Primary cilia project in a single copy from the surface of most vertebrate cell types; they detect and transmit extracellular cues to regulate diverse cellular processes during development and to ...maintain tissue homeostasis. The sensory capacity of primary cilia relies on the coordinated trafficking and temporal localization of specific receptors and associated signal transduction modules in the cilium. The canonical Hedgehog (HH) pathway, for example, is a bona fide ciliary signalling system that regulates cell fate and self-renewal in development and tissue homeostasis. Specific receptors and associated signal transduction proteins can also localize to primary cilia in a cell type-dependent manner; available evidence suggests that the ciliary constellation of these proteins can temporally change to allow the cell to adapt to specific developmental and homeostatic cues. Consistent with important roles for primary cilia in signalling, mutations that lead to their dysfunction underlie a pleiotropic group of diseases and syndromic disorders termed ciliopathies, which affect many different tissues and organs of the body. In this Review, we highlight central mechanisms by which primary cilia coordinate HH, G protein-coupled receptor, WNT, receptor tyrosine kinase and transforming growth factor-β (TGFβ)/bone morphogenetic protein (BMP) signalling and illustrate how defects in the balanced output of ciliary signalling events are coupled to developmental disorders and disease progression.
Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene strongly predispose to development of gastro-intestinal tumors. Central to the tumorigenic events in APC mutant cells is the ...uncontrolled stabilization and transcriptional activation of the protein β-catenin. Many questions remain as to how APC controls β-catenin degradation. Remarkably, the large C-terminal region of APC, which spans over 2000 amino acids and includes critical regions in downregulating β-catenin, is predicted to be natively unfolded. Here we discuss how this uncommonly large disordered region may help to coordinate the multiple cellular functions of APC. Recently, a significant number of germline and somatic missense mutations in the central region of APC were linked to tumorigenesis in the colon as well as extra-intestinal tissues. We classify and localize all currently known missense mutations in the APC structure. The molecular basis by which these mutations interfere with the function of APC remains unresolved. We propose several mechanisms by which cancer-related missense mutations in the large disordered domain of APC may interfere with tumor suppressor activity. Insight in the underlying molecular events will be invaluable in the development of novel strategies to counter dysregulated Wnt signaling by APC mutations in cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Significance Regulation of differential gene expression by Wnt/glycogen synthase kinase 3 (GSK3) signaling plays a major role in development, but there is accumulating evidence for a ...transcription-independent function of this pathway. In mature oocytes and early embryos, transcription is temporarily silenced, providing an opportunity to study the nontranscriptional role of Wnt signaling. Our study shows that inhibition of Wnt signaling in frog oocytes leads to early cleavage arrest after fertilization and provides evidence that this process occurs by regulating proteins involved in mitosis. Our study indicates that control of early development is a prominent role for nontranscriptional, Wnt/GSK3 signaling.
During Xenopus development, Wnt signaling is thought to function first after midblastula transition to regulate axial patterning via β-catenin–mediated transcription. Here, we report that Wnt/glycogen synthase kinase 3 (GSK3) signaling functions posttranscriptionally already in mature oocytes via Wnt/stabilization of proteins (STOP) signaling. Wnt signaling is induced in oocytes after their entry into meiotic metaphase II and declines again upon exit into interphase. Wnt signaling inhibits Gsk3 and thereby protects proteins from polyubiquitination and degradation in mature oocytes. In a protein array screen, we identify a cluster of mitotic effector proteins that are polyubiquitinated in a Gsk3-dependent manner in Xenopus . Consequently inhibition of maternal Wnt/STOP signaling, but not β-catenin signaling, leads to early cleavage arrest after fertilization. The results support a novel role for Wnt signaling in cell cycle progression independent of β-catenin.
Establishment and maintenance of the primary cilium as a signaling-competent organelle requires a high degree of fine tuning, which is at least in part achieved by a variety of post-translational ...modifications. One such modification is ubiquitination. The small and highly conserved ubiquitin protein possesses a unique versatility in regulating protein function
its ability to build mono and polyubiquitin chains onto target proteins. We aimed to take an unbiased approach to generate a comprehensive blueprint of the ciliary ubiquitinome by deploying a multi-proteomics approach using both ciliary-targeted ubiquitin affinity proteomics, as well as ubiquitin-binding domain-based proximity labelling in two different mammalian cell lines. This resulted in the identification of several key proteins involved in signaling, cytoskeletal remodeling and membrane and protein trafficking. Interestingly, using two different approaches in IMCD3 and RPE1 cells, respectively, we uncovered several novel mechanisms that regulate cilia function. In our IMCD3 proximity labeling cell line model, we found a highly enriched group of ESCRT-dependent clathrin-mediated endocytosis-related proteins, suggesting an important and novel role for this pathway in the regulation of ciliary homeostasis and function. In contrast, in RPE1 cells we found that several structural components of caveolae (CAV1, CAVIN1, and EHD2) were highly enriched in our cilia affinity proteomics screen. Consistently, the presence of caveolae at the ciliary pocket and ubiquitination of CAV1 specifically, were found likely to play a role in the regulation of ciliary length in these cells. Cilia length measurements demonstrated increased ciliary length in RPE1 cells stably expressing a ubiquitination impaired CAV1 mutant protein. Furthermore, live cell imaging in the same cells revealed decreased CAV1 protein turnover at the cilium as the possible cause for this phenotype. In conclusion, we have generated a comprehensive list of cilia-specific proteins that are subject to regulation
ubiquitination which can serve to further our understanding of cilia biology in health and disease.
Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 ...and retrograde cytoplasmic dynein-2 motors. Nematodes additionally employ a cell-type-specific kinesin-3 motor, KLP-6, which moves within cilia independently of IFT and regulates ciliary content and function. Here, we provide evidence that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured immortalized human retinal pigment epithelial (hTERT-RPE1) cells. Anterograde and retrograde intraciliary velocities of KIF13B were similar to those of IFT (as assayed using IFT172-eGFP), but intraciliary movement of KIF13B required its own motor domain and appeared to be cell-type specific. Our work provides the first demonstration of motor-driven, intraciliary movement by a vertebrate kinesin other than kinesin-2 motors.
The kinesin-3 motor KIF13B functions in endocytosis, vesicle transport, and regulation of ciliary length and signaling. Direct binding of the membrane-associated guanylate kinase (MAGUK) DLG1 to ...KIF13B's MAGUK-binding stalk (MBS) domain relieves motor autoinhibition and promotes microtubule plus end-directed cargo transport. Here we characterize Angiomotin isoform 2 (Ap80) as a novel KIF13B interactor that promotes binding of another MAGUK, the polarity protein and Crumbs complex component PALS1, to KIF13B. Live-cell imaging analysis indicated that Ap80 is concentrated at and recruits PALS1 to the base of primary cilia, but is not itself a cargo of KIF13B. Consistent with a ciliary function for Ap80, its depletion led to elongated primary cilia and reduced agonist-induced ciliary accumulation of SMO, a key component of the Hedgehog signaling pathway, while Ap80 overexpression caused ciliary shortening. Our results suggest that Ap80 activates KIF13B cargo binding at the base of primary cilia to regulate ciliary length, composition and signaling.
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.
Synopsis
The Scribble polarity complex protein DLG1 regulates ciliary length and protein composition in kidney epithelial cells. A Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant may be associated with ciliary dysfunction.
Loss of DLG1 causes ciliary elongation in kidney epithelial cells.
Loss of DLG1 impairs targeting of SDCCAG3, IFT20 and PC2 to the primary cilium of kidney epithelial cells.
The CAKUT-associated p.T489R DLG1 fails to rescue ciliary defects of
Dlg1
-/-
cells, indicating a possible ciliary involvement in CAKUT disease etiology.
The Scribble polarity complex protein DLG1 regulates ciliary length and protein composition in kidney epithelial cells. A Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant may be associated with ciliary dysfunction.
DuringXenopusdevelopment, Wnt signaling is thought to function first after midblastula transition to regulate axial patterning via β-catenin–mediated transcription. Here, we report that Wnt/glycogen ...synthase kinase 3 (GSK3) signaling functions posttranscriptionally already in mature oocytes via Wnt/stabilization of proteins (STOP) signaling. Wnt signaling is induced in oocytes after their entry into meiotic metaphase II and declines again upon exit into interphase. Wnt signaling inhibits Gsk3 and thereby protects proteins from polyubiquitination and degradation in mature oocytes. In a protein array screen, we identify a cluster of mitotic effector proteins that are polyubiquitinated in a Gsk3-dependent manner inXenopus. Consequently inhibition of maternal Wnt/STOP signaling, but not β-catenin signaling, leads to early cleavage arrest after fertilization. The results support a novel role for Wnt signaling in cell cycle progression independent of β-catenin.
Signaling cascades depend on scaffold proteins that regulate the assembly of multiprotein complexes. Missense mutations in scaffold proteins are frequent in human cancer, but their relevance and mode ...of action are poorly understood. Here we show that cancer point mutations in the scaffold protein Axin derail Wnt signaling and promote tumor growth in vivo through a gain-of-function mechanism. The effect is conserved for both the human and Drosophila proteins. Mutated Axin forms nonamyloid nanometer-scale aggregates decorated with disordered tentacles, which 'rewire' the Axin interactome. Importantly, the tumor-suppressor activity of both the human and Drosophila Axin cancer mutants is rescued by preventing aggregation of a single nonconserved segment. Our findings establish a new paradigm for misregulation of signaling in cancer and show that targeting aggregation-prone stretches in mutated scaffolds holds attractive potential for cancer treatment.
PDF
