Intraflagellar Transport Research Articles

IFT27 regulates the long-term maintenance of photoreceptor outer segments in zebrafish

Approximately a quarter of Retinitis Pigmentosa (RP) is caused by mutations in transport-related genes in cilia. IFT27 (Intraflagellar Transport 27), a core component of the ciliary intraflagellar transport (IFT) system, has been implicated as a significant pathogenic gene in RP. The pathogenic mechanisms and subsequent pathology related to IFT27 mutations in RP are largely obscure. Here, we utilized TALEN technology to create an ift27 knockout (ift27-/-) zebrafish model. Electroretinography (ERG) detection showed impaired vision in this model. Histopathological examinations disclosed that ift27 mutations cause progressive degeneration of photoreceptors in zebrafish, and this degeneration was late-onset. Immunofluorescence labeling of outer segments showed that rods degenerated before cones, aligning with the conventional characterization of RP. In cultured human retinal pigment epithelial cells, we found that IFT27 was involved in maintaining ciliary morphology. Furthermore, decreased IFT27 expression resulted in the inhibition of the Hedgehog (Hh) signaling pathway, including decreased expression of key factors in the Hh pathway and abnormal localization of the ciliary mediator Gli2. In summary, we generated an ift27-/- zebrafish line with retinal degeneration which mimicked the symptoms of RP patients, highlighting IFT27′s integral role in the long-term maintenance of cilia via the Hh signaling pathway. This work may furnish new insights into the treatment or delay of RP caused by IFT27 mutations.

IFT80 promotes early bone healing of tooth sockets through the activation of TAZ/RUNX2 pathway.

Intraflagellar transport (IFT) proteins have been reported to regulate cell growth and differentiation as the essential functional component of primary cilia. The effects of IFT80 on early bone healing of extraction sockets have not been well studied. To investigate whether deletion of Ift80 in alveolar bone-derived mesenchymal stem cells (aBMSCs) affected socket bone healing, we generated a mouse model of specific knockout of Ift80 in Prx1 mesenchymal lineage cells (Prx1Cre;IFT80f/f). Our results demonstrated that deletion of IFT80 in Prx1 lineage cells decreased the trabecular bone volume, ALP-positive osteoblastic activity, TRAP-positive osteoclastic activity, and OSX-/COL I-/OCN-positive areas in tooth extraction sockets of Prx1Cre; IFT80f/f mice compared with IFT80f/f littermates. Furthermore, aBMSCs from Prx1Cre; IFT80f/f mice showed significantly decreased osteogenic markers and downregulated migration and proliferation capacity. Importantly, the overexpression of TAZ recovered significantly the expressions of osteogenic markers and migration capacity of aBMSCs. Lastly, the local administration of lentivirus for TAZ enhanced the expression of RUNX2 and OSX and promoted early bone healing of extraction sockets from Prx1Cre; IFT80f/f mice. Thus, IFT80 promotes osteogenesis and early bone healing of tooth sockets through the activation of TAZ/RUNX2 pathway.

Inpp5e Regulated the Cilium-Related Genes Contributing to the Neural Tube Defects Under 5-Fluorouracil Exposure.

Primary cilia are crucial for neurogenesis, and cilium-related genes are involved in the closure of neural tubes. Inositol polyphosphate-5-phosphatase (Inpp5e) was enriched in primary cilia and closely related to the occurrence of neural tube defects (NTDs). However, the role of Inpp5e in the development of NTDs is not well-known. To investigate whether Inpp5e gene is associated with the neural tube closure, we established a mouse model of NTDs by 5-fluorouracil (5-FU) exposure at gestational day 7.5 (GD7.5). The Inpp5e knockdown (Inpp5e-/-) mouse embryonic stem cells (mESCs) were produced by CRISPR/Cas9 system. The expressions of Inpp5e and other cilium-related genes including intraflagellar transport 80 (Ift80), McKusick-Kaufman syndrome (Mkks), and Kirsten rat sarcoma viral oncogene homolog (Kras) were determined, utilizing quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), western blot, PCR array, and immunofluorescence staining. The result showed that the incidence of NTDs was 37.10% (23 NTDs/62 total embryos) and significantly higher than that in the control group (P < 0.001). The neuroepithelial cells of neural tubes were obviously disarranged in NTD embryos. The mRNA and protein levels of Inpp5e, Ift80, Mkks, and Kras were significantly decreased in NTD embryonic brain tissues, compared to the control (P < 0.05). Knockdown of the Inpp5e (Inpp5e-/-) reduced the expressions of Ift80, Mkks, and Kras in mESCs. Furthermore, the levels of α-tubulin were significantly reduced in NTD embryonic neural tissue and Inpp5e-/- mESCs. These results suggested that maternal 5-FU exposure inhibited the expression of Inpp5e, which resulted in the downregulation of cilium-related genes (Ift80, Mkks, and Kras), leading to the impairment of primary cilium development, and ultimately disrupted the neural tube closure.

Small noncoding RNAs and sperm nuclear basic proteins reflect the environmental impact on germ cells

BackgroundMolecular techniques can complement conventional spermiogram analyses to provide new information on the fertilizing potential of spermatozoa and to identify early alterations due to environmental pollution.MethodsHere, we present a multilevel molecular profiling by small RNA sequencing and sperm nuclear basic protein analysis of male germ cells from 33 healthy young subjects residing in low and high-polluted areas.ResultsAlthough sperm motility and sperm concentration were comparable between samples from the two sites, those from the high-pollution area had a higher concentration of immature/immune cells, a lower protamine/histone ratio, a reduced ability of sperm nuclear basic proteins to protect DNA from oxidative damage, and an altered copper/zinc ratio in sperm. Sperm levels of 32 microRNAs involved in intraflagellar transport, oxidative stress response, and spermatogenesis were different between the two areas. In parallel, a decrease of Piwi-interacting RNA levels was observed in samples from the high-polluted area.ConclusionsThis comprehensive analysis provides new insights into pollution-driven epigenetic alterations in sperm not detectable by spermiogram.Graphical

Joubert syndrome causing mutation in C2 domain of CC2D2A affects structural integrity of cilia and cellular signaling molecules.

Cilia are organelles extend from cells to sense external signals for tuning intracellular signaling for optimal cellular functioning. They have evolved sensory and motor roles in various cells for tissue organization and homeostasis in development and post-development. More than a thousand genes are required for cilia function. Mutations in them cause multisystem disorders termed ciliopathies. The null mutations in CC2D2A result in Meckel syndrome (MKS), which is embryonic lethal, whereas patients who have missense mutations in the C2 domain of CC2D2A display Joubert syndrome (JBTS). They survive with blindness and mental retardation. How C2 domain defects cause disease conditions is not understood. To answer this question, C2 domain of Cc2d2a (mice gene) was knocked down (KD) in IMCD-3 cells by shRNA. This resulted in defective cilia morphology observed by immunofluorescence analysis. To further probe the cellular signaling alteration in affected cells, gene expression profiling was done by RNAseq and compared with the controls. Bioinformatics analysis revealed that the differentially expressed genes (DEGs) have functions in cilia. Among the 61 cilia DEGs identified, 50 genes were downregulated and 11 genes were upregulated. These cilia genes are involved in cilium assembly, protein trafficking to the cilium, intraflagellar transport (IFT), cellular signaling like polarity patterning, and Hedgehog signaling pathway. This suggests that the C2 domain of CC2D2A plays a critical role in cilia assembly and molecular signaling hosted in cilia for cellular homeostasis. Taken together, the missense mutations in the C2 domain of CC2D2A seen in JBTS might have affected cilia-mediated signaling in neurons of the retina and brain.

The relationship between intraflagellar transport and upstream protein trafficking pathways and macrocyclic lactone resistance in Caenorhabditis elegans.

Parasitic nematodes are globally important and place a heavy disease burden on infected humans, crops, and livestock, while commonly administered anthelmintics used for treatment are being rendered ineffective by increasing levels of resistance. It has recently been shown in the model nematode Caenorhabditis elegans that the sensory cilia of the amphid neurons play an important role in resistance toward macrocyclic lactones such as ivermectin (an avermectin) and moxidectin (a milbemycin) either through reduced uptake or intertissue signaling pathways. This study interrogated the extent to which ciliary defects relate to macrocyclic lactone resistance and dye-filling defects using a combination of forward genetics and targeted resistance screening approaches and confirmed the importance of intraflagellar transport in this process. This approach also identified the protein trafficking pathways used by the downstream effectors and the components of the ciliary basal body that are required for effector entry into these nonmotile structures. In total, 24 novel C. elegans anthelmintic survival-associated genes were identified in this study. When combined with previously known resistance genes, there are now 46 resistance-associated genes that are directly involved in amphid, cilia, and intraflagellar transport function.

Osm-5p- driven fluorophores are differentially expressed in ccpp-1Δ and nekl-4Δ mutant ciliated neurons.

Intraflagellar transport (IFT) involves the coordinated transport of molecular motors and other proteins and is required for ciliogenesis and ciliary maintenance. The C. elegans IFT protein OSM-5 /IFT88 is expressed in a majority of the ciliated neurons in the animal, and osm-5 mutants exhibit structurally defective cilia. The osm-5 promoter is commonly used to express genetic constructs in the ciliated neurons. In this study, we show that brightness of osm-5p- driven constructs is altered in mutants of the tubulin deglutamylase ccpp-1 and the NIMA-related kinase nekl-4 . This raises the possibility that osm-5 expression levels may be regulated by ccpp-1 and nekl-4 .

Deciphering vesicle-assisted transport mechanisms in cytoplasm to cilium trafficking.

The cilium, a pivotal organelle crucial for cell signaling and proper cell function, relies on meticulous macromolecular transport from the cytoplasm for its formation and maintenance. While the intraflagellar transport (IFT) pathway has traditionally been the focus of extensive study concerning ciliogenesis and ciliary maintenance, recent research highlights a complementary and alternative mechanism-vesicle-assisted transport (VAT) in cytoplasm to cilium trafficking. Despite its potential significance, the VAT pathway remains largely uncharacterized. This review explores recent studies providing evidence for the dynamics of vesicle-related diffusion and transport within the live primary cilium, employing high-speed super-resolution light microscopy. Additionally, we analyze the spatial distribution of vesicles in the cilium, mainly relying on electron microscopy data. By scrutinizing the VAT pathways that facilitate cargo transport into the cilium, with a specific emphasis on recent advancements and imaging data, our objective is to synthesize a comprehensive model of ciliary transport through the integration of IFT-VAT mechanisms.

The emerging functions of intraflagellar transport 52 in ciliary transport and ciliopathies.

Ciliary transport in eukaryotic cells is an intricate and conserved process involving the coordinated assembly and functioning of a multiprotein intraflagellar transport (IFT) complex. Among the various IFT proteins, intraflagellar transport 52 (IFT52) plays a crucial role in ciliary transport and is implicated in various ciliopathies. IFT52 is a core component of the IFT-B complex that facilitates movement of cargoes along the ciliary axoneme. Stable binding of the IFT-B1 and IFT-B2 subcomplexes by IFT52 in the IFT-B complex regulates recycling of ciliary components and maintenance of ciliary functions such as signal transduction and molecular movement. Mutations in the IFT52 gene can disrupt ciliary trafficking, resulting in dysfunctional cilia and affecting cellular processes in ciliopathies. Such ciliopathies caused by IFT52 mutations exhibit a wide range of clinical features, including skeletal developmental abnormalities, retinal degeneration, respiratory failure and neurological abnormalities in affected individuals. Therefore, IFT52 serves as a promising biomarker for the diagnosis of various ciliopathies, including short-rib thoracic dysplasia 16 with or without polydactyly. Here, we provide an overview of the IFT52-mediated molecular mechanisms underlying ciliary transport and describe the IFT52 mutations that cause different disorders associated with cilia dysfunction.

Chlamydomonas as a model system to study cilia and flagella using genetics, biochemistry, and microscopy.

The unicellular green alga, Chlamydomonas reinhardtii, has played a central role in discovering much of what is currently known about the composition, assembly, and function of cilia and flagella. Chlamydomonas combines excellent genetics, such as the ability to grow cells as haploids or diploids and to perform tetrad analysis, with an unparalleled ability to detach and isolate flagella in a single step without cell lysis. The combination of genetics and biochemistry that is possible in Chlamydomonas has allowed many of the key components of the cilium to be identified by looking for proteins that are missing in a defined mutant. Few if any other model organisms allow such a seamless combination of genetic and biochemical approaches. Other major advantages of Chlamydomonas compared to other systems include the ability to induce flagella to regenerate in a highly synchronous manner, allowing the kinetics of flagellar growth to be measured, and the ability of Chlamydomonas flagella to adhere to glass coverslips allowing Intraflagellar Transport to be easily imaged inside the flagella of living cells, with quantitative precision and single-molecule resolution. These advantages continue to work in favor of Chlamydomonas as a model system going forward, and are now augmented by extensive genomic resources, a knockout strain collection, and efficient CRISPR gene editing. While Chlamydomonas has obvious limitations for studying ciliary functions related to animal development or organ physiology, when it comes to studying the fundamental biology of cilia and flagella, Chlamydomonas is simply unmatched in terms of speed, efficiency, cost, and the variety of approaches that can be brought to bear on a question.

Homozygous CCDC146 mutation causes oligoasthenoteratozoospermia in humans and mice.

Infertility represents a significant health concern, with sperm quantity and quality being crucial determinants of male fertility. Oligoasthenoteratozoospermia (OAT) is characterized by reduced sperm motility, lower sperm concentration, and morphological abnormalities in sperm heads and flagella. Although variants in several genes have been implicated in OAT, its genetic etiologies and pathogenetic mechanisms remain inadequately understood. In this study, we identified a homozygous nonsense mutation (c.916C>T, p.Arg306*) in the coiled-coil domain containing 146 ( CCDC146) gene in an infertile male patient with OAT. This mutation resulted in the production of a truncated CCDC146 protein (amino acids 1-305), retaining only two out of five coiled-coil domains. To validate the pathogenicity of the CCDC146 mutation, we generated a mouse model ( Ccdc146 mut/mut ) with a similar mutation to that of the patient. Consistently, the Ccdc146 mut/mut mice exhibited infertility, characterized by significantly reduced sperm counts, diminished motility, and multiple defects in sperm heads and flagella. Furthermore, the levels of axonemal proteins, including DNAH17, DNAH1, and SPAG6, were significantly reduced in the sperm of Ccdc146 mut/mut mice. Additionally, both human and mouse CCDC146 interacted with intraflagellar transport protein 20 (IFT20), but this interaction was lost in the mutated versions, leading to the degradation of IFT20. This study identified a novel deleterious homozygous nonsense mutation in CCDC146 that causes male infertility, potentially by disrupting axonemal protein transportation. These findings offer valuable insights for genetic counseling and understanding the mechanisms underlying CCDC146 mutant-associated infertility in human males.

Extraciliary OFD1 Is Involved in Melanocyte Survival through Cell Adhesion to ECM via Paxillin.

Primary cilia play a significant role in influencing cell fate, including apoptosis in multiple cell types. In the lesional epidermis of vitiligo patients, a reduced number of ciliated cells was observed. Our study also revealed a downregulation of oral-facial digital syndrome type 1 (OFD1) in the affected skin of vitiligo patients. However, it remains unknown whether primary cilia are involved in the control of melanocyte apoptosis. While both intraflagellar transport 88 (IFT88) and retinitis pigmentosa GTPase regulator-interacting protein-1 like (RPGRIP1L) are associated with ciliogenesis in melanocytes, only the knockdown of OFD1, but not IFT88 and RPGRIP1L, resulted in increased melanocyte apoptosis. OFD1 knockdown led to a decrease in the expression of proteins involved in cell-extracellular matrix (ECM) interactions, including paxillin. The OFD1 amino acid residues 601-1012 interacted with paxillin, while the amino acid residues 1-601 were associated with ciliogenesis, suggesting that the OFD1 domains responsible for paxillin binding are distinct from those involved in ciliogenesis. OFD1 knockdown, but not IFT88 knockdown, inhibited melanocyte adhesion to the ECM, a defect that was restored by paxillin overexpression. In summary, our findings indicate that the downregulation of OFD1 induces melanocyte apoptosis, independent of any impairment in ciliogenesis, by reducing melanocyte adhesion to the ECM via paxillin.

Autonomous and non-cell autonomous role of cilia in structural birth defects in mice.

Ciliopathies are associated with wide spectrum of structural birth defects (SBDs), indicating important roles for cilia in development. Here, we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140, an intraflagellar transport (IFT) protein regulating ciliogenesis. Ift140-deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula (TEF), randomized heart looping, congenital heart defects (CHDs), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAGGCre-ER deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD were not observed with 4 Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest-mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathies.

The cilium like region of the Drosophila bifurca spermatocyte: Elongation of a giant axoneme without intraflagellar transport

AbstractThe growth of the ciliary axonemes mainly depends on the evolutionary conserved intraflagellar transport (IFT) machinery. However, insect spermatocytes are characterized by cilium‐like regions (CLRs) that elongate in the absence of IFT. It is generally believed that the dynamics of these structures relies on the free diffusion of soluble tubulin from the cytoplasm. However, this passive process could allow the elongation of short ciliary axonemes, but it is unclear whether simple diffusion of tubulin molecules can ensure the correct assembly of elongated ciliary structures. To decipher this point we analyzed the assembly of the CLRs held by the primary spermatocytes of Drosophila bifurca. These ciliary structures consist of a very elongated axoneme that grows without IFT and, therefore, could represent a good model in which to evaluate the role played by the free diffusion of soluble tubulin. The observation of wavy microtubules in the axonemal lumen of fully elongated CLRs of D. bifurca may be consistent with the diffusion of tubulin within the axonemal lumen. Progressive consumption of soluble tubulin used for axoneme growth at the apical tip of the CLRs could result in a gradient sufficient to move tubulin from the cytoplasm to the apical end of the forming ciliary structure. When the axoneme reaches its full length, tubulin molecules are not drawn to the tip of the CLRs and accumulate at the base of the axoneme, where its concentration may exceed the threshold need for microtubule polymerization. The presence of γ‐TuRCs at the proximal ends of the supernumerary microtubules could enhance their nucleation.

Phosphorylation of MIF by PIP4K2a is necessary for cilia biogenesis

Primary cilia are microtubule-based organelles that play important roles in development and tissue homeostasis. Macrophage migration inhibitory factor (MIF) has long been recognized as a secreted cytokine in the pathogenesis of various human diseases, including cancer and autosomal dominant polycystic kidney disease (ADPKD). Unlike other cytokines, unique functional characteristics of intracellular MIF have emerged. In this study, we show that MIF is localized and formed a ring like structure at the proximal end of centrioles, where it regulates cilia biogenesis through affecting 1) the recruitment of TTBK2 to basal body and the removal of CP110 from mother centriole, 2) the accumulation of CEP290 at centriolar satellites, and 3) the trafficking of intraflagellar transport (IFT) related proteins. We also show that MIF functions as a novel transcriptional factor to regulate the expression of genes related to ciliogenesis via binding on the promotors of those genes. MIF also binds chromatin and regulates transcription of genes involved in diverse homeostatic signaling pathways. We identify phosphatidylinositol-5-phosphate 4-kinase type 2 alpha (PIP4K2a) as an upstream regulator of MIF, which interacts with and phosphorylates MIF at S91 to increase its interaction with 14-3-3ζ, resulting in its nuclear translocation and transcription regulation. This study suggests that MIF is a key player in cilia biogenesis and a novel transcriptional regulator in homeostasis, which forward our understanding of how MIF is able to carry out several nonoverlapping functions.

CCDC146 is required for sperm flagellum biogenesis and male fertility in mice.

Multiple morphological abnormalities of the flagella (MMAF) is a severe disease of male infertility, while the pathogenetic mechanisms of MMAF are still incompletely understood. Previously, we found that the deficiency of Ccdc38 might be associated with MMAF. To understand the underlying mechanism of this disease, we identified the potential partner of this protein and found that the coiled-coil domain containing 146 (CCDC146) can interact with CCDC38. It is predominantly expressed in the testes, and the knockout of this gene resulted in complete infertility in male mice but not in females. The knockout of Ccdc146 impaired spermiogenesis, mainly due to flagellum and manchette organization defects, finally led to MMAF-like phenotype. Furthermore, we demonstrated that CCDC146 could interact with both CCDC38 and CCDC42. It also interacts with intraflagellar transport (IFT) complexes IFT88 and IFT20. The knockout of this gene led to the decrease of ODF2, IFT88, and IFT20 protein levels, but did not affect CCDC38, CCDC42, or ODF1 expression. Additionally, we predicted and validated the detailed interactions between CCDC146 and CCDC38 or CCDC42, and built the interaction models at the atomic level. Our results suggest that the testis predominantly expressed gene Ccdc146 is essential for sperm flagellum biogenesis and male fertility, and its mutations might be associated with MMAF in some patients.

Dyneins

Dyneins are a family of motor proteins that carry out motility and force generation functions towards the minus end of microtubule filaments. Cytoplasmic dynein (dynein-1) is responsible for transporting intracellular cargos in the retrograde direction in the cytoplasm, anchoring several organelles to the microtubule network, driving nuclear migration in developing neurons, and orienting the mitotic spindle in dividing cells. All other dyneins are localized to cilia. Similar to dynein-1, dynein-2 walks along microtubules and drives intraflagellar transport in the retrograde direction. Other ciliary dyneins are positioned between adjacent microtubule doublets of the axoneme and power ciliary beating by sliding microtubules relative to each other. In this primer, we first highlight the structure, mechanism, and regulation of dynein-1, which is the best-characterized member of the dynein motor family, and then describe the unique features and cellular roles of other dyneins. We also discuss accessory proteins that regulate the activation and motility of dynein motors in different cellular contexts.

ANKS1A-Deficiency Aberrantly Increases the Entry of the Protein Transport Machinery into the Ependymal Cilia.

In this study, we examine whether a change in the protein levels for FOP in Ankyrin repeat and SAM domain-containing protein 1A (ANKS1A)-deficient ependymal cells affects the intraflagellar transport (IFT) protein transport system in the multicilia. Three distinct abnormalities are observed in the multicilia of ANKS1A-deficient ependymal cells. First, there were a greater number of IFT88-positive trains along the cilia from ANKS1A deficiency. The results are similar to each isolated cilium as well. Second, each isolated cilium contains a significant increase in the number of extracellular vesicles (ECVs) due to the lack of ANKS1A. Third, Van Gogh-like 2 (Vangl2), a ciliary membrane protein, is abundantly detected along the cilia and in the ECVs attached to them for ANKS1A-deficient cells. We also use primary ependymal culture systems to obtain the ECVs released from the multicilia. Consequently, we find that ECVs from ANKS1A-deficient cells contain more IFT machinery and Vangl2. These results indicate that ANKS1A deficiency increases the entry of the protein transport machinery into the multicilia and as a result of these abnormal protein transports, excessive ECVs form along the cilia. We conclude that ependymal cells make use of the ECV-based disposal system in order to eliminate excessively transported proteins from basal bodies.

Paralog-specific TTC30 regulation of Sonic hedgehog signaling

The intraflagellar transport (IFT) machinery is essential for cilia assembly, maintenance, and trans-localization of signaling proteins. The IFT machinery consists of two large multiprotein complexes, one of which is the IFT-B. TTC30A and TTC30B are integral components of this complex and were previously shown to have redundant functions in the context of IFT, preventing the disruption of IFT-B and, thus, having a severe ciliogenesis defect upon loss of one paralog. In this study, we re-analyzed the paralog-specific protein complexes and discovered a potential involvement of TTC30A or TTC30B in ciliary signaling. Specifically, we investigated a TTC30A-specific interaction with protein kinase A catalytic subunit α, a negative regulator of Sonic hedgehog (Shh) signaling. Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which, intriguingly, is also linked to a rare TTC30 variant. For an in-depth analysis of this unique interaction and the influence on Shh, TTC30A or B single- and double-knockout hTERT-RPE1 were employed, as well as rescue cells harboring wildtype TTC30 or the corresponding mutation. We could show that mutant TTC30A inhibits the ciliary localization of Smoothened. This observed effect is independent of Patched1 but associated with a distinct phosphorylated PKA substrate accumulation upon treatment with forskolin. This rather prominent phenotype was attenuated in mutant TTC30B. Mass spectrometry analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in protein complex patterns and identified an impaired TTC30A–IFT57 interaction as the possible link leading to synpolydactyly. We could observe no impact on cilia assembly, leading to the hypothesis that a slight decrease in IFT-B binding can be compensated, but mild phenotypes, like synpolydactyly, can be induced by subtle signaling changes. Our systematic approach revealed the paralog-specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA and the uptake of Smoothened into the cilium, resulting in a downregulation of Shh. This downregulation, combined with interactome alterations, suggests a potential mechanism of how mutant TTC30A is linked to synpolydactyly.

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of Bbs1.

Bardet-Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate.