Meckel-Gruber Syndrome Proteins Research Articles

Meckel-Gruber Syndrome: An Update on Diagnosis, Clinical Management, and Research Advances.

Meckel–Gruber syndrome (MKS) is a lethal autosomal recessive congenital anomaly syndrome caused by mutations in genes encoding proteins that are structural or functional components of the primary cilium. Conditions that are caused by mutations in ciliary genes are collectively termed the ciliopathies, and MKS represents the most severe condition in this group of disorders. The primary cilium is a microtubule-based organelle, projecting from the apical surface of vertebrate cells. It acts as an “antenna” that receives and transduces chemosensory and mechanosensory signals, but also regulates diverse signaling pathways, such as Wnt and Shh, that have important roles during embryonic development. Most MKS proteins localize to a distinct ciliary compartment called the transition zone (TZ) that regulates the trafficking of cargo proteins or lipids. In this review, we provide an up-to-date summary of MKS clinical features, molecular genetics, and clinical diagnosis. MKS has a highly variable phenotype, extreme genetic heterogeneity, and displays allelism with other related ciliopathies such as Joubert syndrome, presenting significant challenges to diagnosis. Recent advances in genetic technology, with the widespread use of multi-gene panels for molecular testing, have significantly improved diagnosis, genetic counseling, and the clinical management of MKS families. These include the description of some limited genotype–phenotype correlations. We discuss recent insights into the molecular basis of disease in MKS, since the functions of some of the relevant ciliary proteins have now been determined. A common molecular etiology appears to be disruption of ciliary TZ structure and function, affecting essential developmental signaling and the regulation of secondary messengers.

Drosophila sensory cilia lacking MKS proteins exhibit striking defects in development but only subtle defects in adults.

ABSTRACTCilia are conserved organelles that have important motility, sensory and signalling roles. The transition zone (TZ) at the base of the cilium is crucial for cilia function, and defects in several TZ proteins are associated with human congenital ciliopathies such as nephronophthisis (NPHP) and Meckel–Gruber syndrome (MKS). In several species, MKS and NPHP proteins form separate complexes that cooperate with Cep290 to assemble the TZ, but flies seem to lack core components of the NPHP module. We show that MKS proteins in flies are spatially separated from Cep290 at the TZ, and that flies mutant for individual MKS genes fail to recruit other MKS proteins to the TZ, whereas Cep290 seems to be recruited normally. Although there are abnormalities in microtubule and membrane organisation in developing MKS mutant cilia, these defects are less apparent in adults, where sensory cilia and sperm flagella seem to function quite normally. Thus, localising MKS proteins to the cilium or flagellum is not essential for viability or fertility in flies.

A Migrating Ciliary Gate Compartmentalizes the Site of Axoneme Assembly in Drosophila Spermatids

In most cells, the cilium is formed within a compartment separated from the cytoplasm. Entry into the ciliary compartment is regulated by a specialized gate located at the base of the cilium in a region known as the transition zone. The transition zone is closely associated with multiple structures of the ciliary base, including the centriole, axoneme, and ciliary membrane. However, the contribution of these structures to the ciliary gate remains unclear. Here we report that, in Drosophila spermatids, a conserved module of transition zone proteins mutated in Meckel-Gruber syndrome (MKS), including Cep290, Mks1, B9d1, and B9d2, comprise a ciliary gate that continuously migrates away from the centriole to compartmentalize the growing axoneme tip. We show that Cep290 is essential for transition zone composition, compartmentalization of the axoneme tip, and axoneme integrity and find that MKS proteins also delimit a centriole-independent compartment in mouse spermatids. Our findings demonstrate that the ciliary gate can migrate away from the base of the cilium, thereby functioning independently of the centriole and of a static interaction with the axoneme to compartmentalize the site of axoneme assembly.

ARL13B, PDE6D, and CEP164 form a functional network for INPP5E ciliary targeting

Mutations affecting ciliary components cause a series of related genetic disorders in humans, including nephronophthisis (NPHP), Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS), and Bardet-Biedl syndrome (BBS), which are collectively termed "ciliopathies." Recent protein-protein interaction studies combined with genetic analyses revealed that ciliopathy-related proteins form several functional networks/modules that build and maintain the primary cilium. However, the precise function of many ciliopathy-related proteins and the mechanisms by which these proteins are targeted to primary cilia are still not well understood. Here, we describe a protein-protein interaction network of inositol polyphosphate-5-phosphatase E (INPP5E), a prenylated protein associated with JBTS, and its ciliary targeting mechanisms. INPP5E is targeted to the primary cilium through a motif near the C terminus and prenyl-binding protein phosphodiesterase 6D (PDE6D)-dependent mechanisms. Ciliary targeting of INPP5E is facilitated by another JBTS protein, ADP-ribosylation factor-like 13B (ARL13B), but not by ARL2 or ARL3. ARL13B missense mutations that cause JBTS in humans disrupt the ARL13B-INPP5E interaction. We further demonstrate interactions of INPP5E with several ciliary and centrosomal proteins, including a recently identified ciliopathy protein centrosomal protein 164 (CEP164). These findings indicate that ARL13B, INPP5E, PDE6D, and CEP164 form a distinct functional network that is involved in JBTS and NPHP but independent of the ones previously defined by NPHP and MKS proteins.

NPHP and MKS proteins and the ciliary transition zone

The cilium has emerged as an important signaling and sensory organelle needed for mammalian development and adult tissue homeostasis. It mediates these effects by locating specific receptors, channels, and other signaling factors needed for pathway regulation along the cilium membrane and axoneme. To establish the cilia signaling complement of proteins distinct from that of the cytosol or cell membrane there is a domain called the transition zone (TZ) located at the base that is believed to function as a diffusion barrier, restricting protein entry or exit from the cilium. The structure of the TZ has been well defined through EM analysis, but the proteins required to generate the TZ are only now being identified. Using C. elegans models, we have shown that many of the proteins involved in human ciliopathies such as Meckel Gruber syndrome (MKS) and Nephronophthisis (NPHP) grossly localize to the TZ. We are currently defining where these proteins localize on structures within the TZ using higher resolution EM approaches. Further, the NPHP and MKS proteins form two genetically interacting complexes in C. elegans that when disrupted result in the loss of structures in the TZ, inappropriate ciliary localization of nonciliary proteins, and severe cilia assembly/positioning defects. Based on the synthetic cilia phenotype observed in all mks;nphp double mutants that we generated thus far, we conducted an EMS enhancer screen using nphp-4 single mutants. The goal of the screen was to generate secondary mutations that would genetically interact with the initial nphp-4 mutation. Our prediction is that this would yield novel mutations in proteins that function with the MKS or NPHP complexes and would thus provide ideal candidates for novel loci involved in human ciliopathies patients. Using whole genome sequence analysis, we have begun identifying the affected genes. As predicted, three of the alleles were in known MKS genes. Several other mutations in cilia genes have been identified but have not previously been associated with either NPHP or MKS. The role of these genes and the interaction with the NPHP and MKS complexes are being further explored in both C. elegans and mouse models. Understanding how the TZ forms and how mutations in TZ proteins disrupt the diffusion barrier will provide important insights into the ciliopathies and how this organelle serves its diverse signaling and sensory functions.

MKS proteins regulate centrosome positioning during cell migration

Meckel-Gruber syndrome (MKS) is a severe ciliopathy, but how the underlying mutations lead to the disease is poorly understood. MKS proteins form complexes at the ciliary transition zone, and are also needed for centrosome re-orientation and docking with the plasma membrane during ciliogenesis. However, MKS-mutant cells also have an unexplained cell migration defect: cells can migrate, but the rate is much slower than usually observed. As correct centrosome orientation is vital for many migrating cells, we examined centrosome appearance and position in migrating patient fibroblasts with mutations in MKS2 and MKS3. In control cells, the centrosome and Golgi apparatus were located between the nucleus and the leading edge, but these organelles were randomly positioned in patient fibroblasts. To distinguish between failure and delay in centrosome polarisation, we assessed centrosome position during the first two hours of cell migration. We found that mutant fibroblasts failed to stably re-orient the centrosome over the time-course of the assay, suggesting this may be a contributing factor to the migration defect. Finally, we asked if a cilium is needed for correct centrosome polarisation. Wild-type fibroblasts re-oriented their centrosome in the absence of a cilium, indicating that direction sensing by the primary cilium is not needed for initial centrosome polarisation in these cells. As MKS cells are defective in centrosome positioning during migration as well as ciliogenesis, we conclude that the MKS ciliogenesis defect may not be the primary cause of disease, but a downstream consequence of the failure to correctly position the centrosome.

The functional relationships between BBS and MKS and their role in regulating trafficking of proteins in the primary cilium

Bardet-Biedl syndrome (BBS )a nd Meckel-Gruber syndrome (MKS) are genetic disorders caused by disruption of primary cilia. The BBS and MKS proteins are found in two distinct protein complexes but the functional relationships between these two complexes are not clear. Here we analyze double mutant mouse embryos to define these relationships. Bbs4;Mks1 double mutant embryos show a stronger phenotype than either single mutant, with completely penetrant micropthalmia and polydactyly, loss of ventral neural cell types, and a reduced number of short bulbous primary cilia. This additive phenotype suggests that BBS and MKS function in parallel pathways. We detected accumulation of Smo and abnormal Gli2 localization in Bbs4;Mks1 double mutant cilia, suggesting that the double mutants have a defect in retrograde trafficking. To define a role of BBS4 or MKS1 in ciliary trafficking, we have generated Bbs4 or Mks1 mice that carry mutations in the IFT machinery. Absence of BBS4 did not affect the phenotype of a weak IFT-B mutant or of embryos that lack the retrograde dynein motor, whereas the Mks1 phenotype was stronger when either IFT-B or dynein were mutant. This implies that BBS works together with IFT to regulate ciliary trafficking whereas MKS1 has a parallel role in ciliary function. Together these data suggest that BBS and MKS both have roles in regulating movement of Hh components in the cilium however the way in which they do this is distinct from each other.

The ciliary transitional zone and nephrocystins

Loss of cilia and ciliary protein causes various abnormalities (called ciliopathy), including situs inversus, renal cystic diseases, polydactyly and dysgenesis of the nervous system. Renal cystic diseases are the most frequently observed symptoms in ciliopathies. Cilia are microtubule-based organelles with the following regions: a ciliary tip, shaft, transitional zone and basal body/mother centriole. Joubert syndrome (JBTS), Meckel Gruber syndrome (MKS) and Nephronophthisis (NPHP) are overlapping syndromes. Recent studies show that JBST and MKS responsible gene products are localized in the transitional zone of the cilia, where they function as a diffusion barrier, and control protein sorting and ciliary membrane composition. Nephrocystins are gene products of NPHP responsible genes, and at least 11 genes have been identified. Although some nephrocystins interact with JBST and MKS proteins, proteomic analysis suggests that they do not form a single complex. Localization analysis reveals that nephrocystins can be divided into two groups. Group I nephrocystins are localized in the transitional zone, whereas group II nephrocystins are localized in the Inv compartment. Homologs of group I nephrocystins, but not group II nephrocystins, have been reported in C. reinhardtii and C. elegans. In this review, we summarize the structure of the ciliary base of C. reinhardtii, C. elegans and mammalian primary cilia, and discuss function of nephrocystins. We also propose a new classification of nephrocystins.

Defects in non-canonical Wnt signalling and actin cytoskeleton remodelling as pathogenic mechanisms in Meckel–Gruber syndrome

Primary cilia are mechano- and chemosensory organelles that have a fundamental role in regulating embryogenesis. Inherited disorders that involve aberrant ciliary structure or function are now known as “ciliopathies”, and they invariably present with cystic kidney dysplasia. Meckel–Gruber syndrome (MKS) is a pleiotropic ciliopathy characterized by severe defects in neurodevelopment that include occipital encephalocele, hydrocephaly and severe neural tube defects. Several MKS genes are now known, including MKS1 and MKS3/TMEM67, encoding the proteins MKS1 and meckelin, a novel receptor. Remarkably, MKS is allelic and overlaps in phenotype with the neurodevelopmental disorder Joubert syndrome (JS), with some of the causative genes implicated in regulation of the Hedgehog signalling pathway. However, our recent work has suggested a role for meckelin and some other MKS proteins in modulating non-canonical Wnt signalling and remodelling the actin cytoskeleton. Meckelin is localized at the apical cell surface, basal bodies and ciliary axoneme of ciliated cell lines and tissues, but also interacts with other MKS proteins and the actin-binding proteins nesprin-2 and filamin A. Loss of expression of MKS genes following RNAi-mediated knockdown or in MKS patient fibroblasts: (1) prevents the movement of the basal body to the apical cell surface prior to ciliogenesis; (2) causes hyperactivation of the small GTPase RhoA and Dishevelled, both implicated in the control of apical docking of basal bodies and planar polarization of epithelial cells; and (3) remodels the actin cytoskeleton. These findings are reiterated in the Mks3/Tmem67 knock-out mouse model of MKS/JS. In contrast, MKS1 is implicated in constraining canonical Wnt signalling. These findings therefore underline the critical role of MKS proteins in ciliogenesis and regulation of Wnt signalling, through interactions with apical cell surface proteins associated with the actin cytoskeleton and implicated in basal body docking.

Normal Ciliogenesis Requires Synergy between the Cystic Kidney Disease Genes MKS-3 and NPHP-4

Cilia dysfunction contributes to renal cyst formation in multiple human syndromes including nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), Joubert syndrome (JBTS), and Bardet-Beidl syndrome (BBS). Although genetically heterogeneous, these diseases share several loci that affect cilia and/or basal body proteins, but the functions and interactions of these gene products are incompletely understood. Here, we report that the ciliated sensory neurons (CSNs) of C. elegans express the putative transmembrane protein MKS-3, which localized to the distal end of their dendrites and to the cilium base but not to the cilium itself. Localization of MKS-3 and other known MKS and NPHP proteins partially overlapped. By analyzing mks-3 mutants, we found that ciliogenesis did not require MKS-3; instead, cilia elongated and cilia-mediated chemoreception was abnormal. Genetic analysis indicated that mks-3 functions in a pathway with other mks genes. Furthermore, mks-1 and mks-3 genetically interacted with a separate pathway (involving nphp-1 and nphp-4) to influence proper positioning, orientation, and formation of cilia. Combined disruption of nphp and mks pathways had cell nonautonomous effects on C. elegans sensilla. Taken together, these data demonstrate the importance of mutational load on the presentation and severity of ciliopathies and expand the understanding of the interactions between ciliopathy genes.

Nesprin-2 interacts with meckelin and mediates ciliogenesis via remodelling of the actin cytoskeleton

Meckel-Gruber syndrome (MKS) is a severe autosomal recessively inherited disorder caused by mutations in genes that encode components of the primary cilium and basal body. Here we show that two MKS proteins, MKS1 and meckelin, that are required for centrosome migration and ciliogenesis interact with actin-binding isoforms of nesprin-2 (nuclear envelope spectrin repeat protein 2, also known as Syne-2 and NUANCE). Nesprins are important scaffold proteins for maintenance of the actin cytoskeleton, nuclear positioning and nuclear-envelope architecture. However, in ciliated-cell models, meckelin and nesprin-2 isoforms colocalized at filopodia prior to the establishment of cell polarity and ciliogenesis. Loss of nesprin-2 and nesprin-1 shows that both mediate centrosome migration and are then essential for ciliogenesis, but do not otherwise affect apical-basal polarity. Loss of meckelin (by siRNA and in a patient cell-line) caused a dramatic remodelling of the actin cytoskeleton, aberrant localization of nesprin-2 isoforms to actin stress-fibres and activation of RhoA signalling. These findings further highlight the important roles of the nesprins during cellular and developmental processes, particularly in general organelle positioning, and suggest that a mechanistic link between centrosome positioning, cell polarity and the actin cytoskeleton is required for centrosomal migration and is essential for early ciliogenesis.

The Meckel–Gruber Syndrome proteins MKS1 and meckelin interact and are required for primary cilium formation

Meckel-Gruber syndrome (MKS) is an autosomal recessive lethal malformation syndrome characterized by renal cystic dysplasia, central nervous system malformations (typically, posterior occipital encephalocele), and hepatic developmental defects. Two MKS genes, MKS1 and MKS3, have been identified recently. The present study describes the cellular, sub-cellular and functional characterization of the novel proteins, MKS1 and meckelin, encoded by these genes. In situ hybridization studies for MKS3 in early human embryos showed transcript localizations in agreement with the tissue phenotype of MKS patients. Both MKS proteins predominantly localized to epithelial cells, including proximal renal tubules and biliary epithelial cells. MKS1 localized to basal bodies, while meckelin localized both to the primary cilium and to the plasma membrane in ciliated cell-lines and primary cells. Meckelin protein with the Q376P missense mutation was unable to localize at the cell membrane. siRNA-mediated reduction of Mks1 and Mks3 expression in a ciliated epithelial cell-line blocked centriole migration to the apical membrane and consequent formation of the primary cilium. Co-immunoprecipitation experiments show that wild-type meckelin and MKS1 interact and, in three-dimensional tissue culture assays, epithelial branching morphogenesis was severely impaired. These results suggest that MKS proteins mediate a fundamental developmental stage of ciliary formation and epithelial morphogenesis.