Abstract
Polarized epithelia assemble into sheets that compartmentalize organs and generate tissue barriers by integrating apical surfaces into a single, unified structure. This tissue organization is shared across organs, species, and developmental stages. The processes that regulate development and maintenance of apical epithelial surfaces are, however, undefined. Here, using an intestinal epithelial-specific knockout (KO) mouse and cultured epithelial cells, we show that the tight junction scaffolding protein zonula occludens-1 (ZO-1) is essential for development of unified apical surfaces in vivo and in vitro We found that U5 and GuK domains of ZO-1 are necessary for proper apical surface assembly, including organization of microvilli and cortical F-actin; however, direct interactions with F-actin through the ZO-1 actin-binding region (ABR) are not required. ZO-1 lacking the PDZ1 domain, which binds claudins, rescued apical structure in ZO-1-deficient epithelia, but not in cells lacking both ZO-1 and ZO-2, suggesting that heterodimerization with ZO-2 restores PDZ1-dependent ZO-1 interactions that are vital to apical surface organization. Pharmacologic F-actin disruption, myosin II motor inhibition, or dynamin inactivation restored apical epithelial structure in vitro and in vivo, indicating that ZO-1 directs epithelial organization by regulating actomyosin contraction and membrane traffic. We conclude that multiple ZO-1-mediated interactions contribute to coordination of epithelial actomyosin function and genesis of unified apical surfaces.
Highlights
Polarized epithelia assemble into sheets that compartmentalize organs and generate tissue barriers by integrating apical surfaces into a single, unified structure
We found that U5 and GuK domains of zonula occludens-1 (ZO-1) are necessary for proper apical surface assembly, including organization of microvilli and cortical F-actin; direct interactions with F-actin through the ZO-1 actin-binding region (ABR) are not required
Expression of free EGFP had no effect on apical F-actin accumulation or membrane distensions, but EGFP-tagged full-length ZO-1 localized to normal-appearing tight junctions and restored both F-actin organization and a uniform apical surface (Fig. 3, B and C)
Summary
In vivo ZO-1 knockout disrupts apical cell structure and apical tissue architecture. The obligate role of ZO-1 during embryogenesis [20] has limited in vivo study of ZO-1 to date. Expression of free EGFP had no effect on apical F-actin accumulation or membrane distensions, but EGFP-tagged full-length ZO-1 localized to normal-appearing tight junctions and restored both F-actin organization and a uniform apical surface (Fig. 3, B and C) This was confirmed by SEM, which demonstrated restoration of normal microvillus brush border structure (Fig. 3D). Direct comparisons show a direct correlation between increased EGFP fluorescence intensity and increased cell height (Fig. 4, A and C) These data suggest that, did ZO-1⌬U5-GuK fail to restore cortical F-actin and apical tissue architecture, but it exacerbated the defect induced by ZO-1 KD. ZO-1⌬GuK localized to the tight junction and restored cortical F-actin, microvillus architecture, cell height, and apical surface in a manner similar to full-length ZO-1 F, diagram depicting regular apical surface in a WT cell (left), abnormal F-actin accumulation and apical distension in a ZO-1 KD cell (middle), and restoration of normal apical architecture upon pharmacologic disruption of F-actin in a ZO-1 KD cell (right)
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