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Both inherited and sporadic mutations can give rise to a plethora of human diseases. Through myriad diverse cellular processes, sporadic mutations can arise through a failure to accurately replicate the genetic code or by inaccurate separation of duplicated chromosomes into daughter cells. The human genome has therefore evolved to encode a large number of proteins that work together with regulators of the cell cycle to ensure that it remains error-free. This is collectively known as the DNA damage response (DDR), and genome stability mechanisms involve a complex network of signalling and processing factors that ensure redundancy and adaptability of these systems. The importance of genome stability mechanisms is best illustrated by the dramatic increased risk of cancer in individuals with underlying disruption to genome maintenance mechanisms. Cilia are microtubule-based sensory organelles present on most vertebrate cells, where they facilitate transduction of external signals into the cell. When not embedded within the specialised ciliary membrane, components of the primary cilium’s basal body help form the microtubule organising centre that controls cellular trafficking and the mitotic segregation of chromosomes. Ciliopathies are a collection of diseases associated with functional disruption to cilia function through a variety of different mechanisms. Ciliopathy phenotypes can vary widely, and although some cellular overgrowth phenotypes are prevalent in a subset of ciliopathies, an increased risk of cancer is not noted as a clinical feature. However, recent studies have identified surprising genetic and functional links between cilia-associated proteins and genome maintenance factors. The purpose of this mini-review is to therefore highlight some of these discoveries and discuss their implications with regards to functional crosstalk between the DDR and ciliogenesis pathways, and how this may impact on the development of human disease.

Cilia are microtubule based organelles enveloped by a specialised extension of plasma membrane. Serving as cellular antennae, cilia are essential for perceiving extracellular cues and mediating downstream signal transduction crucial for normal cellular physiology and development. These functions are mediated in large part by the specialised ciliary membrane, which differs in composition from the plasma membrane, housing many of the receptors, channels and effectors that mediate cilium-based signalling. However, the molecular mechanisms underlying the establishment, maintenance and regulation of the ciliary membrane remain poorly understood. In this project we are seeking to identify and functionally characterize genes with ciliogenic roles in ciliary membrane transport and organisation. Specifically, we are taking a candidate gene approach using the Caenorhabditis elegans ciliated sensory neuronal model, employing assays for cilium integrity (dye filling assay) and functionality (osmotic avoidance assay; foraging). Using predicted loss-of-function alleles, we screened 55 mutants of evolutionarily conserved membrane organising and trafficking genes and identified 4 rab-5-related endocytic gene mutants with defects in cilium integrity and function. One of these, rabs-5, encodes a RAB-5 effector, which we found is expressed mainly in ciliated cells. rabs-5 mutants possess short cilia and an expanded periciliary membrane compartment, indicative of defects in ciliary membrane homeostasis. Transgenic expression of a rabs-5(WT) construct rescues the cilium integrity and foraging defects of rabs-5 mutants, confirming the association of rabs-5 with cilia-related functions. By taking a reverse genetics candidate gene approach in C. elegans, a number of RAB-5-dependent downstream effectors have been identified as potential regulators of ciliogenesis and ciliary membrane homeostasis.

Background Cilia are vital to various cellular and sensory functions. The pathway by which ciliary membrane proteins translocate through the transition zone is not well understood. Direct morphological characterization of ciliary cargoes in transit remains lacking. In the vertebrate photoreceptor, rhodopsin is synthesized and transported from the inner segment to the disc membranes of the outer segment (OS), which is a modified cilium. To date, the membrane topology of the basal OS and the mechanisms by which rhodopsin is transported through the transition zone (i.e., connecting cilium) and by which nascent disc membranes are formed remain controversial. Results Using an antibody recognizing its cytoplasmic C-terminus, we localize rhodopsin on both the plasma membrane and lumen of the connecting cilium by immuno-electron microscopy (EM). We also use transmission EM to visualize the electron-dense enzymatic products derived from the rhodopsin-horseradish peroxidase (HRP) fusion in transfected rodent rods. In the connecting cilium, rhodopsin is not only expressed in the plasma membrane but also in the lumen on two types of membranous carriers, long smooth tubules and small, coated, filament-bound vesicles. Additionally, membrane-bound rhodopsin carriers are also found in close proximity to the nascent discs at the basal OS axoneme and in the distal inner segment. This topology-indicative HRP-rhodopsin reporter shows that the nascent basalmost discs and the mature discs have the same membrane topology, with no indication of evagination or invagination from the basal OS plasma membranes. Serial block face and focus ion beam scanning EM analyses both indicate that the transport carriers enter the connecting cilium lumen from either the basal body lumen or cytoplasmic space between the axonemal microtubules and the ciliary plasma membrane. Conclusions Our results suggest the existence of multiple ciliary gate entry pathways in rod photoreceptors. Rhodopsin is likely transported across the connecting cilium on the plasma membrane and through the lumens on two types of tubulovesicular carriers produced in the inner segment. Our findings agree with a previous model that rhodopsin carriers derived from the cell body may fuse directly onto nascent discs as they grow and mature. Electronic supplementary material The online version of this article (doi:10.1186/s13630-015-0013-1) contains supplementary material, which is available to authorized users.

The two dimensional ultrastructure of connective tissue primary cilia shows a physical continuum between the biomechanically functional extracellular matrix, the mechanosensory primary cilium, and the polarized Golgi apparatus required to secrete an extracellular matrix. Our objective was to create an ultrastructurally accurate, tomographic model of the Matrix - Cilium - Golgi - Continuum using optimally fixed chick embryo sternal chondrocytes as a model connective tissue. Proteoglycan granules consisted of multiple minor aggrecan precipitates interconnected three-dimensionally by collagen fibers and linked directly to the ciliary membrane. These receptor-mediated attachments were matched by protein complexes linking the ciliary membrane to axonemal microtubular doublets, each coated with numerous microtubule-associated proteins. Doublets varied in length and position, and all were twisted or bent relative to the basal centriole. Transitional fibers linked the distal end of the basal centriole to the membrane junction between cell and ciliary membranes, while basal feet proteins spanned three triplets of the basal centriole and anchored microtubules radiating from the centrosome. The proximal centriole lacked appendages, but formed protein linkages with nuclear pores. The cis, medial and trans Golgi compartments were polarised by the orientation of the cilium. We have developed an integrated, anatomically accurate, tomographic model of the matrix-cilium-Golgi continuum in chondrocytes that provides a new interactive tool to examine the structural relationship between primary cilia, the ciliary proteome and matrix biomechanics. It will be used to investigate the functional relationship between the mechanosensory primary cilium of connective tissue cells and the mechanically responsive extracellular matrix they produce.

Ciliopathies are a group of autosomal recessive disorders, characterized by defect in central nervous system, that include several, partially overlapping, syndromes such Meckel-Gruber syndrome and COACH syndrome. Ciliopathies are caused by mutations in genes encoding protein of the primary cilium, as Jouberin and Meckelin (TMEM67). Meckelin is a receptor localized to the ciliary membrane and at the cell surface of polarized cells; it interacts with citoskeletal protein participating in ciliogenesis via remodelling of cytoskeleton. Histone Deacetylase (HDAC) 6 is a cytoplasmic deacetylase, that localize to microtubules in a variety of cultured cells, and participate in the deacetylation of the major component of the cilia axoneme, alpha-tubulin, a process that leads to reduced microtubule stability. Moreover, HDAC6 has also been implicated both in cilia resorption and disassembly, through its interaction with Aurora A kinase, that localize to basal bodies. In the present work we show that MKS3 interacts with HDAC6 in MKS3 overexpressing HEK293 cells and in ciliated IMCD3 cells. Moreover, we confirmed this interaction in mouse embryonic stem cells, used as an in vitro model of neurogenesis. In this cellular context, deacetylase activity assays performed on MKS3 immunoprecipitated complexes showed high HDAC activity, which was lost in the presence of a specific HDAC6 inhibitor. Altogether these results reveal an unpredictable interaction between Meckelin and HDAC6, shedding light on a putative, novel role of Meckelin in controlling ciliary microtubules acetylation/deacetylation and primary cilia stability.

Transmission electron microscopy (TEM) is a valuable tool for elucidating cilium ultrastructure and the effects of ciliary and ciliopathy gene mutations on cilium integrity. We perform TEM on amphid channel sensory cilia in C. elegans, enabling us to visualise cilium compartments, namely the axoneme, the transition zone (TZ) and the transition fiber/basal body region. In worms, cilia extend from the dendritic tips of 60 sensory neurons. Here we show how TEM can be used to identify ultrastructural defects observed at the TZ of a number of ciliopathy disease gene mutants. In WT worms, the ~1 micron TZ is the most proximal part of the axoneme that extends from the far distal dendrite membrane, and is defined by a drawing together of the 9 doublet microtubules (MT) and Y-links that connect the doublet MTs with the ciliary membrane. In worms with mutations in Meckel-Gruber Syndrome (MKS) and Nephronophthisis (NPHP) gene homologues, we have observed a range of TZ abnormalities. These include TZs with Y-link defects (missing arms, reduced numbers) and TZs that fail to dock at the tip of the dendritic compartment. In most instances, TZ defects are observed only in worms where both MKS and NPHP functional modules are genetically disrupted and not in worms where only one module is abrogated. Together, our observations indicate redundant roles for multiple TZ-associated ciliopathy proteins in establishing physical connections between the TZ and the ciliary membrane.

Ciliogenesis involves coordinated assembly of a microtubule-based axoneme from the mother centriole and vesicular membrane transport and fusion forming a ciliary membrane around the developing axoneme. We, and others have reported that a Rab11-Rab8 cascade functions in ciliogenesis. Using live high-resolution fluorescence microscopy imaging we show that ciliary membrane assembly proceeds following Rabin8 (a Rab8 activator) binding to Rab11 membranes. Rabin8 transport via Rab11 vesicles to the centrosome is observed resulting in localized activation of Rab8 and leads to initiation of ciliary membrane assembly. Using proteomics approaches, we have discovered that Rabin8 binds to the TRAPPII tethering complex and find that this interaction is important for Rabin8 centrosomal targeting during ciliogenesis. Our work suggests that Rabin8 membrane transport is a highly regulated process controlled by serum-dependent and serum-independent signaling. Interestingly, following ciliogenesis Rabin8 centrosomal localization is lost resulting in reduced Rab8 activation at the ciliary membrane. This finding along with a previous report describing Rabin8 association with Bardet-Biedl syndrome (BBS) proteins has led us to hypothesize that regulation of centrosomal Rabin8 levels is important for establishing the length of primary cilium, an important factor in ciliary signaling. Finally, we describe the discovery of additional factors associated with the Rab11-Rab8 trafficking pathway that function in organizing membrane structure during ciliogenesis.

Background Changes in genes coding for ciliary proteins contribute to complex human syndromes called ciliopathies, such as Bardet-Biedl Syndrome (BBS). We used the model organism Paramecium to focus on ciliary ion channels that affect the beat form and sensory function of motile cilia and evaluate the effects of perturbing BBS proteins on these channels. Methods We used immunoprecipitations and mass spectrometry to explore whether Paramecium proteins interact as in mammalian cells. We used RNA interference (RNAi) and swimming behavior assays to examine the effects of BBS depletion on ciliary ion channels that control ciliary beating. Combining RNA interference and epitope tagging, we examined the effects of BBS depletion of BBS 7, 8 and 9 on the location of three channels and a chemoreceptor in cilia. Results We found 10 orthologs of 8 BBS genes in P. tetraurelia. BBS1, 2, 4, 5, 7, 8 and 9 co-immunoprecipitate. While RNAi reduction of BBS 7 and 9 gene products caused loss and shortening of cilia, RNAi for all BBS genes except BBS2 affected patterns of ciliary motility that are governed by ciliary ion channels. Swimming behavior assays pointed to loss of ciliary K+ channel function. Combining RNAi and epitope tagged ciliary proteins we demonstrated that a calcium activated K+ channel was no longer located in the cilia upon depletion of BBS 7, 8 or 9, consistent with the cells’ swimming behavior. The TRPP channel PKD2 was also lost from the cilia. In contrast, the ciliary voltage gated calcium channel was unaffected by BBS depletion, consistent with behavioral assays. The ciliary location of a chemoreceptor for folate was similarly unperturbed by the depletion of BBS 7, 8 or 9. Conclusions The co-immunoprecipitation of BBS 1,2,4,5,7,8, and 9 suggests a complex of BBS proteins. RNAi for BBS 7, 8 or 9 gene products causes the selective loss of K+ and PKD2 channels from the cilia while the critical voltage gated calcium channel and a peripheral receptor protein remain undisturbed. These channels govern ciliary beating and sensory function. Importantly, in P. tetraurelia we can combine studies of ciliopathy protein function with behavior and location and control of ciliary channels.

The primary cilium has been much celebrated for its central role as a sensory organelle. Whether we consider odorants binding to odorant receptors in olfactory cilia or light stimulating rhodopsin in the retinal outer segment, core ciliary structures and G-protein coupled receptors like rhodopsin allow our senses to function. The importance of ciliary pathways in sensation is well illustrated in patients with Bardet-Biedl syndrome, whose ciliary defects are linked to anosmia and loss of retinal function [1]. In addition to sensation, other ciliary GPCRs may bind specific hormones, like serotonin or somatostatin, to control physiological signals. Other physical means of ciliary signalling have been considered, including flow or mechanosensory sensing, binding of exosomes, and even lateral contacts with supporting cells for neurons in chordotonal organs. But can cilia reach out and touch one another? A report from the Lippincott-Schwartz lab, in the launch issue of Cilia, suggests that not only do cilia touch, they may even stick together for a long time [2]. Cilia are organized around the axoneme, with the centriole organizing the ciliary base [see [1,3], for reviews]. Around the axoneme, the ciliary membrane forms an extended sheath, organizing ciliary receptors and other membrane proteins. At the base of the cilium, the membrane typically shows an infolding or ciliary pocket, which can be a site for endocytosis. At the distal end of the cilium, the ciliary tip is a specialized segment of membrane that extends from the axoneme, and may serve a specialized function in signalling. The ciliary tip may also be a site where exosomes pinch off. A scanning electron microscopy study from the LaRusso lab observed exosomes in close apposition to cholangiocytes in ciliated bile ducts, and showed that isolated exosomes containing polycystin-1, -2, and fibrocystin can efficiently bind to cholangiocyte cilia [4,5]. The study suggested that membrane bound structures can associate with the ciliary membrane, and that specific paracrine effects of exosomes might be mediated by binding to cilia. A study from the Kernan lab in chordotonal organs of Drosophila suggested that a specialized ciliary diliation, a narrowing of the cilia, was organized by the intraflagellar transport (IFT-A) complex, and this ciliary specialization interacted with supporting cells in the chordotonal organ and was required for sensory function [6]. Finally, a number of studies show that the distal end of the retinal outer segment is in close juxtaposition to invaginations in retinal pigment epithelial cells (RPE), and that sloughing of outer segments triggers local phagocytosis by the RPE [7]. Finally, in Chylamydomonas induced to mating, the cilia can form agglutination structures critical to their mating function [8]. So cilia appear to interact purposefully with cells and exosomes from their surrounding cellular neighbourhood. But what about touching each other? Lippincott-Schwartz and colleagues consider cilia-cilia pairing, first looking into the retina and liver. In the retina, they observe that the ciliary membrane of rod cells, enveloping both the connecting cilium and outer segment, are closely packed side-by- side in the photoreceptor layer. The close juxtaposition of outer segments suggested possible adhesion sites. In the liver cholangiocytes, cilia extend into the bile duct and may participate in bile flow and signalling. Some cilia appeared to participate in cilia-cilia contacts, especially in younger animals. Moving to a cell culture model, the authors find that when MDCK cells are induced to form 3D cysts, the cilia are oriented on the apical surface towards the lumen, and indeed form cilia-cilia contacts. In MDCK cell monolayers grown on filters, the cilia are longer (815) and cells are close packed (10 spacing). Here the cilia can form continuous arcs, or point contacts, and even cilia-cilia networks. In other cells, less tightly packed and with short cilia, the cilia-cilia contacts are less apparent. If the cilia-cilia contacts are important, a first guess is that they would be enduring, rather than transient. Indeed, the authors find that by filming of cells, that most showed contacts persisting for 2 hours, and over half of cell for up to 20 hours or longer. So cilia-cilia contacts can be stable; a first estimate is that 13% of cilia make contact. What sets the percentage and whether there are cell mechanisms that favor ciliary pairing would help us understand when and where the phenomena is important. One might guess that the density and organization of cells would be important to favor ciliary pairing. Some data suggest that ciliary length may be regulated once preciliated cells reach confluence and establish adherens junctions, possibly requiring the Par3-Par6-aPKC pathway [9,10]. We could consider a contact-dependent, ciliary extension model, where cells establish cell-cell contacts, completing the formation of adherens junctions or tight junctions, which would then trigger ciliary extension to allow cilia to reach out and establish cilia-cilia communication. An intriguing possibility is suggested by molecular properties of the NPHP1-4-8 network of nephrocystins, a group of genes defective in the nephronophthisis, a paediatric ciliopathy with renal insufficiency. The NPHP1-4-8 proteins have roles in both cell-cell and tight junction formation, and in the formation of the ciliary transition zone [11,12]. These proteins would be interesting candidates to communicate between cell-cell junctions and the cilium. In terms of how the ciliary arcs are formed, the authors consider whether a single cilium might bend into an arc; or if cilia from adjacent cells adhere; or if cilia from adjacent cells actually fuse! Using time-lapse microscopy, it became clear that ciliary contacts are established first, and then zipped up to form a stable pairing. Electron micrographs of the interaction was reminiscent of an adhesive pairing, so the authors considered whether known molecular pathways for adhesion were required. Using EGTA to chelate calcium, and DTT to dissolve disulphide bonds, neither disrupted ciliary pairing, suggesting that neither calcium-dependent cadherins nor disulphide-requiring Ig domains were strongly required. In contrast, treating cells with the mannosidase II inhibitor swainsonine to block oligosaccharide formation, the authors did observe a loss of the ciliary pairing phenomena. Other drugs perturbing glycoprotein formation like neuraminidase treatment or elastase treated failed to block pairing, but these experiments were technically less conclusive. Considering the ever-important cell cycle, it was clear that as cilated cells enter mitosis, they also showed a loss of ciliary pairing, further supporting the potential biological relevance of the pairing phenomena. Because Chylamydomonas also uses glycoproteins for ciliary pairing, there is now question of which ciliary membrane protein(s) may mediate pairing. Inspection of the ciliary or FABB proteome may be a good place to look for candidates [13]. The studies here provide a provocative first glimpse of how glycoprotein contacts may link cilia-cilia pairings in cells, which could proceed from initial synaptic pairing to more stable, zipped-up associations. Additional genetic or molecular studies could inform and support the importance of these pairing for ciliary function. But the larger question is why cilia would use such contacts. Among the potential roles, a general function in intercellular communication between closely apposed cells or intracellular communication in tissues would be important. A somewhat similar mechanism may be provided by cytonemes, specific filopodial intercellular links [14], and much remains to be understood about the various physical connections that allow paracrine signalling in tissues. Indeed, aside from secreted factors, cilia could serve as a second important physical means to have cells communicate. Because ciliary signalling presumably predates neural signalling in evolution, we can imagine the intraciliary contacts would form to regulate a primitive synaptic system communicating local exocytic or cilia-bound signals to bind and trigger signalling to cilia on nearby cells. In highly organized structures like the retina, these cilia-cilia contacts may be important to also serve structural or architectural roles. It may be surprising that new ultrastructure can be discovered today, but as the present study illustrates, we first have to open our minds to the possibility of new modes of cell-cell communication, and then start to look for the evidence that informs us of how these pathways work. With the excitement around cilia, we are being continually surprised. Maybe some labs already have the sense that cilia-cilia pairings directly mediate cellular communication and can now see a path to publishing that data to help the rest of us.

Cilia are hair-like protrusions found at the surface of most eukaryotic cells. They can be divided into two types, motile and non-motile. Motile cilia are found in a restricted number of cell types, are generally present in large numbers, and beat in a coordinated fashion to generate fluid flow or locomotion. Non-motile or primary cilia, on the other hand, are detected in many different cell types, appear once per cell, and primarily function to transmit signals from the extracellular milieu to the cell nucleus. Defects in cilia formation, function, or maintenance are known to cause a bewildering set of human diseases, or ciliopathies, typified by retinal degeneration, renal failure and cystic kidneys, obesity, liver dysfunction, and neurological disorders. A common denominator between motile and primary cilia is their structural similarity, as both types of cilia are composed of an axoneme, the ciliary backbone that is made up of microtubules emanating from a mother centriole/basal body anchored to the cell membrane, surrounded by a ciliary membrane continuous with the plasma membrane. This structural similarity is indicative of a universal mechanism of cilia assembly involving a common set of molecular players and a sophisticated, highly regulated series of molecular events. In this review, we will mainly focus on recent advances in our understanding of the regulatory mechanisms underlying cilia assembly, with special attention paid to the centriolar protein, CP110, its interacting partner Cep290, and the various downstream molecular players and events leading to intraflagellar transport (IFT), a process that mediates the bidirectional movement of protein cargos along the axoneme and that is essential for cilia formation and maintenance.