The vertebrate primary cilium in development, homeostasis, and disease

Abstract

Cilia are complex structures that have garnered interest because of their roles in vertebrate development and their involvement in human genetic disorders. In contrast to multicellular invertebrates in which cilia are restricted to specific cell types, these organelles are found almost ubiquitously in vertebrate cells, where they serve a diverse set of signaling functions. Here, we highlight properties of vertebrate cilia, with particular emphasis on their relationship with other subcellular structures, and explore the physiological consequences of ciliary dysfunction.

Figure 1. Ciliary Structure in Vertebrates

(A) Microtubule structure of the vertebrate cilium. Cilia are tethered to the apical aspect of the cell at the basal body, surrounded by pericentriolar material. Nine radially organized microtubule triplets protrude from the basal body to the transition zone, and then extend as microtubule doublets in the ciliary axoneme. Electron micrograph images of each portion of the cilium are shown to the left. Images are from Kramer-Zucker et al. (2005) and Feistel and Blum (2006), reprinted with permission from John Wiley & Sons, Inc. from Developmental Dynamics, Vol. 235, 2006, p. 3348–3358. Copyright (2006) John Wiley & Sons, Inc., and Fromherz et al. (2004), reproduced with permission from Journal of Cell Science. (B) Diversification of ciliary architecture in vertebrate species. Whereas invertebrate models such as C. elegans and D. melanogaster have cilia restricted to certain cell types, nearly all vertebrate cells are ciliated. Examples shown from zebrafish, mouse, and rabbit demonstrate the possible expansion of ciliary roles in vertebrates. This expansion is exemplified by populations of cilia in vertebrate tissues that are architecturally heterogeneous, such as the rabbit notochordal plate and the mouse kinocilium. Moreover, there are exceptions to the general association between the central microtubule pair and motile cilia, such as motile ependymal cilia (9+0) in the zebrafish spinal cord and nonmotile sensory kinocilia (9+2) in the mouse inner ear.

Figure 2. Vesicular Transport and Intraflagellar Transport

Accumulating evidence indicates that vesicular transport of ciliary proteins from the trans-Golgi network is critical for proper cilium formation and may be facilitated, at least in part, by the intraflagellar transport (IFT) particle protein IFT20. Furthermore, Rabin8 interacts directly with Bardet-Biedl Syndrome protein BBS1, complexed with the BBSome in the basal body. Rabin8 is required for Rab8 GDP/GTP exchange. Rab8-GTP is proposed to be transported in vesicles to the base of the cilium, where it may dock with the ciliary membrane and/or the IFT complex. Upon targeting to the base of the cilium, ciliary proteins are transported toward the ciliary tip with the kinesin-2 motor in cooperation with IFT complex B proteins (listed on the left; shown in orange). TRAF3IP1/elipsa has been shown to function as an accessory anterograde IFT protein that binds directly with IFT20. Recycling of proteins toward the basal body is mediated by a cytoplasmic dynein and IFT complex A proteins (listed on the right; shown in blue).

Figure 3. Control of Ciliogenesis

(A) Ciliary proteins localize to cell junctions and polarity proteins localize to the primary cilium. Inversin localizes to three separate cellular protein pools: the primary cilium, cell junctions, and the nucleus. Two different polarity complexes, Par3/Par6/aPKCζ and CRB3/PATJ1/PALS, as well as the polarity protein 14-3-3η, localize to cell junctions and the primary cilium. Galectin-3 also marks a membrane region of increased lipid density surrounding the cilium. (B) The transcription factors Noto, FoxJ1, and Rfx3 play a role in ciliogenesis. Noto acts downstream of Foxa2 and T (brachyury) and upstream of FoxJ1 and Rfx3. Embryonic node and nodal cilium in E7.5 wild-type and Noto-deficient mice (Beckers et al., 2007) reveal structurally abnormal and stunted cilia. Images are reproduced with permission from PNAS, Vol. 104, p. 15765–15770. Copyright (2007) National Academy of Sciences, U.S.A. Scanning electron micrographs of explanted trachea show multiciliated cells covering most of the apical surface in wild-type trachea. These are absent from mice that are Foxj1 deficient, whereas primary cilia are unaffected. Scanning micrographs of embryonic nodes of Theiler stage 11c of wild-type and Rfx3 deficient mice. Cilia in Rfx3 deficient mice are shorter and misoriented compared to the wild-type (Bonnafe et al., 2004). Images reproduced from Molecular and Cellular Biology, 2004, Vol. 24, p. 4417–4427, with permission from the American Society for Microbiology. (C) A complex of Cep97 and CP110 suppresses ciliogenesis throughout the cell cycle. Mitotic entry is regulated by the G2/M transition through CDC2-Cyclin B, which activates several kinases, including the Nek family and AurA. In murine inner medullary collecting duct (IMCD3) cells, NEK5 localizes to the centrosome and the distal tip of the basal body. In human retinal pigment epithelial cells, a pool of AurA localizes to the basal body.

Figure 4. The Primary Cilium in Wnt and Shh Signaling

(A) In the absence of Wnt stimuli, β-catenin is constitutively phosphorylated by the β-catenin destruction complex consisting of Axin, APC, and GSK3β. Phosphorylated β-catenin is targeted for degradation. Wnt targets are repressed by a complex of TCF/LEF1 and TLE. In canonical Wnt signaling, the ligand binds to a complex of the Frizzled receptor and LRP5/6 coreceptor, which then binds to Axin and Dishevelled (DVL), leading to stabilization of β-catenin in the cytoplasm. β-catenin migrates into the nucleus, replaces TLE, and activates transcription ofβ-catenin/TCF/LEF1-responsive genes. In noncanonical signaling, activated DVL is targeted to the membrane and activates downstream targets. (B) Effects of ciliary and basal body disruption on Wnt signaling. In ciliated cells, noncanonical Wnt signals antagonize canonical Wnt signaling, leading to proteasomal β-catenin degradation and membrane targeting of DVL. In unciliated cells, canonical Wnt signaling stabilizes cytoplasmic β-catenin and DVL, leading to increased nuclear levels of both DVL and β-catenin, initiating transcription of TCF/LEF1-responsive genes. Disruption of ciliary or basal body components leads to loss of noncanonical Wnt signaling and stabilization of both DVL and β-catenin in the cytoplasm and nucleus, resembling the status of nonciliated cells. (C) In the absence of sonic hedgehog (Shh) ligand, Patched (Ptch) represses Smoothened (Smo). Repressive forms of the Gli proteins bind to the promoter regions of Shh targets and inhibit transcription. Stimulation with Shh ligand recruits Smo and Ptch1 to the primary cilium. Gli2 and Gli3 are localized to both the cilium and the nucleus, and in response to the ligand stimulus they are proteasomally processed into their activator Gli2/3A isoforms, activating transcription of Shh targets. (D) Differential role of ciliary and basal body proteins in Gli2/3 processing. Perturbation of anterograde intraflagellar transport (IFT) leads to loss of Gli2 and Gli3 processing and Shh signaling. Loss of structural integrity of the primary cilium by disruption of Arl13b leads to constitutively active Gli2, but leaves Gli3 unaffected. Loss of retrograde IFT triggers both Gli2 and Gli3 proteasomal activation along with transcriptional upregulation of Gli3. Finally, disruption of the basal body does not affect Gli2 processing but increases both expression and proteasomal activation of Gli3.