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.
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.
Bardet-Biedl syndrome (BBS) is a well-known ciliopathy with mutations reported in 18 different genes. Most of the protein products of the BBS genes localize at or near the primary cilium and the centrosome. Near the centrosome, BBS proteins interact with centriolar satellite proteins, and the BBSome (a complex of seven BBS proteins) is believed to play a role in transporting ciliary membrane proteins. However, the precise mechanism by which BBSome ciliary trafficking activity is regulated is not fully understood. Here, we show that a centriolar satellite protein, AZI1 (also known as CEP131), interacts with the BBSome and regulates BBSome ciliary trafficking activity. Furthermore, we show that AZI1 interacts with the BBSome through BBS4. AZI1 is not involved in BBSome assembly, but accumulation of the BBSome in cilia is enhanced upon AZI1 depletion. Under conditions in which the BBSome does not normally enter cilia, such as in BBS3 or BBS5 depleted cells, knock down of AZI1 with siRNA restores BBSome trafficking to cilia. Finally, we show that azi1 knockdown in zebrafish embryos results in typical BBS phenotypes including Kupffer's vesicle abnormalities and melanosome transport delay. These findings associate AZI1 with the BBS pathway. Our findings provide further insight into the regulation of BBSome ciliary trafficking and identify AZI1 as a novel BBS candidate gene.
Bardet-Biedl syndrome (BBS) is a genetically heterogeneous autosomal recessive ciliopathy with 18 causative genes reported to date. The syndrome is characterized by obesity, polydactyly, renal defects, hypogenitalism and retinal degeneration. Previous work has illustrated a role for BBS proteins in the trafficking of ciliary cargo proteins including MCHR1, SSTR3, and dopamine receptor 1. In addition, interaction of BBS proteins with other centriolar satellite proteins has been reported. In order to identify novel BBS interacting proteins and novel BBS candidate genes we generated a transgenic BBS4 mouse. In this study, we utilized the transgenic mice to identify a novel BBSome (a complex of eight BBS proteins) interacting protein, AZI1. We show that AZI1 physically binds to the BBSome via BBS4. We also suggest a negative role of AZI1 in ciliary trafficking of the BBSome: when AZI1 is depleted, more BBSome localizes to cilia. Using zebrafish as a model, we show that azi1 morphants are similar to bbs morphants, a finding that further implicates AZI1 with the BBS pathway. Our findings provide further insight into the regulation of BBSome ciliary trafficking and identify AZI1 as a BBS candidate gene.
It is well understood that apicomplexan parasites, such as the malaria pathogen Plasmodium, are descended from free-living algae, and maintain a vestigial chloroplast that has secondarily lost all genes of photosynthetic function. Recently, two fully photosynthetic relatives of parasitic apicomplexans have been identified, the ‘chromerid’ algae Chromera velia and Vitrella brassicaformis, which retain photosynthesis genes within their chloroplasts. Elucidating the processes governing gene expression in chromerid chloroplasts might provide valuable insights into the origins of parasitism in the apicomplexans. We have characterised chloroplast transcript processing pathways in C. velia, V. brassicaformis and P. falciparum with a focus on the addition of an unusual, 3′ poly(U) tail. We demonstrate that poly(U) tails in chromerids are preferentially added to transcripts that encode proteins that are directly involved in photosynthetic electron transfer, over transcripts for proteins that are not involved in photosynthesis. To our knowledge, this represents the first chloroplast transcript processing pathway to be associated with a particular functional category of genes. In contrast, Plasmodium chloroplast transcripts are not polyuridylylated. We additionally present evidence that poly(U) tail addition in chromerids is involved in the alternative processing of polycistronic precursors covering multiple photosynthesis genes, and appears to be associated with high levels of transcript abundance. We propose that changes to the chloroplast transcript processing machinery were an important step in the loss of photosynthesis in ancestors of parasitic apicomplexans.
Chloroplasts contain their own genomes, containing two broad functional types of gene: genes encoding proteins directly involved in photosynthesis, and genes with a non-photosynthesis function, such as cofactor biosynthesis, assembly of protein complexes, or expression of the chloroplast genome. Thus far, to our knowledge, no chloroplast gene expression pathways in any lineage have been found to target one functional category of gene specifically. Here, we show that a chloroplast RNA processing pathway – the addition of a 3′ poly(U) tail – is specifically associated with photosynthesis genes in two species of algae, the ‘chromerids’ Chromera and Vitrella. The addition of the poly(U) tail enables the precise processing of mature photosynthesis gene transcripts from precursor RNA, and is likely to be essential for expression of the chromerid photosynthesis machinery. The chromerid algae are the closest photosynthetic relatives of a parasitic group of eukaryotes, the apicomplexans, which include the malaria pathogen Plasmodium. Apicomplexans are descended from algae, and retain a reduced chloroplast, which contains genes only of non-photosynthesis function. We have confirmed that 3′ poly(U) tails are not added to Plasmodium chloroplast transcripts. The expression pathways associated with photosynthesis genes have therefore been lost in the evolution of the apicomplexan chloroplast, and this loss could potentially have driven the transition from photosynthesis to parasitism.
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.
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.
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.
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.
Whole genome sequencing is a powerful tool in the discovery of single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) among mutant strains, which simplifies forward genetics approaches. However, identification of the causative mutation among a large number of non-causative SNPs in a mutant strain remains a big challenge. In the unicellular biflagellate green alga Chlamydomonas reinhardtii, we generated a SNP/indel library that contains over 2 million polymorphisms from four wild-type strains, one highly polymorphic strain that is frequently used in meiotic mapping, ten mutant strains that have flagellar assembly or motility defects, and one mutant strain, imp3, which has a mating defect. A comparison of polymorphisms in the imp3 strain and the other 15 strains allowed us to identify a deletion of the last three amino acids, Y313F314L315, in a protein phosphatase 2A catalytic subunit (PP2A3) in the imp3 strain. Introduction of a wild-type HA-tagged PP2A3 rescues the mutant phenotype, but mutant HA-PP2A3 at Y313 or L315 fail to rescue. Our immunoprecipitation results indicate that the Y313, L315, or YFLΔ mutations do not affect the binding of PP2A3 to the scaffold subunit, PP2A-2r. In contrast, the Y313, L315, or YFLΔ mutations affect both the stability and the localization of PP2A3. The PP2A3 protein is less abundant in these mutants and fails to accumulate in the basal body area as observed in transformants with either wild-type HA-PP2A3 or a HA-PP2A3 with a V310T change. The accumulation of HA-PP2A3 in the basal body region disappears in mated dikaryons, which suggests that the localization of PP2A3 may be essential to the mating process. Overall, our results demonstrate that the terminal YFL tail of PP2A3 is important in the regulation on Chlamydomonas mating.
Whole genome sequencing is a powerful tool to detect changes in genomic DNA. However, how to identify a causative mutation from over 20,000 changes remains a big challenge. For the unicellular green alga Chlamydomonas, we built a library that consists of over 2 million changes from 16 strains. A comparison of changes found in a mutant strain with a mating defect, imp3, to 15 other strains, leads to the identification of a three amino acid deletion in the catalytic subunit of a protein phosphatase 2A (PP2A3). The mating defect of imp3 is rescued by an HA-tagged PP2A3 gene. Introduction of the HA-tagged PP2A3 gene with various mutations in these three amino acids reveals that they play a key role in stabilizing and ensuring the proper localization of PP2A3. The ubiquitous enzyme PP2A is involved in diverse cellular processes. Our discovery that PP2A3 is involved in the Chlamydomonas mating signaling pathway, which also contains the polycystin2 homolog (PKD2), makes Chlamydomonas mating an excellent model to study ciliary/flagellar signaling. Since both PP2A and PKD2 play important roles in human health, further investigation of the relationship between these two proteins in Chlamydomonas will facilitate better understanding of their functions.
Cilia are architecturally complex organelles that protrude from the cell membrane and have signalling, sensory and motility functions that are central to normal tissue development and homeostasis. There are two broad categories of cilia; motile and non-motile, or primary, cilia. The central role of primary cilia in health and disease has become prominent in the past decade with the recognition of a number of human syndromes that result from defects in the formation or function of primary cilia. This rapidly growing class of conditions, now known as ciliopathies, impact the development of a diverse range of tissues including the neural axis, craniofacial structures, skeleton, kidneys, eyes and lungs. The broad impact of cilia dysfunction on development reflects the pivotal position of the primary cilia within a signalling nexus involving a growing number of growth factor systems including Hedgehog, Pdgf, Fgf, Hippo, Notch and both canonical Wnt and planar cell polarity. We have identified a novel ENU mutant allele of Ift140, which causes a mid-gestation embryonic lethal phenotype in homozygous mutant mice. Mutant embryos exhibit a range of phenotypes including exencephaly and spina bifida, craniofacial dysmorphism, digit anomalies, cardiac anomalies and somite patterning defects. A number of these phenotypes can be attributed to alterations in Hedgehog signalling, although additional signalling systems are also likely to be involved. We also report the identification of a homozygous recessive mutation in IFT140 in a Jeune syndrome patient. This ENU-induced Jeune syndrome model will be useful in delineating the origins of dysmorphology in human ciliopathies.
Skeletal ciliopathies are an emerging field of human disease in which skeletal birth defects arise due to abnormal communication between cells. This failure in communication arises following mutation in components of the primary cilia, a hair-like structure present on every cell. The skeletal ciliopathies are debilitating and in severe cases lead to death in early infancy. However, the mechanisms by which these malformations come about remains unclear. Mouse models are often used to delineate the causes of human birth defects and we have identified a model that mimics one of these conditions known as Jeune syndrome. It is the first mouse model with a mutation in the Ift140 gene, and these mice exhibit phenotypes that are often seen in this set of human syndromes. We have complimented this study with the discovery of a patient that presents with Jeune Syndrome resulting from mutation of human IFT140. This model will allow us to explore the role of IFT140 and the primary cilia in normal human development and provide insight into the field of human skeletal ciliopathies.
Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating.
Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion.
A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells.
Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.
Cilia are microtubule based organelles that project from cells. Cilia are found on almost every cell type of the human body and numerous diseases, collectively termed ciliopathies, are associated with defects in cilia, including respiratory infections, male infertility, situs inversus, polycystic kidney disease, retinal degeneration, and Bardet-Biedl Syndrome. Here we show that Illumina-based whole-genome transcriptome analysis in the biflagellate green alga Chlamydomonas reinhardtii identifies 1850 genes up-regulated during ciliogenesis, 4392 genes down-regulated, and 4548 genes with no change in expression during ciliogenesis. We examined four genes up-regulated and not previously known to be involved with cilia (ZMYND10, NXN, GLOD4, SPATA4) by knockdown of the human orthologs in human retinal pigment epithelial cells (hTERT-RPE1) cells to ask whether they are involved in cilia-related processes that include cilia assembly, cilia length control, basal body/centriole numbers, and the distance between basal bodies/centrioles. All of the genes have cilia-related phenotypes and, surprisingly, our data show that knockdown of GLOD4 and SPATA4 also affects the cell cycle. These results demonstrate that whole-genome transcriptome analysis during ciliogenesis is a powerful tool to gain insight into the molecular mechanism by which centrosomes and cilia are assembled.
flagella; deflagellation; ZMYND10; NXN; SPATA4; GLOD4
Despite recent progress in defining the ciliome, the genetic basis for many cases of primary ciliary dyskinesia (PCD) remains elusive. We evaluated five children from two unrelated, consanguineous Palestinian families who had PCD with typical clinical features, reduced nasal nitric oxide concentrations, and absent dynein arms. Linkage analyses revealed a single common homozygous region on chromosome 8 and one candidate was conserved in organisms with motile cilia. Sequencing revealed a single novel mutation in LRRC6 (Leucine-rich repeat containing protein 6) that fit the model of autosomal recessive genetic transmission, leading to a change of a highly conserved amino acid from aspartic acid to histidine (Asp146His). LRRC6 was localized to the cytoplasm and was up-regulated during ciliogenesis in human airway epithelial cells in a Foxj1-dependent fashion. Nasal epithelial cells isolated from affected individuals and shRNA-mediated silencing in human airway epithelial cells, showed reduced LRRC6 expression, absent dynein arms, and slowed cilia beat frequency. Dynein arm proteins were either absent or mislocalized to the cytoplasm in airway epithelial cells from a primary ciliary dyskinesia subject. These findings suggest that LRRC6 plays a role in dynein arm assembly or trafficking and when mutated leads to primary ciliary dyskinesia with laterality defects.
Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA–mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency.
Meier-Gorlin syndrome (MGS) is a rare disorder conferring small head circumference, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Our previous findings suggest that impaired DNA replication could cause the developmental defects in these disorders. Here we expand on those findings by showing that ORC1-deficient cells from MGS patients and depletion of origin licensing proteins also confer impaired centrosome and centriole copy number. Unexpectedly, we show that they also cause a striking defect in the rate of formation and function of primary cilia, hair-like mechano-, and chemo-sensory organelles. Finally we show that defects in cilia function in this context are associated with impaired cartilage formation in a model system. Our findings support the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency.
Centrioles and basal bodies are essential for a variety of cellular processes that include the recruitment of proteins to these structures for both centrosomal and ciliary function. This recruitment is compromised when centriole/basal body assembly is defective. Mutations that cause basal body assembly defects confer supersensitivity to Taxol. These include bld2, bld10, bld12, uni3, vfl1, vfl2, and vfl3. Flagellar motility mutants do not confer sensitivity with the exception of mutations in the p60 (pf19) and p80 (pf15) subunits of the microtubule severing protein katanin. We have identified additional pf15 and bld2 (ε-tubulin) alleles in screens for Taxol sensitivity. Null pf15 and bld2 alleles are viable and are not essential genes in Chlamydomonas. Analysis of double mutant strains with the pf15-3 and bld2-6 null alleles suggests that basal bodies in Chlamydomonas may recruit additional proteins beyond katanin that affect spindle microtubule stability. The bld2-5 allele is a hypomorphic allele and its phenotype is modulated by nutritional cues. Basal bodies in bld2-5 cells are missing proximal ends. The basal body mutants show aberrant localization of an epitope-tagged p80 subunit of katanin. Unlike IFT proteins, katanin p80 does not localize to the transition fibers of the basal bodies based on an analysis of the uni1 mutant as well as the lack of colocalization of katanin p80 with IFT74. We suggest that the triplet microtubules are likely to play a key role in katanin p80 recruitment to the basal body of Chlamydomonas rather than the transition fibers that are needed for IFT localization.
A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2) as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT) proteins and delivers a specific set of effector (cargo) proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein–protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.
A greater understanding of the mechanism of male fertility is essential in order to address the medical needs of the 1 in 20 men of reproductive age who are infertile. Conversely, there remains a critical need for additional contraceptive options, including those that target male gametes. Towards the aim of filling these knowledge gaps, we have used random mutagenesis to produce the Mot mouse line and to identify RABL2 as an essential regulator of male fertility. Mice carrying a mutant Rabl2 gene are sterile as a consequence of severely compromised sperm motility. Using biochemical approaches we have revealed that RABL2 binds to components of the intraflagellar transport machinery and have identified a number of RABL2 binding (effector) proteins. The presence of the Mot mutation in RABL2 leads to a significantly compromised ability to deliver binding proteins into the sperm tail. RABL2 is predominantly produced in male germ cells; however, lower levels are notably produced in organs that contain motile cilia (hair like structures involved in fluid/cell movement), thus raising the possibility that RABL2 may be involved in a broader set of human diseases collectively known as primary cilia dyskinesia.
The formation and function of eukaryotic cilia/flagella require the action of a large array of dynein microtubule motor complexes. Due to genetic, biochemical, and microscopic tractability, Chlamydomonas reinhardtii has become the premier model system in which to dissect the role of dyneins in flagellar assembly, motility, and signaling. Currently, fifty-four proteins have been described as components of various Chlamydomonas flagellar dyneins or as factors required for their assembly in the cytoplasm and/or transport into the flagellum; orthologues of nearly all these components are present in other ciliated organisms including humans. For historical reasons, the nomenclature of these diverse dynein components and their corresponding genes, mutant alleles and orthologues has become extraordinarily confusing. Here, we unify Chlamydomonas dynein gene nomenclature and establish a systematic classification scheme based on structural properties of the encoded proteins. Furthermore, we provide detailed tabulations of the various mutant alleles and protein aliases that have been used and explicitly define the correspondence with orthologous components in other model organisms and humans.
Chlamydomonas; Cilia; Dynein; Flagella; Microtubule
Because of the highly conserved nature of the ciliary axoneme, researchers studying the structure and function of cilia have used many different model systems. Each system has advantages and disadvantages, but all provide important information relevant to the understanding and treatment of the ciliopathies. For example, Chlamydomonas is easy to grow and amenable to rapid genetic manipulation and therefore is excellent for motility studies and studies of the structural components of the axoneme. However, this organism cannot be used to study developmental defects or physiological abnormalities that occur in higher organisms (e.g., mucociliary clearance). Human cilia have the advantage of being obtained directly from the tissue of interest but are obtainable only in limited quantities and are difficult to manipulate. Mouse models of ciliopathies are more difficult to study than Chlamydomonas but can be useful to elucidate more aspects of the human diseases. In this review, the overlap between the structure of primary and motile cilia is discussed, and recent advancements in our understanding of cilia structure and function using these three different model systems are presented. Potential therapeutic approaches, based on fundamental knowledge gained from work in these model systems, are also presented.
proteomics; suppressor screens; heterotaxy; congenital heart disease
Analysis of Chlamydomonas axonemes revealed that the protein phosphatase, PP2A, is localized to the outer doublet microtubules and is implicated in regulation of dynein-driven motility. We tested the hypothesis that PP2A is localized to the axoneme by a specialized, highly conserved 55-kDa B-type subunit identified in the Chlamydomonas flagellar proteome. The B-subunit gene is defective in the motility mutant pf4. Consistent with our hypothesis, both the B- and C-subunits of PP2A fail to assemble in pf4 axonemes, while the dyneins and other axonemal structures are fully assembled in pf4 axonemes. Two pf4 intragenic revertants were recovered that restore PP2A to the axonemes and re-establish nearly wild-type motility. The revertants confirmed that the slow-swimming Pf4 phenotype is a result of the defective PP2A B-subunit. These results demonstrate that the axonemal B-subunit is, in part, an anchor protein required for PP2A localization and that PP2A is required for normal ciliary motility.
Cilia; flagella; dynein; axonemes; protein phosphatases; microtubules
During spermiogenesis, haploid round spermatids undergo dramatic cell differentiation and morphogenesis to give rise to mature spermatozoa for fertilization, including nuclear elongation, chromatin remodeling, acrosome formation, and development of flagella. The molecular mechanisms underlining these fundamental processes remain poorly understood. Here, we report that MNS1, a coiled-coil protein of unknown function, is essential for spermiogenesis. We find that MNS1 is expressed in the germ cells in the testes and localizes to sperm flagella in a detergent-resistant manner, indicating that it is an integral component of flagella. MNS1–deficient males are sterile, as they exhibit a sharp reduction in sperm production and the remnant sperm are immotile with abnormal short tails. In MNS1–deficient sperm flagella, the characteristic arrangement of “9+2” microtubules and outer dense fibers are completely disrupted. In addition, MNS1–deficient mice display situs inversus and hydrocephalus. MNS1–deficient tracheal motile cilia lack some outer dynein arms in the axoneme. Moreover, MNS1 monomers interact with each other and are able to form polymers in cultured somatic cells. These results demonstrate that MNS1 is essential for spermiogenesis, the assembly of sperm flagella, and motile ciliary functions.
Cilia are microtubule-based structures present in virtually all cells in vertebrates. Cilia have diverse functions in development, growth, signaling, and fertilization. Primary ciliary dyskinesia (PCD) affects one in 16,000 individuals. PCD is characterized by bronchiectasis and chronic sinusitis, and is often associated with situs inversus and male infertility. The genetic cause of PCD is heterogeneous. Some cases of PCD in humans and animals are caused by single genic mutations such as mutations in genes encoding microtubule-based dynein arm components. We have characterized a protein called MNS1 and found that it plays an essential role in ciliary functions in mice. MNS1 is a novel and integral component of sperm flagella. Mice lacking MNS1 exhibit male sterility as evidenced by abnormal assembly of sperm flagella. MNS1–deficient mice also display defects in left–right asymmetry patterning of internal organs and hydrocephalus. Therefore, mutations in MNS1 may contribute to male infertility and PCD in humans.
Whole-genome sequencing (WGS) provides a new platform for the identification of mutations that produce a mutant phenotype. We used Illumina sequencing to identify the mutational profile of three Chlamydomonas reinhardtii mutant strains. The three strains have more than 38,000 changes from the reference genome. NG6 is aflagellate and maps to 269 kb with only one nonsynonymous change; the V12E mutation falls in the FLA8 gene. Evidence that NG6 is a fla8 allele comes from swimming revertants that are either true or pseudorevertants. NG30 is aflagellate and maps to 458 kb that has six nonsynonomous changes. Evidence that NG30 has a causative nonsense allele in IFT80 comes from rescue of the nonswimming phenotype with a fragment bearing only this gene. This gene has been implicated in Jeune asphyxiating thoracic dystrophy. Electron microscopy of ift80-1 (NG30) shows a novel basal body phenotype. A bar or cap is observed over the distal end of the transition zone, which may be an intermediate in preparing the basal body for flagellar assembly. In the acetate-requiring mutant ac17, we failed to find a nonsynonymous change in the 676 kb mapped region, which is incompletely assembled. In these strains, 43% of the changes occur on two of the 17 chromosomes. The excess on chromosome 6 surrounds the mating-type locus, which has numerous rearrangements and suppressed recombination, and the changes extend beyond the mating-type locus. Unexpectedly, chromosome 16 shows an unexplained excess of single nucleotide polymorphisms and indels. Overall, WGS in combination with limited mapping allows fast and accurate identification of point mutations in Chlamydomonas.
intraflagellar transport (IFT); flagellar assembly; mating-type; basal bodies; mapping
Mutations in the IC138 regulatory subunit of I1 dynein alter dynein motor activity and the flagellar waveform but do not affect phototaxis.
I1 dynein, or dynein f, is a highly conserved inner arm isoform that plays a key role in the regulation of flagellar motility. To understand how the IC138 IC/LC subcomplex modulates I1 activity, we characterized the molecular lesions and motility phenotypes of several bop5 alleles. bop5-3, bop5-4, and bop5-5 are null alleles, whereas bop5-6 is an intron mutation that reduces IC138 expression. I1 dynein assembles into the axoneme, but the IC138 IC/LC subcomplex is missing. bop5 strains, like other I1 mutants, swim forward with reduced swimming velocities and display an impaired reversal response during photoshock. Unlike mutants lacking the entire I1 dynein, however, bop5 strains exhibit normal phototaxis. bop5 defects are rescued by transformation with the wild-type IC138 gene. Analysis of flagellar waveforms reveals that loss of the IC138 subcomplex reduces shear amplitude, sliding velocities, and the speed of bend propagation in vivo, consistent with the reduction in microtubule sliding velocities observed in vitro. The results indicate that the IC138 IC/LC subcomplex is necessary to generate an efficient waveform for optimal motility, but it is not essential for phototaxis. These findings have significant implications for the mechanisms by which IC/LC complexes regulate dynein motor activity independent of effects on cargo binding or complex stability.
Sequence similarity based protein clustering methods organize proteins into families of similar sequences, a task that continues to be critical for automated protein characterization. However, many protein families cannot be automatically characterized further because little is known about the function of any protein in a family of similar sequences. We present a novel phylogenetic profile comparison (PPC) method called Automated Protein Annotation by Coordinate Evolution (APACE) that facilitates the automated characterization of proteins beyond their homology to other similar sequences. Our method implements a new approach for the normalization of similarity scores among multiple species and automates the characterization of proteins by their patterns of co-evolution with other proteins that do not necessarily share a similar sequence. We demonstrate that our method is able to recapitulate the topology of the latest, unresolved, composite deep eukaryotic phylogeny and is able to quantify the as yet unresolved branch lengths. We further demonstrate that our method is able to detect more functionally related proteins, given the same starting data, than existing methods. Finally, we demonstrate that our method can be successfully applied to much larger comparative genomic problem instances where existing methods often fail.
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by
formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2,
encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function
in primary cilia, but it is not well understood how these and other proteins are
targeted to cilia. Here, we provide the first genetic and biochemical link
between polycystins and the exocyst, a highly-conserved eight-protein membrane
trafficking complex. We show that knockdown of exocyst component Sec10 yields
cellular phenotypes associated with ADPKD, including loss of flow-generated
calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10
knockdown in zebrafish phenocopies many aspects of polycystin-2
knockdown—including curly tail up, left-right patterning defects,
glomerular expansion, and MAPK activation—suggesting that the exocyst is
required for pkd2 function in vivo. We observe
a synergistic genetic interaction between zebrafish sec10 and
pkd2 for many of these cilia-related phenotypes.
Importantly, we demonstrate a biochemical interaction between Sec10 and the
ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the
exocyst and polycystin-2 at the primary cilium. Our work supports a model in
which the exocyst is required for the ciliary localization of polycystin-2, thus
allowing for polycystin-2 function in cellular processes.
ADPKD, the most common potentially lethal monogenetic disorder, is caused by
mutations in PKD1 and PKD2. We are beginning to appreciate the important roles
these gene products, and others, play in cilia, which are thin rod-like
organelles projecting from the cell surface. Defects in cilia function are
associated with a variety of human diseases, including all variants of
polycystic kidney disease. Despite intense study of cilia and how they influence
disease, it is not understood how proteins are targeted and delivered to cilia.
Our work provides the first link between the exocyst, a conserved eight-protein
complex involved in protein localization, and a disease gene, PKD2. Knockdown of
the exocyst protein Sec10 results in a number of cellular- and cilia-related
phenotypes that are also seen upon pkd2 loss—both in
kidney cells and zebrafish. We then demonstrate specific genetic and biochemical
interactions between sec10 and pkd2. We
further show that Sec10 interacts with other ciliary proteins, such as IFT20 and
IFT88. From this work, we propose that the exocyst is involved in bringing
multiple types of ciliary proteins to the cilium. Given that the exocyst is
required for cilia structure and function, the exocyst may play a role in
cilia-related human diseases.
Cellular sophistication is not exclusive to multicellular organisms, and unicellular eukaryotes can resemble differentiated animal cells in their complex network of membrane-bound structures. These comparisons can be illuminated by genome-wide surveys of key gene families. We report a systematic analysis of Rabs in a complex unicellular Ciliate, including gene prediction and phylogenetic clustering, expression profiling based on public data, and Green Fluorescent Protein (GFP) tagging. Rabs are monomeric GTPases that regulate membrane traffic. Because Rabs act as compartment-specific determinants, the number of Rabs in an organism reflects intracellular complexity. The Tetrahymena Rab family is similar in size to that in humans and includes both expansions in conserved Rab clades as well as many divergent Rabs. Importantly, more than 90% of Rabs are expressed concurrently in growing cells, while only a small subset appears specialized for other conditions. By localizing most Rabs in living cells, we could assign the majority to specific compartments. These results validated most phylogenetic assignments, but also indicated that some sequence-conserved Rabs were co-opted for novel functions. Our survey uncovered a rare example of a nuclear Rab and substantiated the existence of a previously unrecognized core Rab clade in eukaryotes. Strikingly, several functionally conserved pathways or structures were found to be associated entirely with divergent Rabs. These pathways may have permitted rapid evolution of the associated Rabs or may have arisen independently in diverse lineages and then converged. Thus, characterizing entire gene families can provide insight into the evolutionary flexibility of fundamental cellular pathways.
Single-celled organisms appear simple compared to multicellular organisms, but this may not be true at the level of the individual cell. In fact, microscopic observations suggest that protists can possess networks of organelles just as elaborate as those in animal cells. Consistent with this idea, recent analysis has identified large families of genes in protists that are predicted to act as determinants for complex membrane networks. To test these predictions and to probe relationships between cellular structures across a wide swath of evolution, we focused on one gene family in the single-celled organism Tetrahymena. These genes control the traffic between organelles, with each gene controlling a single step in this traffic. We asked three questions about each of 56 genes in the family. First, what is the gene related to in humans? Second, under what conditions is the gene being used in Tetrahymena? Third, what is the role of each gene? The results provide insights into both the dynamics and evolution of membrane traffic, including the finding that some pathways appearing both structurally and functionally similar in protists and animals are likely to have arisen independently in the two lineages.
The type III secretion system of the Salmonella flagellum consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. However, in some other type III secretion systems, a homologue of FliO is apparently absent, suggesting it has a specialized role. Deleting the fliO gene from the chromosome of a motile strain of Salmonella resulted in a drastic decrease of motility. Incubation of the ΔfliO mutant strain in motility agar, gave rise to pseudorevertants containing extragenic bypass mutations in FliP at positions R143H or F190L. Using membrane topology prediction programs, and alkaline phosphatase or GFPuv chimeric protein fusions into the FliO protein, we demonstrated that FliO is bitopic with its N-terminus in the periplasm and C-terminus in the cytoplasm. Truncation analysis of FliO demonstrated that overexpression of FliO43–125 or FliO1–95 was able to rescue motility of the ΔfliO mutant. Further, residue leucine 91 in the cytoplasmic domain was identified to be important for function. Based on secondary structure prediction, the cytoplasmic domain, FliO43–125, should contain beta-structure and alpha-helices. FliO43–125-Ala was purified and studied using circular dichroism spectroscopy; however, this domain was disordered, and its structure was a mixture of beta-sheet and random coil. Coexpression of full-length FliO with FliP increased expression levels of FliP, but coexpression with the cytoplasmic domain of FliO did not enhance FliP expression levels. Overexpression of the cytoplasmic domain of FliO further rescued motility of strains deleted for the fliO gene expressing bypass mutations in FliP. These results suggest FliO maintains FliP stability through transmembrane domain interaction. The results also demonstrate that the cytoplasmic domain of FliO has functionality, and it presumably becomes structured while interacting with its binding partners.
The propeller-like flagella, which some bacteria use to swim, possess a specialized secretion apparatus, which is imbedded in the cell membrane for their formation. The components are highly conserved among flagella systems and also to the Type III secretion apparatus used by some bacteria in conjunction with virulence-associated needle complexes. The ubiquity of these secretion apparatuses and their function as intricate nanomachines has made them fascinating for biologists. The most studied flagellar system is that of Salmonella enterica, which consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. Among these proteins, FliO shows a sporadic distribution in bacteria, and its function is unknown, suggesting it might have a specialized role to play where it is present. In this study, we show that FliO has an important role in maintaining stability of FliP, which is a highly conserved member of the secretion apparatus. We have characterized the important regions of FliO through mutagenesis. We have shown that it is possible to bypass the effect of not producing the FliO protein, by encoding mutations within FliP or by overexpressing the cytoplasmic domain of FliO only.
The essential coenzyme nicotinamide adenine dinucleotide (NAD+) plays important roles in metabolic reactions and cell regulation in all organisms. Bacteria, fungi, plants, and animals use different pathways to synthesize NAD+. Our molecular and genetic data demonstrate that in the unicellular green alga Chlamydomonas NAD+ is synthesized from aspartate (de novo synthesis), as in plants, or nicotinamide, as in mammals (salvage synthesis). The de novo pathway requires five different enzymes: L-aspartate oxidase (ASO), quinolinate synthetase (QS), quinolate phosphoribosyltransferase (QPT), nicotinate/nicotinamide mononucleotide adenylyltransferase (NMNAT), and NAD+ synthetase (NS). Sequence similarity searches, gene isolation and sequencing of mutant loci indicate that mutations in each enzyme result in a nicotinamide-requiring mutant phenotype in the previously isolated nic mutants. We rescued the mutant phenotype by the introduction of BAC DNA (nic2-1 and nic13-1) or plasmids with cloned genes (nic1-1 and nic15-1) into the mutants. NMNAT, which is also in the de novo pathway, and nicotinamide phosphoribosyltransferase (NAMPT) constitute the nicotinamide-dependent salvage pathway. A mutation in NAMPT (npt1-1) has no obvious growth defect and is not nicotinamide-dependent. However, double mutant strains with the npt1-1 mutation and any of the nic mutations are inviable. When the de novo pathway is inactive, the salvage pathway is essential to Chlamydomonas for the synthesis of NAD+. A homolog of the human SIRT6-like gene, SRT2, is upregulated in the NS mutant, which shows a longer vegetative life span than wild-type cells. Our results suggest that Chlamydomonas is an excellent model system to study NAD+ metabolism and cell longevity.
Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme. NAD+ is necessary for electron transfer in many metabolic reactions. NAD+ functions as a substrate for several enzymes, one of which is sirtuin, an enzyme involved in gene regulation and aging. NAD+ can be synthesized either from amino acids (de novo) or metabolites (salvage). Given the importance of NAD+, enzymes involved in NAD+ synthesis are targets for drug discovery. In the unicellular green alga Chlamydomonas we investigated both the de novo and salvage NAD+ biosynthetic pathways. Mutations in the plant-like de novo synthesis pathway lead to a nicotinamide-requiring phenotype. We identified an insertional mutation in the first enzyme in the mammal-like salvage pathway; it has no growth defect in cells with an active de novo synthesis pathway but causes lethality when the de novo synthesis pathway is inactive. Coupled with NAD+ biosynthesis, sirtuin is involved in NAD+ consumption. Our study links upregulation of a sirtuin gene with extended life span in the nic13-1 mutant strain, which has a defective de novo synthesis pathway and suggests that Chlamydomonas is an excellent genetic model to study NAD+ metabolism and cell longevity.