The complex relationship of Wnt-signaling pathways and cilia.

Wnt family proteins are secreted glycolipoproteins that signal through multitude of signal transduction pathways. The Wnt-pathways are conserved and critical in all metazoans. They are essential for embryonic development, organogenesis and homeostasis, and associated with many diseases when defective or deregulated. Wnt signaling pathways comprise the canonical Wnt pathway, best known for its stabilization of ß-catenin and associated nuclear ß-catenin activity in gene regulation, and several non-canonical signaling branches. Wnt-Planar Cell Polarity (PCP) signaling has received the most attention among the non-canonical Wnt pathways. The relationship of cilia to Wnt-signaling is complex. While it was suggested that canonical Wnt signaling requires cilia this notion was always challenged by results suggesting the opposite. Recent developments provide insight and clarification to the relationship of Wnt signaling pathways and cilia. First, it has been now demonstrated that while ciliary proteins, in particular the IFT-A complex, are required for canonical Wnt/ß-catenin signaling, the cilium as a structure is not. In contrast, recent work has defined a diverged canonical signaling branch (not affecting ß-catenin) to be required for ciliary biogenesis and cilia function. Furthermore, the non-canonical Wnt-PCP pathway does not affect cilia biogenesis per se, but it regulates the position of cilia within cells in many cell types, possibly in all cells where it is active, with cilia being placed near the side of the cell that has the Frizzled-Dishevelled complex. This Wnt/PCP feature is conserved with both centrioles and basal bodies/cilia being positioned accordingly, and it is also used to align mitotic spindles within the Wnt-PCP polarization axis. It also coordinates the alignment of cilia in multiciliated cells. This article addresses these new insights and different links and relationships between cilia and Wnt signaling.

Multi-color live-cell fluorescence imaging of primary ciliary membrane assembly and dynamics.

The ciliary membrane is continuous with the plasma membrane but has distinct lipid and protein composition, which is key to defining the function of the primary cilium. Ciliary membranes dynamically assemble and disassemble in association with the cell cycle and directly transmit signals and molecules through budding membranes. Various imaging approaches have greatly advanced the understanding of the ciliary membrane function. In particular, fluorescence live-cell imaging has revealed important insights into the dynamics of ciliary membrane assembly by monitoring the changes of fluorescent-tagged ciliary proteins. Protein dynamics can be tracked simultaneously using multi-color live cell imaging by coupling ciliary-associated factors with different colored fluorescent tags. Ciliary membrane and membrane associated-proteins such as Smoothened, 5-HTr6, SSTR3, Rab8a, and Arl13b have been used to track ciliary membranes and centriole proteins like Centrin1/2, CEP164, and CEP83 are often used to mark the ciliary basal body. Here, we describe a method for studying ciliogenesis membrane dynamics using spinning disk confocal live-cell imaging.

Role of Primary Cilia in Bone and Cartilage.

The primary cilium is a nonmotile microtubule-based organelle in most vertebrate cell types. The primary cilium plays a critical role in tissue development and homeostasis by sensing and transducing various signaling pathways. Ciliary proteins such as intraflagellar transport (IFT) proteins as well as ciliary motor proteins, kinesin and dynein, comprise a bidirectional intraflagellar transport system needed for cilia formation and function. Mutations in ciliary proteins that lead to loss or dysfunction of primary cilia cause ciliopathies such as Jeune syndrome and Ellis-van Creveld syndrome and cause abnormalities in tooth development. These diseases exhibit severe skeletal and craniofacial dysplasia, highlighting the significance of primary cilia in skeletal development. Cilia are necessary for the propagation of hedgehog, transforming growth factor ß, platelet-derived growth factor, and fibroblast growth factor signaling during osteogenesis and chondrogenesis. Ablation of ciliary proteins such as IFT80 or IFT20 blocks cilia formation, which inhibits osteoblast differentiation, osteoblast polarity, and alignment and reduces bone formation. Similarly, cilia facilitate chondrocyte differentiation and production of a cartilage matrix. Cilia also play a key role in mechanosensing and are needed for increased bone formation in response to mechanical forces.

A Ciliary View of the Immunological Synapse.

The primary cilium has gone from being a vestigial organelle to a crucial signaling hub of growing interest given the association between a group of human disorders, collectively known as ciliopathies, and defects in its structure or function. In recent years many ciliogenesis proteins have been observed at extraciliary sites in cells and likely perform cilium-independent functions ranging from regulation of the cytoskeleton to vesicular trafficking. Perhaps the most striking example is the non-ciliated T lymphocyte, in which components of the ciliary machinery are repurposed for the assembly and function of the immunological synapse even in the absence of a primary cilium. Furthermore, the specialization traits described at the immunological synapse are similar to those seen in the primary cilium. Here, we review common regulators and features shared by the immunological synapse and the primary cilium that document the remarkable homology between these structures.

Ciliary dyslexia candidate genes DYX1C1 and DCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs.

DYX1C1, DCDC2, and KIAA0319 are three of the most replicated dyslexia candidate genes (DCGs). Recently, these DCGs were implicated in functions at the cilium. Here, we investigate the regulation of these DCGs by Regulatory Factor X transcription factors (RFX TFs), a gene family known for transcriptionally regulating ciliary genes. We identify conserved X-box motifs in the promoter regions of DYX1C1, DCDC2, and KIAA0319 and demonstrate their functionality, as well as the ability to recruit RFX TFs using reporter gene and electrophoretic mobility shift assays. Furthermore, we uncover a complex regulation pattern between RFX1, RFX2, and RFX3 and their significant effect on modifying the endogenous expression of DYX1C1 and DCDC2 in a human retinal pigmented epithelial cell line immortalized with hTERT (hTERT-RPE1). In addition, induction of ciliogenesis increases the expression of RFX TFs and DCGs. At the protein level, we show that endogenous DYX1C1 localizes to the base of the cilium, whereas DCDC2 localizes along the entire axoneme of the cilium, thereby validating earlier localization studies using overexpression models. Our results corroborate the emerging role of DCGs in ciliary function and characterize functional noncoding elements, X-box promoter motifs, in DCG promoter regions, which thus can be targeted for mutation screening in dyslexia and ciliopathies associated with these genes.-Tammimies, K., Bieder, A., Lauter, G., Sugiaman-Trapman, D., Torchet, R., Hokkanen, M.-E., Burghoorn, J., Castrén, E., Kere, J., Tapia-Páez, I., Swoboda, P. Ciliary dyslexia candidate genes DYX1C1 and DCDC2 are regulated by Regulatory Factor (RF) X transcription factors through X-box promoter motifs.

Primary Cilia and Intraflagellar Transport Proteins in Bone and Cartilage.

Primary cilia, present on most mammalian cells, function as a sensor to sense the environment change and transduce signaling. Loss of primary cilia causes a group of human pleiotropic syndromes called Ciliopathies. Some of the ciliopathies display skeletal dysplasias, implying the important role of primary cilia in skeletal development and homeostasis. Emerging evidence has shown that loss or malfunction of primary cilia or ciliary proteins in bone and cartilage is associated with developmental and function defects. Intraflagellar transport (IFT) proteins are essential for cilia formation and/or function. In this review, we discuss the role of primary cilia and IFT proteins in the development of bone and cartilage, as well as the differentiation and mechanotransduction of mesenchymal stem cells, osteoblasts, osteocytes, and chondrocytes. We also include the role of primary cilia in tooth development and highlight the current advance of primary cilia and IFT proteins in the pathogenesis of cartilage diseases, including osteoarthritis, osteosarcoma, and chondrosarcoma.