Epithelial cells adhere to each other by tight junctions (TJs) to form cell sheets, which is a critical step in epithelial barrier creation and the morphogenesis of vertebrate tissues (Fleming et al. 2000; Tsukita et al. 2001; Anderson et al. 2004; Furuse and Moriwaki 2009; Van Itallie and Anderson 2014; Tanaka et al. 2017). The apical surface of an epithelial cell sheet faces the outer environment, such as the lumen in the intestinal tract or the environment outside the skin surface. In the sheet, the cells’ apical membranes are regarded as a continuous, connected surface, in which the cell-cell adhesion sites are cemented by TJs. Notably, each epithelial cell exhibits basolateral polarity, and therefore the apical surface of an epithelial cell sheet differs from the basolateral one, and possesses specific features that relate to its roles in a particular biological functional system (Nelson 2009; Apodaca 2017). Apical differentiation is a popular topic of study, and includes microvilli, cilia, circumferential rings (Nelson 2009; Apodaca 2017), and ratchet structures, which have been described in Drosophila but not yet in vertebrates (Martin et al. 2009). Consistent with the critical and varied roles of the apical surface, we recently identified a 3-layered cytoskeletal network of actin filaments, intermediate filaments, and microtubules that exists just below the apical membrane of epithelial cell sheets. The apical cytoskeletons are presumably organized under the control of the TJs and regulate epithelial morphogenesis and barrier functions in conjunction with TJ formation and TJ-based cell signaling. Thus, we propose to define this set of structures as a system called the “TJ-apical complex” (Yano et al. 2017).
A natural question is why this apical cytoskeletal system was not discovered until recently. It is a very thin-layered structure mainly composed of actin filaments, intermediate filaments, and microtubules, which appear as a continuous structure extending horizontally just beneath the apical plasma membrane. We discovered this network using an advanced imaging system, including ultrahigh voltage electron microscopic tomography and confocal super-resolution microscopy, that we developed (Kunimoto et al. 2012; Yano et al. 2013; Tateishi et al. 2017). Although the molecular mechanisms and physiological roles of the “TJ-apical complex” remain to be elucidated, we recently identified four TJMAPs (TJ Microtubule-associated Proteins; previously called J-MAPs), including cingulin which are TJ-associated proteins that bind to the apical cytoskeletons (Yano et al. 2013). During the morphogenesis of epithelial cell sheets, apical constriction and the size of the apical area defined by TJs must be kept in balance. Our recent data suggest that TJMAPs may constitute a platform on which the apical cytoskeletons and TJs associate to organize biological systems, although more study is needed to examine this possibility (Fig. 4.1).
Regarding the functions of the “TJ-apical complex,” multiciliated cells represent a highly specific case in which the apical microtubules are particularly highly developed. In tracheal multiciliated cells (MCCs), the apical microtubules have a specific role in establishing the regular arrangement of basal bodies (BBs), which generate cilia in the apical membranes (Kunimoto et al. 2012; Herawati et al. 2016; Tateishi et al. 2017). To explore the differentiation mechanism leading to the regular BB alignment, we examined tracheal MCCs expressing GFP-centrin, a BB-associated protein, using our new long-term, high-resolution, live-imaging system. The microtubule-dependent regular arrangement of BBs is critical for the synchronous beating and metachronal waves of hundreds of motile cilia on the apical membrane of MCCs (Guirao and Joanny 2007; Elgeti and Gompper 2013). Thus, the physiological role of the apical cytoskeleton that we revealed in this case is likely to be essential for tissue function (Fig. 4.2).
By comparing the findings for various epithelial cells, both the common and unique characteristics of the apical cytoskeletal structures are being revealed. In the following sections, we discuss the roles of the apical cytoskeletons, especially microtubules, in epithelial cell sheets that have specific functional relevance (Fig. 4.3).