Formation and function of multiciliated cells

Abstract

In vertebrates, multiciliated cells (MCCs) are terminally differentiated cells that line the airway tracts, brain ventricles, and reproductive ducts. Each MCC contains dozens to hundreds of motile cilia that beat in a synchronized manner to drive fluid flow across epithelia, the dysfunction of which is associated with a group of human diseases referred to as motile ciliopathies, such as primary cilia dyskinesia. Given the dynamic and complex process of multiciliogenesis, the biological events essential for forming multiple motile cilia are comparatively unelucidated. Thanks to advancements in genetic tools, omics technologies, and structural biology, significant progress has been achieved in the past decade in understanding the molecular mechanism underlying the regulation of multiple motile cilia formation. In this review, we discuss recent studies with ex vivo culture MCC and animal models, summarize current knowledge of multiciliogenesis, and particularly highlight recent advances and their implications.

Figure 1.

Regulation of MCC cell fate determination. The Notch signaling pathway inhibits the differentiation of MCC progenitor cells that express P63 and SOX2. Upon the activation of MCC differentiation, miR-34/449 can inhibit the Notch signaling pathway, which leads to the expression of key regulators, including TRRAP, GEMC1, and MCIDAS. GEMC1 induces cell cycle exit through the P53-P21 pathway and acts upstream of MCIDAS and C-MYB to control the expression of centriole amplification-related regulators such as DEUP1, CCNO, and CDC20B. On the other hand, GEMC1, C-MYB, and P73 can regulate the expression of motile cilia-related master regulators such as FOXJ1, RFX2, and RFX3.

Figure 2.

Formation of multiple motile cilia. (A) Parental centriole dependent centriole amplification. The CEP57–CEP63–CEP152 cascade mediates the initiation of parental centriole-dependent centriole amplification. In differentiating MCCs, both parental centrioles can generate multiple procentrioles. Note that the daughter centriole of parental centrioles can accumulate DEUP1 and promote the deuterosome assembly. (B) Deuterosome-dependent centriole amplification. DEUP1 and CEP152 mediate the initiation of deuterosome-dependent centriole amplification. PCM1 and other fibrogranular material (FGM) components form fibrous granules, which can similarly enrich DEUP1. Regional concentrated DEUP1 can self-assemble into macromolecular deuterosomes. Both centriole amplification pathways utilize common downstream regulators such as PLK4, SASS6, and STIL to generate procentrioles. (C) Centriole dissociation and deuterosome disassembly. As procentrioles grow and mature, CDK1-cyclin B phosphorylates SASS6, which in turn destabilizes the cartwheel structure, and Separase cooperates with other regulators such as PLK1 and CDC20B to release the maturing centrioles from their nucleating platforms, including parental centrioles and deuterosomes. Meanwhile, CCNO downregulates the expression of centriole amplification-related genes and collaborates with EMI2 and PLK1 to accomplish the deuterosome disassembly or clearance. (D) Assembly of multiple motile cilia. As centrioles gain the distal and subdistal appendages (DAs and SDAs), they conduct polarized migration with the help of IFT and the actin–myosin network. MCCs may adopt either extracellular or intracellular pathways to form motile cilia, although CBY1-mediated distal appendage vesicle (DAV) accumulation is involved in multicilia formation. Once docked to the plasm membrane, basal bodies converted from mature centrioles are fastened to the actin network by a ciliary adhesion complex. The centriole number in MCCs is calibrated to the apical surface area via PIEZO1.

Figure 3.

Structure of the motile cilia axoneme. A motile cilium comprises a basal body (BB), a transition zone (TZ), a centriolar microtubule extended axoneme, and a ciliary membrane. Compared with primary cilia, motile cilia display a 9+2 axonemal architecture with a central pair (CP) of microtubule-singlets (C1 and C2) surrounded by nine doublet microtubules (DMTs). The central apparatus (CA) distinguishes motile cilia from primary cilia, which includes the CP microtubules and their associating proteinaceous projections. The CP foot or basal plate is located at the proximal end of the CP and adheres to the distal end of the basal body, where WDR47 and Camsaps form a scaffold to nucleate the CP microtubules. The DMTs of motile cilia are decorated with T-shaped radial spokes (RSs), the nexin-dynein regulatory complexes (N-DRCs), and rows of axonemal dynein complexes (IDA and ODA). The dynein complexes are proposed to be preassembled in a cytoplasmic compartment (dynein assembly particle; DynAP) with the help of axonemal dynein assembly factors (DNAAFs) and transported into cilia via the IFT machinery. Structural biology studies reveal that a basic axonemal building block is 96 nm in length and each contains four ODAs (ODA1-4), one two-headed IDA (IDAf), six single-headed IDAs (IDAa/b/c/e/g/d), three RSs (RS1-3), and one N-DRC. These motility-related complexes may be anchored to the DMT through the interplay with microtubule inner proteins (MIPs).

Figure 4.

Different types of polarities in multiciliated tissues. (A) Planar cell polarity (PCP). The PCP is established before the formation of multiple motile cilia, a process relying on external mechanical forces and the apical microtubule network. Core PCP proteins such as Disheveled (DVL), Frizzled (FZD), Prickle (PRIC), and VANGL2 are asymmetrically distributed along the planar axis. The proximal (VANGL2 and PRIC) and distal (FZD and DVL) complexes segregate to opposite sides of the cell and interact with the opposite complex of the neighboring cell. CELSR1, a center component of the PCP system, is symmetrically distributed to both sides to stabilize the PCP complexes. (B) Rotational and translational polarities. The beat of newly developed motile cilia is in random directions and progressively aligned within each MCC (the rotational polarity). Different from the even distribution of basal bodies in MCCs of Xenopus epidermis and mammalian airway and reproductive tracts, basal bodies in matured ventricular MCCs are unidirectionally aligned within each cell (the rotational polarity) and uniquely clustered on one side of the apical surface (the translational polarity). (C) Axonemal and basal body (BB) orientations. Axonemal and BB orientations are used to assess the relationship between cilia movement and tissue axis. As shown, the nine ciliary doublet microtubules (DMTs) are numbered (D I-IX) and a unique structural feature exists between D V and D VI. The axonemal orientation is orthogonal to the CP plane defined by the line connecting the CP singlets, which runs through D I and across the space between D V and D VI. In MCCs, a cone-like basal foot is formed on the lateral side of the BB, which can occupy three of the nine triplet microtubules. The BB orientation is defined by the direction from the center of the BB to the tip of the basal foot. The axonemal orientation is aligned with the beat direction of motile cilia, while the BB orientation is aligned with the tissue axis.