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1.
Methods Mol Biol ; 2510: 193-216, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35776326

RESUMO

The long intracellular P2X7 C-terminus accounts for diverse downstream effects of P2X7 activation. Although the recent determination of the cryo-EM structure of the full-length P2X7 receptor finally revealed the structure and several unexpected features of the large cytoplasmic domain, its molecular function remains enigmatic. Incorporation of unnatural amino acids (UAA) via an amber Stop codon has been a powerful tool for structure-function analysis of proteins. Voltage clamp fluorometry (VCF) with the fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) provides a means to study intracellular domain movements of ion channel receptors. In the Xenopus laevis oocyte expression system, site-specific introduction of this environment-sensitive fluorophore can be achieved by the nuclear injection of cDNA encoding an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair and subsequent cytoplasmic injection of ANAP together with the respective cRNA containing the amber Stop codon. Here, we describe this protocol for expression of ANAP-labeled P2X7. In addition, we provide a simplified alternative protocol, in which we coinject cRNAs encoding the tRNA synthetase and mutant P2X7 together with the synthesized amber suppressor tRNA and ANAP in one step into the cytosol. We found that the new protocol yielded more reproducible results and was less harmful for the oocytes. By selective fluorescence labeling of the ANAP-labeled P2X7 protein in the oocyte plasma membrane and VCF recordings, we show that this method results in comparable levels of functional ANAP-labeled P2X7 protein.


Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Aminoácidos/química , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Animais , Códon de Terminação , Oócitos/metabolismo , RNA de Transferência/genética , Receptores Purinérgicos P2X7/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo
2.
Methods Mol Biol ; 2510: 157-192, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35776325

RESUMO

P2X7 receptors (P2X7Rs) are fast ATP4--gated ion channels that, like other members of the P2X receptor family, function as homotrimers. A high-resolution cryo-EM structure of the full-length rat P2X7R is available. Using voltage-clamp experiments in Xenopus laevis oocytes, even the earliest steps of P2X7R activation can be quantitatively recorded in the millisecond range. Site-directed mutagenesis combined with voltage-clamp recordings can reveal residues and domains of the P2X7R involved in ATP4- binding, gating (i.e., opening and closing of the channel pore) and ion selectivity. We present here proven voltage-clamp protocols that take into account requirements that are important at the levels of cDNA and vector sequences, cRNA synthesis, and Xenopus laevis oocyte isolation for reliable results.


Assuntos
Oócitos , Receptores Purinérgicos P2X7 , Trifosfato de Adenosina/metabolismo , Animais , Oócitos/metabolismo , RNA Complementar , Ratos , Receptores Purinérgicos P2X7/metabolismo , Xenopus laevis/metabolismo
3.
Elife ; 112022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35838349

RESUMO

In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.


Assuntos
Origem de Replicação , Proteínas de Ligação a Telômeros , Animais , Replicação do DNA , Período de Replicação do DNA , Fase S/genética , Proteínas de Ligação a Telômeros/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo
4.
Aquat Toxicol ; 249: 106227, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35767922

RESUMO

The transition to include in vitro-based data in chemical hazard assessment has resulted in the development and implementation of screening assays to cover a diversity of biological pathways, including recently added assays to interrogate chemical disruption of proteins relevant to thyroid signaling pathways. Iodotyrosine deiodinase (IYD), the iodide recycling enzyme, is one such thyroid-relevant endpoint for which a human-based screening assay has recently been developed and used to screen large libraries of chemicals. Presented here is the development of an amphibian IYD inhibition assay and its implementation to conduct a cross-species comparison between chemical inhibition of mammalian and non-mammalian IYD enzyme activity. The successful development of an amphibian IYD inhibition assay was based on demonstration of sufficient IYD enzyme activity in several tissues collected from larval Xenopus laevis. With this new assay, 154 chemicals were tested in concentration-response to provide a basis for comparison of relative chemical potency to results obtained from the human IYD assay. Most chemicals exhibited similar inhibition in both assays, with less than 25% variation in median inhibition for 120 of 154 chemicals and 85% concordance in categorization of "active" (potential IYD inhibitor) versus "inactive". For chemicals that produced 50% or greater inhibition in both assays, rank-order potency was similar, with the majority of the IC50s varying by less than 2-fold (and all within an order of magnitude). Most differences resulted from greater maximum inhibition or higher chemical potency observed with human IYD. This strong cross-species agreement suggests that results from the human-based assay would be conservatively predictive of chemical effects on amphibian IYD.


Assuntos
Iodeto Peroxidase , Poluentes Químicos da Água , Animais , Humanos , Iodeto Peroxidase/metabolismo , Iodetos/metabolismo , Iodetos/farmacologia , Mamíferos/metabolismo , Glândula Tireoide , Poluentes Químicos da Água/toxicidade , Xenopus laevis/metabolismo
5.
Neuropharmacology ; 216: 109173, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35772522

RESUMO

Smokers report particular appreciation for coffee with their first cigarettes of the day. We investigated with voltage-clamp experiments, effects of aqueous extracts (coffees) of unroasted and roasted coffee beans on the activity of human brain nicotinic acetylcholine receptor (nAChR) subtypes expressed in Xenopus oocytes, looking at complex brews, low molecular weight (LMW) fractions, and specific compounds present in coffee. When co-applied with PNU-120596, a positive allosteric modulator (PAM), the coffees stimulated currents from cells expressing α7 nAChR that were larger than ACh controls. The PAM-dependent responses to green bean coffee were three-fold greater than those to dark roasted coffee, consistent with α7 receptor activation by choline, a component of coffee that is partially degraded in the roasting process. Coffees were tested on both high sensitivity (HS) and low sensitivity (LS) forms of α4ß2 nAChR, which are associated with nicotine addiction. To varying degrees, these receptors were both activated and inhibited by the coffees and LMW extracts. We also examined the activity of nine small molecules present in coffee. Only two compounds, 1-methylpyridinium and 1-1-dimethylpiperidium, produced during the process of roasting coffee beans, showed significant effects on nAChR. The compounds were competitive antagonists of the HS α4ß2 receptors, but were PAMs for LS α4ß2 receptors. HS receptors in smokers are likely to progressively desensitize through a day of smoking but may be hypersensitive in the mornings when brain nicotine levels are low. A smoker's first cup of coffee may therefore balance the effects of the day's first cigarette in the brain.


Assuntos
Receptores Nicotínicos , Produtos do Tabaco , Animais , Biomarcadores , Humanos , Nicotina/farmacologia , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Xenopus laevis/metabolismo , Receptor Nicotínico de Acetilcolina alfa7
6.
Curr Opin Cell Biol ; 77: 102105, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35716530

RESUMO

Multiciliated cells (MCC) are evolutionary conserved, highly specialized cell types that contain dozens to hundreds of motile cilia that they use to propel fluid directionally. To template these cilia, each MCC produces between 30 and 500 basal bodies via a process termed centriole amplification. Much progress has been made in recent years in understanding the pathways involved in MCC fate determination, differentiation, and ciliogenesis. Recent studies using mammalian cell culture systems, mice, Xenopus, and other model organisms have started to uncover the mechanisms involved in centriole and cilia biogenesis. Yet, how MCC progenitor cells regulate the precise number of centrioles and cilia during their differentiation remains largely unknown. In this review, we will examine recent findings that address this fundamental question.


Assuntos
Centríolos , Cílios , Animais , Diferenciação Celular , Centríolos/metabolismo , Cílios/metabolismo , Mamíferos , Camundongos , Xenopus laevis/metabolismo
7.
Science ; 376(6598): eabm9326, 2022 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-35679401

RESUMO

INTRODUCTION The nuclear pore complex (NPC) is the molecular conduit in the nuclear membrane of eukaryotic cells that regulates import and export of biomolecules between the nucleus and the cytosol, with vertebrate NPCs ~110 to 125 MDa in molecular mass and ~120 nm in diameter. NPCs are organized into four main rings: the cytoplasmic ring (CR) at the cytosolic side, the inner ring and the luminal ring on the plane of the nuclear membrane, and the nuclear ring facing the nucleus. Each ring possesses an approximate eightfold symmetry and is composed of multiple copies of different nucleoporins. NPCs have been implicated in numerous biological processes, and their dysfunctions are associated with a growing number of serious human diseases. However, despite pioneering studies from many groups over the past two decades, we still lack a full understanding of NPCs' organization, dynamics, and complexity. RATIONALE We used the Xenopus laevis oocyte as a model system for the structural characterization because each oocyte possesses a large number of NPC particles that can be visualized on native nuclear membranes without the aid of detergent extraction. We used single-particle cryo-electron microscopy (cryo-EM) analysis on data collected at different stage tilt angles for three-dimensional reconstruction and structure prediction with AlphaFold for model building. RESULTS We reconstructed the CR map of X. laevis NPC at 6.9 and 6.7 Å resolutions for the full CR protomer and a core region, respectively, and predicted the structures of the individual nucleoporins using AlphaFold because no high-resolution models of X. laevis Nups were available. For any ambiguous subunit interactions, we also predicted complex structures, which further guided model fitting of the CR protomer. We placed the nucleoporin or complex structures into the CR density to obtain an almost full CR atomic model, composed of the inner and outer Y-complexes, two copies of Nup205, two copies of the Nup214-Nup88-Nup62 complex, one Nup155, and five copies of Nup358. In particular, we predicted the largest protein in the NPC, Nup358, as having an S-shaped globular domain, a coiled-coil domain, and a largely disordered C-terminal region containing phenylalanine-glycine (FG) repeats previously shown to form a gel-like condensate phase for selective cargo passage. Four of the Nup358 copies clamp around the inner and outer Y-complexes to stabilize the CR, and the fifth Nup358 situates in the center of the cluster of clamps. AlphaFold also predicted a homo-oligomeric, likely specifically pentameric, coiled-coil structure of Nup358 that may provide the avidity for Nup358 recruitment to the NPC and for lowering the threshold for Nup358 condensation in NPC biogenesis. CONCLUSION Our studies offer an example of integrative cryo-EM and structure prediction as a general approach for attaining more precise models of megadalton protein complexes from medium-resolution density maps. The more accurate and almost complete model of the CR presented here expands our understanding of the molecular interactions in the NPC and represents a substantial step forward toward the molecular architecture of a full NPC, with implications for NPC function, biogenesis, and regulation. [Figure: see text].


Assuntos
Inteligência Artificial , Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Proteínas de Xenopus , Animais , Microscopia Crioeletrônica , Citosol/metabolismo , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Oócitos , Conformação Proteica , Subunidades Proteicas/metabolismo , Software , Proteínas de Xenopus/química , Xenopus laevis/metabolismo
8.
Int J Mol Sci ; 23(11)2022 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-35682736

RESUMO

The α-, ß- and γ-synucleins are small soluble proteins expressed in the nervous system of mammals and evolutionary conserved in vertebrates. After being discovered in the cartilaginous fish Torpedo californica, synucleins have been sequenced in all vertebrates, showing differences in the number of genes and splicing isoforms in different taxa. Although α-, ß- and γ-synucleins share high homology in the N-terminal sequence, suggesting their evolution from a common ancestor, the three isoforms also differ in molecular characteristics, expression levels and tissue distribution. Moreover, their functions have yet to be fully understood. Great scientific interest on synucleins mainly derives from the involvement of α-synuclein in human neurodegenerative diseases, collectively named synucleinopathies, which involve the accumulation of amyloidogenic α-synuclein inclusions in neurons and glia cells. Studies on synucleinopathies can take advantage of the development of new vertebrate models other than mammals. Moreover, synuclein expression in non-mammalian vertebrates contribute to clarify the physiological role of these proteins in the evolutionary perspective. In this paper, gene expression levels of α-, ß- and γ-synucleins have been analysed in the main organs of adult Xenopus laevis by qRT-PCR. Moreover, recombinant α-, ß- and γ-synucleins were produced to test the specificity of commercial antibodies against α-synuclein used in Western blot and immunohistochemistry. Finally, the secondary structure of Xenopus synucleins was evaluated by circular dichroism analysis. Results indicate Xenopus as a good model for studying synucleinopathies, and provide a useful background for future studies on synuclein functions and their evolution in vertebrates.


Assuntos
Sinucleinopatias , alfa-Sinucleína , Animais , Mamíferos/metabolismo , Isoformas de Proteínas/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , beta-Sinucleína/genética , beta-Sinucleína/metabolismo , gama-Sinucleína/genética , gama-Sinucleína/metabolismo
9.
Mol Biol Evol ; 39(7)2022 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-35763822

RESUMO

Most vertebrate sex-determining genes (SDGs) emerge as neofunctionalized genes through duplication and/or mutation of ancestral genes that are involved with sexual differentiation. We previously demonstrated dm-W to be the SDG in the African clawed frog Xenopus laevis and found that a portion of this gene emerged from the masculinization gene dmrt1 after allotetraploidization by interspecific hybridization between two ancestral species around 17-18 Ma. dm-W has four exons consisting of a noncoding exon 1, dmrt1-derived exons 2 and 3, and an orphan exon 4 (Ex4) of unknown origin that includes coding sequence (CDS). In this study, we searched for the origin of Ex4 and investigated the function of the CDS of this exon. We found that the Ex4-CDS is derived from a noncoding portion of the hAT-10 family of DNA transposon. Evolutionary analysis of transposons and determination of the Ex4 sequences from three other species indicated that Ex4 was generated before the diversification of most or all extant allotetraploid species in subgenus Xenopus, during which time we hypothesize that transposase activity of this hAT superfamily was active. Using DNA-protein binding and transfection assays, we further demonstrate that the Ex4-encoded amino acid sequence increases the DNA-binding ability and transrepression activity of DM-W. These findings suggest that the conversion of the noncoding transposon sequence to the CDS of dm-W contributed to neofunctionalization of a new chimeric SDG in the ancestor of the allotetraploid Xenopus species, offering new insights into de novo origin and functional evolution of chimerical genes.


Assuntos
Elementos de DNA Transponíveis , Processos de Determinação Sexual , Animais , Elementos de DNA Transponíveis/genética , Cromossomos Sexuais , Processos de Determinação Sexual/genética , Fatores de Transcrição/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo
10.
Science ; 376(6598): eabl8280, 2022 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-35679404

RESUMO

INTRODUCTION The nuclear pore complex (NPC) resides on the nuclear envelope (NE) and mediates nucleocytoplasmic cargo transport. As one of the largest cellular machineries, a vertebrate NPC consists of cytoplasmic filaments, a cytoplasmic ring (CR), an inner ring, a nuclear ring, a nuclear basket, and a luminal ring. Each NPC has eight repeating subunits. Structure determination of NPC is a prerequisite for understanding its functional mechanism. In the past two decades, integrative modeling, which combines x-ray structures of individual nucleoporins and subcomplexes with cryo-electron tomography reconstructions, has played a crucial role in advancing our knowledge about the NPC. The CR has been a major focus of structural investigation. The CR subunit of human NPC was reconstructed by cryo-electron tomography through subtomogram averaging to an overall resolution of ~20 Å, with local resolution up to ~15 Å. Each CR subunit comprises two Y-shaped multicomponent complexes known as the inner and outer Y complexes. Eight inner and eight outer Y complexes assemble in a head-to-tail fashion to form the proximal and distal rings, respectively, constituting the CR scaffold. To achieve higher resolution of the CR, we used single-particle cryo-electron microscopy (cryo-EM) to image the intact NPC from the NE of Xenopus laevis oocytes. Reconstructions of the core region and the Nup358 region of the X. laevis CR subunit had been achieved at average resolutions of 5 to 8 Å, allowing identification of secondary structural elements. RATIONALE Packing interactions among the components of the CR subunit were poorly defined by all previous EM maps. Additional components of the CR subunit are strongly suggested by the EM maps of 5- to 8-Å resolution but remain to be identified. Addressing these issues requires improved resolution of the cryo-EM reconstruction. Therefore, we may need to enhance sample preparation, optimize image acquisition, and develop an effective data-processing strategy. RESULTS To reduce conformational heterogeneity of the sample, we spread the opened NE onto the grids with minimal force and used the chemical cross-linker glutaraldehyde to stabilize the NPC. To alleviate orientation bias of the NPC, we tilted sample grids and imaged the sample with higher electron dose at higher angles. We improved the image-processing protocol. With these efforts, the average resolutions for the core and the Nup358 regions have been improved to 3.7 and 4.7 Å, respectively. The highest local resolution of the core region reaches 3.3 Å. In addition, a cryo-EM structure of the N-terminal α-helical domain of Nup358 has been resolved at 3.0-Å resolution. These EM maps allow the identification of five copies of Nup358, two copies of Nup93, two copies of Nup205, and two copies of Y complexes in each CR subunit. Relying on the EM maps and facilitated by AlphaFold prediction, we have generated a final model for the CR of the X. laevis NPC. Our model of the CR subunit includes 19,037 amino acids in 30 nucleoporins. A previously unknown C-terminal fragment of Nup160 was found to constitute a key part of the vertex, in which the short arm, long arm, and stem of the Y complex meet. The Nup160 C-terminal fragment directly binds the ß-propeller proteins Seh1 and Sec13. Two Nup205 molecules, which do not contact each other, bind the inner and outer Y complexes through distinct interfaces. Conformational elasticity of the two Nup205 molecules may underlie their versatility in binding to different nucleoporins in the proximal and distal CR rings. Two Nup93 molecules, each comprising an N-terminal extended helix and an ACE1 domain, bridge the Y complexes and Nup205. Nup93 and Nup205 together play a critical role in mediating the contacts between neighboring CR subunits. Five Nup358 molecules, each in the shape of a shrimp tail and named "the clamp," hold the stems of both Y complexes. The innate conformational elasticity allows each Nup358 clamp to adapt to a distinct local environment for optimal interactions with neighboring nucleoporins. In each CR subunit, the α-helical nucleoporins appear to provide the conformational elasticity; the 12 ß-propellers may strengthen the scaffold. CONCLUSION Our EM map-based model of the X. laevis CR subunit substantially expands the molecular mass over the reported composite models of vertebrate CR subunit. In addition to the Y complexes, five Nup358, two Nup205, and two Nup93 molecules constitute the key components of the CR. The improved EM maps reveal insights into the interfaces among the nucleoporins of the CR. [Figure: see text].


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Proteínas de Xenopus , Xenopus laevis , Animais , Microscopia Crioeletrônica , Citoplasma/metabolismo , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Conformação Proteica , Proteínas de Xenopus/química , Xenopus laevis/metabolismo
11.
Mol Cell Proteomics ; 21(7): 100250, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35618225

RESUMO

As a key structural component of the chromatin of higher eukaryotes, linker histones (H1s) are involved in stabilizing the folding of extended nucleosome arrays into higher-order chromatin structures and function as a gene-specific regulator of transcription in vivo. The H1 C-terminal domain (CTD) is essential for high-affinity binding of linker histones to chromatin and stabilization of higher-order chromatin structure. Importantly, the H1 CTD is an intrinsically disordered domain that undergoes a drastic condensation upon binding to nucleosomes. Moreover, although phosphorylation is a prevalent post-translational modification within the H1 CTD, exactly where this modification is installed and how phosphorylation influences the structure of the H1 CTD remains unclear for many H1s. Using novel mass spectrometry techniques, we identified six phosphorylation sites within the CTD of the archetypal linker histone Xenopus H1.0. We then analyzed nucleosome-dependent CTD condensation and H1-dependent linker DNA organization for H1.0 in which the phosphorylated serine residues were replaced by glutamic acid residues (phosphomimics) in six independent mutants. We find that phosphomimetics at residues S117E, S155E, S181E, S188E, and S192E resulted in a significant reduction in nucleosome-bound H1.0 CTD condensation compared with unphosphorylated H1.0, whereas S130E did not alter CTD structure. Furthermore, we found distinct effects among the phosphomimetics on H1-dependent linker DNA trajectory, indicating unique mechanisms by which this modification can influence H1 CTD condensation. These results bring to light a novel role for linker histone phosphorylation in directly altering the structure of nucleosome-bound H1 and a potential novel mechanism for its effects on chromatin structure and function.


Assuntos
Histonas , Nucleossomos , Animais , Cromatina , DNA/química , Histonas/metabolismo , Fosforilação , Xenopus laevis/metabolismo
12.
J Vis Exp ; (182)2022 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-35532271

RESUMO

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.


Assuntos
Proteômica , Análise de Célula Única , Animais , Linhagem da Célula , Espectrometria de Massas , Proteoma/metabolismo , Proteômica/métodos , Análise de Célula Única/métodos , Xenopus laevis/metabolismo
13.
Dev Biol ; 488: 81-90, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35598626

RESUMO

Pre-placodal ectoderm (PPE), a horseshoe-shaped narrow region formed during early vertebrate development, gives rise to multiple types of sensory organs and ganglia. For PPE induction, a certain level of FGF signal activation is required. However, it is difficult to reproducibly induce the narrow region with variations in gene expression, including FGF, among individuals. An intracellular regulatory factor of FGF signaling, Dusp6, is expressed by FGF signal activation and inactivates a downstream regulator, ERK1/2, in adult tissues; however, its role in early development is not well known. Here, we reveal that Dusp6 is expressed in an FGF-dependent manner in Xenopus PPE. Gain- and loss-of-function experiments showed that Dusp6 is required for expression of a PPE gene, Six1, and patterning of adjacent regions, neural plate, and neural crest. To reveal the importance of Dusp6 in variable FGF production, we performed Dusp6 knockdown with FGF-bead implantation, which resulted in varying Six1 expression patterns. Taken together, these results suggest that Dusp6 is required for PPE formation and that it contributes to the robust patterning of PPE by mediating FGF signaling.


Assuntos
Ectoderma , Placa Neural , Animais , Fosfatase 6 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/metabolismo , Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Crista Neural/metabolismo , Placa Neural/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo
14.
Dev Growth Differ ; 64(5): 243-253, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35581155

RESUMO

Investigating cell lineage requires genetic tools that label cells in a temporal and tissue-specific manner. The bacteriophage-derived Cre-ERT2 /loxP system has been developed as a genetic tool for lineage tracing in many organisms. We recently reported a stable transgenic Xenopus line with a Cre-ERT2 /loxP system driven by the mouse Prrx1 (mPrrx1) enhancer to trace limb fibroblasts during the regeneration process (Prrx1:CreER line). Here we describe the detailed technological development and characterization of such line. Transgenic lines carrying a CAG promoter-driven Cre-ERT2 /loxP system showed conditional labeling of muscle, epidermal, and interstitial cells in both the tadpole tail and the froglet leg upon 4-hydroxytamoxifen (4OHT) treatment. We further improved the labeling efficiency in the Prrx1:CreER lines from 12.0% to 32.9% using the optimized 4OHT treatment regime. Careful histological examination showed that Prrx1:CreER lines also sparsely labeled cells in the brain, spinal cord, head dermis, and fibroblasts in the tail. This work provides the first demonstration of conditional, tissue-specific cell labeling with the Cre-ERT2 /loxP system in stable transgenic Xenopus lines.


Assuntos
Integrases , Animais , Animais Geneticamente Modificados , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Xenopus laevis/genética , Xenopus laevis/metabolismo
15.
Pflugers Arch ; 474(7): 681-697, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35525869

RESUMO

How phosphorylation of the epithelial sodium channel (ENaC) contributes to its regulation is incompletely understood. Previously, we demonstrated that in outside-out patches ENaC activation by serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) was abolished by mutating a serine residue in a putative SGK1 consensus motif RXRXX(S/T) in the channel's α-subunit (S621 in rat). Interestingly, this serine residue is followed by a highly conserved proline residue rather than by a hydrophobic amino acid thought to be required for a functional SGK1 consensus motif according to in vitro data. This suggests that this serine residue is a potential phosphorylation site for the dual-specificity tyrosine phosphorylated and regulated kinase 2 (DYRK2), a prototypical proline-directed kinase. Its phosphorylation may prime a highly conserved preceding serine residue (S617 in rat) to be phosphorylated by glycogen synthase kinase 3 ß (GSK3ß). Therefore, we investigated the effect of DYRK2 on ENaC activity in outside-out patches of Xenopus laevis oocytes heterologously expressing rat ENaC. DYRK2 included in the pipette solution significantly increased ENaC activity. In contrast, GSK3ß had an inhibitory effect. Replacing S621 in αENaC with alanine (S621A) abolished the effects of both kinases. A S617A mutation reduced the inhibitory effect of GKS3ß but did not prevent ENaC activation by DYRK2. Our findings suggest that phosphorylation of S621 activates ENaC and primes S617 for subsequent phosphorylation by GSK3ß resulting in channel inhibition. In proof-of-concept experiments, we demonstrated that DYRK2 can also stimulate ENaC currents in microdissected mouse distal nephron, whereas GSK3ß inhibits the currents.


Assuntos
Canais Epiteliais de Sódio , Proteínas Serina-Treonina Quinases , Animais , Canais Epiteliais de Sódio/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Oócitos/metabolismo , Fosforilação , Prolina/metabolismo , Ratos , Serina/metabolismo , Xenopus laevis/metabolismo
16.
J Headache Pain ; 23(1): 59, 2022 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-35614383

RESUMO

BACKGROUND: The clinical use of calcitonin gene-related peptide receptor (CGRP-R) antagonists and monoclonal antibodies against CGRP and CGRP-R has offered new treatment possibilities for migraine patients. CGRP activates both the CGRP-R and structurally related amylin 1 receptor (AMY1-R). The relative effect of erenumab and the small-molecule CGRP-R antagonist, rimegepant, towards the CGRP-R and AMY-R needs to be further characterized. METHODS: The effect of CGRP and two CGRP-R antagonists were examined in Xenopus laevis oocytes expressing human CGRP-R, human AMY1-R and their subunits. RESULTS: CGRP administered to receptor expressing oocytes induced a concentration-dependent increase in current with the order of potency CGRP-R> > AMY1-R > calcitonin receptor (CTR). There was no effect on single components of the CGRP-R; calcitonin receptor-like receptor and receptor activity-modifying protein 1. Amylin was only effective on AMY1-R and CTR. Inhibition potencies (pIC50 values) for erenumab on CGRP induced currents were 10.86 and 9.35 for CGRP-R and AMY1-R, respectively. Rimegepant inhibited CGRP induced currents with pIC50 values of 11.30 and 9.91 for CGRP-R and AMY1-R, respectively. CONCLUSION: Our results demonstrate that erenumab and rimegepant are potent antagonists of CGRP-R and AMY1-R with 32- and 25-times preference for the CGRP-R over the AMY1-R, respectively. It is discussed if this difference in affinity between the two receptors is the likely reason why constipation is a common and serious adverse effect during CGRP-R antagonism but less so with CGRP binding antibodies.


Assuntos
Anticorpos Monoclonais Humanizados , Peptídeo Relacionado com Gene de Calcitonina , Piperidinas , Piridinas , Receptores de Peptídeo Relacionado com o Gene de Calcitonina , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Oócitos/metabolismo , Piperidinas/farmacologia , Piridinas/farmacologia , Receptores da Calcitonina/química , Receptores da Calcitonina/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Xenopus laevis/metabolismo
17.
Hypertension ; 79(7): 1385-1394, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35510563

RESUMO

BACKGROUND: Elevated expression and increased activity of vascular epithelial sodium channel (ENaC) can result in vascular dysfunction in small animal models. However, there is limited or no knowledge on expression and function of ENaC channels in human vasculature. Hence, this study explored the expression and function of ENaC in human arteries and their association with hypertension. METHODS: Human internal mammary artery (IMA) and aorta were obtained from cardiovascular patients undergoing coronary artery bypass graft surgery. Expression of the ENaC subunit was analyzed by polymerase chain reaction, Western blot, and immunohistochemistry. ENaC function was observed by patch-clamp electrophysiology in endothelial cells isolated from IMA. Levels of ENaC subunit expression levels were compared between arteries from normotensive, uncontrolled hypertensive, and controlled hypertensive patients. RESULTS: For the first time, expression of α, ß, γ, and δ was detected at mRNA and protein levels in human IMA and aorta. Single-channel patch-clamp recordings identified both αßγ- and δßγ-like channel conductance in primary endothelial cells isolated and cultured from IMA. Reduced expression of the δ subunit was observed in controlled hypertensive IMA, whereas reduced expression of γ-ENaC was observed in controlled hypertensive aorta. CONCLUSIONS: These data suggest that functional ENaC channels are expressed in human arteries and their expression levels are associated with hypertension.


Assuntos
Canais Epiteliais de Sódio , Hipertensão , Animais , Artérias/metabolismo , Células Endoteliais/metabolismo , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Humanos , Hipertensão/genética , Xenopus laevis/metabolismo
18.
Molecules ; 27(10)2022 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-35630748

RESUMO

THz spectroscopy is important for the study of ion channels because it directly addresses the low frequency collective motions relevant for their function. Here we used THz spectroscopy to investigate the inhibition of the epithelial sodium channel (ENaC) by its specific blocker, amiloride. Experiments were performed on A6 cells' suspensions, which are cells overexpressing ENaC derived from Xenopus laevis kidney. THz spectra were investigated with or without amiloride. When ENaC was inhibited by amiloride, a substantial increase in THz absorption was noticed. Molecular modeling methods were used to explain the observed spectroscopic differences. THz spectra were simulated using the structural models of ENaC and ENaC-amiloride complexes built here. The agreement between the experiment and the simulations allowed us to validate the structural models and to describe the amiloride dynamics inside the channel pore. The amiloride binding site validated using THz spectroscopy agrees with previous mutagenesis studies. Altogether, our results show that THz spectroscopy can be successfully used to discriminate between native and inhibited ENaC channels and to characterize the dynamics of channels in the presence of their specific antagonist.


Assuntos
Amilorida , Canais Epiteliais de Sódio , Amilorida/metabolismo , Amilorida/farmacologia , Animais , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Oócitos/metabolismo , Análise Espectral , Xenopus laevis/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-35569782

RESUMO

Salinization and pollution are two main environmental stressors leading deterioration to water quality and degradation of aquatic ecosystems. Amphibians are a highly sensitive group of vertebrates to environmental disturbance of aquatic ecosystems. However, studies on the combined effect of salinization and pollution on the physiology of amphibians are limited. In this study, we measured the standard metabolic rate (SMR) and biochemical parameters of adult males of the invasive frog Xenopus laevis after 45 days of exposure to contrasting salinity environments (400 and 150 mOsm NaCl) with either 1.0 µg/L of the organophosphate pesticide chlorpyrifos (CPF) or pesticide-free medium. Our results revealed a decrease in SMR of animals exposed to the pesticide and in the ability to concentrate the plasma in animals exposed simultaneously to both stressors. The lack of ability to increase plasma concentration in animals exposed to both salt water and CPF, suggests that osmoregulatory response is decreased by pesticide exposure. In addition, we found an increase of liver citrate synthase activity in response to salt stress. Likewise, the liver acetylcholinesterase (AChE) activity decreased by 50% in frogs exposed to salt water and CPF and 40% in those exposed only to CPF, which suggest an additive effect of salinity on inhibition of AChE. Finally, oxidative stress increased as shown by the higher lipid peroxidation and concentration of aqueous peroxides found in the group exposed to salt water and pesticide. Thus, our results revealed that X. laevis physiology is compromised by salinization and pesticide exposure to both environmental stressors join.


Assuntos
Clorpirifos , Inseticidas , Praguicidas , Acetilcolinesterase/metabolismo , Animais , Clorpirifos/toxicidade , Ecossistema , Inseticidas/toxicidade , Praguicidas/toxicidade , Xenopus laevis/metabolismo
20.
Development ; 149(10)2022 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-35451459

RESUMO

Apical constriction, or a reduction in size of the apical domain, underlies many morphogenetic events during development. Actomyosin complexes play an essential role in apical constriction; however, the detailed analysis of molecular mechanisms is still pending. Here, we show that Lim domain only protein 7 (Lmo7), a multidomain adaptor at apical junctions, promotes apical constriction in the Xenopus superficial ectoderm, whereas apical domain size increases in Lmo7-depleted cells. Lmo7 is primarily localized at apical junctions and promotes the formation of the dense circumferential actomyosin belt. Strikingly, Lmo7 binds non-muscle myosin II (NMII) and recruits it to apical junctions and the apical cortex. This NMII recruitment is essential for Lmo7-mediated apical constriction. Lmo7 knockdown decreases NMIIA localization at apical junctions and delays neural tube closure in Xenopus embryos. Our findings suggest that Lmo7 serves as a scaffold that regulates actomyosin contractility and apical domain size.


Assuntos
Actomiosina , Ectoderma , Actomiosina/metabolismo , Animais , Ectoderma/metabolismo , Morfogênese/fisiologia , Cadeias Pesadas de Miosina , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Xenopus laevis/metabolismo
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