DLG1 functions upstream of SDCCAG3 and IFT20 to control ciliary targeting of polycystin-2.

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney causes ciliary elongation and cystogenesis, and cell-based proximity labeling proteomics and fluorescence microscopy show alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20, and polycystin-2 (PC2) are reduced in the cilia of DLG1-deficient cells compared to control cells. This phenotype is recapitulated in vivo and rescuable by re-expression of wild-type DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggest that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

Interactome Analysis Reveals a Link of the Novel ALMS1-CEP70 Complex to Centrosomal Clusters.

Alström syndrome (ALMS) is a very rare autosomal-recessive disorder, causing a broad range of clinical defects most notably retinal degeneration, type 2 diabetes, and truncal obesity. The ALMS1 gene encodes a complex and huge ∼0.5 MDa protein, which has hampered analysis in the past. The ALMS1 protein is localized to the centrioles and the basal body of cilia and is involved in signaling processes, for example, TGF-ß signaling. However, the exact molecular function of ALMS1 at the basal body remains elusive and controversial. We recently demonstrated that protein complex analysis utilizing endogenously tagged cells provides an excellent tool to investigate protein interactions of ciliary proteins. Here, CRISPR/Cas9-mediated endogenously tagged ALMS1 cells were used for affinity-based protein complex analysis. Centrosomal and microtubule-associated proteins were identified, which are potential regulators of ALMS1 function, such as the centrosomal protein 70 kDa (CEP70). Candidate proteins were further investigated in ALMS1-deficient hTERT-RPE1 cells. Loss of ALMS1 led to shortened cilia with no change in structural protein localization, for example, acetylated and É£-tubulin, Centrin-3, or the novel interactor CEP70. Conversely, reduction of CEP70 resulted in decreased ALMS1 at the ciliary basal body. Complex analysis of CEP70 revealed domain-specific ALMS1 interaction involving the TPR-containing C-terminal (TRP-CT) fragment of CEP70. In addition to ALMS1, several ciliary proteins, including CEP135, were found to specifically bind to the TPR-CT domain. Data are available via ProteomeXchange with the identifier PXD046401. Protein interactors identified in this study provide candidate lists that help to understand ALMS1 and CEP70 function in cilia-related protein modification, cell death, and disease-related mechanisms.

WeSA: a web server for improving analysis of affinity proteomics data.

Protein-protein interaction experiments still yield many false positive interactions. The socioaffinity metric can distinguish true protein-protein interactions from noise based on available data. Here, we present WeSA (Weighted SocioAffinity), which considers large datasets of interaction proteomics data (IntAct, BioGRID, the BioPlex) to score human protein interactions and, in a statistically robust way, flag those (even from a single experiment) that are likely to be false positives. ROC analysis (using CORUM-PDB positives and Negatome negatives) shows that WeSA improves over other measures of interaction confidence. WeSA shows consistently good results over all datasets (up to: AUC = 0.93 and at best threshold: TPR = 0.84, FPR = 0.11, Precision = 0.98). WeSA is freely available without login (wesa.russelllab.org). Users can submit their own data or look for organized information on human protein interactions using the web server. Users can either retrieve available information for a list of proteins of interest or calculate scores for new experiments. The server outputs either pre-computed or updated WeSA scores for the input enriched with information from databases. The summary is presented as a table and a network-based visualization allowing the user to remove those nodes/edges that the method considers spurious.

Defective airway intraflagellar transport underlies a combined motile and primary ciliopathy syndrome caused by IFT74 mutations.

Ciliopathies are inherited disorders caused by defective cilia. Mutations affecting motile cilia usually cause the chronic muco-obstructive sinopulmonary disease primary ciliary dyskinesia (PCD) and are associated with laterality defects, while a broad spectrum of early developmental as well as degenerative syndromes arise from mutations affecting signalling of primary (non-motile) cilia. Cilia assembly and functioning requires intraflagellar transport (IFT) of cargos assisted by IFT-B and IFT-A adaptor complexes. Within IFT-B, the N-termini of partner proteins IFT74 and IFT81 govern tubulin transport to build the ciliary microtubular cytoskeleton. We detected a homozygous 3-kb intragenic IFT74 deletion removing the exon 2 initiation codon and 40 N-terminal amino acids in two affected siblings. Both had clinical features of PCD with bronchiectasis, but no laterality defects. They also had retinal dysplasia and abnormal bone growth, with a narrowed thorax and short ribs, shortened long bones and digits, and abnormal skull shape. This resembles short-rib thoracic dysplasia, a skeletal ciliopathy previously linked to IFT defects in primary cilia, not motile cilia. Ciliated nasal epithelial cells collected from affected individuals had reduced numbers of shortened motile cilia with disarranged microtubules, some misorientation of the basal feet, and disrupted cilia structural and IFT protein distributions. No full-length IFT74 was expressed, only truncated forms that were consistent with N-terminal deletion and inframe translation from downstream initiation codons. In affinity purification mass spectrometry, exon 2-deleted IFT74 initiated from the nearest inframe downstream methionine 41 still interacts as part of the IFT-B complex, but only with reduced interaction levels and not with all its usual IFT-B partners. We propose that this is a hypomorphic mutation with some residual protein function retained, which gives rise to a primary skeletal ciliopathy combined with defective motile cilia and PCD.

Recessively Inherited Deficiency of Secreted WFDC2 (HE4) Causes Nasal Polyposis and Bronchiectasis.

Rationale: Bronchiectasis is a pathological dilatation of the bronchi in the respiratory airways associated with environmental or genetic causes (e.g., cystic fibrosis, primary ciliary dyskinesia, and primary immunodeficiency disorders), but most cases remain idiopathic. Objectives: To identify novel genetic defects in unsolved cases of bronchiectasis presenting with severe rhinosinusitis, nasal polyposis, and pulmonary Pseudomonas aeruginosa infection. Methods: DNA was analyzed by next-generation or targeted Sanger sequencing. RNA was analyzed by quantitative PCR and single-cell RNA sequencing. Patient-derived cells, cell cultures, and secretions (mucus, saliva, seminal fluid) were analyzed by Western blotting and immunofluorescence microscopy, and mucociliary activity was measured. Blood serum was analyzed by electrochemiluminescence immunoassay. Protein structure and proteomic analyses were used to assess the impact of a disease-causing founder variant. Measurements and Main Results: We identified biallelic pathogenic variants in WAP four-disulfide core domain 2 (WFDC2) in 11 individuals from 10 unrelated families originating from the United States, Europe, Asia, and Africa. Expression of WFDC2 was detected predominantly in secretory cells of control airway epithelium and also in submucosal glands. We demonstrate that WFDC2 is below the limit of detection in blood serum and hardly detectable in samples of saliva, seminal fluid, and airway surface liquid from WFDC2-deficient individuals. Computer simulations and deglycosylation assays indicate that the disease-causing founder variant p.Cys49Arg structurally hampers glycosylation and, thus, secretion of mature WFDC2. Conclusions: WFDC2 dysfunction defines a novel molecular etiology of bronchiectasis characterized by the deficiency of a secreted component of the airways. A commercially available blood test combined with genetic testing allows its diagnosis.

The kinesin motor KIF1C is a putative transporter of the exon junction complex in neuronal cells.

Neurons critically depend on regulated RNA localization and tight control of spatio-temporal gene expression to maintain their morphological and functional integrity. Mutations in the kinesin motor protein gene KIF1C cause Hereditary Spastic Paraplegia, an autosomal recessive disease leading to predominant degeneration of the long axons of central motoneurons. In this study we aimed to gain insight into the molecular function of KIF1C and understand how KIF1C dysfunction contributes to motoneuron degeneration. We used affinity proteomics in neuronally differentiated neuroblastoma cells (SH-SY5Y) to identify the protein complex associated with KIF1C in neuronal cells; candidate interactions were then validated by immunoprecipitation and mislocalization of putative KIF1C cargoes was studied by immunostainings. We found KIF1C to interact with all core components of the exon junction complex (EJC); expression of mutant KIF1C in neuronal cells leads to loss of the typical localization distally in neurites. Instead, EJC core components accumulate in the pericentrosomal region, here co-localizing with mutant KIF1C. These findings suggest KIF1C as a neuronal transporter of the EJC. Interestingly, the binding of KIF1C to the EJC is RNA-mediated, as treatment with RNAse prior to immunoprecipitation almost completely abolishes the interaction. Silica-based solid-phase extraction of UV-crosslinked RNA-protein complexes furthermore supports direct interaction of KIF1C with RNA, as recently also demonstrated for kinesin heavy chain. Taken together, our findings are consistent with a model where KIF1C transports mRNA in an EJC-bound and therefore transcriptionally silenced state along neurites, thus providing the missing link between the EJC and mRNA localization in neurons.

The FOXJ1 target Cfap206 is required for sperm motility, mucociliary clearance of the airways and brain development.

Cilia are complex cellular protrusions consisting of hundreds of proteins. Defects in ciliary structure and function, many of which have not been characterised molecularly, cause ciliopathies: a heterogeneous group of human syndromes. Here, we report on the FOXJ1 target gene Cfap206, orthologues of which so far have only been studied in Chlamydomonas and Tetrahymena In mouse and Xenopus, Cfap206 was co-expressed with and dependent on Foxj1 CFAP206 protein localised to the basal body and to the axoneme of motile cilia. In Xenopus crispant larvae, the ciliary beat frequency of skin multiciliated cells was enhanced and bead transport across the epidermal mucociliary epithelium was reduced. Likewise, Cfap206 knockout mice revealed ciliary phenotypes. Electron tomography of immotile knockout mouse sperm flagella indicated a role in radial spoke formation reminiscent of FAP206 function in Tetrahymena Male infertility, hydrocephalus and impaired mucociliary clearance of the airways in the absence of laterality defects in Cfap206 mutant mice suggests that Cfap206 may represent a candidate for the subgroup of human primary ciliary dyskinesias caused by radial spoke defects.

Dimethyl fumarate induces ferroptosis and impairs NF-κB/STAT3 signaling in DLBCL.

Despite the development of novel targeted drugs, the molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) still poses a substantial therapeutic challenge. DLBCL can be classified into at least 2 major subtypes (germinal center B cell [GCB]-like and activated B cell [ABC]-like DLBCL), each characterized by specific gene expression profiles and mutation patterns. Here we demonstrate a broad antitumor effect of dimethyl fumarate (DMF) on both DLBCL subtypes, which is mediated by the induction of ferroptosis, a form of cell death driven by the peroxidation of phospholipids. As a result of the high expression of arachidonate 5-lipoxygenase in concert with low glutathione and glutathione peroxidase 4 levels, DMF induces lipid peroxidation and thus ferroptosis, particularly in GCB DLBCL. In ABC DLBCL cells, which are addicted to NF-κB and STAT3 survival signaling, DMF treatment efficiently inhibits the activity of the IKK complex and Janus kinases. Interestingly, the BCL-2-specific BH3 mimetic ABT-199 and an inhibitor of ferroptosis suppressor protein 1 synergize with DMF in inducing cell death in DLBCL. Collectively, our findings identify the clinically approved drug DMF as a promising novel therapeutic option in the treatment of both GCB and ABC DLBCLs.

PCARE and WASF3 regulate ciliary F-actin assembly that is required for the initiation of photoreceptor outer segment disk formation.

The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare-/- mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.

Aberrant NOVA1 function disrupts alternative splicing in early stages of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.

The Na+-activated K+ channel Slack contributes to synaptic development and plasticity.

Human mutations of the Na+-activated K+ channel Slack (KCNT1) are associated with epilepsy and intellectual disability. Accordingly, Slack knockout mice (Slack-/-) exhibit cognitive flexibility deficits in distinct behavioral tasks. So far, however, the underlying causes as well as the role of Slack in hippocampus-dependent memory functions remain enigmatic. We now report that infant (P6-P14) Slack-/- lack both hippocampal LTD and LTP, likely due to impaired NMDA receptor (NMDAR) signaling. Postsynaptic GluN2B levels are reduced in infant Slack-/-, evidenced by lower amplitudes of NMDAR-meditated excitatory postsynaptic potentials. Low GluN2B affected NMDAR-mediated Ca2+-influx, rendering cultured hippocampal Slack-/-neurons highly insensitive to the GluN2B-specific inhibitor Ro 25-6981. Furthermore, dephosphorylation of the AMPA receptor (AMPAR) subunit GluA1 at S845, which is involved in AMPAR endocytosis during homeostatic and neuromodulator-regulated plasticity, is reduced after chemical LTD (cLTD) in infant Slack-/-. We additionally detect a lack of mGluR-induced LTD in infant Slack-/-, possibly caused by upregulation of the recycling endosome-associated small GTPase Rab4 which might accelerate AMPAR recycling from early endosomes. Interestingly, LTP and mGluR LTD, but not LTD and S845 dephosphorylation after cLTD are restored in adult Slack-/-. This together with normalized expression levels of GluN2B and Rab4 hints to developmental "restoration" of LTP expression despite Slack ablation, whereas in infant and adult brain, NMDAR-dependent LTD induction depends on this channel. Based on the present findings, NMDAR and vesicular transport might represent novel targets for the therapy of intellectual disability associated with Slack mutations. Consequently, careful modulation of hippocampal Slack activity should also improve learning abilities.

Affinity Proteomics Identifies Interaction Partners and Defines Novel Insights into the Function of the Adhesion GPCR VLGR1/ADGRV1.

The very large G-protein-coupled receptor 1 (VLGR1/ADGRV1) is the largest member of the adhesion G-protein-coupled receptor (ADGR) family. Mutations in VLGR1/ADGRV1 cause human Usher syndrome (USH), a form of hereditary deaf-blindness, and have been additionally linked to epilepsy. In the absence of tangible knowledge of the molecular function and signaling of VLGR1, the pathomechanisms underlying the development of these diseases are still unknown. Our study aimed to identify novel, previously unknown protein networks associated with VLGR1 in order to describe new functional cellular modules of this receptor. Using affinity proteomics, we have identified numerous new potential binding partners and ligands of VLGR1. Tandem affinity purification hits were functionally grouped based on their Gene Ontology terms and associated with functional cellular modules indicative of functions of VLGR1 in transcriptional regulation, splicing, cell cycle regulation, ciliogenesis, cell adhesion, neuronal development, and retinal maintenance. In addition, we validated the identified protein interactions and pathways in vitro and in situ. Our data provided new insights into possible functions of VLGR1, related to the development of USH and epilepsy, and also suggest a possible role in the development of other neuronal diseases such as Alzheimer's disease.

CFAP43 modulates ciliary beating in mouse and Xenopus.

Malfunctions of motile cilia cause a variety of developmental defects and diseases in humans and animal model organisms. Defects include impaired mucociliary clearance of the airways, sperm immotility, hydrocephalus and organ laterality. Here, we characterize the evolutionary conserved Cfap43 gene by loss-of-function experiments in the mouse and the frog Xenopus laevis. Cfap43 is expressed in tissues carrying motile cilia and acts as a target gene of the transcription factor FOXJ1, which is essential for the induction of motile ciliogenesis. We show that CFAP43, a protein of unknown biochemical function, localizes to the ciliary axoneme. CFAP43 is involved in the regulation of the beating frequency of tracheal cilia and loss of CFAP43 causes severe mucus accumulation in the nasal cavity. Likewise, morphant and crispant frog embryos revealed impaired function of motile cilia of the larval epidermis, a model for airway mucociliary epithelia. CFAP43 participates in the formation of flagellar axonemes during spermatogenesis as mice mutant for Cfap43 display male infertility, consistent with observations in male sterile patients. In addition, mice mutant for Cfap43 display early onset hydrocephalus. Together, these results confirm the role of CFAP43 in the male reproductive tract and pinpoint additional functions in airway epithelia mucus clearance and brain development.

DNAAF1 links heart laterality with the AAA+ ATPase RUVBL1 and ciliary intraflagellar transport.

DNAAF1 (LRRC50) is a cytoplasmic protein required for dynein heavy chain assembly and cilia motility, and DNAAF1 mutations cause primary ciliary dyskinesia (PCD; MIM 613193). We describe four families with DNAAF1 mutations and complex congenital heart disease (CHD). In three families, all affected individuals have typical PCD phenotypes. However, an additional family demonstrates isolated CHD (heterotaxy) in two affected siblings, but no clinical evidence of PCD. We identified a homozygous DNAAF1 missense mutation, p.Leu191Phe, as causative for heterotaxy in this family. Genetic complementation in dnaaf1-null zebrafish embryos demonstrated the rescue of normal heart looping with wild-type human DNAAF1, but not the p.Leu191Phe variant, supporting the conserved pathogenicity of this DNAAF1 missense mutation. This observation points to a phenotypic continuum between CHD and PCD, providing new insights into the pathogenesis of isolated CHD. In further investigations of the function of DNAAF1 in dynein arm assembly, we identified interactions with members of a putative dynein arm assembly complex. These include the ciliary intraflagellar transport protein IFT88 and the AAA+ (ATPases Associated with various cellular Activities) family proteins RUVBL1 (Pontin) and RUVBL2 (Reptin). Co-localization studies support these findings, with the loss of RUVBL1 perturbing the co-localization of DNAAF1 with IFT88. We show that RUVBL1 orthologues have an asymmetric left-sided distribution at both the mouse embryonic node and the Kupffer's vesicle in zebrafish embryos, with the latter asymmetry dependent on DNAAF1. These results suggest that DNAAF1-RUVBL1 biochemical and genetic interactions have a novel functional role in symmetry breaking and cardiac development.

Functional analyses of Pericentrin and Syne-2 interaction in ciliogenesis.

Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human PCNT gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor-connecting cilium is unknown. We downregulated Pcnt in the retina ex vivo and in vivo via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9-mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt-Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration.

CRISPR/Cas9-mediated Genomic Editing of Cluap1/IFT38 Reveals a New Role in Actin Arrangement.

CRISPR/Cas9-mediated gene editing allows manipulation of a gene of interest in its own chromosomal context. When applied to the analysis of protein interactions and in contrast to exogenous expression of a protein, this can be studied maintaining physiological stoichiometry, topology, and context. We have used CRISPR/Cas9-mediated genomic editing to investigate Cluap1/IFT38, a component of the intraflagellar transport complex B (IFT-B). Cluap1 has been implicated in human development as well as in cancer progression. Cluap1 loss of function results in early developmental defects with neural tube closure, sonic hedgehog signaling and left-right defects. Herein, we generated an endogenously tagged Cluap1 for protein complex analysis, which was then correlated to the corresponding interactome determined by ectopic expression. Besides IFT-B complex components, new interacting proteins like Ephrin-B1 and TRIP6, which are known to be involved in cytoskeletal arrangement and protein transport, were identified. With the identification of platelet-derived growth factor A (PDGFA) and coiled-coil domain-containing protein 6 (CCDC6) two new interactions were discovered, which link Cluap1 to ciliogenesis and cancer development. The CRISPR/Cas9-mediated knockout of Cluap1 revealed a new phenotype affecting the actin cytoskeleton. Together, these data provide first evidence for a role of Cluap1 not only for cilia assembly and maintenance but also for cytoskeletal rearrangement and intracellular transport processes.

The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium.

RAB28, a member of the RAS oncogene family, is a ubiquitous, farnesylated, small GTPase of unknown function present in photoreceptors and the retinal pigmented epithelium (RPE). Nonsense mutations of the human RAB28 gene cause recessive cone-rod dystrophy 18 (CRD18), characterized by macular hyperpigmentation, progressive loss of visual acuity, RPE atrophy, and severely attenuated cone and rod electroretinography (ERG) responses. In an attempt to elucidate the disease-causing mechanism, we generated Rab28-/- mice by deleting exon 3 and truncating RAB28 after exon 2. We found that Rab28-/- mice recapitulate features of the human dystrophy (i.e. they exhibited reduced cone and rod ERG responses and progressive retina degeneration). Cones of Rab28-/- mice extended their outer segments (OSs) to the RPE apical processes and formed enlarged, balloon-like distal tips before undergoing degeneration. The visual pigment content of WT and Rab28-/- cones was comparable before the onset of degeneration. Cone phagosomes were almost absent in Rab28-/- mice, whereas rod phagosomes displayed normal levels. A protein-protein interaction screen identified several RAB28-interacting proteins, including the prenyl-binding protein phosphodiesterase 6 δ-subunit (PDE6D) and voltage-gated potassium channel subfamily J member 13 (KCNJ13) present in the RPE apical processes. Of note, the loss of PDE6D prevented delivery of RAB28 to OSs. Taken together, these findings reveal that RAB28 is required for shedding and phagocytosis of cone OS discs.

Liver cyst gene knockout in cholangiocytes inhibits cilium formation and Wnt signaling.

Mutations in the PRKCSH, SEC63 and LRP5 genes cause autosomal dominant polycystic liver disease (ADPLD). The proteins products of PRKCSH (alias GIIB) and SEC63 function in protein quality control and processing in the endoplasmic reticulum (ER), while LRP5 is implicated in Wnt/ß-catenin signaling. To identify common denominators in the PLD pathogenesis, we mapped the PLD interactome by affinity proteomics, employing both HEK293T cells and H69 cholangiocytes. Identification of known complex members, such as glucosidase IIA (GIIA) for PRKCSH, and SEC61A1 and SEC61B for SEC63, confirmed the specificity of the analysis. GANAB, encoding GIIA, was very recently identified as an ADPLD gene. The presence of GIIA in the LRP5 complex pinpoints a potential functional connection with PRKCSH. Interestingly, all three PLD-associated protein complexes included filamin A (FLNA), a multifunctional protein described to play a role in ciliogenesis as well as canonical Wnt signalling. As ciliary dysfunction may also contribute to hereditary liver cyst formation, we evaluated the requirement of PRKCSH and SEC63 for ciliogenesis and Wnt signaling. By CRISPR/Cas9 induced knockdown of both ADPLD genes in HEK293T cells and H69 cholangiocytes, we identified that their depletion results in defective ciliogenesis. However, only H69 knockouts displayed reduced Wnt3a activation. Our results suggest that loss of PRKCSH and SEC63 leads to general defects in ciliogenesis, while quenching of the Wnt signaling cascade is cholangiocyte-restricted. Interactions of all three PLD-associated protein complexes with FLNA may mark a common link between the ADPLD proteins and the cystogenic processes driving this disease.

Transcriptional and metabolic rewiring of colorectal cancer cells expressing the oncogenic KRASG13D mutation.

BACKGROUND: Activating mutations in KRAS frequently occur in colorectal cancer (CRC) patients, leading to resistance to EGFR-targeted therapies. METHODS: To better understand the cellular reprogramming which occurs in mutant KRAS cells, we have undertaken a systems-level analysis of four CRC cell lines which express either wild type (wt) KRAS or the oncogenic KRASG13D allele (mtKRAS). RESULTS: RNAseq revealed that genes involved in ribosome biogenesis, mRNA translation and metabolism were significantly upregulated in mtKRAS cells. Consistent with the transcriptional data, protein synthesis and cell proliferation were significantly higher in the mtKRAS cells. Targeted metabolomics analysis also confirmed the metabolic reprogramming in mtKRAS cells. Interestingly, mtKRAS cells were highly transcriptionally responsive to EGFR activation by TGFα stimulation, which was associated with an unexpected downregulation of genes involved in a range of anabolic processes. While TGFα treatment strongly activated protein synthesis in wtKRAS cells, protein synthesis was not activated above basal levels in the TGFα-treated mtKRAS cells. This was likely due to the defective activation of the mTORC1 and other pathways by TGFα in mtKRAS cells, which was associated with impaired activation of PKB signalling and a transient induction of AMPK signalling. CONCLUSIONS: We have found that mtKRAS cells are substantially rewired at the transcriptional, translational and metabolic levels and that this rewiring may reveal new vulnerabilities in oncogenic KRAS CRC cells that could be exploited in future.

Mutations in CEP78 Cause Cone-Rod Dystrophy and Hearing Loss Associated with Primary-Cilia Defects.

Cone-rod degeneration (CRD) belongs to the disease spectrum of retinal degenerations, a group of hereditary disorders characterized by an extreme clinical and genetic heterogeneity. It mainly differentiates from other retinal dystrophies, and in particular from the more frequent disease retinitis pigmentosa, because cone photoreceptors degenerate at a higher rate than rod photoreceptors, causing severe deficiency of central vision. After exome analysis of a cohort of individuals with CRD, we identified biallelic mutations in the orphan gene CEP78 in three subjects from two families: one from Greece and another from Sweden. The Greek subject, from the island of Crete, was homozygous for the c.499+1G>T (IVS3+1G>T) mutation in intron 3. The Swedish subjects, two siblings, were compound heterozygotes for the nearby mutation c.499+5G>A (IVS3+5G>A) and for the frameshift-causing variant c.633delC (p.Trp212Glyfs(∗)18). In addition to CRD, these three individuals had hearing loss or hearing deficit. Immunostaining highlighted the presence of CEP78 in the inner segments of retinal photoreceptors, predominantly of cones, and at the base of the primary cilium of fibroblasts. Interaction studies also showed that CEP78 binds to FAM161A, another ciliary protein associated with retinal degeneration. Finally, analysis of skin fibroblasts derived from affected individuals revealed abnormal ciliary morphology, as compared to that of control cells. Altogether, our data strongly suggest that mutations in CEP78 cause a previously undescribed clinical entity of a ciliary nature characterized by blindness and deafness but clearly distinct from Usher syndrome, a condition for which visual impairment is due to retinitis pigmentosa.