RESUMO
BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.
Assuntos
Cisteína , Proteínas Proto-Oncogênicas B-raf , Humanos , Cisteína/genética , Proteínas Proto-Oncogênicas B-raf/genética , Domínios Proteicos , Mutação , SíndromeRESUMO
Motile cilia on the cell surface generate movement and directional fluid flow that is crucial for various biological processes. Dysfunction of these cilia causes human diseases such as sinopulmonary disease and infertility. Here, we show that Ccdc108, a protein linked to male infertility, has an evolutionarily conserved requirement in motile multiciliation. Using Xenopus laevis embryos, Ccdc108 is shown to be required for the migration and docking of basal bodies to the apical membrane in epidermal multiciliated cells (MCCs). We demonstrate that Ccdc108 interacts with the IFT-B complex, and the ciliation requirement for Ift74 overlaps with Ccdc108 in MCCs. Both Ccdc108 and IFT-B proteins localize to migrating centrioles, basal bodies, and cilia in MCCs. Importantly, Ccdc108 governs the centriolar recruitment of IFT while IFT licenses the targeting of Ccdc108 to the cilium. Moreover, Ccdc108 is required for the centriolar recruitment of Drg1 and activated RhoA, factors that help establish the apical actin network in MCCs. Together, our studies indicate that Ccdc108 and IFT-B complex components cooperate in multiciliogenesis.
Assuntos
Corpos Basais , Infertilidade Masculina , Proteínas de Membrana , Proteínas de Ligação a RNA , Animais , Corpos Basais/metabolismo , Centríolos/metabolismo , Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Humanos , Infertilidade Masculina/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , Xenopus laevisRESUMO
The deubiquitinating enzyme USP37 is known to contribute to timely onset of S phase and progression of mitosis. However, it is not clear if USP37 is required beyond S-phase entry despite expression and activity of USP37 peaking within S phase. We have utilized flow cytometry and microscopy to analyze populations of replicating cells labeled with thymidine analogs and monitored mitotic entry in synchronized cells to determine that USP37-depleted cells exhibited altered S-phase kinetics. Further analysis revealed that cells depleted of USP37 harbored increased levels of the replication stress and DNA damage markers γH2AX and 53BP1 in response to perturbed replication. Depletion of USP37 also reduced cellular proliferation and led to increased sensitivity to agents that induce replication stress. Underlying the increased sensitivity, we found that the checkpoint kinase 1 is destabilized in the absence of USP37, attenuating its function. We further demonstrated that USP37 deubiquitinates checkpoint kinase 1, promoting its stability. Together, our results establish that USP37 is required beyond S-phase entry to promote the efficiency and fidelity of replication. These data further define the role of USP37 in the regulation of cell proliferation and contribute to an evolving understanding of USP37 as a multifaceted regulator of genome stability.
Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Endopeptidases/metabolismo , Fase S , Quinase 1 do Ponto de Checagem/genética , Dano ao DNA , Replicação do DNA , Endopeptidases/genética , Estabilidade Enzimática , Instabilidade Genômica , Células HCT116 , Células HeLa , Histonas , Humanos , Células MCF-7 , UbiquitinaçãoRESUMO
Collective cell migration is required for normal embryonic development and contributes to various biological processes, including wound healing and cancer cell invasion. The M-Ras GTPase and its effector, the Shoc2 scaffold, are proteins mutated in the developmental RASopathy Noonan syndrome, and, here, we report that activated M-Ras recruits Shoc2 to cell surface junctions where M-Ras/Shoc2 signaling contributes to the dynamic regulation of cell-cell junction turnover required for collective cell migration. MCF10A cells expressing the dominant-inhibitory M-RasS27N variant or those lacking Shoc2 exhibited reduced junction turnover and were unable to migrate effectively as a group. Through further depletion/reconstitution studies, we found that M-Ras/Shoc2 signaling contributes to junction turnover by modulating the E-cadherin/p120-catenin interaction and, in turn, the junctional expression of E-cadherin. The regulatory effect of the M-Ras/Shoc2 complex was mediated at least in part through the phosphoregulation of p120-catenin and required downstream ERK cascade activation. Strikingly, cells rescued with the Noonan-associated, myristoylated-Shoc2 mutant (Myr-Shoc2) displayed a gain-of-function (GOF) phenotype, with the cells exhibiting increased junction turnover and reduced E-cadherin/p120-catenin binding and migrating as a faster but less cohesive group. Consistent with these results, Noonan-associated C-Raf mutants that bypass the need for M-Ras/Shoc2 signaling exhibited a similar GOF phenotype when expressed in Shoc2-depleted MCF10A cells. Finally, expression of the Noonan-associated Myr-Shoc2 or C-Raf mutants, but not their WT counterparts, induced gastrulation defects indicative of aberrant cell migration in zebrafish embryos, further demonstrating the function of the M-Ras/Shoc2/ERK cascade signaling axis in the dynamic control of coordinated cell movement.
Assuntos
Adesão Celular/genética , Movimento Celular/genética , Desenvolvimento Embrionário/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Animais , Caderinas/genética , Mutação com Ganho de Função/genética , Gastrulação/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Síndrome de Noonan/genética , Síndrome de Noonan/fisiopatologia , Ligação Proteica , Peixe-Zebra/genéticaRESUMO
The primary cilium is a cellular sensor that detects light, chemicals, and movement and is important for morphogen and growth factor signaling. The small GTPase Rab11-Rab8 cascade is required for ciliogenesis. Rab11 traffics the guanine nucleotide exchange factor (GEF) Rabin8 to the centrosome to activate Rab8, needed for ciliary growth. Rabin8 also requires the transport particle protein complex (TRAPPC) proteins for centrosome recruitment during ciliogenesis. Here, using an MS-based approach for identifying Rabin8-interacting proteins, we identified C7orf43 (also known as microtubule-associated protein 11 (MAP11)) as being required for ciliation both in human cells and zebrafish embryos. We find that C7orf43 directly binds to Rabin8 and that C7orf43 knockdown diminishes Rabin8 preciliary centrosome accumulation. Interestingly, we found that C7orf43 co-sediments with TRAPPII complex subunits and directly interacts with TRAPPC proteins. Our findings establish that C7orf43 is a TRAPPII-specific complex component, referred to here as TRAPPC14. Additionally, we show that TRAPPC14 is dispensable for TRAPPII complex integrity but mediates Rabin8 association with the TRAPPII complex. Finally, we demonstrate that TRAPPC14 interacts with the distal appendage proteins Fas-binding factor 1 (FBF1) and centrosomal protein 83 (CEP83), which we show here are required for GFP-Rabin8 centrosomal accumulation, supporting a role for the TRAPPII complex in tethering preciliary vesicles to the mother centriole during ciliogenesis. In summary, our findings have revealed an uncharacterized TRAPPII-specific component, C7orf43/TRAPPC14, that regulates preciliary trafficking of Rabin8 and ciliogenesis and support previous findings that the TRAPPII complex functions as a membrane tether.
Assuntos
Centríolos/metabolismo , Cílios/metabolismo , Vesículas Citoplasmáticas/metabolismo , Quinases do Centro Germinativo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Centríolos/genética , Cílios/genética , Vesículas Citoplasmáticas/genética , Quinases do Centro Germinativo/genética , Humanos , Proteínas Associadas aos Microtúbulos/genética , Morfogênese , Ligação Proteica , Peixe-ZebraRESUMO
BACKGROUND: KIF17, a kinesin-2 motor that functions in intraflagellar transport, can regulate the onset of photoreceptor outer segment development. However, the function of KIF17 in a mature photoreceptor remains unclear. Additionally, the ciliary localization of KIF17 is regulated by a C-terminal consensus sequence (KRKK) that is immediately adjacent to a conserved residue (mouse S1029/zebrafish S815) previously shown to be phosphorylated by CaMKII. Yet, whether this phosphorylation can regulate the localization, and thus function, of KIF17 in ciliary photoreceptors remains unknown. RESULTS: Using transgenic expression in zebrafish photoreceptors, we show that phospho-mimetic KIF17 has enhanced localization along the cone outer segment. Importantly, expression of phospho-mimetic KIF17 is associated with greatly enhanced turnover of the photoreceptor outer segment through disc shedding in a cell-autonomous manner, while genetic mutants of kif17 in zebrafish and mice have diminished disc shedding. Lastly, cone expression of constitutively active tCaMKII leads to a kif17-dependent increase in disc shedding. CONCLUSIONS: Taken together, our data support a model in which phosphorylation of KIF17 promotes its photoreceptor outer segment localization and disc shedding, a process essential for photoreceptor maintenance and homeostasis. While disc shedding has been predominantly studied in the context of the mechanisms underlying phagocytosis of outer segments by the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding.
Assuntos
Cinesinas/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Cílios/metabolismo , Humanos , Cinesinas/química , Camundongos Endogâmicos C57BL , Mutação/genética , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Fosforilação , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Proteínas de Peixe-Zebra/químicaRESUMO
WDR44 prevents ciliogenesis initiation by regulating RAB11-dependent vesicle trafficking. Here, we describe male patients with missense and nonsense variants within the WD40 repeats (WDR) of WDR44, an X-linked gene product, who display ciliopathy-related developmental phenotypes that we can model in zebrafish. The patient phenotypic spectrum includes developmental delay/intellectual disability, hypotonia, distinct craniofacial features and variable presence of brain, renal, cardiac and musculoskeletal abnormalities. We demonstrate that WDR44 variants associated with more severe disease impair ciliogenesis initiation and ciliary signaling. Because WDR44 negatively regulates ciliogenesis, it was surprising that pathogenic missense variants showed reduced abundance, which we link to misfolding of WDR autonomous repeats and degradation by the proteasome. We discover that disease severity correlates with increased RAB11 binding, which we propose drives ciliogenesis initiation dysregulation. Finally, we discover interdomain interactions between the WDR and NH2-terminal region that contains the RAB11 binding domain (RBD) and show patient variants disrupt this association. This study provides new insights into WDR44 WDR structure and characterizes a new syndrome that could result from impaired ciliogenesis.
Assuntos
Ciliopatias , Genes Ligados ao Cromossomo X , Repetições WD40 , Animais , Humanos , Masculino , Encéfalo , Ciliopatias/genética , Cognição , Peixe-Zebra/genéticaRESUMO
In absence of their natural ligand, 11-cis-retinal, cone opsin G-protein-coupled receptors fail to traffic normally, a condition associated with photoreceptor degeneration and blindness. We created a mouse with a point mutation (F81Y) in cone S-opsin. As expected, cones with this knock-in mutation respond to light with maximal sensitivity red-shifted from 360 to 420 nm, consistent with an altered interaction between the apoprotein and ligand, 11-cis-retinal. However, cones expressing F81Y S-opsin showed an â¼3-fold reduced absolute sensitivity that was associated with a corresponding reduction in S-opsin protein expression. The reduced S-opsin expression did not arise from decreased S-opsin mRNA or cone degeneration, but rather from enhanced endoplasmic reticulum (ER)-associated degradation of the nascent protein. Exogenously increased 11-cis-retinal restored F81Y S-opsin protein expression to normal levels, suggesting that ligand binding in the ER facilitates proper folding. Immunohistochemistry and electron microscopy of normal retinas showed that Mueller cells, which synthesize a precursor of 11-cis-retinal, are closely adjoined to the cone ER, so they could deliver the ligand to the site of opsin synthesis. Together, these results suggest that the binding of 11-cis-retinal in the ER is important for normal folding during cone opsin biosynthesis.
Assuntos
Opsinas/biossíntese , Opsinas/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Retinaldeído/fisiologia , Algoritmos , Animais , Animais Geneticamente Modificados , Western Blotting , Fenômenos Eletrofisiológicos , Retículo Endoplasmático/metabolismo , Imunofluorescência , Imuno-Histoquímica , Imunoprecipitação , Luz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Mutação/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismoRESUMO
Intraflagellar transport (IFT) provides a mechanism for the transport of cilium-specific proteins, but the mechanisms for linkage of cargo and IFT proteins have not been identified. Using the sensory outer segments (OS) of photoreceptors, which are derived from sensory cilia, we have identified IFT-cargo complexes containing IFT proteins, kinesin 2 family proteins, two photoreceptor-specific membrane proteins, guanylyl cyclase 1 (GC1, Gucy2e) and rhodopsin (RHO), and the chaperones, mammalian relative of DNAJ, DnajB6 (MRJ), and HSC70 (Hspa8). Analysis of these complexes leads to a model in which MRJ through its binding to IFT88 and GC1 plays a critical role in formation or stabilization of the IFT-cargo complexes. Consistent with the function of MRJ in the activation of HSC70 ATPase activity, Mg-ATP enhances the co-IP of GC1, RHO, and MRJ with IFT proteins. Furthermore, RNAi knockdown of MRJ in IMCD3 cells expressing GC1-green fluorescent protein (GFP) reduces cilium membrane targeting of GC1-GFP without apparent effect on cilium elongation.
Assuntos
Guanilato Ciclase/metabolismo , Chaperonas Moleculares/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Rodopsina/metabolismo , Sequência de Aminoácidos , Animais , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Técnicas do Sistema de Duplo-HíbridoRESUMO
Kinesin 2 family members are involved in transport along ciliary microtubules. In Caenorhabditis elegans channel cilia, kinesin II and OSM-3 cooperate along microtubule doublets of the axoneme middle segment, whereas OSM-3 alone works on microtubule singlets to elongate the distal segment. Among sensory cilia, vertebrate photoreceptors share a similar axonemal structure with C. elegans channel cilia, and deficiency in either kinesin II or KIF17, the homologue of OSM-3, results in disruption of photoreceptor organization. However, direct comparison of the two effects is confounded by the use of different species and knockdown strategies in prior studies. Here, we directly compare the effects of dominant-negative kinesin II and KIF17 expression in zebrafish cone photoreceptors. Our data indicate that dominant-negative kinesin II disrupts function at the level of the inner segment and synaptic terminal and results in cell death. In contrast, dominant-negative KIF17 has no obvious effect on inner segment or synaptic organization but has an immediate impact on outer segment assembly.
Assuntos
Cinesinas/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/metabolismo , Animais , Western Blotting , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Imuno-Histoquímica , Imunoprecipitação , Cinesinas/genética , Cinesinas/metabolismo , Cinesinas/farmacologia , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/farmacologiaRESUMO
Sensory cilia and intraflagellar transport (IFT), a pathway essential for ciliogenesis, play important roles in embryonic development and cell differentiation. In vertebrate photoreceptors IFT is required for the early development of ciliated sensory outer segments (OS), an elaborate organelle that sequesters the many proteins comprising the phototransduction machinery. As in other cilia and flagella, heterotrimeric members of the kinesin 2 family have been implicated as the anterograde IFT motor in OS. However, in Caenorhabditis elegans, OSM-3, a homodimeric kinesin 2 motor, plays an essential role in some, but not all sensory cilia. Kif17, a vertebrate OSM-3 homologue, is known for its role in dendritic trafficking in neurons, but a function in ciliogenesis has not been determined. We show that in zebrafish Kif17 is widely expressed in the nervous system and retina. In photoreceptors Kif17 co-localizes with IFT proteins within the OS, and co-immunoprecipitates with IFT proteins. Knockdown of Kif17 has little if any effect in early embryogenesis, including the formation of motile sensory cilia in the pronephros. However, OS formation and targeting of the visual pigment protein is severely disrupted. Our analysis shows that Kif17 is essential for photoreceptor OS development, and suggests that Kif17 plays a cell type specific role in vertebrate ciliogenesis.
Assuntos
Cinesinas/metabolismo , Segmento Externo da Célula Bastonete/embriologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Axonema/metabolismo , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Cílios/fisiologia , Cinesinas/antagonistas & inibidores , Cinesinas/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Transporte Proteico , Segmento Externo da Célula Bastonete/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
In the original version of this Article, the fifth sentence of the abstract incorrectly read 'Remarkably, we show that PACSIN1 and EHD1 assemble membrane t7ubules from the developing intracellular cilium that attach to the plasma membrane, creating an extracellular membrane channel (EMC) to the outside of the cell.', and should have read 'Remarkably, we show that PACSIN1 and EHD1 assemble membrane tubules from the developing intracellular cilium that attach to the plasma membrane, creating an extracellular membrane channel (EMC) to the outside of the cell.'. This has been corrected in both the PDF and HTML versions of the Article.
RESUMO
The intracellular ciliogenesis pathway requires membrane trafficking, fusion, and reorganization. Here, we demonstrate in human cells and zebrafish that the F-BAR domain containing proteins PACSIN1 and -2 play an essential role in ciliogenesis, similar to their binding partner and membrane reorganizer EHD1. In mature cilia, PACSINs and EHDs are dynamically localized to the ciliary pocket membrane (CPM) and transported away from this structure on membrane tubules along with proteins that exit the cilium. PACSINs function early in ciliogenesis at the ciliary vesicle (CV) stage to promote mother centriole to basal body transition. Remarkably, we show that PACSIN1 and EHD1 assemble membrane t7ubules from the developing intracellular cilium that attach to the plasma membrane, creating an extracellular membrane channel (EMC) to the outside of the cell. Together, our work uncovers a function for F-BAR proteins and membrane tubulation in ciliogenesis and explains how the intracellular cilium emerges from the cell.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Corpos Basais/metabolismo , Cílios/metabolismo , Células Epiteliais/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Corpos Basais/ultraestrutura , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Centríolos/metabolismo , Centríolos/ultraestrutura , Cílios/ultraestrutura , Embrião não Mamífero , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica , Humanos , Fusão de Membrana , Camundongos , Células NIH 3T3 , Ligação Proteica , Domínios Proteicos , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Peixe-ZebraRESUMO
Serum starvation stimulates cilia growth in cultured cells, yet serum factors associated with ciliogenesis are unknown. Previously, we showed that starvation induces rapid Rab11-dependent vesicular trafficking of Rabin8, a Rab8 guanine-nucleotide exchange factor (GEF), to the mother centriole, leading to Rab8 activation and cilium growth. Here, we demonstrate that through the LPA receptor 1 (LPAR1), serum lysophosphatidic acid (LPA) inhibits Rab11a-Rabin8 interaction and ciliogenesis. LPA/LPAR1 regulates ciliogenesis initiation via downstream PI3K/Akt activation, independent of effects on cell cycle. Akt stabilizes Rab11a binding to its effector, WDR44, and a WDR44-pAkt-phosphomimetic mutant blocks ciliogenesis. WDR44 depletion promotes Rabin8 preciliary trafficking and ciliogenesis-initiating events at the mother centriole. Our work suggests disruption of Akt signaling causes a switch from Rab11-WDR44 to the ciliogenic Rab11-FIP3-Rabin8 complex. Finally, we demonstrate that Akt regulates downstream ciliogenesis processes associated with Rab8-dependent cilia growth. Together, this study uncovers a mechanism whereby serum mitogen signaling regulates Rabin8 preciliary trafficking and ciliogenesis initiation.
Assuntos
Cílios/fisiologia , Regulação da Expressão Gênica , Quinase I-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Humanos , Quinase I-kappa B/genética , Fosfatidilinositol 3-Quinases/genética , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/genética , Peixe-Zebra , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11-Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11-Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby EHD1 and EHD3 reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth.
Assuntos
Proteínas de Transporte/genética , Cílios/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/genética , Animais , Axonema/metabolismo , Axonema/ultraestrutura , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Linhagem Celular , Centríolos/metabolismo , Centríolos/ultraestrutura , Cílios/ultraestrutura , Embrião não Mamífero , Células Epiteliais/ultraestrutura , Humanos , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/ultraestrutura , Camundongos , Morfogênese/genética , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/ultraestrutura , Transdução de Sinais , Vesículas Transportadoras/ultraestrutura , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteínas de Transporte Vesicular/metabolismo , Peixe-Zebra , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: The central tenet of cilia function is sensing and transmitting information. The capacity to directly contact extracellular surfaces would empower primary cilia to probe the environment for information about the nature and location of nearby surfaces. It has been well established that flagella and other motile cilia perform diverse cellular functions through adhesion. We hypothesized that mammalian primary cilia also interact with the extracellular environment through direct physical contact. METHODS: We identified cilia in rod photoreceptors and cholangiocytes in fixed mouse tissues and examined the structures that these cilia contact in vivo. We then utilized an MDCK cell culture model to characterize the nature of the contacts we observed. RESULTS: In retina and liver tissue, we observed that cilia from nearby cells touch one another. Using MDCK cells, we found compelling evidence that these contacts are stable adhesions that form bridges between two cells, or networks between many cells. We examined the nature and duration of the cilia-cilia contacts and discovered primary cilia movements that facilitate cilia-cilia encounters. Stable adhesions form as the area of contact expands from a single point to a stretch of tightly bound, adjacent cilia membranes. The cilia-cilia contacts persisted for hours and were resistant to several harsh treatments such as proteases and DTT. Unlike many other cell adhesion mechanisms, calcium was not required for the formation or maintenance of cilia adhesion. However, swainsonine, which blocks maturation of N-linked glycoproteins, reduced contact formation. We propose that cellular control of adhesion maintenance is active because cilia adhesion did not prevent cell division; rather, contacts dissolved during mitosis as cilia were resorbed. CONCLUSIONS: The demonstration that mammalian primary cilia formed prolonged, direct, physical contacts supports a novel paradigm: that mammalian primary cilia detect features of the extracellular space, not just as passive antennae, but also through direct physical contact. We present a model for the cycle of glycoprotein-dependent contact formation, maintenance, and termination, and discuss the implications for potential physiological functions of cilia-cilia contacts.
RESUMO
Mouse cone photoreceptors, like those of most mammals including humans, express cone opsins derived from two ancient families: S-opsin (gene Opn1sw) and M-opsin (gene Opn1mw). Most C57Bl/6 mouse cones co-express both opsins, but in dorso-ventral counter-gradients, with M-opsin dominant in the dorsal retina and S-opsin in the ventral retina, and S-opsin 4-fold greater overall. We created a mouse lacking S-opsin expression by the insertion of a Neomycin selection cassette between the third and fourth exons of the Opn1sw gene (Opn1sw(Neo/Neo)). In strong contrast to published results characterizing mice lacking rhodopsin (Rhoâ»/â») in which retinal rods undergo cell death by 2.5 months, cones of the Opn1sw(Neo/Neo) mouse remain viable for at least 1.5 yrs, even though many ventral cones do not form outer segments, as revealed by high resolution immunohistochemistry and electron microscopy. Suction pipette recordings revealed that functional ventral cones of the Opn1sw(Neo/Neo) mouse not only phototransduce light with normal kinetics, but are more sensitive to mid-wavelength light than their WT counterparts. Quantitative Western blot analysis revealed the basis of the heightened sensitivity to be increased M-opsin expression. Because S- and M-opsin transcripts must compete for the same translational machinery in cones where they are co-expressed, elimination of S-opsin mRNA in ventral Opn1sw(Neo/Neo) cones likely increases M-opsin expression by relieving competition for translational machinery, revealing an important consequence of eliminating a dominant transcript. Overall, our results reveal a striking capacity for cone photoreceptors to function with much reduced opsin expression, and to remain viable in the absence of an outer segment.
Assuntos
Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Opsinas de Bastonetes/deficiência , Opsinas de Bastonetes/metabolismo , Animais , Western Blotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Opsinas de Bastonetes/imunologiaRESUMO
BACKGROUND: Photoreceptors of the retina are highly compartmentalized cells that function as the primary sensory neurons for receiving and initiating transmission of visual information. Proper morphogenesis of photoreceptor neurons is essential for their normal function and survival. We have characterized a zebrafish mutation, cannonball, that completely disrupts photoreceptor morphogenesis. RESULTS: Analysis revealed a non-sense mutation in cytoplasmic dynein heavy chain 1 (dync1h1), a critical subunit in Dynein1, to underlie the cannonball phenotypes. Dynein1 is a large minus-end directed, microtubule motor protein complex that has been implicated in multiple, essential cellular processes. In photoreceptors, Dynein1 is thought to mediate post-Golgi vesicle trafficking, while Dynein2 is thought to be responsible for outer segment maintenance. Surprisingly, cannonball embryos survive until larval stages, owing to wild-type maternal protein stores. Retinal photoreceptor neurons, however, are significantly affected by loss of Dync1h1, as transmission electron microscopy and marker analyses demonstrated defects in organelle positioning and outer segment morphogenesis and suggested defects in post-Golgi vesicle trafficking. Furthermore, dosage-dependent antisense oligonucleotide knock-down of dync1h1 revealed outer segment abnormalities in the absence of overt inner segment polarity and trafficking defects. Consistent with a specific function of Dync1h1 within the outer segment, immunolocalization showed that this protein and other subunits of Dynein1 and Dynactin localized to the ciliary axoneme of the outer segment, in addition to their predicted inner segment localization. However, knock-down of Dynactin subunits suggested that this protein complex, which is known to augment many Dynein1 activities, is only essential for inner segment processes as outer segment morphogenesis was normal. CONCLUSIONS: Our results indicate that Dynein1 is required for multiple cellular processes in photoreceptor neurons, including organelle positioning, proper outer segment morphogenesis, and potentially post-Golgi vesicle trafficking. Titrated knock-down of dync1h1 indicated that outer segment morphogenesis was affected in photoreceptors that showed normal inner segments. These observations, combined with protein localization studies, suggest that Dynein1 may have direct and essential functions in photoreceptor outer segments, in addition to inner segment functions.
Assuntos
Dineínas do Citoplasma/genética , Dineínas/genética , Mutação/genética , Células Fotorreceptoras/metabolismo , Retina/crescimento & desenvolvimento , Retina/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Animais , Axonema/metabolismo , Axonema/ultraestrutura , Diferenciação Celular/genética , Dineínas do Citoplasma/antagonistas & inibidores , Dineínas do Citoplasma/metabolismo , Dineínas/antagonistas & inibidores , Regulação da Expressão Gênica no Desenvolvimento/genética , Microscopia Eletrônica de Transmissão , Neurogênese/fisiologia , Oligonucleotídeos Antissenso/farmacologia , Células Fotorreceptoras/ultraestrutura , Retina/ultraestrutura , Vesículas Transportadoras/fisiologia , Vesículas Transportadoras/ultraestruturaRESUMO
The photoreceptor outer segment (OS), a well-defined sensory cilium, provides an important context for the study of intraflagellar transport (IFT). The early phases of OS development involve successive events that are common to virtually all cilia. Additionally, intense protein trafficking occurs through the cilium and relies on IFT to maintain proper cellular morphology and optimize the photosensitive function. In the past decade, progress has been made in the characterization of photoreceptor OS trafficking in murine and amphibian models. Recently, powerful and cost-effective molecular tools and techniques for zebrafish have opened new opportunities to study photoreceptor IFT. Studies using zebrafish take advantage of its rapid embryogenesis to characterize the early events involved in photoreceptor ciliogenesis and OS assembly. In this overview, we describe phenotypes associated with knockdown strategies or genetic mutations of IFT components in zebrafish and detail a general experimental approach that has enabled us to study the function of the two anterograde IFT motors, KIF17 and kinesin II, in zebrafish cone photoreceptors.