Tracheal motile cilia in mice require CAMSAP3 for the formation of central microtubule pair and coordinated beating.

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body (BB), which are linked by a "transition zone" (TZ). The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a protein that can stabilize the minus-end of a microtubule, concentrates at multiple sites of the cilium-BB complex, including the upper region of the TZ or the axonemal basal plate (BP) where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the BP, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme and thereby supports the coordinated motion of multicilia in airway epithelial cells.

Cyst formation in proximal renal tubules caused by dysfunction of the microtubule minus-end regulator CAMSAP3.

Epithelial cells organize an ordered array of non-centrosomal microtubules, the minus ends of which are regulated by CAMSAP3. The role of these microtubules in epithelial functions, however, is poorly understood. Here, we show that the kidneys of mice in which Camsap3 is mutated develop cysts at the proximal convoluted tubules (PCTs). PCTs were severely dilated in the mutant kidneys, and they also exhibited enhanced cell proliferation. In these PCTs, epithelial cells became flattened along with perturbation of microtubule arrays as well as of certain subcellular structures such as interdigitating basal processes. Furthermore, YAP and PIEZO1, which are known as mechanosensitive regulators for cell shaping and proliferation, were activated in these mutant PCT cells. These observations suggest that CAMSAP3-mediated microtubule networks are important for maintaining the proper mechanical properties of PCT cells, and its loss triggers cell deformation and proliferation via activation of mechanosensors, resulting in the dilation of PCTs.

Nanopore Formation in the Cuticle of an Insect Olfactory Sensillum.

Nanometer-level patterned surface structures form the basis of biological functions, including superhydrophobicity, structural coloration, and light absorption [1-3]. In insects, the cuticle overlying the olfactory sensilla has multiple small (50- to 200-nm diameter) pores [4-8], which are supposed to function as a filter that admits odorant molecules, while preventing the entry of larger airborne particles and limiting water loss. However, the cellular processes underlying the patterning of extracellular matrices into functional nano-structures remain unknown. Here, we show that cuticular nanopores in Drosophila olfactory sensilla originate from a curved ultrathin film that is formed in the outermost envelope layer of the cuticle and secreted from specialized protrusions in the plasma membrane of the hair forming (trichogen) cell. The envelope curvature coincides with plasma membrane undulations associated with endocytic structures. The gore-tex/Osiris23 gene encodes an endosomal protein that is essential for envelope curvature, nanopore formation, and odor receptivity and is expressed specifically in developing olfactory trichogen cells. The 24-member Osiris gene family is expressed in cuticle-secreting cells and is found only in insect genomes. These results reveal an essential requirement for nanopores for odor reception and identify Osiris genes as a platform for investigating the evolution of surface nano-fabrication in insects.

FGFR1-mediated protocadherin-15 loading mediates cargo specificity during intraflagellar transport in inner ear hair-cell kinocilia.

The mechanosensory hair cells of the inner ear are required for hearing and balance and have a distinctive apical structure, the hair bundle, that converts mechanical stimuli into electrical signals. This structure comprises a single cilium, the kinocilium, lying adjacent to an ensemble of actin-based projections known as stereocilia. Hair bundle polarity depends on kinociliary protocadherin-15 (Pcdh15) localization. Protocadherin-15 is found only in hair-cell kinocilia, and is not localized to the primary cilia of adjacent supporting cells. Thus, Pcdh15 must be specifically targeted and trafficked into the hair-cell kinocilium. Here we show that kinocilial Pcdh15 trafficking relies on cell type-specific coupling to the generic intraflagellar transport (IFT) transport mechanism. We uncover a role for fibroblast growth factor receptor 1 (FGFR1) in loading Pcdh15 onto kinociliary transport particles in hair cells. We find that on activation, FGFR1 binds and phosphorylates Pcdh15. Moreover, we find a previously uncharacterized role for clathrin in coupling this kinocilia-specific cargo with the anterograde IFT-B complex through the adaptor, DAB2. Our results identify a modified ciliary transport pathway used for Pcdh15 transport into the cilium of the inner ear hair cell and coordinated by FGFR1 activity.

Neural retina-specific Aldh1a1 controls dorsal choroidal vascular development via Sox9 expression in retinal pigment epithelial cells.

VEGF secreted from retinal pigment epithelial (RPE) cells is responsible for the choroidal vascular development; however, the molecular regulatory mechanism is unclear. We found that Aldh1a1-/- mice showed choroidal hypoplasia with insufficient vascularization in the dorsal region, although Aldh1a1, an enzyme that synthesizes retinoic acids (RAs), is expressed in the dorsal neural retina, not in the RPE/choroid complex. The level of VEGF in the RPE/choroid was significantly decreased in Aldh1a1-/- mice, and RA-dependent enhancement of VEGF was observed in primary RPE cells. An RA-deficient diet resulted in dorsal choroidal hypoplasia, and simple RA treatment of Aldh1a1-/- pregnant females suppressed choroid hypoplasia in their offspring. We also found downregulation of Sox9 in the dorsal neural retina and RPE of Aldh1a1-/- mice and RPE-specific disruption of Sox9 phenocopied Aldh1a1-/- choroidal development. These results suggest that RAs produced by Aldh1a1 in the neural retina directs dorsal choroidal vascular development via Sox9 upregulation in the dorsal RPE cells to enhance RPE-derived VEGF secretion.

IKKε inhibits PKC to promote Fascin-dependent actin bundling.

Signaling molecules have pleiotropic functions and are activated by various extracellular stimuli. Protein kinase C (PKC) is activated by diverse receptors, and its dysregulation is associated with diseases including cancer. However, how the undesired activation of PKC is prevented during development remains poorly understood. We have previously shown that a protein kinase, IKKε, is active at the growing bristle tip and regulates actin bundle organization during Drosophila bristle morphogenesis. Here, we demonstrate that IKKε regulates the actin bundle localization of a dynamic actin cross-linker, Fascin. IKKε inhibits PKC, thereby protecting Fascin from inhibitory phosphorylation. Excess PKC activation is responsible for the actin bundle defects in IKKε-deficient bristles, whereas PKC is dispensable for bristle morphogenesis in wild-type bristles, indicating that PKC is repressed by IKKε in wild-type bristle cells. These results suggest that IKKε prevents excess activation of PKC during bristle morphogenesis.

Inheritance of a Nuclear PIWI from Pluripotent Stem Cells by Somatic Descendants Ensures Differentiation by Silencing Transposons in Planarian.

Differentiation of pluripotent stem cells (PSCs) requires transposon silencing throughout the process. PIWIs, best known as key factors in germline transposon silencing, are also known to act in somatic differentiation of planarian PSCs (neoblasts). However, how PIWIs control the latter process remains elusive. Here, using Dugesia japonica, we show that a nuclear PIWI, DjPiwiB, was bound to PIWI-interacting RNAs (generally key mediators of PIWI-dependent transposon silencing), and was detected in not only neoblasts but also their descendant somatic cells, which do not express piwi. In contrast, cytoplasmic DjPiwiA and DjPiwiC were detected only in neoblasts, in accord with their transcription there. DjPiwiB was indispensable for regeneration, but dispensable for transposon silencing in neoblasts. However, transposons were derepressed at the onset of differentiation in DjPiwiB-knockdown planarians. Thus, DjPiwiB appears to be inherited by descendant somatic cells of neoblasts to ensure transposon silencing in those cells, which are unable to produce PIWI proteins.

Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells.

The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs.

CAMSAP3 orients the apical-to-basal polarity of microtubule arrays in epithelial cells.

Polarized epithelial cells exhibit a characteristic array of microtubules that are oriented along the apicobasal axis of the cells. The minus-ends of these microtubules face apically, and the plus-ends face toward the basal side. The mechanisms underlying this epithelial-specific microtubule assembly remain unresolved, however. Here, using mouse intestinal cells and human Caco-2 cells, we show that the microtubule minus-end binding protein CAMSAP3 (calmodulin-regulated-spectrin-associated protein 3) plays a pivotal role in orienting the apical-to-basal polarity of microtubules in epithelial cells. In these cells, CAMSAP3 accumulated at the apical cortices, and tethered the longitudinal microtubules to these sites. Camsap3 mutation or depletion resulted in a random orientation of these microtubules; concomitantly, the stereotypic positioning of the nucleus and Golgi apparatus was perturbed. In contrast, the integrity of the plasma membrane was hardly affected, although its structural stability was decreased. Further analysis revealed that the CC1 domain of CAMSAP3 is crucial for its apical localization, and that forced mislocalization of CAMSAP3 disturbs the epithelial architecture. These findings demonstrate that apically localized CAMSAP3 determines the proper orientation of microtubules, and in turn that of organelles, in mature mammalian epithelial cells.

Absence of Radial Spokes in Mouse Node Cilia Is Required for Rotational Movement but Confers Ultrastructural Instability as a Trade-Off.

Determination of left-right asymmetry in mouse embryos is established by a leftward fluid flow that is generated by clockwise rotation of node cilia. How node cilia achieve stable unidirectional rotation has remained unknown, however. Here we show that brief exposure to the microtubule-stabilizing drug paclitaxel (Taxol) induces randomly directed rotation and changes the ultrastructure of node cilia. In vivo observations and a computer simulation revealed that a regular 9+0 arrangement of doublet microtubules is essential for stable unidirectional rotation of node cilia. The 9+2 motile cilia of the airway, which manifest planar beating, are resistant to Taxol treatment. However, the airway cilia of mice lacking the radial spoke head protein Rsph4a undergo rotational movement instead of planar beating, are prone to microtubule rearrangement, and are sensitive to Taxol. Our results suggest that the absence of radial spokes allows node cilia to rotate unidirectionally but, as a trade-off, renders them ultrastructurally fragile.

Protocadherin-17 mediates collective axon extension by recruiting actin regulator complexes to interaxonal contacts.

In the process of neuronal wiring, axons derived from the same functional group typically extend together, resulting in fascicle formation. How these axons communicate with one another remains largely unknown. Here, we show that protocadherin-17 (Pcdh17) supports this group extension by recruiting actin polymerization regulators to interaxonal contact sites. Pcdh17 is expressed by a subset of amygdala neurons, and it accumulates at axon-axon boundaries because of homophilic binding. Pcdh17 knockout in mice suppressed the extension of these axons. Ectopically expressed Pcdh17 altered the pattern of axon extension. In in-vitro cultures, wild-type growth cones normally migrate along other axons, whereas Pcdh17 null growth cones do not. Pcdh17 recruits the WAVE complex, Lamellipodin, and Ena/VASP to cell-cell contacts, converting these sites into motile structures. We propose that, through these mechanisms, Pcdh17 maintains the migration of growth cones that are in contact with other axons, thereby supporting their collective extension.

Homeostatic epithelial renewal in the gut is required for dampening a fatal systemic wound response in Drosophila.

Effective defense responses involve the entire organism. To maintain body homeostasis after tissue damage, a systemic wound response is induced in which the response of each tissue is tightly orchestrated to avoid incomplete recovery or an excessive, damaging response. Here, we provide evidence that in the systemic response to wounding, an apoptotic caspase pathway is activated downstream of reactive oxygen species in the midgut enterocytes (ECs), cells distant from the wound site, in Drosophila. We show that a caspase-pathway mutant has defects in homeostatic gut cell renewal and that inhibiting caspase activity in fly ECs results in the production of systemic lethal factors after wounding. Our results indicate that wounding remotely controls caspase activity in ECs, which activates the tissue stem cell regeneration pathway in the gut to dampen the dangerous systemic wound reaction.

Joint morphology in the insect leg: evolutionary history inferred from Notch loss-of-function phenotypes in Drosophila.

Joints permit efficient locomotion, especially among animals with a rigid skeleton. Joint morphologies vary in the body of individual animals, and the shapes of homologous joints often differ across species. The diverse locomotive behaviors of animals are based, in part, on the developmental and evolutionary history of joint morphogenesis. We showed previously that strictly coordinated cell-differentiation and cell-movement events within the epidermis sculpt the interlocking ball-and-socket joints in the adult Drosophila tarsus (distal leg). Here, we show that the tarsal joints of various insect species can be classified into three types: ball-and-socket, side-by-side and uniform. The last two probably result from joint formation without the cell-differentiation step, the cell-movement step, or both. Similar morphological variations were observed in Drosophila legs when Notch function was temporarily blocked during joint formation, implying that the independent acquisition of cell differentiation and cell movement underlay the elaboration of tarsal joint morphologies during insect evolution. These results provide a framework for understanding how the seemingly complex morphology of the interlocking joint could have developed during evolution by the addition of simple developmental modules: cell differentiation and cell movement.

Metalloprotease-dependent onset of blood circulation in zebrafish.

The primitive blood circulation requires intravascular plasma flow. However, it remains unclear whether the onset of earliest blood circulation is dependent solely on establishment of a functional circulatory organ or whether it also requires active processes inherent in blood cells. In this study, we present novel mechanisms for the onset of blood circulation by monitoring fluorescently labeled blood precursors and blood vessels in zebrafish. The earliest blood circulation occurs synchronously. This synchrony is achieved by the retention of erythroid precursors on the lumen of the vasculature after their invasion from the subaortic region, and then by simultaneous release of these precursors into the flow. Morphological and biochemical analyses suggest that the onset of blood circulation accompanies disruption of blood cell-to-vessel adhesion and requires metalloprotease-dependent processes. ADAM8, a member of the a disintegrin and metalloprotease (ADAM) family, mediates the onset of blood circulation. In ADAM8-depleted embryos, erythroid cells fail to detach from the vascular lumen and stagnate. Expression of a protease-defective ADAM8 in erythroid cells causes dominant-negative effects on blood circulation, suggesting cell-autonomous roles of ADAM8. Based on these findings, we propose that the first erythroid cells require both flow-dependent passive and proteolysis-dependent active processes to enter the circulation.

Dynamic shape changes of ECM-producing cells drive morphogenesis of ball-and-socket joints in the fly leg.

Animal body shape is framed by the skeleton, which is composed of extracellular matrix (ECM). Although how the body plan manifests in skeletal morphology has been studied intensively, cellular mechanisms that directly control skeletal ECM morphology remain elusive. In particular, how dynamic behaviors of ECM-secreting cells, such as shape changes and movements, contribute to ECM morphogenesis is unclear. Strict control of ECM morphology is crucial in the joints, where opposing sides of the skeleton must have precisely reciprocal shapes to fit each other. Here we found that, in the development of ball-and-socket joints in the Drosophila leg, the two sides of ECM form sequentially. We show that distinct cell populations produce the 'ball' and the 'socket', and that these cells undergo extensive shape changes while depositing ECM. We propose that shape changes of ECM-producing cells enable the sequential ECM formation to allow the morphological coupling of adjacent components. Our results highlight the importance of dynamic cell behaviors in precise shaping of skeletal ECM architecture.

Mammalian Fat and Dachsous cadherins regulate apical membrane organization in the embryonic cerebral cortex.

Compartmentalization of the plasma membrane in a cell is fundamental for its proper functions. In this study, we present evidence that mammalian Fat4 and Dachsous1 cadherins regulate the apical plasma membrane organization in the embryonic cerebral cortex. In neural progenitor cells of the cortex, Fat4 and Dachsous1 were concentrated together in a cell-cell contact area positioned more apically than the adherens junction (AJ). These molecules interacted in a heterophilic fashion, affecting their respective protein levels. We further found that Fat4 associated and colocalized with the Pals1 complex. Ultrastructurally, the apical junctions of the progenitor cells comprised the AJ and a stretch of plasma membrane apposition extending apically from the AJ, which positionally corresponded to the Fat4-Dachsous1-positive zone. Depletion of Fat4 or Pals1 abolished this membrane apposition. These results highlight the importance of the Fat4-Dachsous1-Pals1 complex in organizing the apical membrane architecture of neural progenitor cells.

Non-receptor tyrosine kinase CSK-1 controls pharyngeal muscle organization in Caenorhabditis elegans.

C-terminal Src kinase (Csk) is a non-receptor type of tyrosine kinase, and serves as an essential negative regulator of Src family tyrosine kinases (SFKs) in vertebrates. However, analyses of Csk and SFKs from primitive animals suggest that the Csk-mediated mechanisms regulating SFK activity might diverge between evolutional branches, different tissues or SFK family members. We examined in vivo roles of CSK-1, a Caenorhabditis elegans orthologue of Csk, by generating animals lacking csk-1 function. Although some csk-1 mutants died during embryogenesis, the majority of mutants died during the first stage of larval development. In csk-1 mutants, the function of pharyngeal muscles, the major site of CSK-1 expression, was severely damaged. The pumping of pharyngeal grinder cells became arrhythmic, causing disabled feeding. Electron microscopy showed that pharyngeal muscle filaments were disorientated in the csk-1 mutants. These indicate that CSK-1 is crucial for proper organization of pharyngeal muscles. However, the growth arrest phenotype in csk-1 mutants could not be suppressed by src-1 and/or src-2 mutation, and SRC-1 was not significantly activated in the csk-1 mutants. These results suggest that CSK-1 has an essential function in organization of pharyngeal muscle filaments that does not require C. elegans SFKs.

The preprophase band is a localized center of clathrin-mediated endocytosis in late prophase cells of the onion cotyledon epidermis.

The preprophase band (PPB) marks the site on the plant cell cortex where the cell plate will fuse during the final stage of cytokinesis. Recent studies have shown that several cytoskeletal proteins are depleted at the PPB site, but the processes that bring about these changes are still unknown. We have investigated the membrane systems associated with the PPB regions of epidermal cells of onion cotyledons by means of serial thin sections and electron tomograms. In contrast with specimens preserved by chemical fixatives, our high-pressure frozen cells demonstrated the presence of large numbers of clathrin-coated pits and vesicles in the PPB regions. The vesicles were of two types: clathrin-coated and structurally related, non-coated vesicles. Quantitative analysis of the data revealed that the number of clathrin-coated pits and vesicles is higher in the PPB regions than outside of these regions. Immunofluorescent microscopy using anti-plant clathrin-antibody confirmed this result. In contrast, no differences in secretory activities were observed. We postulate that the removal of membrane proteins by endocytosis plays a role in the formation of PPB 'memory' structures.

Cthrc1 selectively activates the planar cell polarity pathway of Wnt signaling by stabilizing the Wnt-receptor complex.

Vertebrate Wnt proteins activate several distinct pathways. Intrinsic differences among Wnt ligands and Frizzled (Fzd) receptors, and the availability of pathway-specific coreceptors, LRP5/6, and Ror2, affect pathway selection. Here, we show that a secreted glycoprotein, Cthrc1, is involved in selective activation of the planar cell polarity (PCP) pathway by Wnt proteins. Although Cthrc1 null mutant mice appeared normal, the introduction of a heterozygous mutation of a PCP gene, Vangl2, resulted in abnormalities characteristic of PCP mutants. In HEK293T cells, Cthrc1 activated the PCP pathway but suppressed the canonical pathway. Cell-surface-anchored Cthrc1 bound to Wnt proteins, Fzd proteins, and Ror2 and enhanced the interaction of Wnt proteins and Fzd/Ror2 by forming the Cthrc1-Wnt-Fzd/Ror2 complex. Consistent with this, Ror2 mutant mice also showed PCP-related abnormalities in the inner ear. These results suggest that Cthrc1 is a Wnt cofactor protein that selectively activates the Wnt/PCP pathway by stabilizing ligand-receptor interaction.