| Summary: | Epithelia are contiguous sheets of cells that line body surfaces to create barriers between inside and out and allow for compartmentalization of multicellular organisms. To form and renew, epithelia undergo morphogenesis, which entails establishing and maintaining apico-basal polarity and cell-cell junctions. My work has focused on a role for the tight junction scaffolding protein zonula occludens (ZO)-1 in morphogenetic processes in both two- and three-dimensional systems.
In two-dimensional systems, I found that ZO-1 is critical for epithelia to generate a uniform apical surface with organized microvilli both in vitro and in vivo. Apical surfaces of ZO-1 deficient monolayers were characterized as distended with thin, membranous extensions from the apical surface. Apical distensions were accompanied by striking changes in cortical actin both at cell-cell junctions and at the apical membrane. Additionally, delivery of apical membrane components was enhanced in apically-distended ZO-1 deficient monolayers. Mutagenesis studies indicated that multiple domains within the N-terminus of ZO-1 were necessary for ZO-1 to promote formation of a uniform apical surface. Despite controlling cortical actin organization, a direct interaction between ZO-1 and the F-actin cytoskeleton was not necessary for ZO-1 to organize cortical actin or apical cell architecture. Treatment with inhibitors of actomyosin, small GTPases, and vesicle trafficking during apical surface generation revealed a dependence of apical distensions on activity of both the actomyosin cytoskeleton and dynamin II. Interestingly, treatment with inhibitors of actomyosin and vesicle trafficking were each able to rescue both actomyosin organization and excess delivery of apical membrane, suggesting that 1) these processes are tightly linked and 2) that ZO-1 mediates functional crosstalk between them. Thus, ZO-1 orchestrates multiple cellular processes to control apical epithelial architecture.
When studying a role for ZO-1 in three-dimensional morphogenesis, I found that ZO-1 deficient cells were unable to form well-polarized single lumen cysts. Multiple lumen ZO-1 deficient cysts were characterized by a delay in initial lumen formation, proliferative imbalance at later stages of development, and misoriented cell division. Supplementing ZO-1 deficient cells with EGFP-tagged mutants of ZO-1 revealed a partial dispensability for the ZO-1 actin binding region (ABR), and the requirement for the ABR depended on the strength of external polarization cues. These studies also revealed an absolutely critical role for the U5-GuK region in establishing well-polarized, single lumen cysts. Depletion of each of the three known U5-GuK binding partners (Shroom2, alpha-catenin, and occludin) from parental cells revealed that occludin is also necessary for formation of single lumen cysts, which suggests that ZO-1 mediated recruitment of occludin to tight junctions may promote lumen formation.
These studies highlight previously unappreciated roles for tight junctions, and particularly the tight junction scaffolding protein ZO-1, in establishing epithelial architecture in both in vitro and in vivo . These results will direct future work in uncovering the protein-protein interactions that ZO-1 mediates to orchestrate morphogenetic processes and provide a foundation upon which to begin exploring potential roles for ZO-1 in disease pathogenesis.
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