Role In Ciliogenesis Research Articles

The LIM protein Ajuba is required for ciliogenesis and left–right axis determination in medaka

Cilia are microtubule-based organelles that are present on the surfaces of almost all vertebrate cells. Most cilia function as sensory or molecular transport structures. Malfunctions of cilia have been implicated in several diseases of human development. The assembly of cilia is initiated by the centriole (or basal body), and several centrosomal proteins are involved in this process. The mammalian LIM protein Ajuba is a well-studied centrosomal protein that regulates cell division but its role in ciliogenesis is unknown. In this study, we isolated the medaka homolog of Ajuba and showed that Ajuba localizes to basal bodies of cilia in growth-arrested cells. Knockdown of Ajuba resulted in randomized left–right organ asymmetries and altered expression of early genes responsible for left–right body axis determination. At the cellular level, we found that Ajuba function was essential for ciliogenesis in the cells lining Kupffer’s vesicle; it is these cells that induce the asymmetric fluid flow required for left–right axis determination. Taken together, our findings identify a novel role for Ajuba in the regulation of vertebrate ciliogenesis and left–right axis determination.

Intraflagellar transport is required for polarized recycling of the TCR/CD3 complex to the immune synapse

Most eukaryotic cells have a primary cilium which functions as a sensory organelle. Cilia are assembled by intraflagellar transport (IFT), a process mediated by multimeric IFT particles and molecular motors. Here we show that lymphoid and myeloid cells, which lack primary cilia, express IFT proteins. IFT20, an IFT component essential for ciliary assembly, was found to colocalize with both the microtubule organizing centre (MTOC) and Golgi and post-Golgi compartments in T-lymphocytes. In antigen-specific conjugates, IFT20 translocated to the immune synapse. IFT20 knockdown resulted in impaired T-cell receptor/CD3 (TCR/CD3) clustering and signalling at the immune synapse, due to defective polarized recycling. Moreover, IFT20 was required for the inducible assembly of a complex with other IFT components (IFT57 and IFT88) and the TCR. The results identify IFT20 as a new regulator of immune synapse assembly in T cells and provide the first evidence to implicate IFT in membrane trafficking in cells lacking primary cilia, thereby introducing a new perspective on IFT function beyond its role in ciliogenesis.

16-P005 Mutations in the lethal ciliopathy Meckel–Gruber syndrome alter the subcellular distribution of actin-binding proteins and disrupt the actin cytoskeleton

MKS3, encoding a novel trans-membrane receptor, meckelin with similarity to frizzled proteins, is mutated in Meckel–Gruber syndrome (MKS), an autosomal recessive lethal ciliopathy. Meckelin is a ciliary protein, but it also localises to the actin cytoskeleton at baso-lateral and basal cell surfaces. Filamin A (FLNa) and specific isoforms of nesprin-2 are actin-binding proteins (ABP) that interact with meckelin. Both are important for maintenance and remodelling of the actin cytoskeleton at the cell membrane and nuclear envelope. Meckelin and FLNa localised throughout ciliogenesis and in post-mitotic ciliated cells. However, a meckelin–nesprin-2 interaction was seen only during early ciliogenesis prior to the establishment of cell polarity and centriole migration; crucial steps in cilia formation. siRNA knockdown of meckelin caused loss of cilia and failure of centriole migration. This was also seen for FLNa and nesprin-2 indicating that mutation of ABPs could lead, or contribute, to a ciliopathy phenotype. In MKS patient fibroblasts, nuclear membrane architecture was distorted and cell-migration reduced; both indicative of disruption of the actin cytoskeleton. In addition, RhoA-GTPase activity was increased. Small GTPases are key regulators of actin assembly and remodelling pathways and the appearance of actin stress fibres was concurrent with this increased activity. Both FLNA and nesprin-2 were redirected to these fibres showing loss of meckelin altered distribution of ABPs. These novel findings suggest the actin cytoskeleton and actin-binding proteins play a crucial role in ciliogenesis and cilia function. This has important implications for defining the ciliopathy phenotype and determining the multi-functional role of ciliary proteins.

Chronological expression of Ciliated Bronchial Epithelium 1 during pulmonary development

Ciliated Bronchial Epithelium (CBE) 1 is a novel gene, which is expressed in ciliated cells. As cilia are important during embryogenesis, the present authors characterised the murine homologue of CBE1 (Cbe1) and compared its temporal expression during murine and human lung development. Cbe1 cDNA was cloned and characterised using sequencing, standard PCR and Western blotting. Mouse and human embryonic/fetal lungs (HELs) were harvested for mRNA analysis and protein localisation in vivo and in vitro using RT-PCR and immunohistochemistry. The Cbe1 amino acid sequence was >75% identical with CBE1 and its alternative splicing and tissue distribution were highly conserved. Pulmonary expression of Cbe1 mRNA was increased at embryonic day (E)16, 1 day later than Foxj1, which is consistent with a role in ciliogenesis. In HELs, CBE1 mRNA was detectable at 8-9 weeks post-conception and increased in explant culture. CBE1 protein expression was weak at 10 weeks post-conception but strong at 12.3 weeks post-conception, in parallel with cilia formation. Additionally, Cbe1 mRNA was expressed at E11 (4-5 weeks post-conception in HELs) in the absence of Foxj1, implying a distinct role in early development. Chronological regulation of CBE1/Cbe1 expression during pulmonary differentiation suggests involvement in ciliogenesis, with an additional role during early lung development.

Defective ciliogenesis, embryonic lethality and severe impairment of the Sonic Hedgehog pathway caused by inactivation of the mouse complex A intraflagellar transport gene Ift122/Wdr10, partially overlapping with the DNA repair gene Med1/Mbd4

Primary cilia are assembled and maintained by evolutionarily conserved intraflagellar transport (IFT) proteins that are involved in the coordinated movement of macromolecular cargo from the basal body to the cilium tip and back. The IFT machinery is organized in two structural complexes named complex A and complex B. Recently, inactivation in the mouse germline of Ift genes belonging to complex B revealed a requirement of ciliogenesis, or proteins involved in ciliogenesis, for Sonic Hedgehog (Shh) signaling in mammals. Here we report on a complex A mutant mouse, defective for the Ift122 gene. Ift122-null embryos show multiple developmental defects (exencephaly, situs viscerum inversus, delay in turning, hemorrhage and defects in limb development) that result in lethality. In the node, primary cilia were absent or malformed in homozygous mutant and heterozygous embryos, respectively. Impairment of the Shh pathway was apparent in both neural tube patterning (expansion of motoneurons and rostro-caudal level-dependent contraction or expansion of the dorso-lateral interneurons), and limb patterning (ectrosyndactyly). These phenotypes are distinct from both complex B IFT mutant embryos and embryos defective for the ciliary protein hennin/Arl13b, and suggest reduced levels of both Gli2/Gli3 activator and Gli3 repressor functions. We conclude that complex A and complex B factors play similar but distinct roles in ciliogenesis and Shh/Gli3 signaling.

Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development

Mucociliary epithelia are essential for homeostasis of many organs and consist of mucus-secreting goblet cells and ciliated cells. Here, we present the ciliated epidermis of Xenopus embryos as a facile model system for in vivo molecular studies of mucociliary epithelial development. Using an in situ hybridization-based approach, we identified numerous genes expressed differentially in mucus-secreting cells or in ciliated cells. Focusing on genes expressed in ciliated cells, we have identified new candidate ciliogenesis factors, including several not present in the current ciliome. We find that TTC25-GFP is localized to the base of cilia and to ciliary axonemes, and disruption of TTC25 function disrupts ciliogenesis. Mig12-GFP localizes very strongly to the base of cilia and confocal imaging of this construct allows for simple visualization of the planar polarity of basal bodies that underlies polarized ciliary beating. Knockdown of Mig12 disrupts ciliogenesis. Finally, we show that ciliogenesis factors identified in the Xenopus epidermis are required in the midline to facilitate neural tube closure. These results provide further evidence of a requirement for cilia in neural tube morphogenesis and suggest that genes identified in the Xenopus epidermis play broad roles in ciliogenesis. The suites of genes identified here will provide a foundation for future studies, and may also contribute to our understanding of pathological changes in mucociliary epithelia that accompany diseases such as asthma.

THE EXPRESSION OF TUBULIN AND TEKTIN GENES IN DICYEMID MESOZOANS (PHYLUM: DICYEMIDA)

Dicyemid mesozoans (Phylum Dicyemida) are endoparasites (or endosymbionts) that typically are found in the renal sac of benthic cephalopod mollusks such as octopuses and cuttlefishes. Adult dicyemids likely adhere to the renal appendage of hosts via cilia of calotte peripheral cells. These cilia seem to be continuously worn away in the interaction between the dicyemids and the epidermal cells of host renal appendages. We cloned 4 cDNAs and genes, alpha-tubulin, beta-tubulin, tektin B, and tektin C, which are thought to play a key role in ciliogenesis, from Dicyema japonicum, and studied expression patterns of these genes by whole-mount in situ hybridization. We detected coexpression of these genes in the calotte peripheral cells, but not in the trunk peripheral cells. This suggests that regeneration and turnover of cilia continuously occur in the calotte. In vermiform and infusoriform embryos, we also detected coexpression patterns of these genes, which might correlate with ciliogenesis during the embryogenesis. We also predicted the secondary structure and the coiled-coil regions of dicyemid tektins.

Embryonic expression of pericentrin suggests universal roles in ciliogenesis

Pericentrin (Pcnt) is a giant coiled-coil protein known to mediate microtubule organization. It has been recently reported that mitosis-specific centrosomal anchoring of gamma tubulin complexes by Pcnt acts to control mitotic spindle organization, though little is known about the in vivo expression of Pcnt. In this study, we investigated Pcnt expression in mouse embryos. In situ hybridization analysis revealed preferential expression of Pcnt in quiescent G(0) phase cells throughout the embryo with an unexpectedly low expression level in proliferating cells, suggesting that Pcnt might not play an important role in mitotic proliferation. Immunofluorescence analysis confirmed preferential expression of the Pcnt protein in G(0) phase cells. Moreover, Pcnt was shown to be localized to the base of primary cilia in multiple embryonic tissues, in agreement with a recent study demonstrating the involvement of Pcnt in primary cilia formation using cultured mammalian cells.

Role of foxj1 and estrogen receptor alpha in ciliated epithelial cell differentiation of the neonatal oviduct.

Estrogen regulates proliferation and differentiation of epithelial cells in the mammalian oviduct, but pathways for cell-specific differentiation are not well understood. In the epithelial cells of the developing rat oviduct, we found estrogen receptor (ER) alpha is expressed at birth and persists in all cells through neonatal day (ND) 7 when ciliated cells appear. To determine a specific function of ER and foxj1, a transcription factor known to have fundamental roles in ciliogenesis in the lung, in differentiation of the ciliated epithelial cells, we treated newborn rats from ND 0 to 5 with estradiol-17beta (E2) with and without a selective ER antagonist. E2 enhanced the number of proliferating cells and accelerated the process of epithelial cell differentiation resulting in ciliogenesis by ND 5, and co-treatment with an ER antagonist inhibited these changes. Foxj1 was expressed only in the infundibulum and ampulla (INF/AMP). That expression preceded the appearance of cilia and was induced by E2. Cilia were absent in oviducts of foxj1-deficient mice, indicating that foxj1 plays a critical role in oviductal ciliogenesis. However, we found the presence of cilia in the ERalpha-deficient mouse oviduct. The widespread expression of ERalpha in oviductal epithelium, but restriction of cilia to the INF/AMP regions, and importantly, the presence of cilia in the ERalpha-deficient mice, suggested ER signaling is not essential for ciliated epithelial cell differentiation. These observations demonstrate that, although E2 stimulates the differentiation process of ciliated epithelial cells, foxj1 is directly required for epithelial cell ciliogenesis of the neonatal oviduct.