RESUMO
Ciliopathies are characterized by the absence or dysfunction of primary cilia. Despite the fact that cognitive impairments are a common feature of ciliopathies, how cilia dysfunction affects neuronal development has not been characterized in detail. Here, we show that primary cilium-mediated signaling is required cell-autonomously by neurons during neural circuit formation. In particular, a functional primary cilium is crucial during axonal pathfinding for the switch in responsiveness of axons at a choice point or intermediate target. Using different animal models and in vivo, ex vivo and in vitro experiments, we provide evidence for a crucial role of primary cilium-mediated signaling in long-range axon guidance. The primary cilium on the cell body of commissural neurons transduces long-range guidance signals sensed by growth cones navigating an intermediate target. In extension of our finding that Shh is required for the rostral turn of post-crossing commissural axons, we suggest a model implicating the primary cilium in Shh signaling upstream of a transcriptional change of axon guidance receptors, which in turn mediate the repulsive response to floorplate-derived Shh shown by post-crossing commissural axons.
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Cerebral ischemia/reperfusion (CIRI) is a leading cause of death worldwide. A small GTPase known as ADP-ribosylation factor-like protein 13B (ARL13B) is essential in several illnesses. The role of ARL13B in CIRI remains unknown, though. A middle cerebral artery occlusion/reperfusion (MCAO/R) in rats as well as an oxygen-glucose deprivation/reoxygenation (OGD/R) models in PC12 cells were constructed. The neuroprotective effects of ARL13B against MCAO/R were evaluated using neurological scores, TTC staining, rotarod testing, H&E staining, and Nissl staining. To detect the expression of proteins associated with the SHH pathway and apoptosis, western blotting and immunofluorescence were employed. Apoptosis was detected using TUNEL assays and flow cytometry. There was increased expression of ARL13B in cerebral ischemia/reperfusion models. However, ARL13B knockdown aggravated CIRI nerve injury by inhibiting the sonic hedgehog (SHH) pathway. In addition, the use of SHH pathway agonist (SAG) can increased ARL13B expression, reverse the effects of ARL13B knockdown exacerbating CIRI nerve injury. ARL13B alleviated cerebral infarction and pathological injury and played a protective role against MCAO/R. Furthermore, ARL13B significantly increased the expression of SHH pathway-related proteins and the anti-apoptotic protein BCL-2, while decreased the expression of pro-apoptotic protein BAX, thus reducing apoptosis. The results from the OGD/R model in PC12 cells were consistent with those obtained in vivo. Surprisingly, we demonstrated that ARL13B regulates the cell cycle to protect against CIRI nerve injury. Our findings indicate that ARL13B protects against CIRI by reducing apoptosis through SHH-dependent pathway activation, and suggest that ARL13B plays a crucial role in CIRI pathogenesis.
Assuntos
Fatores de Ribosilação do ADP , Proteínas Hedgehog , Traumatismo por Reperfusão , Animais , Masculino , Ratos , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Apoptose/fisiologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Proteínas Hedgehog/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Fármacos Neuroprotetores/farmacologia , Células PC12 , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/patologia , Transdução de Sinais/fisiologiaRESUMO
Tumor Treating Fields (TTFields) extend the survival of glioblastoma (GBM) patients by interfering with a broad range of tumor cellular processes. Among these, TTFields disrupt primary cilia stability on GBM cells. Here we asked if concomitant treatment of TTFields with other agents that interfere with GBM ciliogenesis further suppress GBM cell proliferation in vitro. Aurora kinase A (AURKA) promotes both cilia disassembly and GBM growth. Inhibitors of AURKA, such as Alisertib, inhibit cilia disassembly and increase ciliary frequency in various cell types. However, we found that Alisertib treatment significantly reduced GBM cilia frequency in gliomaspheres across multiple patient derived cell lines, and in patient biopsies treated ex vivo. This effect appeared glioma cell-specific as it did not reduce normal neuronal or glial cilia frequencies. Alisertib-mediated depletion of glioma cilia appears specific to AURKA and not AURKB inhibition, and attributable in part to autophagy pathway activation. Treatment of two different GBM patient-derived cell lines with TTFields and Alisertib resulted in a significant reduction in cell proliferation compared to either treatment alone. However, this effect was not cilia-dependent as the combined treatment reduced proliferation in cilia-depleted cell lines lacking, ARL13B, or U87MG cells which are naturally devoid of ARL13B+ cilia. Thus, Alisertib-mediated effects on glioma cilia may be a useful biomarker of drug efficacy within tumor tissue. Considering Alisertib can cross the blood brain barrier and inhibit intracranial growth, our data warrant future studies to explore whether concomitant Alisertib and TTFields exposure prolongs survival of brain tumor-bearing animals in vivo.
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Primary cilia are hair-like structures found on nearly all mammalian cell types, including cells in the developing and adult brain. A diverse set of receptors and signaling proteins localize within cilia to regulate many physiological and developmental pathways, including the Hedgehog (Hh) pathway. Defects in cilia structure, protein localization, and function lead to genetic disorders called ciliopathies, which present with various clinical features that include several neurodevelopmental phenotypes and hyperphagia-associated obesity. Despite their dysfunction being implicated in several disease states, understanding their roles in central nervous system (CNS) development and signaling has proven challenging. We hypothesize that dynamic changes to ciliary protein composition contribute to this challenge and may reflect unrecognized diversity of CNS cilia. The proteins ARL13B and ADCY3 are established markers of cilia in the brain. ARL13B is a regulatory GTPase important for regulating cilia structure, protein trafficking, and Hh signaling, and ADCY3 is a ciliary adenylyl cyclase. Here, we examine the ciliary localization of ARL13B and ADCY3 in the perinatal and adult mouse brain. We define changes in the proportion of cilia enriched for ARL13B and ADCY3 depending on brain region and age. Furthermore, we identify distinct lengths of cilia within specific brain regions of male and female mice. ARL13B+ cilia become relatively rare with age in many brain regions, including the hypothalamic feeding centers, while ADCY3 becomes a prominent cilia marker in the mature adult brain. It is important to understand the endogenous localization patterns of these proteins throughout development and under different physiological conditions as these common cilia markers may be more dynamic than initially expected. Understanding regional- and developmental-associated cilia protein composition signatures and physiological condition cilia dynamic changes in the CNS may reveal the molecular mechanisms associated with the features commonly observed in ciliopathy models and ciliopathies, like obesity and diabetes.
Assuntos
Ciliopatias , Proteínas Hedgehog , Animais , Feminino , Masculino , Camundongos , Fatores de Ribosilação do ADP/metabolismo , Encéfalo/metabolismo , Proteínas Hedgehog/metabolismo , Mamíferos/metabolismo , ObesidadeRESUMO
Primary cilia are microtubule-based sensory organelles that project from the apical surface of most mammalian cells, including oligodendrocytes, which are myelinating cells of the central nervous system (CNS) that support critical axonal function. Dysfunction of CNS glia is associated with aging-related white matter diseases and neurodegeneration, and ciliopathies are known to affect CNS white matter. To investigate age-related changes in ciliary profile, we examined ciliary length and frequency in the retinogeniculate pathway, a white matter tract commonly affected by diseases of aging but in which expression of cilia has not been characterized. We found expression of Arl13b, a marker of primary cilia, in a small group of Olig2-positive oligodendrocytes in the optic nerve, optic chiasm, and optic tract in young and aged C57BL/6 wild-type mice. While the ciliary length and ciliated oligodendrocyte cells were constant in young mice in the retinogeniculate pathway, there was a significant increase in ciliary length in the anterior optic nerve as compared to the aged animals. Morphometric analysis confirmed a specific increase in the ciliation rate of CC1+ /Olig2+ oligodendrocytes in aged mice compared with young mice. Thus, the prevalence of primary cilia in oligodendrocytes in the visual pathway and the age-related changes in ciliation suggest that they may play important roles in white matter and age-associated optic neuropathies.
Assuntos
Nervo Óptico , Substância Branca , Animais , Camundongos , Camundongos Endogâmicos C57BL , Oligodendroglia , Neuroglia , MamíferosRESUMO
Joubert syndrome (JBTS) is a systematic developmental disorder mainly characterized by a pathognomonic mid-hindbrain malformation. All known JBTS-associated genes encode proteins involved in the function of antenna-like cellular organelle, primary cilium, which plays essential roles in cellular signal transduction and development. Here, we identified four unreported variants in ARL13B in two patients with the classical features of JBTS. ARL13B is a member of the Ras GTPase family and functions in ciliogenesis and cilia-related signaling. The two missense variants in ARL13B harbored the substitutions of amino acids at evolutionarily conserved positions. Using model cell lines, we found that the accumulations of the missense variants in cilia were impaired and the variants showed attenuated functions in ciliogenesis or the trafficking of INPP5E. Overall, these findings expanded the ARL13B pathogenetic variant spectrum of JBTS.
Assuntos
Fatores de Ribosilação do ADP , Anormalidades Múltiplas , Cerebelo , Anormalidades do Olho , Doenças Renais Císticas , Retina , Humanos , Anormalidades Múltiplas/genética , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Cerebelo/anormalidades , Cílios/genética , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Anormalidades do Olho/patologia , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Doenças Renais Císticas/patologia , Monoéster Fosfórico Hidrolases/metabolismo , Retina/metabolismo , Retina/anormalidades , Masculino , Feminino , LactenteRESUMO
A murine osmotic demyelinating syndrome (ODS) model was developed through chronic hyponatremia, induced by desmopressin subcutaneous implants, followed by precipitous sodium restoration. The thalamic ventral posterolateral (VPL) and ventral posteromedial (VPM) relay nuclei were the most demyelinated regions where neuroglial damage could be evidenced without immune response. This report showed that following chronic hyponatremia, 12 h and 48 h time lapses after rebalancing osmolarity, amid the ODS-degraded outskirts, some resilient neuronal cell bodies built up primary cilium and axon hillock regions that extended into axon initial segments (AIS) where ADP-ribosylation factor-like protein 13B (ARL13B)-immunolabeled rod-like shape content was revealed. These AIS-labeled shaft lengths appeared proportional with the distance of neuronal cell bodies away from the ODS damaged epicenter and time lapses after correction of hyponatremia. Fine structure examination verified these neuron abundant transcriptions and translation regions marked by the ARL13B labeling associated with cell neurotubules and their complex cytoskeletal macromolecular architecture. This necessitated energetic transport to organize and restore those AIS away from the damaged ODS core demyelinated zone in the murine model. These labeled structures could substantiate how thalamic neuron resilience occurred as possible steps of a healing course out of ODS.
Assuntos
Segmento Inicial do Axônio , Doenças Desmielinizantes , Hiponatremia , Animais , Camundongos , Fatores de Ribosilação do ADP/metabolismo , Cílios/metabolismo , Neurônios/metabolismo , Doenças Desmielinizantes/metabolismoRESUMO
Cilia are near ubiquitous small, cellular appendages critical for cell-to-cell communication. As such, they are involved in diverse developmental and homeostatic processes, including energy homeostasis. ARL13B is a regulatory GTPase highly enriched in cilia. Mice expressing an engineered ARL13B variant, ARL13BV358A which retains normal biochemical activity, display no detectable ciliary ARL13B. Surprisingly, these mice become obese. Here, we measured body weight, food intake, and blood glucose levels to reveal these mice display hyperphagia and metabolic defects. We showed that ARL13B normally localizes to cilia of neurons in specific brain regions and pancreatic cells but is excluded from these cilia in the Arl13bV358A/V358A model. In addition to its GTPase function, ARL13B acts as a guanine nucleotide exchange factor (GEF) for ARL3. To test whether ARL13B's GEF activity is required to regulate body weight, we analyzed the body weight of mice expressing ARL13BR79Q, a variant that lacks ARL13B GEF activity for ARL3. We found no difference in body weight. Taken together, our results show that ARL13B functions within cilia to control body weight and that this function does not depend on its role as a GEF for ARL3. Controlling the subcellular localization of ARL13B in the engineered mouse model, ARL13BV358A, enables us to define the cilia-specific role of ARL13B in regulating energy homeostasis.
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Photoreceptors are highly polarized sensory neurons. Precise localization of signaling molecules within the ciliary outer segment is critical for photoreceptor function and viability. The small GTPase Arl3 plays a particularly important role in photoreceptors as it regulates outer segment enrichment of lipidated proteins essential for the visual response: transducin-α, transducin-γ, PDEα, PDE ß, and Grk1. Recently, mutations in Arl3 have been identified in human patients with nonsyndromic autosomal recessive and dominant inherited retinal degenerations as well as syndromic Joubert syndrome including retinal dystrophy.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Distrofias Retinianas , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transducina/metabolismo , Cílios/genética , Cílios/metabolismo , Fatores de Ribosilação do ADP/genética , Distrofias Retinianas/genética , MutaçãoRESUMO
ARL13B is a small GTPase enriched in cilia. Deletion of Arl13b in mouse kidney results in renal cysts and an associated absence of primary cilia. Similarly, ablation of cilia leads to kidney cysts. To investigate whether ARL13B functions from within cilia to direct kidney development, we examined kidneys of mice expressing an engineered cilia-excluded ARL13B variant, ARL13BV358A. These mice retained renal cilia and developed cystic kidneys. Because ARL13B functions as a guanine nucleotide exchange factor (GEF) for ARL3, we examined kidneys of mice expressing an ARL13B variant that lacks ARL3 GEF activity, ARL13BR79Q. We found normal kidney development with no evidence of cysts in these mice. Taken together, our results show that ARL13B functions within cilia to inhibit renal cystogenesis during mouse development, and that this function does not depend on its role as a GEF for ARL3.
Assuntos
Doenças Renais Císticas , Rim , Animais , Camundongos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Cílios/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Rim/metabolismo , Doenças Renais Císticas/genéticaRESUMO
Primary cilia are microtubule-based organelles presenting on the surface of most postmitotic mammalian cells. As being signaling hubs and sensory organelles, primary cilia can respond to mechanical and chemical stimuli from the extracellular environment. Arl13b (ADP-ribosylation factor-like 13B), an atypical Arf/Arl family GTPase, was identified in genetic screening as a protein essential for maintaining the integrity of cilia and neural tubes. Previous studies on Arl13b have mostly focused on its role in the development of neural tubes, polycystic kidneys, and tumors, but no role in bone patterns was described. This study reported the essential roles of Arl13b in bone formation and osteogenic differentiation. Arl13b was highly expressed in bone tissues and osteoblasts, positively correlated with osteogenic activity during bone development. Furthermore, Arl13b was essential for primary cilium maintenance and Hedgehog signaling activation in osteoblasts. Arl13b knockdown in osteoblasts decreased the length of primary cilia and the upregulated levels of Gli1, Smo, and Ptch1 upon Smo agonist treatment. Additionally, Arl13b knockdown inhibited cell proliferation and migration. Moreover, Arl13b mediated osteogenesis and cell mechanosensation. Cyclic tension strain upregulated the Arl13b expression. Arl13b knockdown suppressed osteogenesis and mitigated cyclic tension strain-induced osteogenesis. These results suggest that Arl13b have important roles in bone formation and mechanosensation.
Assuntos
Proteínas Hedgehog , Osteogênese , Animais , Osso e Ossos/metabolismo , Cílios/genética , Cílios/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Osteoblastos/metabolismo , Osteogênese/genética , Transdução de Sinais/genética , CamundongosRESUMO
Arl13b, an ARF/Arl-family GTPase, is highly enriched in the cilium. Recent studies have established Arl13b as one of the most crucial regulators for ciliary organization, trafficking, and signaling. The ciliary localization of Arl13b is known to require the RVEP motif. However, its cognate ciliary transport adaptor has been elusive. Here, by imaging the ciliary localization of truncation and point mutations, we defined the ciliary targeting sequence (CTS) of Arl13b as a C-terminal stretch of 17 amino acids containing the RVEP motif. We found Rab8-GDP, but not Rab8-GTP, and TNPO1 simultaneously and directly bind to the CTS of Arl13b in pull-down assays using cell lysates or purified recombinant proteins. Furthermore, Rab8-GDP substantially enhances the interaction between TNPO1 and CTS. Additionally, we determined that the RVEP motif is an essential element as its mutation abolishes the interaction of the CTS with Rab8-GDP and TNPO1 in pull-down and TurboID-based proximity ligation assays. Finally, the knockdown of endogenous Rab8 or TNPO1 decreases the ciliary localization of endogenous Arl13b. Therefore, our results suggest Rab8 and TNPO1 might function together as a ciliary transport adaptor for Arl13b by interacting with its RVEP-containing CTS.
Assuntos
Cílios , GTP Fosfo-Hidrolases , Fatores de Ribosilação do ADP/metabolismo , Cílios/genética , Cílios/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Transdução de Sinais , Humanos , Linhagem CelularRESUMO
Primary cilia provide a specialized subcellular environment favoring ordered and timely interaction and modification of signaling molecules, necessary for the sensing and transduction of extracellular signals and environmental conditions. Crucial to the understanding of ciliary function is the knowledge of the signaling molecules composing the ciliary compartment. While proteomes of primary cilia have been published recently, the selective isolation of primary cilia from specific cell types and whole tissue still proves difficult, and many laboratories instead resort to the analysis of cultured cells, which may introduce experimental artifacts. Here we present a flow cytometry-based method to isolate and characterize primary cilia from the murine ventricular-subventricular zone. After deciliation, primary cilia are immunolabeled with antibodies against ciliary markers. As an example, we here use a double-staining with acetylated tubulin, which stains the ciliary axoneme, and ciliary membrane protein ADP-ribosylation-like factor 13b (Arl13b); additionally, we triple-labeled primary cilia using the ciliary marker adenylate cyclase 3 (AC3). Besides analysis at the single particle level, fluorescence activated cell sorting (FACS) allows collection of pure preparations of primary cilia suited for subsequent proteomic analyses like mass spectrometry or western blot. As an example of analytical application, we performed triple immunostaining and FACS analysis to reveal cilia heterogeneity. Thus, our cilia isolation method, which can readily be applied to other tissues or cell culture, will facilitate the study of this key cellular organelle and shed light on its role in normal conditions and disease.
Assuntos
Cílios , Proteômica , Animais , Camundongos , Cílios/metabolismo , Citometria de Fluxo , Tubulina (Proteína)/metabolismo , Transporte ProteicoRESUMO
ARL13B is a small GTPase enriched in cilia. Deletion of Arl13b in mouse kidney results in renal cysts and an associated absence of primary cilia. Similarly, ablation of cilia leads to kidney cysts. To investigate whether ARL13B functions from within cilia to direct kidney development, we examined kidneys of mice expressing an engineered cilia-excluded ARL13B variant, ARL13BV358A. These mice retained renal cilia and developed cystic kidneys. Because ARL13B functions as a guanine nucleotide exchange factor (GEF) for ARL3, we examined kidneys of mice expressing an ARL13B variant that lacks ARL3 GEF activity, ARL13BR79Q. We found normal kidney development with no evidence of cysts in these mice. Taken together, our results show that ARL13B functions within cilia to inhibit renal cystogenesis during mouse development, and that this function does not depend on its role as a GEF for ARL3.
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Olfactory sensory neurons (OSNs) form embryonically and mature perinatally, innervating glomeruli and extending dendrites with multiple cilia. This process and its timing are crucial for odor detection and perception and continues throughout life. In the olfactory epithelium (OE), differentiated OSNs proceed from an immature (iOSN) to a mature (mOSN) state through well-defined sequential morphological and molecular transitions, but the precise mechanisms controlling OSN maturation remain largely unknown. We have identified that a GTPase, ARL13B, has a transient and maturation state-dependent expression in OSNs marking the emergence of a primary cilium. Utilizing an iOSN-specific Arl13b-null murine model, we examined the role of ARL13B in the maturation of OSNs. The loss of Arl13b in iOSNs caused a profound dysregulation of the cellular homeostasis and development of the OE. Importantly, Arl13b null OSNs demonstrated a delay in the timing of their maturation. Finally, the loss of Arl13b resulted in severe deformation in the structure and innervation of glomeruli. Our findings demonstrate a previously unknown role of ARL13B in the maturation of OSNs and development of the OE.
Assuntos
Fatores de Ribosilação do ADP , GTP Fosfo-Hidrolases , Neurônios Receptores Olfatórios , Animais , Camundongos , Cílios , Neurogênese , Mucosa Olfatória , Fatores de Ribosilação do ADP/genéticaRESUMO
The small GTPase Arl3 is important for the enrichment of lipidated proteins to primary cilia, including the outer segment of photoreceptors. Human mutations in the small GTPase Arl3 cause both autosomal recessive and dominant inherited retinal dystrophies. We discovered that dominant mutations result in increased active G-protein-Arl3-D67V has constitutive activity and Arl3-Y90C is fast cycling-and their expression in mouse rods resulted in a displaced nuclear phenotype due to an aberrant Arl3-GTP gradient. Using multiple strategies, we go on to show that removing or restoring the Arl3-GTP gradient within the cilium is sufficient to rescue the nuclear migration defect. Together, our results reveal that an Arl3 ciliary gradient is involved in proper positioning of photoreceptor nuclei during retinal development.
Assuntos
Fatores de Ribosilação do ADP , Proteínas de Membrana , Células Fotorreceptoras Retinianas Bastonetes , Animais , Humanos , Camundongos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Cílios/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico , Células Fotorreceptoras Retinianas Bastonetes/metabolismoRESUMO
BACKGROUND: Tumor angiogenesis is essential for solid tumor progression, invasion and metastasis. The aim of this study was to identify potential signaling pathways involved in tumor angiogenesis. METHODS: Genetically engineered mouse models were used to investigate the effects of endothelial ARL13B(ADP-ribosylation factor-like GTPase 13B) over-expression and deficiency on retinal and cerebral vasculature. An intracranially transplanted glioma model and a subcutaneously implanted melanoma model were employed to examine the effects of ARL13B on tumor growth and angiogenesis. Immunohistochemistry was used to measure ARL13B in glioma tissues, and scRNA-seq was used to analyze glioma and endothelial ARL13B expression. GST-fusion protein-protein interaction and co-immunoprecipitation assays were used to determine the ARL13B-VEGFR2 interaction. Immunobloting, qPCR, dual-luciferase reporter assay and functional experiments were performed to evaluate the effects of ARL13B on VEGFR2 activation. RESULTS: Endothelial ARL13B regulated vascular development of both the retina and brain in mice. Also, ARL13B in endothelial cells regulated the growth of intracranially transplanted glioma cells and subcutaneously implanted melanoma cells by controlling tumor angiogenesis. Interestingly, this effect was attributed to ARL13B interaction with VEGFR2, through which ARL13B regulated the membrane and ciliary localization of VEGFR2 and consequently activated its downstream signaling in endothelial cells. Consistent with its oncogenic role, ARL13B was highly expressed in human gliomas, which was well correlated with the poor prognosis of glioma patients. Remarkably, ARL13B, transcriptionally regulated by ZEB1, enhanced the expression of VEGFA by activating Hedgehog signaling in glioma cells. CONCLUSIONS: ARL13B promotes angiogenesis and tumor growth by activating VEGFA-VEGFR2 signaling. Thus, targeting ARL13B might serve as a potential approach for developing an anti-glioma or anti-melanoma therapy.
Assuntos
Células Endoteliais , Glioma , Humanos , Camundongos , Animais , Células Endoteliais/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Glioma/patologia , Neovascularização Patológica/metabolismo , Proliferação de Células , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/farmacologiaRESUMO
Primary cilia transduce signals via transmembrane and membrane-associated proteins localized to the ciliary membrane in vertebrate cells. In humans, transmembrane protein 67 (TMEM67), a component of the multiprotein complex functioning as a gatekeeper at the transition zone (TZ) of primary cilia, is mutated in patients suffering from cilia-related pleiotropic diseases, collectively referred to as ciliopathies. The requirement of TMEM67 for the gating function of the TZ that delivers membrane proteins into the ciliary compartment has not been determined. In this study, we established hTERT-RPE1 cells with knockout (KO) of TMEM67 and examined whether cilium formation and TZ gating are affected by its ablation. TMEM67-KO cells displayed impaired ciliogenesis, elongated cilia, perturbed ciliary localization of membrane-associated proteins ARL13B and INPP5E but normal recruitment of TZ proteins CEP290, RPGRIP1L and NPHP5. The exogenous expression of ciliopathy-associated TMEM67 mutants restored ciliary localization of ARL13B and INPP5E but failed to attenuate aberrant cilium elongation in TMEM67-KO cells. Furthermore, we found that TMEM67 localization is not confined to the TZ but extends into the cilium. Our findings indicate that TMEM67 is required not only for ciliogenesis and cilium length regulation but also for the gating function of the TZ independently of RPGRIP1L/CEP290/NPHP5 recruitment to this region. They further suggest that aberrant cilium elongation underlies the pathogenesis of TMEM67-linked ciliopathies.
Assuntos
Cílios , Ciliopatias , Humanos , Cílios/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Ciliopatias/genética , Ciliopatias/metabolismo , Antígenos de Neoplasias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fatores de Ribosilação do ADP/metabolismoRESUMO
The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119-dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119-dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior-Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse.
Assuntos
Ciliopatias , Sinapses , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ciliopatias/metabolismo , Proteínas Correpressoras/metabolismo , Humanos , Fosfoproteínas/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sinapses/metabolismoRESUMO
Loss of retinal ganglion cells (RGCs) underlies several forms of retinal disease including glaucomatous optic neuropathy, a leading cause of irreversible blindness. Several rare genetic disorders associated with cilia dysfunction have retinal degeneration as a clinical hallmark. Much of the focus of ciliopathy associated blindness is on the connecting cilium of photoreceptors; however, RGCs also possess primary cilia. It is unclear what roles RGC cilia play, what proteins and signaling machinery localize to RGC cilia, or how RGC cilia are differentiated across the subtypes of RGCs. To better understand these questions, we assessed the presence or absence of a prototypical cilia marker Arl13b and a widely distributed neuronal cilia marker AC3 in different subtypes of mouse RGCs. Interestingly, not all RGC subtype cilia are the same and there are significant differences even among these standard cilia markers. Alpha-RGCs positive for osteopontin, calretinin, and SMI32 primarily possess AC3-positive cilia. Directionally selective RGCs that are CART positive or Trhr positive localize either Arl13b or AC3, respectively, in cilia. Intrinsically photosensitive RGCs differentially localize Arl13b and AC3 based on melanopsin expression. Taken together, we characterized the localization of gold standard cilia markers in different subtypes of RGCs and conclude that cilia within RGC subtypes may be differentially organized. Future studies aimed at understanding RGC cilia function will require a fundamental ability to observe the cilia across subtypes as their signaling protein composition is elucidated. A comprehensive understanding of RGC cilia may reveal opportunities to understanding how their dysfunction leads to retinal degeneration.