in culture.  Top: WIF-B cells were plated onto tissue culture plastic and grown for >10 days.  The cells have a polygonal appearance and are closely packed.  Phase-lucent "blisters" between neighboring cells  are clearly evident and correspond to the "bilie canalicular" or apical space.  Bottom: A cross-sectional "view" of the cell monolayer taken along the line indicated in the upper panel.  The phase-lucent spaces are closed off from both the substrate and the overlaying medium. 





ZO-1 marks the borders between apical and basolateral domains.  WIF-B cells were double labeled for the tight junction-associated protein ZO-1 and the apical marker HA4.  Schematic drawing of the BC which is marked by an arrow and/or arrowhead (n=nucleus of adjacent cell).  ZO-1 (arrows) was visualized with rat mAb followed by goat FITC-IgG, and HA4 (arrowheads was stained with rabbit pAb followed by goat Texas red-IgG.  The image of ZO-1 is a compiled z-series taken in 1 um steps, and that of HA4 is a 1 um optical x-y section.  Both images were merged to generate the image in C to illustrate the three dimensional BC architecture.  The belt-like presence of ZO-1 around BC between the participating cells suggests the existence of tight-junctional structures separating apical and basolateral domains.  One can see an intercellular "vacuolar compartment" (v) which is positive for HA4 but has no ZO-1 staining.  Bar, 10 um.  See Ihrke, et all (1993), JCB, 1761-1775 for details.