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The V-shaped arrangement of hair bundles on cochlear hair cells is critical for auditory sensing. However, regulation of hair bundle arrangements has not been fully understood. Recently, defects in hair bundle arrangement were reported in postnatal Dishevelled-associating protein (ccdc88c, alias Daple)-deficient mice. In the present study, we found that adult Daple−/− mice exhibited hearing disturbances over a broad frequency range through auditory brainstem response testing. Consistently, distorted patterns of hair bundles were detected in almost all regions, more typically in the basal region of the cochlear duct. In adult Daple−/− mice, apical microtubules were irregularly aggregated, and the number of microtubules attached to plasma membranes was decreased. Similar phenotypes were manifested upon nocodazole treatment in a wild type cochlea culture without affecting the microtubule structure of the kinocilium. These results indicate critical role of Daple in hair bundle arrangement through the orchestration of apical microtubule distribution, and thereby in hearing, especially at high frequencies.

Nakayama et al. show that Daple is responsible for the increased microtubule (MT) density on the Frizzled side of the apical junctional complex (AJC) in multiciliated cells via bundling and stabilizing MTs and mediating interactions between MTs and the AJC., Multiciliated cells (MCCs) in tracheas generate mucociliary clearance through coordinated ciliary beating. Apical microtubules (MTs) play a crucial role in this process by organizing the planar cell polarity (PCP)–dependent orientation of ciliary basal bodies (BBs), for which the underlying molecular basis remains elusive. Herein, we found that the deficiency of Daple, a dishevelled-associating protein, in tracheal MCCs impaired the planar polarized apical MTs without affecting the core PCP proteins, causing significant defects in the BB orientation at the cell level but not the tissue level. Using live-cell imaging and ultra-high voltage electron microscope tomography, we found that the apical MTs accumulated and were stabilized by side-by-side association with one side of the apical junctional complex, to which Daple was localized. In vitro binding and single-molecule imaging revealed that Daple directly bound to, bundled, and stabilized MTs through its dimerization. These features convey a PCP-related molecular basis for the polarization of apical MTs, which coordinate ciliary beating in tracheal MCCs.

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.

The availability of in vitro cell culture derived from local breed provides an opportunity for tackling problems related to the preservation of its genetic materials and can potentially be applied for downstream in vitro-based studies. Here, we established primary fibroblast cell culture from Pelung chicken, then explored its growth characteristic and potential uses for wound healing assay and cytotoxicity tests of medicinal bioactive compounds. Fibroblast cells were isolated from embryonic skin tissue and maintained in DMEM-FBS media. Wound healing assay was performed by creating a “scratch” in the cell monolayer, followed by capturing periodic images of migrating cells. Cell viability was measured using trypan blue dye exclusion assay in various doses of Centella asiatica L. leaf extract. Cells outgrowth from the skin explant revealed a typical morphology of fibroblast-like cells that reached maximum growth at 7.95 × 104 cells/cm2 after 5 days. With continuous passage, the population of the cells became more homogeneous and population doubling time increased. In the wound healing assay, cells migrated towards the wound area within 24 hours, suggesting their ability to normally respond to chemical cues. In cytotoxicity test, cells’ viability corresponded in a dose-dependent manner with the amount of C. asiatica extract tested into the culture.

Herawati et al. developed a long-term and high-resolution live imaging system for cultured mouse tracheal multiciliated cells. Using both experimental and theoretical studies, they reveal the developmental principle of ciliary basal body alignment directed by apical cytoskeletons., 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.