Angiomotin isoform 2 promotes binding of PALS1 to KIF13B at primary cilia and regulates ciliary length and signaling.

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 the MAGUK-binding stalk domain of KIF13B relieves motor autoinhibition and promotes microtubule plus-end-directed cargo transport. Here, we characterize angiomotin (AMOT) isoform 2 (p80, referred to as 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 the primary cilium, but is not a cargo of KIF13B itself. 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, whereas Ap80 overexpression caused ciliary shortening. Our results suggest that Ap80 activates KIF13B cargo binding at the base of the primary cilium to regulate ciliary length, composition and signaling.

CEP78 functions downstream of CEP350 to control biogenesis of primary cilia by negatively regulating CP110 levels.

CEP78 is a centrosomal protein implicated in ciliogenesis and ciliary length control, and mutations in the CEP78 gene cause retinal cone-rod dystrophy associated with hearing loss. However, the mechanism by which CEP78 affects cilia formation is unknown. Based on a recently discovered disease-causing CEP78 p.L150S mutation, we identified the disease-relevant interactome of CEP78. We confirmed that CEP78 interacts with the EDD1-DYRK2-DDB1VPRBP E3 ubiquitin ligase complex, which is involved in CP110 ubiquitination and degradation, and identified a novel interaction between CEP78 and CEP350 that is weakened by the CEP78L150S mutation. We show that CEP350 promotes centrosomal recruitment and stability of CEP78, which in turn leads to centrosomal recruitment of EDD1. Consistently, cells lacking CEP78 display significantly increased cellular and centrosomal levels of CP110, and depletion of CP110 in CEP78-deficient cells restored ciliation frequency to normal. We propose that CEP78 functions downstream of CEP350 to promote ciliogenesis by negatively regulating CP110 levels via an EDD1-dependent mechanism.

RRP7A links primary microcephaly to dysfunction of ribosome biogenesis, resorption of primary cilia, and neurogenesis.

Primary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role. We identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, segregating with MCPH in a consanguineous family with 10 affected individuals. RRP7A is highly expressed in neural stem cells in developing human forebrain, and targeted mutation of Rrp7a leads to defects in neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in ribosomal RNA processing, primary cilia resorption, and cell cycle progression. Analysis of zebrafish embryos supported that the patient mutation in RRP7A causes reduced brain size, impaired neurogenesis and cell proliferation, and defective ribosomal RNA processing. These findings provide novel insight into human brain development and MCPH.

Cellular signalling by primary cilia in development, organ function and disease.

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-ß (TGFß)/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.

Challenges for the Sustainability of University-Run Biobanks.

Most university biobanks begin like other university research projects, that is, with an idea conceived by an individual researcher in pursuit of his/her own research interests, publications, funding, and career. Some biobanks, however, come to have scientific value that goes beyond the projects that were initially responsible for the collection of the samples and data they contain. Such value may derive from among other things the uniqueness of the samples in terms of their sheer volume, the quality of the samples, the ability to link the samples with information retrieved in disease registries, or the fact that the samples represent very rare diseases. This article focuses on biobanks of this kind, and the special obligations that publicly funded universities have to ensure the sustainability of biobanks with continued scientific value. We argue that universities should adopt policies to deal with the various, diverse issues which may arise during the lifecycle of a biobank. The policies should be flexible, accommodate the freedoms of individual researchers, and reflect the multifaceted nature of biobanks. Yet they should be specific enough to provide guidance and robust enough to safeguard legal norms and ethical values. The article sets out concrete recommendations which universities should consider and act upon.

Regulation of ciliary membrane protein trafficking and signalling by kinesin motor proteins.

Primary cilia are antenna-like sensory organelles that regulate a substantial number of cellular signalling pathways in vertebrates, both during embryonic development as well as in adulthood, and mutations in genes coding for ciliary proteins are causative of an expanding group of pleiotropic diseases known as ciliopathies. Cilia consist of a microtubule-based axoneme core, which is subtended by a basal body and covered by a bilayer lipid membrane of unique protein and lipid composition. Cilia are dynamic organelles, and the ability of cells to regulate ciliary protein and lipid content in response to specific cellular and environmental cues is crucial for balancing ciliary signalling output. Here we discuss mechanisms involved in regulation of ciliary membrane protein trafficking and signalling, with main focus on kinesin-2 and kinesin-3 family members.

TSC1 and TSC2 regulate cilia length and canonical Hedgehog signaling via different mechanisms.

Primary cilia are sensory organelles that coordinate multiple cellular signaling pathways, including Hedgehog (HH), Wingless/Int (WNT) and Transforming Growth Factor-ß (TGF-ß) signaling. Similarly, primary cilia have been implicated in regulation of mTOR signaling, in which Tuberous Sclerosis Complex proteins 1 and 2 (TSC1/2) negatively regulate protein synthesis by inactivating the mTOR complex 1 (mTORC1) at energy limiting states. Here we report that TSC1 and TSC2 regulate Smoothened (SMO)-dependent HH signaling in mouse embryonic fibroblasts (MEFs). Reduced SMO-dependent expression of Gli1 was demonstrated in both Tsc1-/- and Tsc2-/- cells, and we found that Tsc1 is required for TGF-ß induced phosphorylation of SMAD2/3 and subsequent expression of the HH signaling effector and transcription factor GLI2. Hedgehog signaling was restored in Tsc1-/- cells after exogenous expression of Gli2, whereas rapamycin restored HH signaling in Tsc2-/- cells. Furthermore, we observed that Tsc1-/- MEFs display significantly elongated cilia, whereas cilia in Tsc2-/- MEFs were shorter than normal. The elongated cilium phenotype of Tsc1-/- MEFs is likely due to increased mTORC1-dependent autophagic flux observed in these cells, as both the autophagic flux and the cilia length phenotype was restored by rapamycin. In addition, ciliary length control in Tsc1-/- MEFs was also influenced by reduced expression of Gli2, which compromised expression of Wnt5a that normally promotes cilia disassembly. In summary, our results support distinct functions of Tsc1 and Tsc2 in cellular signaling as the two genes affect ciliary length control and HH signaling via different mechanisms.

IFT20 modulates ciliary PDGFRα signaling by regulating the stability of Cbl E3 ubiquitin ligases.

Primary cilia have pivotal roles as organizers of many different signaling pathways, including platelet-derived growth factor receptor α (PDGFRα) signaling, which, when aberrantly regulated, is associated with developmental disorders, tumorigenesis, and cancer. PDGFRα is up-regulated during ciliogenesis, and ciliary localization of the receptor is required for its appropriate ligand-mediated activation by PDGF-AA. However, the mechanisms regulating sorting of PDGFRα and feedback inhibition of PDGFRα signaling at the cilium are unknown. Here, we provide evidence that intraflagellar transport protein 20 (IFT20) interacts with E3 ubiquitin ligases c-Cbl and Cbl-b and is required for Cbl-mediated ubiquitination and internalization of PDGFRα for feedback inhibition of receptor signaling. In wild-type cells treated with PDGF-AA, c-Cbl becomes enriched in the cilium, and the receptor is subsequently ubiquitinated and internalized. In contrast, in IFT20-depleted cells, PDGFRα localizes aberrantly to the plasma membrane and is overactivated after ligand stimulation because of destabilization and degradation of c-Cbl and Cbl-b.

Ins and outs of GPCR signaling in primary cilia.

Primary cilia are specialized microtubule-based signaling organelles that convey extracellular signals into a cellular response in most vertebrate cell types. The physiological significance of primary cilia is underscored by the fact that defects in assembly or function of these organelles lead to a range of severe diseases and developmental disorders. In most cell types of the human body, signaling by primary cilia involves different G protein-coupled receptors (GPCRs), which transmit specific signals to the cell through G proteins to regulate diverse cellular and physiological events. Here, we provide an overview of GPCR signaling in primary cilia, with main focus on the rhodopsin-like (class A) and the smoothened/frizzled (class F) GPCRs. We describe how such receptors dynamically traffic into and out of the ciliary compartment and how they interact with other classes of ciliary GPCRs, such as class B receptors, to control ciliary function and various physiological and behavioral processes. Finally, we discuss future avenues for developing GPCR-targeted drug strategies for the treatment of ciliopathies.

Cilia and coordination of signaling networks during heart development.

Primary cilia are unique sensory organelles that coordinate a wide variety of different signaling pathways to control cellular processes during development and in tissue homeostasis. Defects in function or assembly of these antenna-like structures are therefore associated with a broad range of developmental disorders and diseases called ciliopathies. Recent studies have indicated a major role of different populations of cilia, including nodal and cardiac primary cilia, in coordinating heart development, and defects in these cilia are associated with congenital heart disease. Here, we present an overview of the role of nodal and cardiac primary cilia in heart development.

TGF-ß signaling is associated with endocytosis at the pocket region of the primary cilium.

Transforming growth factor ß (TGF-ß) signaling is regulated by clathrin-dependent endocytosis (CDE) for the control of cellular processes during development and in tissue homeostasis. The primary cilium coordinates several signaling pathways, and the pocket surrounding the base and proximal part of the cilium is a site for CDE. We report here that TGF-ß receptors localize to the ciliary tip and endocytic vesicles at the ciliary base in fibroblasts and that TGF-ß stimulation increases receptor localization and activation of SMAD2/3 and ERK1/2 at the ciliary base. Inhibition of CDE reduced TGF-ß-mediated signaling at the cilium, and TGF-ß signaling and CDE activity are reduced at stunted primary cilia in Tg737orpk fibroblasts. Similarly, TGF-ß signaling during cardiomyogenesis correlated with accumulation of TGF-ß receptors and activation of SMAD2/3 at the ciliary base. Our results indicate that the primary cilium regulates TGF-ß signaling and that the ciliary pocket is a compartment for CDE-dependent regulation of signal transduction.

The Na+/H+ exchanger NHE1 is required for directional migration stimulated via PDGFR-alpha in the primary cilium.

We previously demonstrated that the primary cilium coordinates platelet-derived growth factor (PDGF) receptor (PDGFR) alpha-mediated migration in growth-arrested fibroblasts. In this study, we investigate the functional relationship between ciliary PDGFR-alpha and the Na(+)/H(+) exchanger NHE1 in directional cell migration. NHE1 messenger RNA and protein levels are up-regulated in NIH3T3 cells and mouse embryonic fibroblasts (MEFs) during growth arrest, which is concomitant with cilium formation. NHE1 up-regulation is unaffected in Tg737(orpk) MEFs, which have no or very short primary cilia. In growth-arrested NIH3T3 cells, NHE1 is activated by the specific PDGFR-alpha ligand PDGF-AA. In wound-healing assays on growth-arrested NIH3T3 cells and wild-type MEFs, NHE1 inhibition by 5'-(N-ethyl-N-isopropyl) amiloride potently reduces PDGF-AA-mediated directional migration. These effects are strongly attenuated in interphase NIH3T3 cells, which are devoid of primary cilia, and in Tg737(orpk) MEFs. PDGF-AA failed to stimulate migration in NHE1-null fibroblasts. In conclusion, stimulation of directional migration in response to ciliary PDGFR-alpha signals is specifically dependent on NHE1 activity, indicating that NHE1 activation is a critical event in the physiological response to PDGFR-alpha stimulation.