Three-dimensional Organization of Layered Apical Cytoskeletal Networks Associated with Mouse Airway Tissue Development

Abstract

The cytoskeleton is an essential cellular component that enables various sophisticated functions of epithelial cells by forming specialized subcellular compartments. However, the functional and structural roles of cytoskeletons in subcellular compartmentalization are still not fully understood. Here we identified a novel network structure consisting of actin filaments, intermediate filaments, and microtubules directly beneath the apical membrane in mouse airway multiciliated cells and in cultured epithelial cells. Three-dimensional imaging by ultra-high voltage electron microscopy and immunofluorescence revealed that the morphological features of each network depended on the cell type and were spatiotemporally integrated in association with tissue development. Detailed analyses using Odf2 mutant mice, which lack ciliary basal feet and apical microtubules, suggested a novel contribution of the intermediate filaments to coordinated ciliary beating. These findings provide a new perspective for viewing epithelial cell differentiation and tissue morphogenesis through the structure and function of apical cytoskeletal networks.

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

“…97 Moreover, in Eph4 cells, a mouse mammary epithelial cell line, apical microtubules that were not associated with centrosomes were found just beneath the apical membrane; such observations suggest that the extent of the apical microtubule network may depend on the specific cell type. 98,99 Consistent with these observations, microtubules were found to be generally required for the restoration of the cell sheet barrier function after Ca 2+ chelation and repletion. 100 With regard to how these apical microtubules associate with the AJCs, in TJ-enriched fractions, several microtubule-associated proteins have been identified.…”

“…In mouse trachea multiciliated cells, the microtubular network has further been shown to have a specific lattice organization . Moreover, in Eph4 cells, a mouse mammary epithelial cell line, apical microtubules that were not associated with centrosomes were found just beneath the apical membrane; such observations suggest that the extent of the apical microtubule network may depend on the specific cell type . Consistent with these observations, microtubules were found to be generally required for the restoration of the cell sheet barrier function after Ca 2+ chelation and repletion …”

“…Recently, networks of apical IFs have been three‐dimensionally revealed by UHVEMT, which showed that they are closely associated with the apical microtubule network (Fig. ) …”

“…All three types of apical cytoskeleton—actin filaments, IFs, and microtubules—may be involved in the formation, function, and maintenance of the apical compartment. The three‐dimensional relationships among these apical cytoskeletons were recently examined in detail and found to have a layered structure in both multiciliated cells and cultured epithelial cells . These layers are located just beneath the plasma membrane and above the terminal web region.…”

“…2). 99 Studies of certain mutations, as well as knockdown and knockout experiments, have revealed that IFs containing heteropolymerization of type I (K18-K20 and K23) and type II (K7 and K8) keratins have roles in epithelial apical processes, such as transport and barrier functions. 106,107 Such IFs are often also associated with desmosomes, which are instrumental in integration of apical components for cellular functions.…”