A microtubule-LUZP1 association around tight junction promotes epithelial cell apical constriction.

Apical constriction is critical for epithelial morphogenesis, including neural tube formation. Vertebrate apical constriction is induced by di-phosphorylated myosin light chain (ppMLC)-driven contraction of actomyosin-based circumferential rings (CRs), also known as perijunctional actomyosin rings, around apical junctional complexes (AJCs), mainly consisting of tight junctions (TJs) and adherens junctions (AJs). Here, we revealed a ppMLC-triggered system at TJ-associated CRs for vertebrate apical constriction involving microtubules, LUZP1, and myosin phosphatase. We first identified LUZP1 via unbiased screening of microtubule-associated proteins in the AJC-enriched fraction. In cultured epithelial cells, LUZP1 was found localized at TJ-, but not at AJ-, associated CRs, and LUZP1 knockout resulted in apical constriction defects with a significant reduction in ppMLC levels within CRs. A series of assays revealed that ppMLC promotes the recruitment of LUZP1 to TJ-associated CRs, where LUZP1 spatiotemporally inhibits myosin phosphatase in a microtubule-facilitated manner. Our results uncovered a hitherto unknown microtubule-LUZP1 association at TJ-associated CRs that inhibits myosin phosphatase, contributing significantly to the understanding of vertebrate apical constriction.

Vinculin is critical for the robustness of the epithelial cell sheet paracellular barrier for ions.

The paracellular barrier function of tight junctions (TJs) in epithelial cell sheets is robustly maintained against mechanical fluctuations, by molecular mechanisms that are poorly understood. Vinculin is an adaptor of a mechanosensory complex at the adherens junction. Here, we generated vinculin KO Eph4 epithelial cells and analyzed their confluent cell-sheet properties. We found that vinculin is dispensable for the basic TJ structural integrity and the paracellular barrier function for larger solutes. However, vinculin is indispensable for the paracellular barrier function for ions. In addition, TJs stochastically showed dynamically distorted patterns in vinculin KO cell sheets. These KO phenotypes were rescued by transfecting full-length vinculin and by relaxing the actomyosin tension with blebbistatin, a myosin II ATPase activity inhibitor. Our findings indicate that vinculin resists mechanical fluctuations to maintain the TJ paracellular barrier function for ions in epithelial cell sheets.

Apical cytoskeletons and junctional complexes as a combined system in epithelial cell sheets.

Epithelial cell sheet formation is central to many aspects of vertebrate development and function. For example, it is a major principle of differentiation in embryogenesis and regeneration, enables the compartmentalization of tissues, and is the basis for the maintenance of homeostasis throughout the body. A key characteristic of biologically functional epithelial cell sheets is a clear difference between the top and bottom sides owing to the apicobasal polarity of the cells in the sheet, as seen in the simple polar epithelia. Epithelial cell sheets are formed by cell-cell adhesion conferred by junctional complexes, in particular via tight junctions (TJs), which thus create a paracellular barrier. This review focuses on the apical side of the sheet, which serves as the front line. The apical membranes and TJs of the various tissues have specific characteristics that enable them to function and adapt to their biological context: each system must be robust, but also dynamic and flexible to maintain homeostasis. Here, we describe various apical cytoskeletal structures that are critical to the integrity of epithelial cell sheets. We also discuss the association of apical cytoskeletal networks with TJs, which thus forms a combined system, tentatively termed the TJ-apical complex.

Multiciliated cell basal bodies align in stereotypical patterns coordinated by the apical cytoskeleton.

Multiciliated cells (MCCs) promote fluid flow through coordinated ciliary beating, which requires properly organized basal bodies (BBs). Airway MCCs have large numbers of BBs, which are uniformly oriented and, as we show here, align linearly. The mechanism for BB alignment is unexplored. To study this mechanism, we developed a long-term and high-resolution live-imaging system and used it to observe green fluorescent protein-centrin2-labeled BBs in cultured mouse tracheal MCCs. During MCC differentiation, the BB array adopted four stereotypical patterns, from a clustering "floret" pattern to the linear "alignment." This alignment process was correlated with BB orientations, revealed by double immunostaining for BBs and their asymmetrically associated basal feet (BF). The BB alignment was disrupted by disturbing apical microtubules with nocodazole and by a BF-depleting Odf2 mutation. We constructed a theoretical model, which indicated that the apical cytoskeleton, acting like a viscoelastic fluid, provides a self-organizing mechanism in tracheal MCCs to align BBs linearly for mucociliary transport.