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. |