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1.
Methods Mol Biol ; 1957: 271-289, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30919360

RESUMO

Primary cilia (PC) are microtubule-based organelles that behave like a cellular antenna controlling key signaling pathways during development and tissue homeostasis. The ciliary membrane is highly enriched for G protein-coupled receptors (GPCRs), and PC are a crucial signaling compartment for this large receptor family. Downstream effectors of GPCR signaling are also present in cilia, and evidence obtained by our labs and others demonstrated that ß-arrestin (ßarr) family members are differentially recruited to PC and have investigated the role of GPCR activation in this process. In this chapter, we provide methods based on fluorescence microscopy on fixed or live cells suitable for investigating targeting and recruitment of ßarrs at PC.


Assuntos
Corpos Basais/metabolismo , Centrossomo/metabolismo , Cílios/metabolismo , Microscopia de Fluorescência/métodos , beta-Arrestina 2/metabolismo , Animais , Corpos Basais/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Centrossomo/efeitos dos fármacos , Cílios/efeitos dos fármacos , DNA/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Plasmídeos/metabolismo , Somatostatina/farmacologia
2.
Nat Rev Nephrol ; 15(4): 199-219, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30733609

RESUMO

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.

3.
Genesis ; 56(8): e23217, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29806135

RESUMO

The neuropeptide, melanin concentrating hormone (MCH), and its G protein-coupled receptor, melanin concentrating hormone receptor 1 (Mchr1), are expressed centrally in adult rodents. MCH signaling has been implicated in diverse behaviors such as feeding, sleep, anxiety, as well as addiction and reward. While a model utilizing the Mchr1 promoter to drive constitutive expression of Cre recombinase (Mchr1-Cre) exists, there is a need for an inducible Mchr1-Cre to determine the roles for this signaling pathway in neural development and adult neuronal function. Here, we generated a BAC transgenic mouse where the Mchr1 promotor drives expression of tamoxifen inducible CreER recombinase. Many aspects of the Mchr1-Cre expression pattern are recapitulated by the Mchr1-CreER model, though there are also notable differences. Most strikingly, compared to the constitutive model, the new Mchr1-CreER model shows strong expression in adult animals in hypothalamic brain regions involved in feeding behavior but diminished expression in regions involved in reward, such as the nucleus accumbens. The inducible Mchr1-CreER allele will help reveal the potential for Mchr1 signaling to impact neural development and subsequent behavioral phenotypes, as well as contribute to the understanding of the MCH signaling pathway in terminally differentiated adult neurons and the diverse behaviors that it influences.

4.
Dev Cell ; 42(3): 286-300.e4, 2017 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-28787594

RESUMO

Appropriate growth and synaptic integration of GABAergic inhibitory interneurons are essential for functional neural circuits in the brain. Here, we demonstrate that disruption of primary cilia function following the selective loss of ciliary GTPase Arl13b in interneurons impairs interneuronal morphology and synaptic connectivity, leading to altered excitatory/inhibitory activity balance. The altered morphology and connectivity of cilia mutant interneurons and the functional deficits are rescued by either chemogenetic activation of ciliary G-protein-coupled receptor (GPCR) signaling or the selective induction of Sstr3, a ciliary GPCR, in Arl13b-deficient cilia. Our results thus define a specific requirement for primary cilia-mediated GPCR signaling in interneuronal connectivity and inhibitory circuit formation.


Assuntos
Interneurônios/metabolismo , Transdução de Sinais , Sinapses/metabolismo , Potenciais Sinápticos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Células Cultivadas , Cílios/metabolismo , Interneurônios/citologia , Interneurônios/fisiologia , Camundongos , Neurogênese , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Sinapses/fisiologia
5.
Artigo em Inglês | MEDLINE | ID: mdl-28159877

RESUMO

G-protein-coupled receptors (GPCRs) are the largest and most versatile family of signaling receptors in humans. They respond to diverse external signals, such as photons, proteins, peptides, chemicals, hormones, lipids, and sugars, and mediate a myriad of functions in the human body. Signaling through GPCRs can be optimized by enriching receptors and downstream effectors in discrete cellular domains. Many GPCRs have been found to be selectively targeted to cilia on numerous mammalian cell types. Moreover, investigations into the pathophysiology of human ciliopathies have implicated GPCR ciliary signaling in a number of developmental and cellular pathways. Thus, cilia are now appreciated as an increasingly important nexus for GPCR signaling. Yet, we are just beginning to understand the precise signaling pathways mediated by most ciliary GPCRs and how they impact cellular function and mammalian physiology.


Assuntos
Cílios/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Humanos , Modelos Biológicos , Receptores Odorantes/metabolismo , Receptores Odorantes/fisiologia , Transdução de Sinais
6.
Mol Cell Biol ; 36(1): 223-35, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26503786

RESUMO

Primary cilia are essential sensory and signaling organelles present on nearly every mammalian cell type. Defects in primary cilia underlie a class of human diseases collectively termed ciliopathies. Primary cilia are restricted subcellular compartments, and specialized mechanisms coordinate the localization of proteins to cilia. Moreover, trafficking of proteins into and out of cilia is required for proper ciliary function, and this process is disrupted in ciliopathies. The somatostatin receptor subtype 3 (Sstr3) is selectively targeted to primary cilia on neurons in the mammalian brain and is implicated in learning and memory. Here, we show that Sstr3 localization to cilia is dynamic and decreases in response to somatostatin treatment. We further show that somatostatin treatment stimulates ß-arrestin recruitment into Sstr3-positive cilia and this recruitment can be blocked by mutations in Sstr3 that impact agonist binding or phosphorylation. Importantly, somatostatin treatment fails to decrease Sstr3 ciliary localization in neurons lacking ß-arrestin 2. Together, our results implicate ß-arrestin in the modulation of Sstr3 ciliary localization and further suggest a role for ß-arrestin in the mediation of Sstr3 ciliary signaling.


Assuntos
Arrestinas/metabolismo , Cílios/metabolismo , Memória/fisiologia , Neurônios/metabolismo , Receptores de Somatostatina/metabolismo , Animais , Encéfalo/metabolismo , Células Cultivadas , Aprendizagem/fisiologia , Camundongos , Transdução de Sinais/fisiologia , beta-Arrestina 2 , beta-Arrestinas
7.
Proc Natl Acad Sci U S A ; 111(28): 10335-40, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24982149

RESUMO

Most central neurons in the mammalian brain possess an appendage called a primary cilium that projects from the soma into the extracellular space. The importance of these organelles is highlighted by the fact that primary cilia dysfunction is associated with numerous neuropathologies, including hyperphagia-induced obesity, hypogonadism, and learning and memory deficits. Neuronal cilia are enriched for signaling molecules, including certain G protein-coupled receptors (GPCRs), suggesting that neuronal cilia sense and respond to neuromodulators in the extracellular space. However, the impact of cilia on signaling to central neurons has never been demonstrated. Here, we show that the kisspeptin receptor (Kiss1r), a GPCR that is activated by kisspeptin to regulate the onset of puberty and adult reproductive function, is enriched in cilia projecting from mouse gonadotropin-releasing hormone (GnRH) neurons. Interestingly, GnRH neurons in adult animals are multiciliated and the percentage of GnRH neurons possessing multiple Kiss1r-positive cilia increases during postnatal development in a progression that correlates with sexual maturation. Remarkably, disruption of cilia selectively on GnRH neurons leads to a significant reduction in kisspeptin-mediated GnRH neuronal activity. To our knowledge, this result is the first demonstration of cilia disruption affecting central neuronal activity and highlights the importance of cilia for proper GPCR signaling.


Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Kisspeptinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Reprodução/fisiologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Cílios/genética , Cílios/metabolismo , Feminino , Hormônio Liberador de Gonadotropina/genética , Kisspeptinas/genética , Masculino , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Receptores Acoplados a Proteínas-G/genética , Receptores de Kisspeptina-1 , Maturidade Sexual/fisiologia
9.
Cell Mol Life Sci ; 71(11): 2165-78, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24104765

RESUMO

Primary cilia with a diameter of ~200 nm have been implicated in development and disease. Calcium signaling within a primary cilium has never been directly visualized and has therefore remained a speculation. Fluid-shear stress and dopamine receptor type-5 (DR5) agonist are among the few stimuli that require cilia for intracellular calcium signal transduction. However, it is not known if these stimuli initiate calcium signaling within the cilium or if the calcium signal originates in the cytoplasm. Using an integrated single-cell imaging technique, we demonstrate for the first time that calcium signaling triggered by fluid-shear stress initiates in the primary cilium and can be distinguished from the subsequent cytosolic calcium response through the ryanodine receptor. Importantly, this flow-induced calcium signaling depends on the ciliary polycystin-2 calcium channel. While DR5-specific agonist induces calcium signaling mainly in the cilioplasm via ciliary CaV1.2, thrombin specifically induces cytosolic calcium signaling through the IP3 receptor. Furthermore, a non-specific calcium ionophore triggers both ciliary and cytosolic calcium responses. We suggest that cilia not only act as sensory organelles but also function as calcium signaling compartments. Cilium-dependent signaling can spread to the cytoplasm or be contained within the cilioplasm. Our study thus provides the first model to understand signaling within the cilioplasm of a living cell.


Assuntos
Sinalização do Cálcio , Cílios/metabolismo , Células Epiteliais/metabolismo , Mecanotransdução Celular , Canais de Cátion TRPP/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Ionóforos de Cálcio/farmacologia , Cílios/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Imagem Molecular , Cultura Primária de Células , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Reologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Análise de Célula Única , Estresse Mecânico , Suínos , Canais de Cátion TRPP/genética , Trombina/farmacologia
10.
J Neurosci ; 33(6): 2626-38, 2013 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-23392690

RESUMO

The formation of primary cilia is a highly choreographed process that can be disrupted in developing neurons by overexpressing neuromodulatory G-protein-coupled receptors GPCRs or by blocking intraflagellar transport. Here, we examined the effects of overexpressing the ciliary GPCRs, 5HT6 and SSTR3, on cilia structure and the differentiation of neocortical neurons. Neuronal overexpression of 5HT6 and SSTR3 was achieved by electroporating mouse embryo cortex in utero with vectors encoding these receptors. We found that overexpression of ciliary GPCRs in cortical neurons, especially 5HT6, induced the formation of long (>30 µm) and often forked cilia. These changes were associated with increased levels of intraflagellar transport proteins and accelerated ciliogenesis in neonatal neocortex, the induction of which required Kif3a, an anterograde motor critical for cilia protein trafficking and growth. GPCR overexpression also altered the complement of signaling molecules within the cilia. We found that SSTR3 and type III adenylyl cyclase (ACIII), proteins normally enriched in neuronal cilia, were rarely detected in 5HT6-elongated cilia. Intriguingly, the changes in cilia structure were accompanied by changes in neuronal morphology. Specifically, disruption of normal ciliogenesis in developing neocortical neurons, either by overexpressing cilia GPCRs or a dominant-negative form of Kif3a, significantly impaired dendrite outgrowth. Remarkably, coexpression of ACIII with 5HT6 restored ACIII to cilia, normalized cilia structure, and restored dendrite outgrowth, effects that were not observed in neurons coexpressing ACIII and dominant-negative form of Kif3a. Collectively, our data suggest the formation of neuronal dendrites in developing neocortex requires structurally normal cilia enriched with ACIII.


Assuntos
Adenilil Ciclases/fisiologia , Cílios/enzimologia , Dendritos/enzimologia , Neocórtex/enzimologia , Neurônios/enzimologia , Receptores de Serotonina/biossíntese , Animais , Células Cultivadas , Cílios/fisiologia , Feminino , Cinesina/biossíntese , Masculino , Camundongos , Células NIH 3T3 , Neocórtex/embriologia , Neurogênese/fisiologia , Neurônios/citologia , Gravidez
11.
PLoS One ; 7(9): e46304, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23029470

RESUMO

Nearly every cell type in the mammalian body projects from its cell surface a primary cilium that provides important sensory and signaling functions. Defects in the formation or function of primary cilia have been implicated in the pathogenesis of many human developmental disorders and diseases, collectively termed ciliopathies. Most neurons in the brain possess cilia that are enriched for signaling proteins such as G protein-coupled receptors and adenylyl cyclase type 3, suggesting neuronal cilia sense neuromodulators in the brain and contribute to non-synaptic signaling. Indeed, disruption of neuronal cilia or loss of neuronal ciliary signaling proteins is associated with obesity and learning and memory deficits. As the functions of primary cilia are defined by the signaling proteins that localize to the ciliary compartment, identifying the complement of signaling proteins in cilia can provide important insights into their physiological roles. Here we report for the first time that different GPCRs can colocalize within the same cilium. Specifically, we found the ciliary GPCRs, melanin-concentrating hormone receptor 1 (Mchr1) and somatostatin receptor 3 (Sstr3) colocalizing within cilia in multiple mouse brain regions. In addition, we have evidence suggesting Mchr1 and Sstr3 form heteromers. As GPCR heteromerization can affect ligand binding properties as well as downstream signaling, our findings add an additional layer of complexity to neuronal ciliary signaling.


Assuntos
Encéfalo/fisiologia , Cílios/metabolismo , Neurônios/metabolismo , Receptores de Somatostatina/metabolismo , Animais , Encéfalo/citologia , Células Cultivadas , Cílios/genética , Expressão Gênica , Células HEK293 , Humanos , Camundongos , Neurônios/citologia , Multimerização Proteica , Receptores de Somatostatina/genética , Transdução de Sinais/fisiologia
12.
Cell Mol Life Sci ; 68(17): 2951-60, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21152952

RESUMO

Primary cilia are nearly ubiquitous cellular appendages that provide important sensory and signaling functions. Ciliary dysfunction underlies numerous human diseases, collectively termed ciliopathies. Primary cilia have distinct functions on different cell types and these functions are defined by the signaling proteins that localize to the ciliary membrane. Neurons throughout the mammalian brain possess primary cilia upon which certain G protein-coupled receptors localize. Yet, the precise signaling proteins present on the vast majority of neuronal cilia are unknown. Here, we report that dopamine receptor 1 (D1) localizes to cilia on mouse central neurons, thereby implicating neuronal cilia in dopamine signaling. Interestingly, ciliary localization of D1 is dynamic, and the receptor rapidly translocates to and from cilia in response to environmental cues. Notably, the translocation of D1 from cilia requires proteins mutated in the ciliopathy Bardet-Biedl syndrome (BBS), and we find that one of the BBS proteins, Bbs5, specifically interacts with D1.


Assuntos
Proteínas de Transporte/metabolismo , Cílios/metabolismo , Receptores de Dopamina D1/metabolismo , Animais , Síndrome de Bardet-Biedl/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Humanos , Camundongos , Camundongos Knockout , Neurônios/citologia , Proteínas/metabolismo , Receptores de Dopamina D1/análise , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais
13.
Cell Mol Life Sci ; 67(19): 3287-97, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20544253

RESUMO

Primary cilia are a class of cilia that are typically solitary, immotile appendages present on nearly every mammalian cell type. Primary cilia are believed to perform specialized sensory and signaling functions that are important for normal development and cellular homeostasis. Indeed, primary cilia dysfunction is now linked to numerous human diseases and genetic disorders. Collectively, primary cilia disorders are termed as ciliopathies and present with a wide range of clinical features, including cystic kidney disease, retinal degeneration, obesity, polydactyly, anosmia, intellectual disability, and brain malformations. Although significant progress has been made in elucidating the functions of primary cilia on some cell types, the precise functions of most primary cilia remain unknown. This is particularly true for primary cilia on neurons throughout the mammalian brain. This review will introduce primary cilia and ciliary signaling pathways with a focus on neuronal cilia and their putative functions and roles in human diseases.


Assuntos
Neurônios/fisiologia , Transdução de Sinais , Cílios/metabolismo , Cílios/fisiologia , Doenças Genéticas Inatas/metabolismo , Homeostase , Humanos , Doenças Renais Císticas/metabolismo , Neurônios/citologia , Doenças Renais Policísticas/metabolismo , Polidactilia/metabolismo , Degeneração Retiniana/metabolismo
14.
Methods Cell Biol ; 91: 111-21, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20409783

RESUMO

Primary cilia were first detected on neurons in the mammalian brain over 40 years ago using electron microscopy. However, this approach is very labor intensive and has inherent limitations that restrict its utility for studying neuronal cilia. While the study of cilia in other tissues was greatly facilitated by the identification of specific ciliary markers, historically there have been no markers for neuronal cilia. Fortunately, recent developments make the study of neuronal cilia more practical. First, specific proteins have been shown to selectively localize to neuronal cilia and can serve as markers by immunolabeling. Second, neurons have been shown to possess cilia in culture, which allows for the use of additional approaches, such as live-cell imaging of neuronal cilia. This chapter provides an overview of the current techniques for visualizing neuronal cilia in tissue as well as fixed and living cells. These approaches allow for the identification of additional neuronal ciliary proteins and provide a basis for future functional studies.


Assuntos
Biomarcadores/metabolismo , Cílios , Neurônios , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Cílios/metabolismo , Cílios/ultraestrutura , Imuno-Histoquímica/métodos , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem/métodos
15.
Proc Natl Acad Sci U S A ; 105(11): 4242-6, 2008 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-18334641

RESUMO

Primary cilia are ubiquitous cellular appendages that provide important yet not well understood sensory and signaling functions. Ciliary dysfunction underlies numerous human genetic disorders. However, the precise defects in cilia function and the basis of disease pathophysiology remain unclear. Here, we report that the proteins disrupted in the human ciliary disorder Bardet-Biedl syndrome (BBS) are required for the localization of G protein-coupled receptors to primary cilia on central neurons. We demonstrate a lack of ciliary localization of somatostatin receptor type 3 (Sstr3) and melanin-concentrating hormone receptor 1 (Mchr1) in neurons from mice lacking the Bbs2 or Bbs4 gene. Because Mchr1 is involved in the regulation of feeding behavior and BBS is associated with hyperphagia-induced obesity, our results suggest that altered signaling caused by mislocalization of ciliary signaling proteins underlies the BBS phenotypes. Our results also provide a potential molecular mechanism to link cilia defects with obesity.


Assuntos
Síndrome de Bardet-Biedl/metabolismo , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas/metabolismo , Receptores de Somatostatina/metabolismo , Animais , Síndrome de Bardet-Biedl/genética , Células Cultivadas , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Proteínas/genética
16.
Mol Biol Cell ; 19(4): 1540-7, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18256283

RESUMO

Primary cilia are sensory organelles present on most mammalian cells. The functions of cilia are defined by the signaling proteins localized to the ciliary membrane. Certain G protein-coupled receptors (GPCRs), including somatostatin receptor 3 (Sstr3) and serotonin receptor 6 (Htr6), localize to cilia. As Sstr3 and Htr6 are the only somatostatin and serotonin receptor subtypes that localize to cilia, we hypothesized they contain ciliary localization sequences. To test this hypothesis we expressed chimeric receptors containing fragments of Sstr3 and Htr6 in the nonciliary receptors Sstr5 and Htr7, respectively, in ciliated cells. We found the third intracellular loop of Sstr3 or Htr6 is sufficient for ciliary localization. Comparison of these loops revealed a loose consensus sequence. To determine whether this consensus sequence predicts ciliary localization of other GPCRs, we compared it with the third intracellular loop of all human GPCRs. We identified the consensus sequence in melanin-concentrating hormone receptor 1 (Mchr1) and confirmed Mchr1 localizes to primary cilia in vitro and in vivo. Thus, we have identified a putative GPCR ciliary localization sequence and used this sequence to identify a novel ciliary GPCR. As Mchr1 mediates feeding behavior and metabolism, our results implicate ciliary signaling in the regulation of body weight.


Assuntos
Cílios/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Sequência Consenso , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Receptores Acoplados a Proteínas-G/genética , Receptores de Serotonina/química , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Receptores de Somatostatina/química , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos
17.
J Comp Neurol ; 505(5): 562-71, 2007 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-17924533

RESUMO

Solitary primary cilia project from nearly every cell type in the human body. These organelles are considered to have important sensory and signaling functions. Although primary cilia have been detected throughout the mammalian brain, their functions are unknown. The study of primary cilia in the brain is constrained by the scarcity of specific markers for these organelles. We previously demonstrated that type III adenylyl cyclase (ACIII) is a marker for primary cilia on neonatal hippocampal neurons in vivo and in vitro. We further showed that ACIII localizes to cilia on cultured glial cells. Here, we report that ACIII is a marker for primary cilia throughout many regions of the adult mouse brain. Furthermore, we report that ACIII localizes to primary cilia on choroid plexus cells and some astrocytes in the brain, which to our knowledge is the first report of a marker for visualizing cilia on glia in vivo. Overall, our data indicate that ACIII is a prominent marker of primary cilia in the brain and will provide an important tool to facilitate further investigations into the functions of these organelles.


Assuntos
Adenilil Ciclases/metabolismo , Astrócitos/metabolismo , Biomarcadores/metabolismo , Encéfalo/metabolismo , Cílios/enzimologia , Camundongos Endogâmicos/metabolismo , Adenilil Ciclases/imunologia , Fatores Etários , Animais , Anticorpos , Astrócitos/ultraestrutura , Encéfalo/citologia , Plexo Corióideo/citologia , Plexo Corióideo/metabolismo , Imunofluorescência , Isoenzimas/imunologia , Isoenzimas/metabolismo , Camundongos , Coelhos
19.
Nephron Exp Nephrol ; 106(3): e88-96, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17519557

RESUMO

BACKGROUND: Bardet-Biedl syndrome (BBS) is a heterogeneous genetic disorder that comprises numerous features, including renal cystic disease. Twelve BBS genes have been identified (BBS1-12). Although the exact functions of the BBS proteins are unknown, evidence suggests that they are involved in cilia assembly, maintenance and/or function. Renal primary cilia dysfunction can lead to cystic kidney disease. To test whether lacking Bbs4 affects cilia assembly and structure, we analyzed primary cilia in Bbs4-null (Bbs4(-/-)) mice. METHODS: Renal tubule cultures from wild-type (Bbs4(+/+)) and Bbs4(-/-) mice were examined by immunocytochemistry and scanning and transmission electron microscopy. RESULTS: Our culture conditions generated ciliated epithelial cells that were mostly of collecting duct origin. The microtubule ultrastructure of cilia and basal bodies did not appear disrupted in Bbs4(-/-) cells. In control cells, cilia length was maximal at 7 days in culture. In cells cultured from Bbs4(-/-) mice, cilia were shorter initially, but surpassed the length of control cilia by 10 days. Renal primary cilia were also longer in Bbs4(-/-) kidneys. CONCLUSIONS: Lacking Bbs4 does not lead to aberrant cilia or basal body structure. However, the dynamics of cilia assembly is altered in Bbs4(-/-) cells, suggesting a role for Bbs4 in the regulation of ciliary assembly.


Assuntos
Síndrome de Bardet-Biedl/patologia , Cílios/ultraestrutura , Túbulos Renais/patologia , Animais , Síndrome de Bardet-Biedl/fisiopatologia , Células Cultivadas , Cílios/fisiologia , Modelos Animais de Doenças , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica , Túbulos Renais/fisiopatologia , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura
20.
J Neurosci Res ; 85(5): 1095-100, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17304575

RESUMO

Primary cilia are cellular appendages that provide important sensory functions and defects in primary ciliary signaling have been implicated in the pathophysiology of human diseases and developmental abnormalities. Almost all human cell types possess a primary cilium. Neurons throughout the brain possess primary cilia on which certain receptors localize, suggesting that neurons possess cilia-mediated signaling. However, the functional significance of neuronal cilia is unknown. Although there is a great deal of interest in understanding the functions of neuronal cilia, their study is hampered by the lack of an in vitro model system. We report that the majority of hippocampal neurons cultured from postnatal mice possess primary cilia in vitro. Further, we describe cilia proteins that can be labeled to readily visualize neuronal primary cilia in culture. These findings are the first characterization of neuronal primary cilia in vitro and should greatly facilitate further investigations into the function of these organelles.


Assuntos
Cílios/ultraestrutura , Hipocampo/citologia , Neurônios/citologia , Adenilil Ciclases/análise , Adenilil Ciclases/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/análise , Biomarcadores/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Forma Celular/fisiologia , Células Cultivadas , Cílios/metabolismo , Hipocampo/metabolismo , Imuno-Histoquímica/métodos , Isoenzimas/análise , Isoenzimas/metabolismo , Camundongos , Neurônios/metabolismo , Receptores de Somatostatina/análise , Receptores de Somatostatina/metabolismo
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