Apical Cytoskeletons Help Define the Barrier Functions of Epithelial Cell Sheets in Biological Systems
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Epithelial cell sheets cover every compartment of the vertebrate body, from tiny capillaries to the entire body surface. We found that specific apical cytoskeletons are associated with the cell sheet’s apical membranes and tight junctions (TJs). Thus, we defined this set of structures as a system called the “TJ-apical complex.” These structures presumably determine the transcellular and paracellular barrier characteristics of the cell sheet, and thus its overall epithelial barrier function. We recently explored the specific function of the apical cytoskeletons in tracheal multiciliated cells. During the differentiation of these cells, the basal bodies are regularly aligned just beneath the apical membrane, which ultimately lead to cilia that beat in a coordinated manner to move the surrounding fluid. To examine the dynamic features of the basal bodies in these cells, we developed a novel high-resolution, long-term, live-imaging system. We also performed a biotheoretical analysis that revealed the role of the apical microtubules in aligning the basal bodies in the apical plane which also includes the TJs. Here we discuss the molecular composition and physiological roles of the TJ-associated apical cytoskeleton in both general and multiciliated epithelial cell sheets.
KeywordsEpithelium Tight junction Cell-cell adhesion Cytoskeleton Microtubule Cilia Live imaging Multiciliated cell
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).
4.2 The Apical Cytoskeletons in General Epithelial Cells
In confluent epithelial cell sheets, each cell is highly polarized in the apico-basal direction, and the cells’ apical membranes are regarded as a continuous surface connected by TJs. Although the mechanism by which TJs are positioned at the most apical part of the lateral membrane is not understood, the TJs determine the edges of each cell’s apical membrane when the cell sheet is viewed from the top (the apical view). Distinct, specific differentiation mechanisms occur in the apical area. Well-known classical examples of apical differentiation are the circumferential ring at cell-cell boundaries and the terminal web (Leblond et al. 1960; Hull and Staehelin 1979; Owaribe et al. 1981; Burgess 1982; Keller et al. 1985). In Drosophila epithelial cells, a “ratchet structure” consisting of actin filaments exists in the medial area of apical membranes (Martin et al. 2009), although no similar structure has been clearly identified in mammalian epithelial cells. By applying our super-resolution microscopy system to cultured epithelial cell sheets, we discovered the detailed structure of the apical cytoskeleton, which was previously unknown and uniquely distributed beneath the apical membrane like a shell (Yano et al. 2013). This location led us to propose that the apical cytoskeletal network is associated with TJs, which are located at the most apical part of the lateral membrane, and that these structures form a system called the “TJ-apical complex.” However, to establish this new point of view, we needed to acquire evidence at the molecular level for the association of the apical cytoskeleton with the TJs.
We further examined the apical cytoskeleton in detail, by performing ultra-high voltage electron microscopy experiments in which the microtubules and intermediate filaments were tracked. In general epithelial cells in culture, the apical microtubules and intermediate filaments were distributed in the apical plane in rather uniformly scattered patterns and partly overlapped each other, without any specific patterns in their distribution (Tateishi et al. 2017). In this respect, it is notable that we identified four microtubule-associated proteins, which also associate with TJs, in our recent findings, TJ-associated microtubule-binding proteins. We believe that analyses of the TJ-associated microtubule-binding proteins, which may form the platform for associations among the apical cytoskeletons, TJs, and cell signaling molecules, represent a unique direction for studying epithelial cell sheets and epithelial barriers.
4.3 The Apical Cytoskeletons in Multiciliated Cells, a Possible Extreme Example of a “TJ-Apical Complex” with a Clear Function
MCCs drive fluid transport through coordinated ciliary beating, the direction of which is established by the BB orientation of hundreds of cilia on one cell (Salathe 2007). In airway MCCs, the BBs are uniformly oriented and linearly aligned by an unknown mechanism. To explore the mechanism for BB alignment, we observed GFP-centrin2-labeled BBs in mouse tracheal MCCs in primary culture using our long-term, high-resolution, live-imaging method (Herawati et al. 2016). We found that the differentiating BB arrays sequentially adopted four stereotyped patterns: a clustering “Floret,” “Scatter,” “Partial alignment,” and linear “Alignment” pattern. During this acquisition of regularity, we particularly noted that the patterns and densities of microtubules in the apical plane of the MCCs were well correlated with the BB patterning. In addition, the BB alignment was perturbed by disrupting the apical microtubules with nocodazole or by a basal foot (BF)-depleting Odf2 mutation. Based on these experimental results, we explored the development of BB alignment from random to the final well-ordered pattern biotheoretically. We found that the self-organization could be explained by applying hydrodynamic theories in which the apical cytoskeletons were treated as a two-dimensional viscous fluid that underwent a contractile force mediated by cytoskeletal motors and filament polymerization (Marchetti et al. 2013; Prost et al. 2015; Herawati et al. 2016). These results revealed the functional importance of the cytoskeletal components that exist in the apical plane of the epithelial cell sheet in tracheal MCCs. Although the relationship between the apical cytoskeletons and TJs remains to be elucidated, their locations and binding molecules suggest that they are closely related both physically and functionally in MCCs. How the apical cytoskeleton is built by the TJ and its related signaling is another critical issue that remains to be explored.
Epithelial cell sheets with a differentiated apical side are formed and organized by mechanisms involving apicobasal polarity, the details of which have been well addressed in other reviews (Shin et al. 2006; Nelson 2009; Rodriguez-Boulan and Macara 2014; Apodaca 2017). In general, to establish the apicobasal polarization in epithelial cell sheets, polarity proteins generate asymmetric membrane domains that form the basis for establishing the cell–cell adhesive TJs and adherens junctions (AJs), which combine to form apical junctional complexes (AJCs). In addition, planar cell polarity (PCP), which forms in the apical plane of epithelial cell sheets, is arranged perpendicular to the basolateral polarity. The actin filaments of the circumferential ring also lie horizontally along the apical membrane at cell boundaries. The “TJ-apical complex” expands horizontally below the apical membrane in an almost evenly scattered pattern. Since it includes TJs, it is likely to play a role in the paracellular barrier. On the other hand, since it is directly or indirectly associated with the apical membranes of epithelial cells, it probably also has a role in the transcellular barrier. Thus, the epithelial barrier is created and regulated by the combination of paracellular and transcellular barriers, which are determined, at least in part, by the “TJ-apical complex.” Our continued investigation of the “TJ-apical complex” in ciliated and non-ciliated epithelial cells is expected to unveil its physiological significance in a variety of biological epithelial barrier systems.
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