Brian J. Mitchell

Dishevelled controls apical docking and planar polarization of basal bodies in ciliated epithelial cells

The planar cell polarity (PCP) signaling system governs many aspects of polarized cell behavior. Here, we use an in vivo model of vertebrate mucociliary epithelial development to show that Dishevelled (Dvl) is essential for the apical positioning of basal bodies. We find that Dvl and Inturned mediate the activation of the Rho GTPase specifically at basal bodies, and that these three proteins together mediate the docking of basal bodies to the apical plasma membrane. Moreover, we find that this docking involves a Dvl-dependent association of basal bodies with membrane-bound vesicles and the vesicle-trafficking protein, Sec8. Once docked, basal bodies again require Dvl and Rho for the planar polarization that underlies directional beating of cilia. These results demonstrate previously undescribed functions for PCP signaling components and suggest that a common signaling apparatus governs both apical docking and planar polarization of basal bodies.

Strange as it may seem: the many links between Wnt signaling, planar cell polarity, and cilia

Cilia are important cellular structures that have been implicated in a variety of signaling cascades. In this review, we discuss the current evidence for and against a link between cilia and both the canonical Wnt/β-catenin pathway and the noncanonical Wnt/planar cell polarity (PCP) pathway. Furthermore, we address the evidence implicating a role for PCP components in ciliogenesis. Given the lack of consensus in the field, we use new data on the control of ciliary protein localization as a basis for proposing new models by which cell type-specific regulation of ciliary components via differential transport, regulated entry and exit, or diffusion barriers might generate context-dependent functions for cilia.

A positive feedback mechanism governs the polarity and motion of motile cilia

Ciliated epithelia produce fluid flow in many organ systems, ranging from the respiratory tract where it clears mucus to the ventricles of the brain where it transports cerebrospinal fluid. Human diseases that disable ciliary flow, such as primary ciliary dyskinesia, can compromise organ function or the ability to resist pathogens, resulting in recurring respiratory infections, otitis, hydrocephaly and infertility. To create a ciliary flow, the cilia within each cell need to be polarized coordinately along the planar axis of the epithelium, but how polarity is established in any ciliated epithelia is not known. Here we analyse the developmental mechanisms that polarize cilia, using the ciliated cells in the developing Xenopus larval skin as a model system. We show that cilia acquire polarity through a sequence of events, beginning with a polar bias set by tissue patterning, followed by a refinement phase. Our results indicate that during refinement, fluid flow is both necessary and sufficient in determining cilia polarity. These findings reveal a novel mechanism in which tissue patterning coupled with fluid flow act in a positive feedback loop to direct the planar polarity of cilia.

The PCP Pathway Instructs the Planar Orientation of Ciliated Cells in the Xenopus Larval Skin

Planar cell polarity (PCP) is a property of epithelial tissues where cellular structures coordinately orient along a two-dimensional plane lying orthogonal to the axis of apical-basal polarity. PCP is particularly striking in tissues where multiciliate cells generate a directed fluid flow, as seen, for example, in the ciliated epithelia lining the respiratory airways or the ventricles of the brain. To produce directed flow, ciliated cells orient along a common planar axis in a direction set by tissue patterning, but how this is achieved in any ciliated epithelium is unknown. Here, we show that the planar orientation of Xenopus multiciliate cells is disrupted when components in the PCP-signaling pathway are altered non-cell-autonomously. We also show that wild-type ciliated cells located at a mutant clone border reorient toward cells with low Vangl2 or high Frizzled activity and away from those with high Vangl2 activity. These results indicate that the PCP pathway provides directional non-cell-autonomous cues to orient ciliated cells as they differentiate, thus playing a critical role in establishing directed ciliary flow.

Reversible centriole depletion with an inhibitor of Polo-like kinase 4

Giving an old organelle the old heave-ho Centrioles are ancient cellular organelles that build centrosomes, the major microtubule-organizing centers in animal cells. Duplication of centrioles is tightly controlled to ensure that each dividing cell has precisely two centrosomes. Human cancer cells often have extra centrosomes, which has been hypothesized to confer a proliferative advantage. Wong et al. developed small molecules (centrinones) that allowed them to reversibly “delete” centrioles from cells (see the Perspective by Stearns). Surprisingly, cancer cells continued to divide in the absence of centrosomes, whereas normal cells stopped dividing. Science, this issue p. 1155; see also p. 1091 An “organelle knockout” strategy reveals that cancer cells but not normal cells can divide in the absence of centrosomes. [Also see Perspective by Stearns] Centrioles are ancient organelles that build centrosomes, the major microtubule-organizing centers of animal cells. Extra centrosomes are a common feature of cancer cells. To investigate the importance of centrosomes in the proliferation of normal and cancer cells, we developed centrinone, a reversible inhibitor of Polo-like kinase 4 (Plk4), a serine-threonine protein kinase that initiates centriole assembly. Centrinone treatment caused centrosome depletion in human and other vertebrate cells. Centrosome loss irreversibly arrested normal cells in a senescence-like G1 state by a p53-dependent mechanism that was independent of DNA damage, stress, Hippo signaling, extended mitotic duration, or segregation errors. In contrast, cancer cell lines with normal or amplified centrosome numbers could proliferate indefinitely after centrosome loss. Upon centrinone washout, each cancer cell line returned to an intrinsic centrosome number “set point.” Thus, cells with cancer-associated mutations fundamentally differ from normal cells in their response to centrosome loss.

Actin and microtubules drive differential aspects of planar cell polarity in multiciliated cells

Actin dynamics are required for proper cilia spacing, global coordination of cilia polarity, and coordination of metachronic cilia beating, whereas cytoplasmic microtubule dynamics are required for local coordination of polarity between neighboring cilia.

ZMYND10 Is Mutated in Primary Ciliary Dyskinesia and Interacts with LRRC6

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.

The hydrolethalus syndrome protein HYLS-1 links core centriole structure to cilia formation

Centrioles are subcellular organelles composed of a ninefold symmetric microtubule array that perform two important functions: (1) They build centrosomes that organize the microtubule cytoskeleton, and (2) they template cilia, microtubule-based projections with sensory and motile functions. We identified HYLS-1, a widely conserved protein, based on its direct interaction with the core centriolar protein SAS-4. HYLS-1 localization to centrioles requires SAS-4 and, like SAS-4, HYLS-1 is stably incorporated into the outer centriole wall. Unlike SAS-4, HYLS-1 is dispensable for centriole assembly and centrosome function in cell division. Instead, HYLS-1 plays an essential role in cilia formation that is conserved between Caenorhabditis elegans and vertebrates. A single amino acid change in human HYLS1 leads to a perinatal lethal disorder termed hydrolethalus syndrome, and we show that this mutation impairs HYLS-1 function in ciliogenesis. HYLS-1 is required for the apical targeting/anchoring of centrioles at the plasma membrane but not for the intraflagellar transport-dependent extension of the ciliary axoneme. These findings classify hydrolethalus syndrome as a severe human ciliopathy and shed light on the dual functionality of centrioles, defining the first stably incorporated centriolar protein that is not required for centriole assembly but instead confers on centrioles the capacity to initiate ciliogenesis.

Understanding ciliated epithelia: The power of Xenopus

Ciliated epithelia are important in a wide variety of biological contexts where they generate directed fluid flow. Here we address the fundamental advances in understanding ciliated epithelia that have been achieved using Xenopus as a model system. Xenopus embryos are covered with a ciliated epithelium that propels fluid unidirectionally across their surface. The external nature of this tissue, coupled with the molecular tools available in Xenopus and the ease of microscopic analysis on intact animals has thrust Xenopus to the forefront of ciliated epithelia biology. We discuss advances in understanding the molecular regulators of ciliated epithelia cell fate as well as basic aspects of ciliated epithelia cell biology including ciliogenesis and cell polarity. genesis 50:176–185, 2012.

c21orf59/kurly Controls Both Cilia Motility and Polarization

Cilia are microtubule-based projections that function in the movement of extracellular fluid. This requires cilia to be: (1) motile and driven by dynein complexes and (2) correctly polarized on the surface of cells, which requires planar cell polarity (PCP). Few factors that regulate both processes have been discovered. We reveal that C21orf59/Kurly (Kur), a cytoplasmic protein with some enrichment at the base of cilia, is needed for motility; zebrafish mutants exhibit characteristic developmental abnormalities and dynein arm defects. kur was also required for proper cilia polarization in the zebrafish kidney and the larval skin of Xenopus laevis. CRISPR/Cas9 coupled with homologous recombination to disrupt the endogenous kur locus in Xenopus resulted in the asymmetric localization of the PCP protein Prickle2 being lost in mutant multiciliated cells. Kur also makes interactions with other PCP components, including Disheveled. This supports a model wherein Kur plays a dual role in cilia motility and polarization.