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The primary cilium is a hair-like immotile organelle with specific membrane receptors, including the receptor of Hedgehog signaling, smoothened. The cilium organized in preosteoblasts promotes differentiation of the cells into osteoblasts (osteoblast differentiation) by mediating Hedgehog signaling to achieve bone formation. Notably, 4.1G is a plasma membrane-associated cytoskeletal protein that plays essential roles in various tissues, including the peripheral nervous system, testis, and retina. However, its function in the bone remains unexplored. In this study, we identified 4.1G expression in the bone. We found that, in the 4.1G-knockout mice, calcium deposits and primary cilium formation were suppressed in the trabecular bone, which is preosteoblast-rich region of the newborn tibia, indicating that 4.1G is a prerequisite for osteoblast differentiation by organizing the primary cilia in preosteoblasts. Next, we found that the primary cilium was elongated in the differentiating mouse preosteoblast cell line MC3T3-E1, whereas the knockdown of 4.1G suppressed its elongation. Moreover, 4.1G-knockdown suppressed the induction of the cilia-mediated Hedgehog signaling and subsequent osteoblast differentiation. These results demonstrate a new regulatory mechanism of 4.1G in bone formation that promotes the primary ciliogenesis in the differentiating preosteoblasts and induction of cilia-mediated osteoblast differentiation, resulting in bone formation at the newborn stage.

We report a complete 3D structural model of typical epithelial primary cilia based on structural maps of full-length primary cilia obtained by serial section electron tomography. Our data demonstrate the architecture of primary cilia differs extensively from the commonly acknowledged 9+0 paradigm. The axoneme structure is relatively stable but gradually evolves from base to tip with a decreasing number of microtubule complexes (MtCs) and a reducing diameter. The axonemal MtCs are cross-linked by previously unrecognized fibrous protein networks. Such an architecture explains why primary cilia can elastically withstand liquid flow for mechanosensing. The nine axonemal MtCs in a cilium are found to differ significantly in length indicating intraflagellar transport processes in primary cilia may be more complicated than that reported for motile cilia. The 3D maps of microtubule doublet–singlet transitions generally display longitudinal gaps at the inner junction between the A- and B-tubules, which indicates the inner junction protein is a major player in doublet–singlet transitions. In addition, vesicles releasing from kidney primary cilia were observed in the structural maps, supporting that ciliary vesicles budding may serve as ectosomes for cell–cell communication.

The primary cilium is a solitary, nonmotile and transitory appendage that is present in virtually all mammalian cells. Our knowledge of its ultrastructure and function is the result of more than fifty years of research that has dramatically changed our perspectives on the primary cilium. The mutual regulation between ciliogenesis and the cell cycle is now well-recognized, as well as the function of the primary cilium as a cellular “antenna” for perceiving external stimuli, such as light, odorants, and fluids. By displaying receptors and signaling molecules, the primary cilium is also a key coordinator of signaling pathways that converts extracellular cues into cellular responses. Given its critical tasks, any defects in primary cilium formation or function lead to a wide spectrum of diseases collectively called “ciliopathies”. An emerging role of primary cilium is in the regulation of cancer development. In this review, we seek to describe the current knowledge about the influence of the primary cilium in cancer progression, with a focus on some of the events that cancers need to face to sustain survival and growth in hypoxic microenvironment: the cancer hallmarks.

The presence of single, non-motile \"primary\" cilia on the surface of epithelial cells has been well described since the 1960s. However, for decades these organelles were believed to be vestigial, with no remaining function, having lost their motility. It wasn't until 2003, with the discovery that proteins responsible for transport along the primary cilium are essential for hedgehog signaling in mice, that the fundamental importance of primary cilia in signal transduction was realized. Little more than a decade later, it is now clear that the vast majority of signaling pathways in vertebrates function through the primary cilium. This has led to the adoption of the term \"the cells's antenna\" as a description for the primary cilium. Primary cilia are particularly important during development, playing fundamental roles in embryonic patterning and organogenesis, with a suite of inherited developmental disorders known as the \"ciliopathies\" resulting from mutations in genes encoding cilia proteins. This review summarizes our current understanding of the role of these fascinating organelles in a wide range of signaling pathways.

Previously we proposed that transmission of the hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.

The primary cilium permits compartmentalization of specific signaling pathways, including elements of the Hedgehog (Hh) pathway. Hh transcriptional activity is thought to be negatively regulated by constitutively high ciliary cAMP maintained by the Gα(s)-coupled GPCR, GPR161. However, cilia also sequester many other Gα(s)-coupled GPCRs with unknown potential to regulate Hh. Here we used biosensors optimized for ciliary cAMP and strategies to isolate signals in the cilium from the cell body and neighboring cells. We found that ciliary cAMP was not elevated relative to cellular cAMP, inconsistent with constitutive cAMP production. Gα(s)-coupled GPCRs (e.g., the 5-HT₆ serotonin and D1R dopamine receptor) had reduced ability to generate cAMP upon trafficking to the ciliary membrane. However, activation of the Hh pathway restored or amplified GPCR function to permit cAMP elevation selectively in the cilium. Hh therefore enables its own local GPCR-dependent cAMP regulatory circuit. Considering that GPCRs comprise much of the druggable genome, these data suggest alternative strategies to modify Hh signaling.

The balance between neural stem cell proliferation and neuronal differentiation is paramount for the appropriate development of the nervous system. Sonic hedgehog (Shh) is known to sequentially promote cell proliferation and specification of neuronal phenotypes, but the signaling mechanisms responsible for the developmental switch from mitogenic to neurogenic have remained unclear. Here, we show that Shh enhances Ca2+ activity at the neural cell primary cilium of developing Xenopus laevis embryos through Ca2+ influx via transient receptor potential cation channel subfamily C member 3 (TRPC3) and release from intracellular stores in a developmental stage-dependent manner. This ciliary Ca2+ activity in turn antagonizes canonical, proliferative Shh signaling in neural stem cells by down-regulating Sox2 expression and up-regulating expression of neurogenic genes, enabling neuronal differentiation. These discoveries indicate that the Shh-Ca2+-dependent switch in neural cell ciliary signaling triggers the switch in Shh action from canonical-mitogenic to neurogenic. The molecular mechanisms identified in this neurogenic signaling axis are potential targets for the treatment of brain tumors and neurodevelopmental disorders.

Stable cell lines and animal models expressing tagged proteins are important tools for studying behaviors of cells and molecules. Several molecular biology technologies have been applied with varying degrees of success and efficiencies to establish cell lines expressing tagged proteins. Here we applied CRISPR/Cas9 for the knock-in of tagged proteins into the 5’UTR of the endogenous gene loci. With this 5’UTR-targeting knock-in strategy, stable cell lines expressing Arl13b-Venus, Reep6-HA, and EGFP-alpha-tubulin were established with high efficiencies ranging from 50 to 80% in antibiotic selected cells. The localization of the knock-in proteins were identical to that of the endogenous proteins in wild-type cells and showed homogenous expression. Moreover, the expression of knock-in EGFP-alpha-tubulin from the endogenous promoter was stable over long-term culture. We further demonstrated that the fluorescent signals were enough for a long time time-lapse imaging. The fluorescent signals were distinctly visible during the whole duration of the time-lapse imaging and showed specific subcellular localizations. Altogether, our strategy demonstrates that 5’UTR is an amenable site to generate cell lines for the stable expression of tagged proteins from endogenous loci in mammalian cells. Key words: CRISPR/Cas9, NHEJ, Knock-in, Primary Cilium, UTR, Tubulin

Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein–protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein–protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.

Endometrial decidualization plays a pivotal role during early pregnancy. Compromised decidualization has been tightly associated with recurrent implantation failure (RIF). Primary cilium is an antenna-like sensory organelle and acts as a signaling nexus to mediate Hh, Wnt, TGFβ, BMP, FGF, and Notch signaling. However, whether primary cilium is involved in human decidualization is still unknown. In this study, we found that primary cilia are present in human endometrial stromal cells. The ciliogenesis and cilia length are increased by progesterone during in vitro and in vivo decidualization. Primary cilia are abnormal in the endometrium of RIF patients. Based on data from both assembly and disassembly of primary cilia, it has been determined that primary cilium is essential to human decidualization. Trichoplein (TCHP)-Aurora A signaling mediates cilia disassembly during human in vitro decidualization. Mechanistically, primary cilium modulates human decidualization through PTEN-PI3K-AKT-FOXO1 signaling. Our study highlights primary cilium as a novel decidualization-related signaling pathway.