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2.
Sci Rep ; 12(1): 20515, 2022 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-36443454

RESUMO

The formins constitute a large class of multi-domain polymerases that catalyze the localization and growth of unbranched actin filaments in cells from yeast to mammals. The conserved FH2 domains form dimers that bind actin at the barbed end of growing filaments and remain attached as new subunits are added. Profilin-actin is recruited and delivered to the barbed end by formin FH1 domains via the binding of profilin to interspersed tracts of poly-L-proline. We present a structural model showing that profilin-actin can bind the FH2 dimer at the barbed end stabilizing a state where profilin prevents its associated actin subunit from directly joining the barbed end. It is only with the dissociation of profilin from the polymerase that an actin subunit rotates and docks into its helical position, consistent with observations that under physiological conditions optimal elongation rates depend on the dissociation rate of profilin, independently of cellular concentrations of actin subunits.


Assuntos
Actinas , Profilinas , Animais , Forminas , Citoesqueleto de Actina , Modelos Estruturais , Nucleotidiltransferases , Polímeros , Saccharomyces cerevisiae , Mamíferos
3.
Sci Signal ; 2(95): ra70, 2009 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-19887680

RESUMO

Regulatory factor X (Rfx) homologs regulate the transcription of genes necessary for ciliogenesis in invertebrates and vertebrates. Primary cilia are necessary for Hedgehog signaling and regulation of the activity of the transcriptional regulators known as Gli proteins, which are targets of Hedgehog signaling. Here, we describe an Rfx4(L298P) mouse mutant with distinct dorsoventral patterning defects in the ventral spinal cord and telencephalon due to aberrant Sonic hedgehog (Shh) signaling and Gli3 activity. We find that Ift172, which encodes an intraflagellar transport protein necessary for ciliogenesis, is a direct transcriptional target of Rfx4, and the decrease in its expression in the developing telencephalon and spinal cord of Rfx4(L298P) mutants correlates with defects in patterning and cilia formation. Our data indicate that Rfx4 is a regionally specific transcriptional regulator of ciliogenesis and thus is also a regionally specific modulator of Shh signaling during development of the central nervous system.


Assuntos
Cílios , Proteínas de Ligação a DNA/fisiologia , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Fatores de Transcrição/fisiologia , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Proteínas de Ligação a DNA/genética , Camundongos , Mutação , Fatores de Transcrição de Fator Regulador X , Medula Espinal , Telencéfalo , Fatores de Transcrição/genética , Transcrição Gênica
4.
Nat Neurosci ; 12(8): 1028-35, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19578380

RESUMO

The hindbrain transcription factors Phox2b and Egr2 (also known as Krox20) are linked to the development of the autonomic nervous system and rhombomere-related regulation of breathing, respectively. Mutations in these proteins can lead to abnormal breathing behavior as a result of an alteration in an unidentified neuronal system. We characterized a bilateral embryonic parafacial (e-pF) population of rhythmically bursting neurons at embryonic day (E) 14.5 in mice. These cells expressed Phox2b, were derived from Egr2-expressing precursors and their development was dependent on the integrity of the Egr2 gene. Silencing or eliminating the e-pF oscillator, but not the putative inspiratory oscillator (preBötzinger complex, preBötC), led to an abnormally slow rhythm, demonstrating that the e-pF controls the respiratory rhythm. The e-pF oscillator, the only one active at E14.5, entrained and then coupled with the preBötC, which emerged independently at E15.5. These data establish the dual organization of the respiratory rhythm generator at the time of its inception, when it begins to drive fetal breathing.


Assuntos
Relógios Biológicos/genética , Tronco Encefálico/embriologia , Tronco Encefálico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Centro Respiratório/embriologia , Centro Respiratório/metabolismo , Animais , Tronco Encefálico/citologia , Diferenciação Celular/genética , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Inalação/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Rede Nervosa/citologia , Rede Nervosa/embriologia , Rede Nervosa/metabolismo , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Periodicidade , Centro Respiratório/citologia , Fenômenos Fisiológicos Respiratórios/genética , Formação Reticular/citologia , Formação Reticular/embriologia , Formação Reticular/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Exp Cell Res ; 312(16): 3108-19, 2006 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16919269

RESUMO

Highly conserved non-coding DNA regions (HCNR) occur frequently in vertebrate genomes, but their functional roles remain unclear. Here, we provide evidence that a large portion of HCNRs are enriched for binding sites for Sox, POU and Homeodomain transcription factors, and such HCNRs can act as cis-regulatory regions active in neural stem cells. Strikingly, these HCNRs are linked to several hundreds of genes expressed in the developing CNS and they may exert locus-wide regulatory effects on multiple genes flanking their genomic location. Moreover, these data imply a unifying transcriptional logic for a large set of CNS-expressed genes in which Sox and POU proteins act as generic promoters of transcription while Homeodomain proteins control the spatial expression of genes through active repression.


Assuntos
Sistema Nervoso Central/metabolismo , Sequência Conservada/genética , Genoma/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica/genética , Animais , Sequência de Bases , Sítios de Ligação , Padronização Corporal/genética , Células Cultivadas , Embrião de Galinha , Regulação para Baixo/genética , Genômica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Íntrons/genética , Camundongos , Dados de Sequência Molecular , Neurônios/metabolismo , Fatores do Domínio POU/metabolismo , Tetraodontiformes/genética
6.
Gene Expr Patterns ; 6(2): 162-70, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16326147

RESUMO

Members of the Nkx family of homeodomain proteins are involved in a variety of developmental processes such as cell fate determination in the CNS and in the pancreas. Here we describe the cloning and developmental expression pattern of Nkx6.3, a new member of the Nkx6 subfamily of homeodomain proteins. Nkx6.3 is expressed in the developing CNS and gastro-intestinal tract. In contrast to Nkx6.1 and Nkx6.2 that are broadly expressed in ventral positions of the developing CNS, Nkx6.3 shows a remarkably selective expression in a subpopulation of differentiating V2 neurons at caudal hindbrain levels. The expression of Nkx6.3 at this level depends on the activity of other Nkx6 proteins. In the gut, Nkx6.3 is expressed in duodenal and glandular stomach endoderm and at the end of gestation Nkx6.3 became restricted to the base of the gastric units in the glandular stomach. The expression of Nkx6.3 overlapped with the expression of Nkx6.2 both in the CNS and in the gut. Transient Nkx6.2 expression was also detected in the developing pancreas. However, analysis of Nkx6.2(-/-) mice did not display any obvious aberrations of pancreatic or stomach development.


Assuntos
Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Trato Gastrointestinal/embriologia , Trato Gastrointestinal/metabolismo , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/deficiência
7.
Dev Biol ; 287(2): 378-89, 2005 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16229832

RESUMO

Sonic Hedgehog (Shh) signals are transduced into nuclear ratios of Gli transcriptional activator versus repressor. The initial part of this process is accomplished by Shh acting through Patched (Ptc) to regulate Smoothened (Smo) activity. The mechanisms by which Ptc regulates Smo, and Smo activity is transduced to processing of Gli proteins remain unclear. Recently, a forward genetic approach in mice identified a role for intraflagellar transport (IFT) genes in Shh signal transduction, downstream of Patched (Ptc) and Rab23. Here, we show that the retrograde motor for IFT is required in the mouse for the phenotypic expression of both Gli activator and repressor function and for effective proteolytic processing of Gli3. Furthermore, we show that the localization of Smo to primary cilia is disrupted in mutants. These data indicate that primary cilia act as specialized signal transduction organelles required for coupling Smo activity to the biochemical processing of Gli3 protein.


Assuntos
Cílios/metabolismo , Dineínas/metabolismo , Fatores de Transcrição Kruppel-Like/fisiologia , Proteínas Motores Moleculares/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Padronização Corporal , Cílios/ultraestrutura , Dineínas/genética , Extremidades/embriologia , Flagelos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Proteínas Motores Moleculares/genética , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/metabolismo , Prosencéfalo/anormalidades , Prosencéfalo/embriologia , Transporte Proteico/genética , Receptor Smoothened , Medula Espinal/anormalidades , Medula Espinal/embriologia , Transativadores/fisiologia , Proteína Gli3 com Dedos de Zinco
8.
Mol Cell Neurosci ; 29(2): 250-8, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15911349

RESUMO

TAF4 is crucial for the activity of many transcription factors, including CREB, RAR and CSL/RBP-Jkappa, but the role for TAF4 in neural development is unknown. Embryonic cortical neural stem cells (NSC) showed strong expression of TAF4 that decreased during neuronal but not glial differentiation. In a protein-protein interaction screen, we identified the intracellular signaling factor RanBPM as a co-factor of TAF4. RanBPM co-localized with TAF4 in a subset of mitotic progenitors in vivo and endogenous TAF4 and RanBPM could be co-immunoprecipitated from NSC extracts. Interestingly, co-transfections of TAF4 and RanBPM led to a significant increase in the number of primary neurite processes but no increase in total neurite length, whereas RanBPM and a TAF4 isoform lacking the RanBPM-interacting domain exerted no significant effect. Our results demonstrate that temporally high expression levels of two factors considered to be relatively general in function can influence very specific events in neuronal differentiation.


Assuntos
Diferenciação Celular/fisiologia , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Células-Tronco/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células Cultivadas , Córtex Cerebral/citologia , Proteínas do Citoesqueleto , Neuritos/metabolismo , Neuritos/ultraestrutura , Neurônios/citologia , Proteínas Nucleares/genética , Estrutura Terciária de Proteína/fisiologia , Ratos , Células-Tronco/citologia , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Transfecção , Proteína ran de Ligação ao GTP/genética
9.
J Cell Sci ; 115(Pt 13): 2735-46, 2002 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12077364

RESUMO

The human BRG1 (brahma-related gene 1) protein is a component of the SWI/SNF family of the ATP-dependent chromatin remodelling complexes. We show here that expression of the BRG1 protein, but not of an ATPase-deficient BRG1 protein, in BRG1-deficient SW13 cells alters the organisation of actin filaments. BRG1 expression induces the formation of thick actin filament bundles resembling stress-fibres, structures that are rarely seen in native SW13 cells. BRG1 expression does not influence the activity state of the RhoA-GTPase, which is involved in stress-fibre formation. We find that RhoA is equally activated by stimuli, such as serum, in BRG1-expressing cells, ATPase-deficient BRG1-expressing cells and native SW13 cells. However, the activation of RhoA by lysophosphatidic acid and serum does not trigger the formation of stress-fibre-like structures in SW13 cells. Activation of the RhoA-GTPase in BRG1-expressing cells induces stress-fibre-like structures, indicating that the BRG1 can couple RhoA activation to stress-fibre formation. At least two downstream effectors are involved in stress-fibre formation, Rho-kinase/ROCK and Dia. BRG1 expression, but not the expression of the ATP-deficient BRG1, increases the protein level of ROCK1, one form of the Rho-kinase/ROCK. That this is of importance is supported by the findings that an increased Rho-kinase/ROCK activity in SW13 cells, obtained by overexpressing wild-type ROCK1 and ROCK2, induces stress-fibre formation. No specificity between the two Rho-kinase/ROCK forms exists. Our results suggest that the BRG1 protein affects the RhoA pathway by increasing the protein level of ROCK1, which allows stress-fibre-like structures to form.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Células Eucarióticas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Tamanho Celular/genética , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , DNA Helicases , Células Eucarióticas/citologia , Imunofluorescência , Regulação da Expressão Gênica/fisiologia , Genes Reguladores/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Tempo de Reação/genética , Transdução de Sinais/genética , Estresse Mecânico , Fatores de Transcrição/genética , Regulação para Cima/genética , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/genética
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