The primary cilium

The primary cilium is an antenna-like organelle that plays a vital role in organ generation and maintenance. It protrudes from the cell surface where it receives signals from the surrounding environment and relays them into the cell. These signals are then integrated to give the required outputs in terms of proliferation, differentiation, migration and polarization that ultimately lead to organ development and homeostasis. Defects in cilia function underlie a wide range of diverse but related human developmental or degenerative diseases. Collectively known as ciliopathies, these disorders present with varying severity and multiple organ involvement. The appreciation of the medical importance of the primary cilium has stimulated a huge effort into studies of the underlying cellular mechanisms. These in turn have revealed that ciliopathies result not only from defective assembly or organization of the primary cilium, but also from impaired ciliary signaling. This special edition of Organogenesis contains a set of review articles that highlight the role of the primary cilium in organ development and homeostasis, much of which has been learnt from studies of the associated human diseases. Here, we provide an introductory overview of our current understanding of the structure and function of the cilium, with a focus on the signaling pathways that are coordinated by primary cilia to ensure proper organ generation and maintenance.     

Heteromerization of Ciliary G Protein-Coupled Receptors in the Mouse Brain

Nearly every cell type in the mammalian body projects from its cell surface a primary cilium that provides important sensory and signaling functions. Defects in the formation or function of primary cilia have been implicated in the pathogenesis of many human developmental disorders and diseases, collectively termed ciliopathies. Most neurons in the brain possess cilia that are enriched for signaling proteins such as G protein-coupled receptors and adenylyl cyclase type 3, suggesting neuronal cilia sense neuromodulators in the brain and contribute to non-synaptic signaling. Indeed, disruption of neuronal cilia or loss of neuronal ciliary signaling proteins is associated with obesity and learning and memory deficits. As the functions of primary cilia are defined by the signaling proteins that localize to the ciliary compartment, identifying the complement of signaling proteins in cilia can provide important insights into their physiological roles. Here we report for the first time that different GPCRs can colocalize within the same cilium. Specifically, we found the ciliary GPCRs, melanin-concentrating hormone receptor 1 (Mchr1) and somatostatin receptor 3 (Sstr3) colocalizing within cilia in multiple mouse brain regions. In addition, we have evidence suggesting Mchr1 and Sstr3 form heteromers. As GPCR heteromerization can affect ligand binding properties as well as downstream signaling, our findings add an additional layer of complexity to neuronal ciliary signaling.     

PDGFRalphaalpha signaling is regulated through the primary cilium in fibroblasts

Recent findings show that cilia are sensory organelles that display specific receptors and ion channels, which transmit signals from the extracellular environment via the cilium to the cell to control tissue homeostasis and function. Agenesis of primary cilia or mislocation of ciliary signal components affects human pathologies, such as polycystic kidney disease and disorders associated with Bardet-Biedl syndrome. Primary cilia are essential for hedgehog ligand-induced signaling cascade regulating growth and patterning. Here, we show that the primary cilium in fibroblasts plays a critical role in growth control via platelet-derived growth factor receptor alpha (PDGFRalpha), which localizes to the primary cilium during growth arrest in NIH3T3 cells and primary cultures of mouse embryonic fibroblasts. Ligand-dependent activation of PDGFRalphaalpha is followed by activation of Akt and the Mek1/2-Erk1/2 pathways, with Mek1/2 being phosphorylated within the cilium and at the basal body. Fibroblasts derived from Tg737(orpk) mutants fail to form normal cilia and to upregulate the level of PDGFRalpha; PDGF-AA fails to activate PDGFRalphaalpha and the Mek1/2-Erk1/2 pathway. Signaling through PDGFRbeta, which localizes to the plasma membrane, is maintained at comparable levels in wild-type and mutant cells. We propose that ciliary PDGFRalphaalpha signaling is linked to tissue homeostasis and to mitogenic signaling pathways.     

PDGF-AA activates AKT and ERK signaling for testicular interstitial Leydig cell growth via primary cilia

Testes control the development of male reproductive system. The testicular interstitial Leydig cells (Leydig cells) synthesize testosterone for promoting spermatogenesis and secondary sexual characteristics. Type A platelet-derived growth factor (PDGF-AA) is one of the most important growth factors in regulating Leydig cell growth and function. Knockout of PDGF-AA or its congenital receptor PDGFR-α leads to poor testicular development caused by reducing Leydig cell numbers, supporting PDGF-AA/PDGFR-α signaling regulates Leydig cell development. Primary cilium is a cellular antenna that functions as an integrative platform to transduce extracellular signaling for proper development and differentiation. Several receptors including PDGFR-α are observed on primary cilia for initiating signaling cascades in distinct cell types. Here we showed that PDGF-AA/PDGFR-α signaling promoted Leydig cells growth, migration, and invasion via primary cilia. Upon PDGF-AA treatment, AKT and ERK signaling were activated to regulate these cellular events. Interestingly, active AKT and ERK were detected around the base of primary cilia. Depletion of ciliary genes (IFT88 and CEP164) alleviated PDGF-AA-activated AKT and ERK, thus reducing Leydig cell growth, migration, and invasion. Thus, our study not only reveals the function of PDGF-AA/PDGFR-α signaling in maintaining testicular physiology but also uncovers the role of primary cilium and downstream signaling in regulating Leydig cell development.     

Role of high-voltage-activated calcium channels in glucose-regulated beta-cell calcium homeostasis and insulin release

High-voltage-activated (HVA) calcium channels are known to be the primary source of calcium for glucose-stimulated insulin secretion. However, few studies have investigated how these channels can be regulated by chronically elevated levels of glucose. In the present study, we determined the level of expression of the four major HVA calcium channels (N-type, P/Q-type, L(C)-type, and L(D)-type) in rat pancreatic beta-cells. Using quantitative real-time PCR (QRT-PCR), we found the expression of all four HVA genes in rat insulinoma cells (INS-1) and in primary isolated rat islet cells. We then determined the role of each channel in insulin secretion by using channel-selective antagonists. Insulin secretion analysis revealed that N- and L-type channels are both involved in immediate glucose-induced insulin secretion. However, L-type was preferentially coupled to secretion at later time points. P/Q-type channels were not found to play a role in insulin secretion at any stage. It was also found that long-term exposure to elevated glucose increases basal calcium in these cells. Interestingly, chronically elevated glucose decreased the mRNA expression of the channels involved with insulin secretion and diminished the level of stimulated calcium influx in these cells. Using whole cell patch clamp, we found that N- and L-type channel currents increase gradually subsequent to lower intracellular calcium perfusion, suggesting that these channels may be regulated by glucose-induced changes in calcium.     

Cilia and polycystic kidney disease,kith and kin

In the past decade, cilia have been found to play important roles in renal cystogenesis. Many genes, such as PKD1 and PKD2 which, when mutated, cause autosomal dominant polycystic kidney disease (ADPKD), have been found to localize to primary cilia. The cilium functions as a sensor to transmit extracellular signals into the cell. Abnormal cilia structure and function are associated with the development of polyscystic kidney disease (PKD). Cilia assembly includes centriole migration to the apical surface of the cell, ciliary vesicle docking and fusion with the cell membrane at the intended site of cilium outgrowth, and microtubule growth from the basal body. This review summarizes the most recent advances in cilia and PKD research, with special emphasis on the mechanisms of cytoplasmic and intraciliary protein transport during ciliogenesis. Birth Defects Research (Part C) 102:174–185, 2014 . © 2014 Wiley Periodicals, Inc .     

The mechanics of the primary cilium: an intricate structure with complex function

The primary cilium is a non-motile singular cellular structure that extends from the surface of nearly every cell in the body. The cilium has been shown to play numerous roles in maintaining tissue homeostasis, through regulating signaling pathways and sensing both biophysical and biochemical changes in the extracellular environment. The structural performance of the cilium is paramount to its function as defective cilia have been linked to numerous pathologies. In particular, the cilium has demonstrated a mechanosensory role in tissues such as the kidney, liver, endothelium and bone, where cilium deflection under mechanical loading triggers a cellular response. Understanding of how cilium structure and subsequent mechanical behavior contributes to the roles that cilium plays in regulating cellular behavior is a compelling question, yet is a relatively untouched research area. Recent advances in biophysical measurements have demonstrated the cilium to be a structurally intricate organelle containing an array of load bearing proteins. Furthermore advances in modeling of this organelle have revealed the importance of these proteins at regulating the cilium's mechanosensitivity. Remarkably, the cilium is capable of adapting its mechanical state, altering its length and possibly it's bending resistance, to regulate its mechanosensitivity demonstrating the importance of cilium mechanics in cellular responses. In this review, we introduce the cilium as a mechanosensor; discuss the advances in the mechanical modeling of cilia; explore the structural features of the cilium, which contribute to its mechanics and finish with possible mechanisms in which alteration in structure may affect ciliary mechanics, consequently affecting ciliary based mechanosensing.     

The contrasting roles of primary cilia and cytonemes in Hh signaling

Hedgehog (Hh) is a paracrine signaling protein with major roles in development and disease. In vertebrates and invertebrates, Hh signal transduction is carried out almost entirely by evolutionarily conserved components, and in both, intercellular movement of Hh is mediated by cytonemes – specialized filopodia that serve as bridges that bring distant cells into contact. A significant difference is the role of the primary cilium, a slender, tubulin-based protuberance of many vertebrate cells. Although the primary cilium is essential for Hh signaling in cells that have one, most Drosophila cells lack a primary cilium. This perspective addresses the roles of primary cilia and cytonemes, and proposes that for Hh signaling, the role of primary cilia is to provide a specialized hydrophobic environment that hosts lipid-modified Hh and other components of Hh signal transduction after Hh has traveled from elsewhere in the cell. Implicit in this model is the idea that initial binding and uptake of Hh is independent of and segregated from the processes of signal transduction and activation.     

Soluble levels of cytosolic tubulin regulate ciliary length control

The primary cilium is an evolutionarily conserved dynamic organelle important for regulating numerous signaling pathways, and, as such, mutations disrupting ciliogenesis result in a variety of developmental abnormalities and postnatal disorders. The length of the cilium is regulated by the cell through largely unknown mechanisms. Normal cilia length is important, as either shortened or elongated cilia have been associated with disease and developmental defects. Here we explore the importance of cytoskeletal dynamics in regulating cilia length. Using pharmacological approaches in different cell types, we demonstrate that actin depolymerization or stabilization and protein kinase A activation result in a rapid elongation of the primary cilium. The effects of pharmacological agents on cilia length are associated with a subsequent increase in soluble tubulin levels and can be impaired by depletion of soluble tubulin with taxol. In addition, subtle nocodazole treatment was able to induce ciliogenesis under conditions in which cilia are not normally formed and also increases cilia length on cells that have already established cilia. Together these data indicate that cilia length can be regulated through changes in either the actin or microtubule network and implicate a possible role for soluble tubulin levels in cilia length control.     

Role of cilia in structural birth defects: Insights from ciliopathy mutant mouse models

Structural birth defect (SBD) is a major cause of morbidity and mortality in the newborn period. Although the etiology of SBD is diverse, a wide spectrum of SBD associated with ciliopathies points to the cilium as having a central role in the pathogenesis of SBDs. Ciliopathies are human diseases arising from disruption of cilia structure and/or function. They are associated with developmental anomalies in one or more organ systems and can involve defects in motile cilia, such as those in the airway epithelia or from defects in nonmotile (primary cilia) that have sensory and cell signaling function. Availability of low cost next generation sequencing has allowed for explosion of new knowledge in genetic etiology of ciliopathies. This has led to the appreciation that many genes are shared in common between otherwise clinically distinct ciliopathies. Further insights into the relevance of the cilium in SBD has come from recovery of pathogenic mutations in cilia‐related genes from many large‐scale mouse forward genetic screens with differing developmental phenotyping focus. Our mouse mutagenesis screen for congenital heart disease (CHD) using noninvasive fetal echocardiography has yielded a marked enrichment for pathogenic mutations in genes required for motile or primary cilia function. These novel mutant mouse models will be invaluable for modeling human ciliopathies and further interrogating the role of the cilium in the pathogenesis of SBD and CHD. Overall, these findings suggest a central role for the cilium in the pathogenesis of a wide spectrum of developmental anomalies associated with CHD and SBDs. Birth Defects Research (Part C) 102:115–125, 2014. © 2014 Wiley Periodicals, Inc.     

Cilia in the CNS: the quiet organelle claims center stage

The primary cilium is a cellular organelle that is almost ubiquitous in eukaryotes, yet its functions in vertebrates have been slow to emerge. The last fifteen years have been marked by accelerating insight into the biology of primary cilia, arising from the synergy of three major lines of research. These research programs describe a specialized mode of protein trafficking in cilia, reveal that genetic disruptions of primary cilia cause complex human disease syndromes, and establish that Sonic hedgehog (Shh) signal transduction requires the primary cilium. New lines of research have branched off to investigate the role of primary cilia in neuronal signaling, adult neurogenesis, and brain tumor formation. We review a fast expanding literature to determine what we now know about the primary cilium in the developing and adult CNS and what new directions should lead to further clarity.     

Wnt and planar cell polarity signaling in cystic renal disease

Cystic kidney diseases can cause end stage renal disease, affecting millions of individuals worldwide. They may arise early or later in life, are characterized by a spectrum of symptoms and can be caused by diverse genetic defects. The primary cilium, a microtubule-based organelle that can serve as a signaling antenna, has been demonstrated to have a significant role in ensuring correct kidney development and function. In the kidney, one of the signaling pathways that requires the cilium for normal development is Wnt signaling. In this review, the roles of primary cilia in relation to canonical and non-canonical Wnt/PCP signaling in cystic renal disease are described. The evidence of the associations between cilia, Wnt signaling and cystic renal disease is discussed and the significance of planar cell polarity-related mechanisms in cystic kidney disease is presented. Although defective Wnt signaling is not the only cause of renal disease, research is increasingly highlighting its importance, encouraging the development of Wnt-associated diagnostic and prognostic tools for cystic renal disease.     

Developmental signaling: Does it bridge the gap between cilia dysfunction and renal cystogenesis?

For more than a decade, evidence has accumulated linking dysfunction of primary cilia to renal cystogenesis, yet molecular mechanisms remain undefined. The pathogenesis of renal cysts is complex, involving multiple cellular aberrations and signaling pathways. Adding to this complexity, primary cilia exhibit multiple roles in a context‐dependent manner. On renal epithelial cells, primary cilia act as mechanosensors and trigger extracellular Ca²⁺ influx in response to laminar fluid flow. During mammalian development, primary cilia mediate the Hedgehog (Hh), Wnt, and Notch pathways, which control cell proliferation and differentiation, and tissue morphogenesis. Further, experimental evidence suggests the developmental state of the kidney strongly influences renal cystic disease. Thus, we review evidence for regulation of Ca²⁺ and cAMP, key molecules in renal cystogenesis, at the primary cilium, the role of Hh, Wnt, and Notch signaling in renal cystic disease, and the interplay between these developmental pathways and Ca²⁺ signaling. Indeed if these developmental pathways influence renal cystogenesis, these may represent novel therapeutic targets that can be integrated into a combination therapy for renal cystic disease. Birth Defects Research (Part C) 102:159–173, 2014. © 2014 Wiley Periodicals, Inc.     

Wnt and planar cell polarity signaling in cystic renal disease

Cystic kidney diseases can cause end stage renal disease, affecting millions of individuals worldwide. They may arise early or later in life, are characterized by a spectrum of symptoms and can be caused by diverse genetic defects. The primary cilium, a microtubule-based organelle that can serve as a signaling antenna, has been demonstrated to have a significant role in ensuring correct kidney development and function. In the kidney, one of the signaling pathways that requires the cilium for normal development is Wnt signaling. In this review, the roles of primary cilia in relation to canonical and non-canonical Wnt/PCP signaling in cystic renal disease are described. The evidence of the associations between cilia, Wnt signaling and cystic renal disease is discussed and the significance of planar cell polarity-related mechanisms in cystic kidney disease is presented. Although defective Wnt signaling is not the only cause of renal disease, research is increasingly highlighting its importance, encouraging the development of Wnt-associated diagnostic and prognostic tools for cystic renal disease.     

Primary Cilia Reconsidered in the Context of Ciliopathies: Extraciliary and Ciliary Functions of Cilia Proteins Converge on a Polarity theme?

Once dismissed as vestigial organelles, primary cilia have garnered the interest of scientists, given their importance in development/signaling, and for their implication in a new disease category known as ciliopathies. However, many, if not all, “cilia” proteins also have locations/functions outside of the primary cilium. These extraciliary functions can complicate the interpretation of a particular ciliopathy phenotype: it may be a result of defects at the cilium and/or at extraciliary locations, and it could be broadly related to a unifying cellular process for these proteins, such as polarity. Assembly of a cilium has many similarities to the development of other polarized structures. This evolutionarily preserved process for the assembly of polarized cell structures offers a perspective on how the cilium may have evolved. We hypothesize that cilia proteins are critical for cell polarity, and that core polarity proteins may have been specialized to form various cellular protrusions, including primary cilia.

     

Centriole is the pivot co-ordinating dynamic signaling for cell proliferation and organization during early development in the vertebrates

Vertebrates have an elaborate and functionally segmented body. It evolves from a single cell by systematic cell proliferation but attains a complex body structure with exquisite precision. This development requires two cellular events: cell cycle and ciliogenesis. For these events, the dynamic molecular signaling is converged at the centriole. The cell cycle helps in cell proliferation and growth of the body and is a highly regulated and integrated process. Its errors cause malignancies and developmental disorders. The cells newly proliferated are organized during organogenesis. For a cellular organization, dedicated signaling hubs are developed in the cells, and most often cilia are utilized. The cilium is generated from one of the centrioles involved in cell proliferation. The developmental signaling pathways hosted in cilia are essential for the elaboration of the body plan. The cilium's compartmental seclusion is ideal for noise-free molecular signaling and is essential for the precision of the body layout. The dysfunctional centrioles and primary cilia distort the development of body layout that manifest as serious developmental disorders. Thus, centriole has a dual role in the growth and cellular organization. It organizes dynamically expressed molecules of cell cycle and ciliogenesis and plays a balancing act to generate new cells and organize them during development. A putative master molecule may regulate and co-ordinate the dynamic gene expression at the centrioles. The convergence of many critical signaling components at the centriole reiterates the idea that centriole is a major molecular workstation involved in elaborating the structural design and complexity in vertebrates.     

Orpk mouse model of polycystic kidney disease reveals essential role of primary cilia in pancreatic tissue organization

Polycystic kidney disease (PKD) includes a group of disorders that are characterized by the presence of cysts in the kidney and other organs, including the pancreas. Here we show that in orpk mice, a model system for PKD that harbors a mutation in the gene that encodes the polaris protein, pancreatic defects start to occur at the end of gestation, with an initial expansion of the developing pancreatic ducts. Ductal dilation continues rapidly after birth and results in the formation of large, interconnected cysts. Expansion of pancreatic ducts is accompanied by apoptosis of neighboring acinar cells, whereas endocrine cell differentiation and islet formation appears to be unaffected. Polaris has been shown to co-localize with primary cilia, and these structures have been implicated in the formation of renal cysts. In the orpk pancreas, cilia numbers are reduced and cilia length is decreased. Expression of polycystin-2, a protein involved in PKD, is mislocalized in orpk mice. Furthermore, the cellular localization of beta-catenin, a protein involved in cell adhesion and Wnt signaling, is altered. Thus, polaris and primary cilia function are required for the maturation and maintenance of proper tissue organization in the pancreas.     

Primary cilia mediate mechanotransduction through control of ATP-induced Ca2+ signaling in compressed chondrocytes

We investigated the role of the chondrocyte primary cilium in mechanotransduction events related to cartilage extracellular matrix synthesis. We generated conditionally immortalized wild-type (WT) and IFT88(orpk) (ORPK) mutant chondrocytes that lack primary cilia and assessed intracellular Ca(2+) signaling, extracellular matrix synthesis, and ATP release in response to physiologically relevant compressive strains in a 3-dimensional chondrocyte culture system. All conditions were compared to unloaded controls. We found that cilia were required for compression-induced Ca(2+) signaling mediated by ATP release, and an associated up-regulation of aggrecan mRNA and sulfated glycosaminosglycan secretion. However, chondrocyte cilia were not the initial mechanoreceptors, since both WT and ORPK cells showed mechanically induced ATP release. Rather, we found that primary cilia were required for downstream ATP reception, since ORPK cells did not elicit a Ca(2+) response to exogenous ATP even though WT and ORPK cells express similar levels of purine receptors. We suggest that purinergic Ca(2+) signaling may be regulated by polycystin-1, since ORPK cells only expressed the C-terminal tail. This is the first study to demonstrate that primary cilia are essential organelles for cartilage mechanotransduction, as well as identifying a novel role for primary cilia not previously reported in any other cell type, namely cilia-mediated control of ATP reception.     

Cilia in cell signaling and human disorders

One of the most widespread cellular organelles in nature is cilium, which is found in many unicellular and multicellular organisms. Formerly thought to be a mostly vestigial organelle, the cilium has been discovered in the past several decades to play critical motile and sensory roles involved in normal organogenesis during development. The role of cilia has also been implicated in an ever increasing array of seemingly unrelated human diseases, including blindness, kidney cysts, neural tube defects and obesity. In this article we review some of the recent developments in research on cilia, and how defects in ciliogenesis and function can give rise to developmental disorders and disease.