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. 2010 Mar;21(3):478–488. doi: 10.1681/ASN.2009070698

Figure 4.

Figure 4.

ZONAB is expressed in peripheral proliferating cells but not in differentiated central cells. Sparsely plated OK cells were cultured for 4 days, allowing individual cell clustering and reorganization into small colonies, with opposite status between peripheral and central cells. Islands were examined by confocal microscopy (A, B, and F; bars = 10 μm) and scanning electron microscopy (C through E; bars = 2 μm). (A) Double immunolabeling for ZO-1 (red) and megalin (green); boxed area at top left is enlarged at bottom. Whereas central cells are equipped by continuous polygonal junctional belts (ZO-1), with little or no detectable ZO-1 in the cytoplasm, junctions of peripheral cells are by necessity interrupted at their outer margin: Their ZO-1 belt seems to be reinforced at the border of the island (arrowheads), then abruptly disappears. Peripheral cells show an additional diffuse cytoplasmic and perinuclear ZO-1 labeling. Note the strong expression of megalin in all central cells, contrasting with its poor labeling in the peripheral cell rim (arrows). (B) Double immunolabeling for megalin and either β-catenin (top) or ZONAB (bottom). Note the strong nuclear labeling in peripheral cells by ZONAB but not by β-catenin (similar pattern to that of ZO-1). The opposite immunolabeling pattern for ZONAB and megalin between adjacent cells is obvious at the merge shown at bottom right. (C through E) Scanning electron microscopy. The low-power image in C shows a peripheral cell with a ruffled membrane at its outer margin (arrowhead) and scattered individual microvilli elsewhere (better seen in D), contrasting with the adjacent polarized cell shown below, where tips of microvilli cluster as asters (better seen in E); emerging primary cilia were not detected in islands after 4 d. (F) Relation between ZONAB and proliferation. OK cells incorporated EdU for 1 hour for monitoring of entry into the S phase, then were immunolabeled for ZONAB. Selective peripheral nuclear ZONAB labeling coincides with EdU incorporation (arrowheads).