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1.  The nphp-2 and arl-13 Genetic Modules Interact to Regulate Ciliogenesis and Ciliary Microtubule Patterning in C. elegans 
PLoS Genetics  2014;10(12):e1004866.
Cilia are microtubule-based cellular organelles that mediate signal transduction. Cilia are organized into several structurally and functionally distinct compartments: the basal body, the transition zone (TZ), and the cilia shaft. In vertebrates, the cystoprotein Inversin localizes to a portion of the cilia shaft adjacent to the TZ, a region termed the “Inversin compartment” (InvC). The mechanisms that establish and maintain the InvC are unknown. In the roundworm C. elegans, the cilia shafts of amphid channel and phasmid sensory cilia are subdivided into two regions defined by different microtubule ultrastructure: a proximal doublet-based region adjacent to the TZ, and a distal singlet-based region. It has been suggested that C. elegans cilia also possess an InvC, similarly to mammalian primary cilia. Here we explored the biogenesis, structure, and composition of the C. elegans ciliary doublet region and InvC. We show that the InvC is conserved and distinct from the doublet region. nphp-2 (the C. elegans Inversin homolog) and the doublet region genes arl-13, klp-11, and unc-119 are redundantly required for ciliogenesis. InvC and doublet region genes can be sorted into two modules—nphp-2+klp-11 and arl-13+unc-119—which are both antagonized by the hdac-6 deacetylase. The genes of this network modulate the sizes of the NPHP-2 InvC and ARL-13 doublet region. Glutamylation, a tubulin post-translational modification, is not required for ciliary targeting of InvC and doublet region components; rather, glutamylation is modulated by nphp-2, arl-13, and unc-119. The ciliary targeting and restricted localization of NPHP-2, ARL-13, and UNC-119 does not require TZ-, doublet region, and InvC-associated genes. NPHP-2 does require its calcium binding EF hand domain for targeting to the InvC. We conclude that the C. elegans InvC is distinct from the doublet region, and that components in these two regions interact to regulate ciliogenesis via cilia placement, ciliary microtubule ultrastructure, and protein localization.
Author Summary
Cilia are sensory organelles that are found on most types of human cells and play essential roles in diverse processes ranging from vision and olfaction to embryonic symmetry breaking and kidney development. Individual cilia are divided into multiple functionally and compositionally distinct compartments, including a proximal “Inversin” compartment, which is located near the base of cilia. We used the nematode C. elegans, a well-defined animal model of cilia biology, to characterize the genetics, components, and defining properties of the proximal cilium. The Inversin compartment is conserved in C. elegans, and is established independent of another proximal ciliary region, the microtubule doublet-based region. We showed how components of both the doublet region and the Inversin compartment genetically interact to regulate many pathways linked to core aspects of cilia biology, including ciliogenesis, cilia placement, cilia ultrastructure, microtubule stability, and the protein composition of ciliary compartments. In addition to expanding and clarifying our knowledge of basic cilia biology, these results also have direct implications for human health research because several of the genes and pathways explored in our work are linked to ciliopathies, a group of diseases caused by dysfunctional cilia.
PMCID: PMC4263411  PMID: 25501555
2.  Evolutionarily Ancient Association of the FoxJ1 Transcription Factor with the Motile Ciliogenic Program 
PLoS Genetics  2012;8(11):e1003019.
It is generally believed that the last eukaryotic common ancestor (LECA) was a unicellular organism with motile cilia. In the vertebrates, the winged-helix transcription factor FoxJ1 functions as the master regulator of motile cilia biogenesis. Despite the antiquity of cilia, their highly conserved structure, and their mechanism of motility, the evolution of the transcriptional program controlling ciliogenesis has remained incompletely understood. In particular, it is presently not known how the generation of motile cilia is programmed outside of the vertebrates, and whether and to what extent the FoxJ1-dependent regulation is conserved. We have performed a survey of numerous eukaryotic genomes and discovered that genes homologous to foxJ1 are restricted only to organisms belonging to the unikont lineage. Using a mis-expression assay, we then obtained evidence of a conserved ability of FoxJ1 proteins from a number of diverse phyletic groups to activate the expression of a host of motile ciliary genes in zebrafish embryos. Conversely, we found that inactivation of a foxJ1 gene in Schmidtea mediterranea, a platyhelminth (flatworm) that utilizes motile cilia for locomotion, led to a profound disruption in the differentiation of motile cilia. Together, all of these findings provide the first evolutionary perspective into the transcriptional control of motile ciliogenesis and allow us to propose a conserved FoxJ1-regulated mechanism for motile cilia biogenesis back to the origin of the metazoans.
Author Summary
Cilia are microtubule-based, hair-like organelles that project from the surfaces of eukaryotic cells. Protists use motile cilia for locomotion as well as for sensory perception. In metazoans, motile cilia also function in fluid transport over epithelia, such as in the mammalian lungs. Most vertebrate and some invertebrate cell-types differentiate non-motile primary cilia, which function exclusively in sensory transduction. It is believed that primary cilia arose from motile cilia through the loss of the motility apparatus. Cilia are complex organelles: a large number of proteins are involved in their assembly and maintenance. FoxJ1, a forkhead-domain transcription factor, is the master regulator of motile ciliogenesis in vertebrates. It is not known to what extent this transcriptional control is conserved and how it may have evolved. Here, we document the existence of FoxJ1 orthologs in several eukaryotic groups besides the vertebrates. FoxJ1 proteins from three representative phyla—Placozoa, Platyhelminthes, and Echinodermata—were able to activate the expression of ciliary genes when mis-expressed in zebrafish embryos. Moreover, inactivation of FoxJ1 in planaria (Platyhelminthes) abolished motile cilia differentiation. These results provide new insights into the transcriptional regulation of motile cilia biogenesis outside the vertebrates and demonstrate a remarkable conservation of the activity of FoxJ1.
PMCID: PMC3493443  PMID: 23144623
3.  A Novel zf-MYND Protein, CHB-3, Mediates Guanylyl Cyclase Localization to Sensory Cilia and Controls Body Size of Caenorhabditis elegans 
PLoS Genetics  2010;6(11):e1001211.
Cilia are important sensory organelles, which are thought to be essential regulators of numerous signaling pathways. In Caenorhabditis elegans, defects in sensory cilium formation result in a small-body phenotype, suggesting the role of sensory cilia in body size determination. Previous analyses suggest that lack of normal cilia causes the small-body phenotype through the activation of a signaling pathway which consists of the EGL-4 cGMP-dependent protein kinase and the GCY-12 receptor-type guanylyl cyclase. By genetic suppressor screening of the small-body phenotype of a cilium defective mutant, we identified a chb-3 gene. Genetic analyses placed chb-3 in the same pathway as egl-4 and gcy-12 and upstream of egl-4. chb-3 encodes a novel protein, with a zf-MYND motif and ankyrin repeats, that is highly conserved from worm to human. In chb-3 mutants, GCY-12 guanylyl cyclase visualized by tagged GFP (GCY-12::GFP) fails to localize to sensory cilia properly and accumulates in cell bodies. Our analyses suggest that decreased GCY-12 levels in the cilia of chb-3 mutants may cause the suppression of the small-body phenotype of a cilium defective mutant. By observing the transport of GCY-12::GFP particles along the dendrites to the cilia in sensory neurons, we found that the velocities and the frequencies of the particle movement are decreased in chb-3 mutant animals. How membrane proteins are trafficked to cilia has been the focus of extensive studies in vertebrates and invertebrates, although only a few of the relevant proteins have been identified. Our study defines a new regulator, CHB-3, in the trafficking process and also shows the importance of ciliary targeting of the signaling molecule, GCY-12, in sensory-dependent body size regulation in C. elegans. Given that CHB-3 is highly conserved in mammal, a similar system may be used in the trafficking of signaling proteins to the cilia of other species.
Author Summary
Caenorhabditis elegans is a 1–2 mm long nematode. Its body size is controlled by sensory inputs; some mutants with defects in sensory perception grow into small size (20%–30% decrease in body volume), although the animals seem to feed normally. The EGL-4 cGMP-dependent protein kinase and the GCY-12 guanylyl cyclase act in sensory neurons to control body size downstream of sensory inputs. GCY-12 is localized to cilia, antenna-like cellular structures of sensory neurons. In the cilia, a number of signaling molecules are localized. Dysfunction of cilia is known to cause several human disorders such as Bardet-Biedl syndrome, illustrating the importance of these organelles. In this study, we identified a novel protein, CHB-3, involved in sensory-dependent body size regulation. Our analyses suggest that CHB-3 protein regulates the trafficking of GCY-12 from the cell bodies to the cilia. Without CHB-3 protein, GCY-12 fails to localize to cilia and body size is not controlled properly. Thus, the cilia are a special place for sensory information processing in body size regulation. Our analyses identified CHB-3 as a novel trafficking regulator of ciliary protein(s).
PMCID: PMC2991246  PMID: 21124861
4.  Coordinated genomic control of ciliogenesis and cell movement by RFX2 
eLife  2014;3:e01439.
The mechanisms linking systems-level programs of gene expression to discrete cell biological processes in vivo remain poorly understood. In this study, we have defined such a program for multi-ciliated epithelial cells (MCCs), a cell type critical for proper development and homeostasis of the airway, brain and reproductive tracts. Starting from genomic analysis of the cilia-associated transcription factor Rfx2, we used bioinformatics and in vivo cell biological approaches to gain insights into the molecular basis of cilia assembly and function. Moreover, we discovered a previously un-recognized role for an Rfx factor in cell movement, finding that Rfx2 cell-autonomously controls apical surface expansion in nascent MCCs. Thus, Rfx2 coordinates multiple, distinct gene expression programs in MCCs, regulating genes that control cell movement, ciliogenesis, and cilia function. As such, the work serves as a paradigm for understanding genomic control of cell biological processes that span from early cell morphogenetic events to terminally differentiated cellular functions.
eLife digest
Cells that have hundreds of tiny hair-like structures called cilia on their surface have important roles in our airways and also in the brain and reproductive system. By beating in a coordinated manner, the cilia cause fluid to flow in a particular direction. The development of these multiciliated cells is a complex process in which genes are expressed as proteins, with this gene expression being regulated by other proteins called transcription factors.
In invertebrates the development of the cilia is controlled by transcription factors from the RFX family, which also appear to be important for development of cilia in vertebrates. However, the details of this process—in particular, the identities of the genes that are involved and how their functions are related—are not well understood in vertebrates.
Chung et al. have sought to remedy this by analyzing the network of genes whose expression is controlled by the transcription factor Rfx2 in vertebrates. The results showed that the genes controlled by Rfx2 were involved in all aspects of cilia, including several genes that are known to be mutated in diseases caused by abnormal cilia. Chung et al. also identified genes that were not previously thought to be relevant to cilia.
As multiciliated cells are developing, but before they can generate cilia, they must first migrate from the bottom of the epithelium, the layer of tissue in which they function, to the top of this layer. Chung et al. found that Rfx2 was also involved in this process.
The approach taken by Chung et al.—which involved a combination of RNA sequence analysis, examination of Rfx2 binding sites on chromosomes, computational predictions of protein interactions and in vivo cellular imaging—could be used to perform similar systems-level analyses of other developmental and biological processes.
PMCID: PMC3889689  PMID: 24424412
cilia; multiciliated cells; mucociliary epithelium; cilia beating; Rfx2; genomics; ttc29; ribc2; nme5; protofilament ribbon; Xenopus
5.  The dynamic cilium in human diseases 
PathoGenetics  2009;2:3.
Cilia are specialized organelles protruding from the cell surface of almost all mammalian cells. They consist of a basal body, composed of two centrioles, and a protruding body, named the axoneme. Although the basic structure of all cilia is the same, numerous differences emerge in different cell types, suggesting diverse functions. In recent years many studies have elucidated the function of 9+0 primary cilia. The primary cilium acts as an antenna for the cell, and several important pathways such as Hedgehog, Wnt and planar cell polarity (PCP) are transduced through it. Many studies on animal models have revealed that during embryogenesis the primary cilium has an essential role in defining the correct patterning of the body. Cilia are composed of hundreds of proteins and the impairment or dysfunction of one protein alone can cause complete loss of cilia or the formation of abnormal cilia. Mutations in ciliary proteins cause ciliopathies which can affect many organs at different levels of severity and are characterized by a wide spectrum of phenotypes. Ciliary proteins can be mutated in more than one ciliopathy, suggesting an interaction between proteins. To date, little is known about the role of primary cilia in adult life and it is tempting to speculate about their role in the maintenance of adult organs. The state of the art in primary cilia studies reveals a very intricate role. Analysis of cilia-related pathways and of the different clinical phenotypes of ciliopathies helps to shed light on the function of these sophisticated organelles. The aim of this review is to evaluate the recent advances in cilia function and the molecular mechanisms at the basis of their activity.
PMCID: PMC2694804  PMID: 19439065
6.  The master cell cycle regulator APC-Cdc20 regulates ciliary length and disassembly of the primary cilium 
eLife  2014;3:e03083.
The primary cilium has an important role in signaling; defects in structure are associated with a variety of human diseases. Much of the most basic biology of this organelle is poorly understood, even basic mechanisms, such as control of growth and resorption. We show that the activity of the anaphase-promoting complex (APC), an E3 that regulates the onset of anaphase, destabilizes axonemal microtubules in the primary cilium. Furthermore, the metaphase APC co-activator, Cdc20, is specifically recruited to the basal body of primary cilia. Inhibition of APC-Cdc20 activity increases the ciliary length, while overexpression of Cdc20 suppresses cilium formation. APC-Cdc20 activity is required for the timely resorption of the cilium after serum stimulation. In addition, APC regulates the stability of axonemal microtubules through targeting Nek1, the ciliary kinase, for proteolysis. These data demonstrate a novel function of APC beyond cell cycle control and implicate critical role of ubiquitin-mediated proteolysis in ciliary disassembly.
eLife digest
The majority of cells in the human body have small hair-like structures that project from the cell surface. These structures, known as primary cilia, are involved in sensing light and touch, and they are also required for an organism to develop normally. Defects in cilia result in a wide range of human diseases that are collectively known as ciliopathies. These include polycystic kidney disease and Bardet–Biedl syndrome. Ciliary disorders can also affect almost every organ in the body leading to blindness, obesity, diabetes, and cancer.
Cilia are dynamic structures that are dis-assembled when cells start to divide and are then re-assembled when cells are quiescent. The anaphase promoting complex (APC) has a critical role during cell division and targets key proteins that need to be degraded at specific times during this process. APC is localized in the basal body, which is found at the bottom of cilia, and it works together with a number of proteins which assist its function.
Wang et al. now report that a complex formed by APC and its co-activator protein Cdc20 has two functions at the basal body: it is needed to maintain the optimal length of the cilia in quiescent cells and to shorten the cilia when cells exit from quiescent stage.
Wang et al. also investigated the role of Nek1, an enzyme that is localised in the basal body. It was found that reducing the level of Nek1 in quiescent cells resulted in the formation of defective cilia, suggesting that this enzyme controls the stability and integrity of cilia. Moreover, when cells undergo division, the APC-Cdc20 complex targets the Nek1 enzyme, causing it to be degraded and allowing the cilia to be disassembled. A detailed understanding of how cells maintain the length of cilia could lead to the development of new approaches for the treatment of human ciliopathies.
PMCID: PMC4135350  PMID: 25139956
primary cilum; ubiquitin; cell cycle; retinal cell culture; None
7.  An Essential Role for DYF-11/MIP-T3 in Assembling Functional Intraflagellar Transport Complexes 
PLoS Genetics  2008;4(3):e1000044.
MIP-T3 is a human protein found previously to associate with microtubules and the kinesin-interacting neuronal protein DISC1 (Disrupted-in-Schizophrenia 1), but whose cellular function(s) remains unknown. Here we demonstrate that the C. elegans MIP-T3 ortholog DYF-11 is an intraflagellar transport (IFT) protein that plays a critical role in assembling functional kinesin motor-IFT particle complexes. We have cloned a loss of function dyf-11 mutant in which several key components of the IFT machinery, including Kinesin-II, as well as IFT subcomplex A and B proteins, fail to enter ciliary axonemes and/or mislocalize, resulting in compromised ciliary structures and sensory functions, and abnormal lipid accumulation. Analyses in different mutant backgrounds further suggest that DYF-11 functions as a novel component of IFT subcomplex B. Consistent with an evolutionarily conserved cilia-associated role, mammalian MIP-T3 localizes to basal bodies and cilia, and zebrafish mipt3 functions synergistically with the Bardet-Biedl syndrome protein Bbs4 to ensure proper gastrulation, a key cilium- and basal body-dependent developmental process. Our findings therefore implicate MIP-T3 in a previously unknown but critical role in cilium biogenesis and further highlight the emerging role of this organelle in vertebrate development.
Author Summary
The transport of protein complexes and associated cargo along microtubule tracks represents an essential eukaryotic process responsible for a multitude of cellular functions, including cell division, vesicle movement to membranes, and trafficking along dendrites, axons, and cilia. The latter organelles are hair-like cellular appendages implicated in cell and fluid motility, sensing and transducing information from their environment, and development. Their biogenesis and maintenance depends on a kinesin- and dynein-mediated motility process termed intraflagellar transport (IFT). In addition to comprising these specialized molecular motors, the IFT machinery consists of large multisubunit complexes whose exact composition and organization has not been fully defined. Here we identify a protein, DYF-11/MIP-T3, that is conserved in all ciliated organisms and is associated with IFT in C. elegans. Disruption of C. elegans DYF-11 results in structurally compromised cilia, likely as a result of IFT motor and subunit misassembly. Animals lacking DYF-11 display chemosensory anomalies, consistent with a role for the protein in cilia-associated sensory processes. In zebrafish, MIP-T3 is essential for gastrulation movements during development, similar to that observed for other ciliary components, including Bardet-Biedl syndrome proteins. In conclusion, we have identified a novel IFT machinery component that is also essential for development in vertebrates.
PMCID: PMC2268012  PMID: 18369462
8.  Arf4 Is Required for Mammalian Development but Dispensable for Ciliary Assembly 
PLoS Genetics  2014;10(2):e1004170.
The primary cilium is a sensory organelle, defects in which cause a wide range of human diseases including retinal degeneration, polycystic kidney disease and birth defects. The sensory functions of cilia require specific receptors to be targeted to the ciliary subdomain of the plasma membrane. Arf4 has been proposed to sort cargo destined for the cilium at the Golgi complex and deemed a key regulator of ciliary protein trafficking. In this work, we show that Arf4 binds to the ciliary targeting sequence (CTS) of fibrocystin. Knockdown of Arf4 indicates that it is not absolutely required for trafficking of the fibrocystin CTS to cilia as steady-state CTS levels are unaffected. However, we did observe a delay in delivery of newly synthesized CTS from the Golgi complex to the cilium when Arf4 was reduced. Arf4 mutant mice are embryonic lethal and die at mid-gestation shortly after node formation. Nodal cilia appeared normal and functioned properly to break left-right symmetry in Arf4 mutant embryos. At this stage of development Arf4 expression is highest in the visceral endoderm but we did not detect cilia on these cells. In the visceral endoderm, the lack of Arf4 caused defects in cell structure and apical protein localization. This work suggests that while Arf4 is not required for ciliary assembly, it is important for the efficient transport of fibrocystin to cilia, and also plays critical roles in non-ciliary processes.
Author Summary
Primary cilia are ubiquitous sensory organelles that play vital roles in an ever-growing class of human diseases termed ciliopathies including obesity, retinal degeneration and polycystic kidney disease. The proper function of the primary cilium relies on a cell's ability to target and concentrate specific receptors to the ciliary membrane – a unique subdomain of the plasma membrane yet little is known about how receptors are trafficked to the primary cilium. Mutations affecting the ciliary localized receptor fibrocystin (PKHD1) cause autosomal recessive polycystic kidney disease, which affects approximately 1∶20,000 individuals. Previously we identified a motif located in the cytoplasmic domain of fibrocystin that is required for its ciliary localization. In this work we demonstrate that the ciliary targeting sequence (CTS) of fibrocystin interacts with the small G protein Arf4 and this interaction is important for the efficient delivery of the CTS to cilia in cultured cells. Disruption of Arf4 in mice results in defects in the non-ciliated visceral endoderm and death at mid-gestation indicating Arf4 has vital functions in addition to ciliary protein trafficking.
PMCID: PMC3930517  PMID: 24586199
9.  Acute Versus Chronic Loss of Mammalian Azi1/Cep131 Results in Distinct Ciliary Phenotypes 
PLoS Genetics  2013;9(12):e1003928.
Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1Gt/Gt). Surprisingly, Azi1Gt/Gt MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease.
Author Summary
Cilia are slender projections from the surface of most mammalian cells and have sensory and sometimes motile functions. They are essential for mammalian development and defects in cilia lead to a group of human diseases, termed ciliopathies, with variable symptoms including embryonic lethality, lung and kidney defects, blindness and infertility. Cilia are complex structures composed of hundreds of components, whose individual functions are poorly understood. We screened for mammalian genes important in building cilia, and identified Azi1/Cep131, a gene previously shown to be required for cilia formation and function in fish and flies. We show that if we acutely reduce levels of Azi1 in mouse cells, fewer cells form cilia, but if we generate cells chronically lacking all Azi1, cilia form normally. In addition, mice without any Azi1 are healthy and viable, confirming normal cilia function. However, in these mice, the highly specialised ciliary structure of the sperm tail does not form, resulting in male infertility. We suggest Azi1 has conserved trafficking roles in both primary cilia and the specialised sperm flagella. Abruptly removing Azi1 results in instability causing the existing cilia network to collapse, whereas chronic deletion of Azi1 allows the system to re-equilibrate, and cilia to form normally.
PMCID: PMC3887133  PMID: 24415959
10.  Active Transport and Diffusion Barriers Restrict Joubert Syndrome-Associated ARL13B/ARL-13 to an Inv-like Ciliary Membrane Subdomain 
PLoS Genetics  2013;9(12):e1003977.
Cilia are microtubule-based cell appendages, serving motility, chemo-/mechano-/photo- sensation, and developmental signaling functions. Cilia are comprised of distinct structural and functional subregions including the basal body, transition zone (TZ) and inversin (Inv) compartments, and defects in this organelle are associated with an expanding spectrum of inherited disorders including Bardet-Biedl syndrome (BBS), Meckel-Gruber Syndrome (MKS), Joubert Syndrome (JS) and Nephronophthisis (NPHP). Despite major advances in understanding ciliary trafficking pathways such as intraflagellar transport (IFT), how proteins are transported to subciliary membranes remains poorly understood. Using Caenorhabditis elegans and mammalian cells, we investigated the transport mechanisms underlying compartmentalization of JS-associated ARL13B/ARL-13, which we previously found is restricted at proximal ciliary membranes. We now show evolutionary conservation of ARL13B/ARL-13 localisation to an Inv-like subciliary membrane compartment, excluding the TZ, in many C. elegans ciliated neurons and in a subset of mammalian ciliary subtypes. Compartmentalisation of C. elegans ARL-13 requires a C-terminal RVVP motif and membrane anchoring to prevent distal cilium and nuclear targeting, respectively. Quantitative imaging in more than 20 mutants revealed differential contributions for IFT and ciliopathy modules in defining the ARL-13 compartment; IFT-A/B, IFT-dynein and BBS genes prevent ARL-13 accumulation at periciliary membranes, whereas MKS/NPHP modules additionally inhibit ARL-13 association with TZ membranes. Furthermore, in vivo FRAP analyses revealed distinct roles for IFT and MKS/NPHP genes in regulating a TZ barrier to ARL-13 diffusion, and intraciliary ARL-13 diffusion. Finally, C. elegans ARL-13 undergoes IFT-like motility and quantitative protein complex analysis of human ARL13B identified functional associations with IFT-B complexes, mapped to IFT46 and IFT74 interactions. Together, these findings reveal distinct requirements for sequence motifs, IFT and ciliopathy modules in defining an ARL-13 subciliary membrane compartment. We conclude that MKS/NPHP modules comprise a TZ barrier to ARL-13 diffusion, whereas IFT genes predominantly facilitate ARL-13 ciliary entry and/or retention via active transport mechanisms.
Author Summary
Protruding from most cells surfaces is a hair-like extension called the primary cilium. This organelle functions as a cellular antenna, receiving physical and chemical signals such as light, odorants, and molecules that coordinate cell growth, differentiation and migration. Underscoring their importance, cilium defects underlie an expanding spectrum of diseases termed ciliopathies, characterised by wide-ranging symptoms such as cystic kidneys, blindness and bone abnormalities. A key question is how ciliary proteins are targeted to and retained within cilia. The best understood system is intraflagellar transport (IFT), thought to ferry proteins between the ciliary base and tip. Also, ciliopathy protein modules organise protein diffusion barriers at the ciliary base transition zone (TZ). Despite major advances, it remains poorly understood how proteins are targeted to cilia, and ciliary membrane subdomains in particular. Here, we investigated how Joubert syndrome-associated ARL13B/ARL-13 is compartmentalized at subciliary membranes. Using C. elegans nematodes and mammalian cell experimental systems, we uncovered differential requirements for sequence motifs, IFT and ciliopathy modules in regulating ARL-13 ciliary restriction, mobility and compartment length. Also, we provide essential insight into how IFT and ciliopathy-associated protein complexes and modules influence ciliary membrane protein transport, diffusion across the TZ, the integrity of the ciliary membrane, and subciliary protein composition.
PMCID: PMC3854969  PMID: 24339792
11.  The Par-PrkC Polarity Complex Is Required for Cilia Growth in Zebrafish Photoreceptors 
PLoS ONE  2014;9(8):e104661.
Specification and development of the apical membrane in epithelial cells requires the function of polarity proteins, including Pard3 and an atypical protein kinase C (PrkC). Many epithelial cells possess microtubule-based organelles, known as cilia, that project from their apical surface and the membrane surrounding the cilium is contiguous with the apical cell membrane. Although cilia formation in cultured cells required Pard3, the in vivo requirement for Pard3 in cilia development remains unknown. The vertebrate photoreceptor outer segment represents a highly specialized cilia structure in which to identify factors necessary for apical and ciliary membrane formation. Pard3 and PrkC localized to distinct domains within vertebrate photoreceptors. Using partial morpholino knockdown, photo-morpholinos, and pharmacological approaches, the function of Pard3 and PrkC were found to be required for the formation of both the apical and ciliary membrane of vertebrate photoreceptors. Inhibition of Pard3 or PrkC activity significantly reduced the size of photoreceptor outer segments and resulted in mislocalization of rhodopsin. Suppression of Pard3 or PrkC also led to a reduction in cilia size and cilia number in Kupffer’s Vesicle, which resulted in left-right asymmetry defects. Thus, the Par-PrkC complex functions in cilia formation in vivo and this likely reflects a general role in specifying non-ciliary and ciliary compartments of the apical domain.
PMCID: PMC4140697  PMID: 25144710
12.  Forkhead Transcription Factor Fd3F Cooperates with Rfx to Regulate a Gene Expression Program for Mechanosensory Cilia Specialization 
Developmental Cell  2012;22(6):1221-1233.
Cilia have evolved hugely diverse structures and functions to participate in a wide variety of developmental and physiological processes. Ciliary specialization requires differences in gene expression, but few transcription factors are known to regulate this, and their molecular function is unclear. Here, we show that the Drosophila Forkhead box (Fox) gene, fd3F, is required for specialization of the mechanosensory cilium of chordotonal (Ch) neurons. fd3F regulates genes for Ch-specific axonemal dyneins and TRPV ion channels, which are required for sensory transduction, and retrograde transport genes, which are required to differentiate their distinct motile and sensory ciliary zones. fd3F is reminiscent of vertebrate Foxj1, a motile cilia regulator, but fd3F regulates motility genes as part of a broader sensory regulation program. Fd3F cooperates with the pan-ciliary transcription factor, Rfx, to regulate its targets directly. This illuminates pathways involved in ciliary specialization and the molecular mechanism of transcription factors that regulate them.
Graphical Abstract
► Fd3F is a Fox transcription factor for mechanosensory ciliary specialization ► Regulates genes for ciliary motility, compartmentalization, and sensory transduction ► Fd3F modulates the activity of the pan-ciliogenic transcription factor, Rfx ► Fd3F is likely related to Foxj1, the vertebrate regulator of motile ciliated cells
Cilia are highly diverse. The motile sensory cilia of Drosophila mechanosensory neurons provide a good model of ciliary structural and functional specialization. Newton et al. show that Fox and Rfx transcription factors work in concert to activate a gene expression program for ciliary specialization in these neurons.
PMCID: PMC3414849  PMID: 22698283
13.  The Mechanics of the Primary Cilium: An Intricate Structure with Complex Function 
Journal of biomechanics  2011;45(1):17-26.
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 how cilium structure and subsequent mechanical behavior contributes to the roles the 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.
PMCID: PMC3242821  PMID: 21899847
Primary Cilium; Mechanics; Cellular mechanosensor; Structure; Model
14.  Evidence for reciliation of RPE1 cells in late G1 phase, and ciliary localisation of cyclin B1☆ 
FEBS Open Bio  2013;3:334-340.
The primary cilium, an organelle that transduces extracellular signals important for development and tissue homeostasis, is typically assembled upon cell cycle exit and disassembled upon cell cycle re-entry. Cilium assembly is thought to be suppressed in cycling cells, however the extent of suppression is not clear. For example, primary cilia are present in certain proliferating cells during development, and a period of reciliation has been reported to occur in late G1 in murine 3T3 cells released from serum starvation-induced quiescence. Human retinal pigmented epithelial (hTERT-RPE1; herein, RPE1) cells are commonly used to investigate pathways regulating cilium disassembly, however the ciliary disassembly profile of these cells remains uncertain. A period of reciliation has not been observed. Here, we analyse the ciliary disassembly profile of RPE1 cells by immunofluorescence microscopy. The results suggest a profile similar to 3T3 cells, including a period of reciliation in late G1 and a second wave of deciliation in S phase. We present evidence that arresting cells in early S phase with hydroxyurea or excess thymidine prevents the second wave of deciliation, and that deciliation is initiated shortly after release from a thymidine block, consistent with coupling to DNA replication. These findings support the often overlooked notion that cilium formation can occur in late G1, and suggest that RPE1 cells could serve as a model system for studying the molecular pathways that direct this process, in addition to those that stimulate cilium disassembly. We also present immunofluorescence data indicating that cyclin B1 localises to primary cilia.
•Ciliary disassembly profile of human RPE1 cells was analysed by immunofluorescence.•After release from serum-starvation, RPE1 cells re-assemble cilia in late G1 phase.•Cilium disassembly is not essential for S phase entry, but is coupled to DNA replication.•Cyclin B1 localises to primary cilia.
PMCID: PMC3821022  PMID: 24251092
Primary cilia; Cyclin B1; CDK1; Aurora A; DNA replication; Cilium disassembly; AurA, Aurora kinase A; BrdU, bromodeoxyuridine; CDK, cyclin-dependent kinase; DAPI, 4’,6-diamidino-2-phenylindole; FBS, fetal bovine serum; HU, hydroxyurea; Mim, mimosine; siRNA, short interfering RNA; SS, serum-starved; Thy, thymidine
15.  The PCP effector Fuzzy controls cilial assembly and signaling by recruiting Rab8 and Dishevelled to the primary cilium 
Molecular Biology of the Cell  2013;24(5):555-565.
During vertebrate development, the PCP pathway controls multiple cellular processes. Loss of the gene for the PCP effector Fuzzy affects formation of primary cilia via mostly unknown mechanisms. We report that Fuzzy localizes to the primary cilia and orchestrates delivery of Rab8 and Dishevelled to the primary cilium; loss of Fuzzy affects cilia-dependent signaling.
The planar cell polarity (PCP) pathway controls multiple cellular processes during vertebrate development. Recently the PCP pathway was implicated in ciliogenesis and in ciliary function. The primary cilium is an apically projecting solitary organelle that is generated via polarized intracellular trafficking. Because it acts as a signaling nexus, defects in ciliogenesis or cilial function cause multiple congenital anomalies in vertebrates. Loss of the PCP effector Fuzzy affects PCP signaling and formation of primary cilia; however, the mechanisms underlying these processes are largely unknown. Here we report that Fuzzy localizes to the basal body and ciliary axoneme and is essential for ciliogenesis by delivering Rab8 to the basal body and primary cilium. Fuzzy appears to control subcellular localization of the core PCP protein Dishevelled, recruiting it to Rab8-positive vesicles and to the basal body and cilium. We show that loss of Fuzzy results in inhibition of PCP signaling and hyperactivation of the canonical WNT pathway. We propose a mechanism by which Fuzzy participates in ciliogenesis and affects both canonical WNT and PCP signaling.
PMCID: PMC3583660  PMID: 23303251
16.  Proteomic Analysis of Mammalian Primary Cilia 
Current Biology  2012;22(5):414-419.
The primary cilium is a microtubule-based organelle that senses extracellular signals as a cellular antenna [1]. Primary cilia are found on many types of cells in our body and play important roles in development and physiology. Defects of primary cilia cause a broad class of human genetic diseases called ciliopathies. To gain new insights into ciliary functions and better understand the molecular mechanisms underlying ciliopathies, it is of high importance to generate a catalog of primary cilia proteins. In this study, we isolated primary cilia from mouse kidney cells by using a calcium shock method and identified 195 candidate primary cilia proteins by MudPIT (multidimensional protein identification technology), protein correlation profiling, and subtractive proteomic analysis. Based on comparisons with other proteomic studies of cilia, around 75% of our candidate primary cilia proteins are shared components with motile or specialized sensory cilia. The remaining 25% of the candidate proteins are possible primary cilia specific proteins. These possible primary cilia specific proteins include Evc2, Inpp5e and Inversin, several of which have been linked to known ciliopathies. We have performed the first reported proteomic analysis of primary cilia from mammalian cells. These results provide new insights into primary cilia structure and function.
PMCID: PMC3298568  PMID: 22326026
17.  Reduced cilia frequencies in human renal cell carcinomas versus neighboring parenchymal tissue 
Cilia  2013;2:2.
Cilia are essential organelles in multiple organ systems, including the kidney where they serve as important regulators of renal homeostasis. Renal nephron cilia emanate from the apical membrane of epithelia, extending into the lumen where they function in flow-sensing and ligand-dependent signaling cascades. Ciliary dysfunction underlies renal cyst formation that is in part caused by deregulation of planar cell polarity and canonical Wnt signaling. Renal cancer pathologies occur sporadically or in heritable syndromes caused by germline mutations in tumor suppressor genes including VHL. Importantly, Von Hippel-Lindau (VHL) patients frequently develop complex renal cysts that can be considered a premalignant stage. One of the well-characterized molecular functions of VHL is its requirement for the maintenance of cilia. In this study, tissue from 110 renal cancer patients who underwent nephrectomy was analyzed to determine if lower ciliary frequency is a common hallmark of renal tumorigenesis by comparing cilia frequencies in both tumor and adjacent parenchymal tissue biopsies from the same kidney.
We stained sections of human renal material using markers for cilia. Preliminary staining was performed using an immunofluorescent approach and a combination of acetylated-α-tubulin and pericentrin antibodies and DAPI. After validation of an alternative, higher throughput approach using acetylated-α-tubulin immunohistochemistry, we continued to manually quantify cilia in all tissues. Nuclei were separately counted in an automated fashion in order to determine ciliary frequencies. Similar staining and scoring for Ki67 positive cells was performed to exclude that proliferation obscures cilia formation potential.
Samples from renal cell carcinoma patients deposited in our hospital tissue bank were previously used to compose a tissue microarray containing three cores of both tumor and parenchymal tissue per patient. Cilia frequencies in a total of eighty-nine clear cell, eight papillary, five chromophobe renal cell carcinomas, two sarcomatoid renal tumors and six oncocytomas were determined. A marked decrease of primary cilia across renal cell carcinoma subtypes was observed compared to adjacent nontumorigenic tissue.
Our study shows that cilia are predominantly lost in renal cell carcinomas compared to tissue of the tumor parenchyma. These results suggest that ciliary loss is common in renal tumorigenesis, possibly participating in the sequence of cellular events leading to malignant tumor development. Future therapies aimed at restoring or circumventing cilia signaling might therefore aid in current treatment efficacy.
PMCID: PMC3564780  PMID: 23369289
Cilia; Kidney; Clear cell renal cell carcinoma; Chromophobe RCC; Papillary RCC; Oncocytoma; Histology
18.  Trafficking in and to the primary cilium 
Cilia  2012;1:4.
Polarized vesicle trafficking is mediated by small GTPase proteins, such as Rabs and Arls/Arfs. These proteins have essential roles in maintaining normal cellular function, in part, through regulating intracellular trafficking. Moreover, these families of proteins have recently been implicated in the formation and function of the primary cilium. The primary cilium, which is found on almost every cell type in vertebrates, is an organelle that protrudes from the surface of the cell and functions as a signaling center. Interestingly, it has recently been linked to a variety of human diseases, collectively referred to as ciliopathies. The primary cilium has an exceptionally high density of receptors on its membrane that are important for sensing and transducing extracellular stimuli. Moreover, the primary cilium serves as a separate cellular compartment from the cytosol, providing for unique spatial and temporal regulation of signaling molecules to initiate downstream events. Thus, functional primary cilia are essential for normal signal transduction. Rabs and Arls/Arfs play critical roles in early cilia formation but are also needed for maintenance of ciliary function through their coordination with intraflagellar transport (IFT), a specialized trafficking system in primary cilia. IFT in cilia is pivotal for the proper movement of proteins into and out of this highly regulated organelle. In this review article, we explore the involvement of polarized vesicular trafficking in cilia formation and function, and discuss how defects in these processes could subsequently lead to the abnormalities observed in ciliopathies.
PMCID: PMC3541539  PMID: 23351793
Primary cilium; Trafficking; Ciliopathies; Intraflagellar transport; Ciliary signaling
19.  Primary Cilia in Planar Cell Polarity Regulation of the Inner Ear 
Primary cilia are essential components of diverse cellular processes. Many of the requirements can be linked to the apparent signaling function of primary cilia. Recent studies have also uncovered a role for primary cilia in planar cell polarity (PCP) signaling. PCP refers to the coordinated orientation of cells along an axis parallel to the plane of the cell sheet. In vertebrates, the inner ear sensory organs display distinctive forms of PCP. One of the inner ear PCP characteristics is the coordinated positioning of a primary cilium eccentrically in every sensory hair cell within each organ. The inner ear, therefore, provides an opportunity to explore the cellular role of primary cilia in PCP signaling. In this chapter, we will introduce the PCP of the inner ear sensory organs, describe the conserved mechanism underlying the establishment of the planar polarity axis in invertebrates and vertebrates, and highlight a unique requirement for primary cilia in PCP regulation in vertebrates. Additionally, we will discuss a potentially ubiquitous role for cilia in cellular polarization in general.
PMCID: PMC4158840  PMID: 19147007
20.  Ciliopathies: an expanding disease spectrum 
Ciliopathies comprise a group of disorders associated with genetic mutations encoding defective proteins, which result in either abnormal formation or function of cilia. As cilia are a component of almost all vertebrate cells, cilia dysfunction can manifest as a constellation of features that include characteristically, retinal degeneration, renal disease and cerebral anomalies. Additional manifestations include congenital fibrocystic diseases of the liver, diabetes, obesity and skeletal dysplasias. Ciliopathic features have been associated with mutations in over 40 genes to date. However, with over 1,000 polypeptides currently identified within the ciliary proteome, several other disorders associated with this constellation of clinical features will likely be ascribed to mutations in other ciliary genes. The mechanisms underlying many of the disease phenotypes associated with ciliary dysfunction have yet to be fully elucidated. Several elegant studies have crucially demonstrated the dynamic ciliary localisation of components of the Hedgehog and Wnt signalling pathways during signal transduction. Given the critical role of the cilium in transducing “outside-in” signals, it is not surprising therefore, that the disease phenotypes consequent to ciliary dysfunction are a manifestation of aberrant signal transduction. Further investigation is now needed to explore the developmental and physiological roles of aberrant signal transduction in the manifestation of ciliopathy phenotypes. Utilisation of conditional and inducible murine models to delete or overexpress individual ciliary genes in a spatiotemporal and organ/cell-specific manner should help clarify some of the functional roles of ciliary proteins in the manifestation of phenotypic features.
Electronic supplementary material
The online version of this article (doi:10.1007/s00467-010-1731-7) contains supplementary material, which is available to authorised users.
PMCID: PMC3098370  PMID: 21210154
Ciliopathy; Renal disease; Retinal disease; Heterogeneous
21.  Primary cilia and signaling pathways in mammalian development, health and disease 
Nephron. Physiology  2009;111(3):p39-p53.
Although first described 1898 and long considered a vestigial organelle of little functional importance, the primary cilium has become one of the hottest research topics in modern cell biology and physiology. Primary cilia are non-motile sensory organelles present in a single copy on the surface of most growth-arrested or differentiated mammalian cells, and defects in their assembly or function are tightly coupled to many developmental defects, diseases and disorders. In normal tissues the primary cilium coordinates a series of signal transduction pathways, including Hedgehog, Wnt, PDGFRα and integrin signaling. In the kidney the primary cilium may function as a mechano-, chemo- and osmosensing unit that probes the extracellular environment and transmits signals to the cell via e.g. polycystins, which depend on ciliary localization for appropriate function. Indeed, hypomorphic mutations in the mouse ift88 (previously called Tg737) gene, which encodes a ciliogenic intraflagellar transport (IFT) protein, result in malformation of primary cilia, and in the collecting ducts of kidney tubules this is accompanied by development of autosomal recessive polycystic kidney disease (PKD; (1)). While PKD was one of the first diseases to be linked to dysfunctional primary cilia, defects in this organelle have subsequently been associated with many other phenotypes, including cancer, obesity, diabetes as well as a number of developmental defects. Collectively, these disorders of the cilium are now referred to as the ciliopathies. In this review we provide a brief overview of the structure and function of primary cilia and some of their roles in coordinating signal transduction pathways in mammalian development, health and disease. This review was written in conjunction with the Takis Anagnostopoulos Symposium on Renal and Epithelial Physiology and Physiopathology at Faculté de Médecine Necker in Paris, June 26-27, 2008.
PMCID: PMC2881330  PMID: 19276629
Primary cilia; cellular GPS; signal transduction; development; tissue homeostasis; diseases; disorders; ciliopathies
22.  Meckelin Is Necessary for Photoreceptor Intraciliary Transport and Outer Segment Morphogenesis 
This study revealed a role for meckelin in the intraciliary trafficking of phototransduction molecules and in the elongation and maintenance of the photoreceptor outer segments.
Cilia, complex structures found ubiquitously in most vertebrate cells, serve a variety of functions ranging from cell and fluid movement, cell signaling, tissue homeostasis, to sensory perception. Meckelin is a component of ciliary and cell membranes and is encoded by Tmem67 (Mks3). In this study, the retinal morphology and ciliary function in a mouse model for Meckel Syndrome Type 3 (MKS3) throughout the course of photoreceptor development was examined.
To study the effects of a disruption in the Mks3 gene on the retina, the authors introduced a functional allele of Pde6b into B6C3Fe a/a-bpck/J mice and evaluated their retinas by ophthalmoscopic, histologic, and ultrastructural examination. In addition, immunofluorescence microscopy was used to assess protein trafficking through the connecting cilium and to examine the localization of ciliary and synaptic proteins in Tmem67bpck mice and controls.
Photoreceptors degenerate early and rapidly in bpck/bpck mutant mice. In addition, phototransduction proteins, such as rhodopsin, arrestin, and transducin, are mislocalized. Ultrastructural examination of photoreceptors reveal morphologically intact connecting cilia but dysmorphic and misoriented outer segment (OS) discs, at the earliest time point examined.
These findings underscore the important role for meckelin in intraciliary transport of phototransduction molecules and their effects on subsequent OS morphogenesis and maintenance.
PMCID: PMC3317434  PMID: 22247471
23.  A high-resolution morphological and ultrastructural map of anterior sensory cilia and glia in Caenorhabditis elegans 
eLife  2014;3:e01948.
Many primary sensory cilia exhibit unique architectures that are critical for transduction of specific sensory stimuli. Although basic ciliogenic mechanisms are well described, how complex ciliary structures are generated remains unclear. Seminal work performed several decades ago provided an initial but incomplete description of diverse sensory cilia morphologies in C. elegans. To begin to explore the mechanisms that generate these remarkably complex structures, we have taken advantage of advances in electron microscopy and tomography, and reconstructed three-dimensional structures of fifty of sixty sensory cilia in the C. elegans adult hermaphrodite at high resolution. We characterize novel axonemal microtubule organization patterns, clarify structural features at the ciliary base, describe new aspects of cilia–glia interactions, and identify structures suggesting novel mechanisms of ciliary protein trafficking. This complete ultrastructural description of diverse cilia in C. elegans provides the foundation for investigations into underlying ciliogenic pathways, as well as contributions of defined ciliary structures to specific neuronal functions.
eLife digest
To survive, animals must constantly gather information about their surroundings and then decide how to respond. Animals rely on cells called sensory neurons to help them perceive and process this information, and these neurons in most animals have smaller structures called cilia that help them to gather this information. The structures of these cilia can range from simple hair-like rods to complex branched arbors. Defective cilia can lead to cell degeneration and death.
Scientists have identified and determined the functions of many of the 60 sensory neurons with cilia in C. elegans, a tiny roundworm with a simple nervous system. These experiments have revealed that the shapes of these cilia are quite diverse, and that the shape determines the type of information the neurons process. Learning more about how cilia are shaped, and how these shapes allow them to perform specific sensory functions, would give scientists a better understanding of how the brain processes sensory information.
Doroquez et al. have now taken advantage of advances in imaging technology to generate highly detailed three-dimensional reconstructions of the cilia on 50 neurons in the nose of C. elegans. The experiments involved rapidly freezing the worms, slowly replacing the frozen water molecules with a preservative solution, and then embedding in resin. This allowed Doroquez et al. to slice the samples into very thin sections—some 1400 times thinner than a sheet of paper—and then image them with transmission electron microscopy and electron tomography. Finally, all these images were combined in a computer to produce 3D models of the cilia.
The models reveal a wide range of cilia structures, including some that have never been examined in detail before. Doroquez et al. were also able to see detailed structures within the cilia, including compartments that determine which proteins should enter into, or be excluded from, an individual cilium. The models, along with the results of previous studies, suggest that cilia are shaped by genetic factors and also by interactions with the environment. This detailed description of diverse cilia structures should now allow researchers to identify the genes that determine their unique shapes, and explore how specific shapes contribute to specific sensory functions.
PMCID: PMC3965213  PMID: 24668170
cilia; electron microscopy; electron tomography; C. elegans
24.  The Golgin GMAP210/TRIP11 Anchors IFT20 to the Golgi Complex 
PLoS Genetics  2008;4(12):e1000315.
Eukaryotic cells often use proteins localized to the ciliary membrane to monitor the extracellular environment. The mechanism by which proteins are sorted, specifically to this subdomain of the plasma membrane, is almost completely unknown. Previously, we showed that the IFT20 subunit of the intraflagellar transport particle is localized to the Golgi complex, in addition to the cilium and centrosome, and hypothesized that the Golgi pool of IFT20 plays a role in sorting proteins to the ciliary membrane. Here, we show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP210/Trip11. Mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction, ventricular septal defects of the heart, omphalocele, and lung hypoplasia. Cells lacking GMAP210 have normal Golgi structure, but IFT20 is no longer localized to this organelle. GMAP210 is not absolutely required for ciliary assembly, but cilia on GMAP210 mutant cells are shorter than normal and have reduced amounts of the membrane protein polycystin-2 localized to them. This work suggests that GMAP210 and IFT20 function together at the Golgi in the sorting or transport of proteins destined for the ciliary membrane.
Author Summary
The primary cilium is a sensory organelle used by cells to monitor the extracellular environment. In mouse, severe defects in primary cilia lead to embryonic lethality while less severe defects cause a pleiotrophic phenotype that includes cystic kidney disease, retinal degeneration, obesity, and hydrocephaly, among others. The sensory functions of cilia rely on proteins localized to the ciliary membrane, which is continuous with the plasma membrane of the cell. Cells have the ability to specifically localize proteins to the ciliary membrane to the exclusion of the rest of the plasma membrane. Little is known about how this is accomplished. In prior work, we showed that the ciliary assembly protein IFT20 is localized to the Golgi complex, in addition to the cilium, and we proposed that it is involved in sorting or transport of membrane proteins to the cilium. In this work, we show that IFT20 is anchored to the Golgi complex by the golgin GMAP210. Mice defective in GMAP210 die at birth with lung and heart defects. Cells from these animals have ciliary defects, suggesting that IFT20 and GMAP210 function together at the Golgi complex in the trafficking of ciliary membrane proteins.
PMCID: PMC2602600  PMID: 19112494
25.  hemingway is required for sperm flagella assembly and ciliary motility in Drosophila 
Molecular Biology of the Cell  2014;25(8):1276-1286.
Motile cilia play important functions in many organisms. In Drosophila, hemingway (hmw) encodes a novel protein conserved in species with motile cilia. hmw-mutant flies are hearing impaired and male sterile. HMW is required for acquisition of motile properties of cilia in the fly sound receiver and stability of the sperm axoneme.
Cilia play major functions in physiology and development, and ciliary dysfunctions are responsible for several diseases in humans called ciliopathies. Cilia motility is required for cell and fluid propulsion in organisms. In humans, cilia motility deficiencies lead to primary ciliary dyskinesia, with upper-airways recurrent infections, left–right asymmetry perturbations, and fertility defects. In Drosophila, we identified hemingway (hmw) as a novel component required for motile cilia function. hmw encodes a 604–amino acid protein characterized by a highly conserved coiled-coil domain also found in the human orthologue, KIAA1430. We show that HMW is conserved in species with motile cilia and that, in Drosophila, hmw is expressed in ciliated sensory neurons and spermatozoa. We created hmw-knockout flies and found that they are hearing impaired and male sterile. hmw is implicated in the motility of ciliated auditory sensory neurons and, in the testis, is required for elongation and maintenance of sperm flagella. Because HMW is absent from mature flagella, we propose that HMW is not a structural component of the motile axoneme but is required for proper acquisition of motile properties. This identifies HMW as a novel, evolutionarily conserved component necessary for motile cilium function and flagella assembly.
PMCID: PMC3982993  PMID: 24554765

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