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Dietary intake of omega-3 polyunsaturated fatty acids (eicosapentaenoic acid, EPA) exerts antiarrhythmic effects, although the mechanisms are poorly understood. Here, we investigated the possible beneficial actions of EPA on saturated fatty acid-induced changes in the L-type Ca2+ channel in cardiomyocytes. Cardiomyocytes were cultured with an oleic acid/palmitic acid mixture (OAPA) in the presence or absence of EPA. Beating rate reduction in cardiomyocytes caused by OAPA were reversed by EPA. EPA also retrieved a reduction in Cav1.2 L-type Ca2+ current, mRNA, and protein caused by OAPA. Immunocytochemical analysis revealed a distinct downregulation of the Cav1.2 channel caused by OAPA with a concomitant decrease in the phosphorylated component of a transcription factor adenosine-3',5'-cyclic monophosphate (cAMP) response element binding protein (CREB) in the nucleus, which were rescued by EPA. A free fatty acid receptor 4 (FFAR4) agonist TUG-891 reversed expression of Cav1.2 and CREB mRNA caused by OAPA, whereas an FFAR4 antagonist AH-7614 abolished the effects of EPA. Excessive reactive oxygen species (ROS) accumulation caused by OAPA decreased Cav1.2 and CREB mRNA expressions, which was reversed by an ROS scavenger. Our data suggest that EPA rescues cellular Cav1.2-Ca2+ channel decline caused by OAPA lipotoxicity and oxidative stresses via both free fatty acid receptor 4-dependent and -independent pathways.
Assuntos
Canais de Cálcio Tipo L , Ácido Eicosapentaenoico , Miócitos Cardíacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Ácido Eicosapentaenoico/farmacologia , Animais , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/genética , Ratos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Ácidos Graxos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células CultivadasRESUMO
The endosomal-lysosomal system represents a crucial degradation pathway for various extracellular substances, and its dysfunction is linked to cardiovascular and neurodegenerative diseases. This degradation process involves multiple steps: (1) the uptake of extracellular molecules, (2) transport of cargos to lysosomes, and (3) digestion by lysosomal enzymes. While cellular uptake and lysosomal function are reportedly regulated by the mTORC1-TFEB axis, the key regulatory signal for cargo transport remains unclear. Notably, our previous study discovered that isorhamnetin, a dietary flavonoid, enhances endosomal-lysosomal proteolysis in the J774.1 cell line independently of the mTORC1-TFEB axis. This finding suggests the involvement of another signal in the mechanism of isorhamnetin. This study analyzes the molecular mechanism of isorhamnetin using transcriptome analysis and reveals that the transcription factor GATA3 plays a critical role in enhanced endosomal-lysosomal degradation. Our data also demonstrate that mTORC2 regulates GATA3 nuclear translocation, and the mTORC2-GATA3 axis alters endosomal formation and maturation, facilitating the efficient transport of cargos to lysosomes. This study suggests that the mTORC2-GATA3 axis might be a novel target for the degradation of abnormal substances.
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Taste recognition mediated by taste receptors is critical for the survival of animals in nature and is an important determinant of nutritional status and quality of life in humans. However, many factors including aging, diabetes, zinc deficiency, infection with influenza or cold viruses, and chemotherapy can trigger dysgeusia, for which a standard treatment has not been established. We here established an engineered strain of medaka (Oryzias latipes) that expresses green fluorescent protein (GFP) from the endogenous taste 1 receptor 3 (T1R3) gene locus with the use of the CRISPR-Cas9 system. This T1R3-GFP knock-in (KI) strain allows direct visualization of expression from this locus by monitoring of GFP fluorescence. The pattern of GFP expression in the T1R3-GFP KI fish thus mimicked that of endogenous T1R3 gene expression. Furthermore, exposure of T1R3-GFP KI medaka to water containing monosodium glutamate or the anticancer agent 5-fluorouracil resulted in an increase or decrease, respectively, in GFP fluorescence intensity, effects that also recapitulated those on T1R3 mRNA abundance. Finally, screening for agents that affect GFP fluorescence intensity in T1R3-GFP KI medaka identified tryptophan as an amino acid that increases T1R3 gene expression. The establishment of this screening system for taste receptor expression in medaka provides a new tool for the development of potential therapeutic agents for dysgeusia.
Assuntos
Oryzias , Animais , Sistemas CRISPR-Cas/genética , Disgeusia/genética , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Oryzias/genética , Qualidade de Vida , PaladarRESUMO
Immune checkpoint inhibitor (ICI) programmed death (PD)-1/PD-ligand 1 (PD-L1) blockade has been approved for various cancers. However, the underlying antitumor mechanisms mediated by ICIs and the predictive biomarkers remain unclear. We report the effects of anti-PD-L1/PD-1 Ab in tumor angiogenesis. In syngeneic mouse models, anti-PD-L1 Ab inhibited tumor angiogenesis and induces net-like hypoxia only in ICI-sensitive cell lines. In tumor tissue and serum of ICI-sensitive cell line-bearing mice, interferon-γ (IFN-γ) inducible angiostatic chemokines CXCL10/11 were upregulated by PD-L1 blockade. In vitro, CXCL10/11 gene upregulation by IFN-γ stimulation in tumor cell lines correlated with the sensitivity of PD-L1 blockade. The CXCL10/11 receptor CXCR3-neutralizing Ab or CXCL11 silencing in tumor cells inhibited the antiangiogenic effect of PD-L1 blockade in vivo. In pretreatment serum of lung carcinoma patients receiving anti-PD-1 Ab, the concentration of CXCL10/11 significantly correlated with the clinical outcome. Our results indicate the antiangiogenic function of PD-1/PD-L1 blockade and identify tumor-derived CXCL10/11 as a potential circulating biomarker of therapeutic sensitivity.
Assuntos
Antígeno B7-H1/metabolismo , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/metabolismo , Neoplasias Experimentais/metabolismo , Neovascularização Patológica/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Animais , Antígeno B7-H1/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Quimiocina CXCL10/genética , Quimiocina CXCL11/genética , Células HEK293 , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Interferon gama/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Nus , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Interferência de RNARESUMO
Transfer entropy in information theory was recently demonstrated [Basak et al., Phys. Rev. E 102, 012404 (2020)] to enable us to elucidate the interaction domain among interacting elements solely from an ensemble of trajectories. Therefore, only pairs of elements whose distances are shorter than some distance variable, termed cutoff distance, are taken into account in the computation of transfer entropies. The prediction performance in capturing the underlying interaction domain is subject to the noise level exerted on the elements and the sufficiency of statistics of the interaction events. In this paper, the dependence of the prediction performance is scrutinized systematically on noise level and the length of trajectories by using a modified Vicsek model. The larger the noise level and the shorter the time length of trajectories, the more the derivative of average transfer entropy fluctuates, which makes the identification of the interaction domain in terms of the position of global minimum of the derivative of average transfer entropy difficult. A measure to quantify the degree of strong convexity at the coarse-grained level is proposed. It is shown that the convexity score scheme can identify the interaction distance fairly well even while the position of the global minimum of the derivative of average transfer entropy does not. We also derive an analytical model to explain the relationship between the interaction domain and the change in transfer entropy that supports our cutoff distance technique to elucidate the underlying interaction domain from trajectories.
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DNA interstrand crosslinks (ICLs) are highly toxic lesions that stall the replication fork to initiate the repair process during the S phase of vertebrates. Proteins involved in Fanconi anemia (FA), nucleotide excision repair (NER), and translesion synthesis (TS) collaboratively lead to homologous recombination (HR) repair. However, it is not understood how ICL-induced HR repair is carried out and completed. Here, we showed that the replicative helicase-related Mcm family of proteins, Mcm8 and Mcm9, forms a complex required for HR repair induced by ICLs. Chicken DT40 cells lacking MCM8 or MCM9 are viable but highly sensitive to ICL-inducing agents, and exhibit more chromosome aberrations in the presence of mitomycin C compared with wild-type cells. During ICL repair, Mcm8 and Mcm9 form nuclear foci that partly colocalize with Rad51. Mcm8-9 works downstream of the FA and BRCA2/Rad51 pathways, and is required for HR that promotes sister chromatid exchanges, probably as a hexameric ATPase/helicase.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Reparo de DNA por Recombinação/genética , Adenosina Trifosfatases/metabolismo , Animais , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Galinhas , Aberrações Cromossômicas/efeitos dos fármacos , DNA/genética , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/genética , Mitomicina/farmacologia , Dados de Sequência Molecular , Troca de Cromátide Irmã/efeitos dos fármacos , Troca de Cromátide Irmã/genéticaRESUMO
BACKGROUND: Social amoeba, Dictyostelium discoideum, is a well-established model organism for studying cellular physiology and developmental pattern formation. Its haploid genome facilitates functional analysis of genes by a single round of mutagenesis including targeted disruption. Although the efficient generation of knockout strains based on an intrinsically high homologous recombination rate has been demonstrated, successful reports for knockin strains have been limited. As social amoeba has an exceptionally high adenine and thymine (A/T)-content, conventional plasmid-based vector construction has been constrained due to deleterious deletion in E. coli. RESULTS: We describe here a simple and efficient strategy to construct GFP-knockin cassettes by using a linear DNA cloning vector derived from N15 bacteriophage. This allows reliable handling of DNA fragments whose A/T-content may be as high as 85 %, and which cannot be cloned into a circular plasmid. By optimizing the length of recombination arms, we successfully generate GFP-knockin strains for five genes involved in cAMP signalling, including a triple-colour knockin strain. CONCLUSIONS: This robust strategy would be useful in handling DNA fragments with biased A/T-contents such as the genome of lower organisms and the promoter/terminator regions of higher organisms.
Assuntos
Sequência Rica em At/genética , DNA de Protozoário/química , DNA de Protozoário/genética , Dictyostelium/genética , Técnicas de Introdução de Genes/métodos , Genoma/genética , Adenina , Clonagem Molecular , DNA de Protozoário/isolamento & purificação , Dictyostelium/citologia , TiminaRESUMO
Genetically encoded Ca2+ indicators (GECIs) are versatile for live imaging of cellular activities. Besides the brightness and dynamic range of signal change of GECIs, Ca2+ affinity is another critical parameter for successful Ca2+ imaging, as the concentration range of Ca2+ dynamics differs from low nanomolar to sub-millimolar depending on the celltype and organism. However, ultrahigh-affinity GECIs, particularly the single fluorescent protein (1FP)-type, are lacking. Here, we report a simple strategy that increases Ca2+ affinity through the linker length optimization in topology mutants of existing 1FP-type GECIs. The resulting ultrahigh-affinity GECIs, CaMPARI-nano, BGECO-nano, and RCaMP-nano (Kd = 17-25 nM), enable unique biological applications, including the detection of low nanomolar Ca2+ dynamics, highlighting active signaling cells, and multi-functional imaging with other second messengers. The linker length optimization in topology mutants could be applied to other 1FP-type indicators of glutamate and potassium, rendering it a widely applicable technique for modulating indicator affinity.
Assuntos
Cálcio , Proteínas Luminescentes , Mutação , Cálcio/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Luminescentes/química , Células HEK293RESUMO
We report ultrasensitive Ca(2+) indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca(2+)-sensing domain of a genetically encoded Ca(2+) indicator, YC2.60 or YC3.60. Their high Ca(2+) affinities (K(d) = 15-140 nM) and large signal change (1,450%) enabled detection of subtle Ca(2+) transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multicellular networks.
Assuntos
Cálcio/análise , Animais , Cálcio/metabolismo , Dictyostelium , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Indicadores e Reagentes/análise , Indicadores e Reagentes/química , Camundongos , Dados de Sequência Molecular , Neurônios/metabolismo , Transdução de Sinais , Peixe-ZebraRESUMO
Advance in the live imaging technology revealed that cells display a variety of Ca(2 +) dynamics, such as the wave, oscillation, blip, puff and spark. Accumulating evidences suggest that not only Ca(2 +) itself, but its spatio-temporal dynamics are the important information carrier in the physiological responses. Here, we will introduce the pattern of Ca(2 +) dynamics along with the latest version of high-performance Ca(2 +) probes.
Assuntos
Cálcio/análise , Citosol/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismo , Sobrevivência Celular , HumanosRESUMO
Collective migration of cells is a fundamental behavior in biology. For the quantitative understanding of collective cell migration, live-cell imaging techniques have been used using e.g., phase contrast or fluorescence images. Particle tracking velocimetry (PTV) is a common recipe to quantify cell motility with those image data. However, the precise tracking of cells is not always feasible. Particle image velocimetry (PIV) is an alternative to PTV, corresponding to Eulerian picture of fluid dynamics, which derives the average velocity vector of an aggregate of cells. However, the accuracy of PIV in capturing the underlying cell motility and what values of the parameters should be chosen is not necessarily well characterized, especially for cells that do not adhere to a viscous flow. Here, we investigate the accuracy of PIV by generating images of simulated cells by the Vicsek model using trajectory data of agents at different noise levels. It was found, using an alignment score, that the direction of the PIV vectors coincides with the direction of nearby agents with appropriate choices of PIV parameters. PIV is found to accurately measure the underlying motion of individual agents for a wide range of noise level, and its condition is addressed.
Assuntos
Hidrodinâmica , Reologia/métodos , Movimento Celular , Velocidade do Fluxo SanguíneoRESUMO
We report a pH-insensitive and photostable ultramarine fluorescent protein, Sirius, with an emission peak at 424 nm, the shortest emission wavelength among fluorescent proteins reported to date. The pH-insensitivity of Sirius allowed prolonged visualization of biological events in an acidic environment. Two fluorescence resonance energy transfer (FRET) pairs, Sirius-mseCFP and Sapphire-DsRed, allowed dual-FRET imaging with single-wavelength excitation, enabling detection of Ca(2+) concentration and caspase-3 activation in the same apoptotic cells.
Assuntos
Proteínas Luminescentes/química , Substituição de Aminoácidos , Animais , Apoptose , Sinalização do Cálcio , Caspase 3/metabolismo , Cor , Dictyostelium/microbiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Mutagênese , Fagocitose , Fotodegradação , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , TransfecçãoRESUMO
Periodic somite segmentation in vertebrate embryos is controlled by the 'segmentation clock', which consists of numerous cellular oscillators. Although the properties of a single oscillator, driven by a hairy negative-feedback loop, have been investigated, the system-level properties of the segmentation clock remain largely unknown. To explore these characteristics, we have examined the response of a normally oscillating clock in zebrafish to experimental stimuli using in vivo mosaic experiments and mathematical simulation. We demonstrate that the segmentation clock behaves as a coupled oscillator, by showing that Notch-dependent intercellular communication, the activity of which is regulated by the internal hairy oscillator, couples neighbouring cells to facilitate synchronized oscillation. Furthermore, the oscillation phase of individual oscillators fluctuates due to developmental noise such as stochastic gene expression and active cell proliferation. The intercellular coupling was found to have a crucial role in minimizing the effects of this noise to maintain coherent oscillation.
Assuntos
Relógios Biológicos/fisiologia , Padronização Corporal/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Mitose , Modelos Biológicos , Receptores Notch/metabolismo , Transdução de Sinais , Somitos/citologia , Somitos/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Bioluminescence imaging of cellular function is a promising strategy. It has advantages over fluorescence imaging such as high sensitivity, no phototoxicity or no autofluorescence, and compatibility to deep-tissue imaging or optogenetics. However, functional imaging of cellular signaling by bioluminescence is not so easy due to the limited availability of bright bioluminescent indicators.Here we describe a detailed strategy to detect cellular cAMP dynamics by using Nano-lantern (cAMP1.6), one of the brightest bioluminescent indicator for cAMP . Both induced and spontaneous cAMP signaling in social amoeba, with a large and small signal change, respectively, were imaged by this method.
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Cálcio , Optogenética , Proteínas Luminescentes/genética , Imagem Óptica , Optogenética/métodosRESUMO
Longitudinal bone growth is achieved by a tightly controlled process termed endochondral bone formation. C-type natriuretic peptide (CNP) stimulates endochondral bone formation through binding to its specific receptor, guanylyl cyclase (GC)-B. However, CNP/GC-B signaling dynamics in different stages of endochondral bone formation have not been fully clarified, especially in terms of the interaction between the cyclic guanine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) pathways. Here, we demonstrated that CNP activates the cAMP/protein kinase A (PKA) pathway and that this activation contributed to the elongation of the hypertrophic zone in the growth plate. Cells of the chondrogenic line ATDC5 were transfected with Förster resonance energy transfer (FRET)-based cGMP and PKA biosensors. Dual-FRET imaging revealed that CNP increased intracellular cGMP levels and PKA activities in chondrocytes. Further, CNP-induced PKA activation was enhanced following differentiation of ATDC5 cells. Live imaging of the fetal growth plate of transgenic mice, expressing a FRET biosensor for PKA, PKAchu mice, showed that CNP predominantly activates the PKA in the hypertrophic chondrocytes. Additionally, histological analysis of the growth plate of PKAchu mice demonstrated that CNP increased the length of the growth plate, but coadministration of a PKA inhibitor, H89, inhibited the growth-promoting effect of CNP only in the hypertrophic zone. In summary, we revealed that CNP-induced cGMP elevation activated the cAMP/PKA pathway, and clarified that this PKA activation contributed to the bone growth-promoting effect of CNP in hypertrophic chondrocytes. These results provide insights regarding the cross-talk between cGMP and cAMP signaling in endochondral bone formation and in the physiological role of the CNP/GC-B system.
Assuntos
Condrócitos/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peptídeo Natriurético Tipo C/farmacologia , Osteogênese/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , GMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Lâmina de Crescimento/crescimento & desenvolvimento , Camundongos , Camundongos Transgênicos , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Synchronized movement of (both unicellular and multicellular) systems can be observed almost everywhere. Understanding of how organisms are regulated to synchronized behavior is one of the challenging issues in the field of collective motion. It is hypothesized that one or a few agents in a group regulate(s) the dynamics of the whole collective, known as leader(s). The identification of the leader (influential) agent(s) is very crucial. This article reviews different mathematical models that represent different types of leadership. We focus on the improvement of the leader-follower classification problem. It was found using a simulation model that the use of interaction domain information significantly improves the leader-follower classification ability using both linear schemes and information-theoretic schemes for quantifying influence. This article also reviews different schemes that can be used to identify the interaction domain using the motion data of agents.
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We designed a new caging group that can be photoactivated only in the presence of a non-endogenous enzyme when exposed to 405 nm light. Because cells or tissues can be genetically tagged by an exogenously expressed enzyme, this novel method can serve as a strategy for adding targeting abilities to photocaged compounds.
Assuntos
Nucleotídeos Cíclicos/síntese química , Células HeLa , Humanos , Luz , Estrutura Molecular , Nucleotídeos Cíclicos/química , Nucleotídeos Cíclicos/genética , Processos Fotoquímicos , Células Tumorais CultivadasRESUMO
Elongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid-liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.
Assuntos
Endossomos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endocitose/fisiologia , Endossomos/fisiologia , Células HEK293 , Células HeLa , Humanos , Proteínas dos Microfilamentos/fisiologia , Ligação Proteica/fisiologia , Transporte Proteico/fisiologia , Junções Íntimas/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
HIV-1 infectivity is achieved through virion maturation. Virus particles undergo structural changes via cleavage of the Gag polyprotein mediated by the viral protease, causing the transition from an uninfectious to an infectious status. The majority of proviruses in people living with HIV-1 treated with combination antiretroviral therapy are defective with large internal deletions. Defective proviral DNA frequently preserves intact sequences capable of expressing viral structural proteins to form virus-like particles whose maturation status is an important factor for chronic antigen-mediated immune stimulation and inflammation. Thus, novel methods to study the maturation capability of defective virus particles are needed to characterize their immunogenicity. To build a quantitative tool to study virion maturation in vitro, we developed a novel single virion visualization technique based on fluorescence resonance energy transfer (FRET). We inserted an optimized intramolecular CFP-YPF FRET donor-acceptor pair bridged with an HIV-1 protease cleavage sequence between the Gag MA-CA domains. This system allowed us to microscopically distinguish mature and immature virions via their FRET signal when the FRET donor and acceptor proteins were separated by the viral protease during maturation. We found that approximately 80% of the FRET labeled virus particles were mature with equivalent infectivity to wild type. The proportion of immature virions was increased by treatment of virus producer cells with a protease inhibitor in a dose-dependent manner, which corresponded to a relative decrease in infectivity. Potential areas of application for this tool are assessing maturation efficiency in different cell type settings of intact or deficient proviral DNA integrated cells. We believe that this FRET-based single-virion imaging platform will facilitate estimating the impact on the immune system of both extracellular intact and defective viruses by quantifying the Gag maturation status.
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Auditory perception in vertebrates depends on transduction of sound into neural signals in the inner ear hair cells (HCs) and on transmission of these signals to the brain through auditory (VIIIth) nerve afferents. To investigate the developmental acquisition of auditory inputs by the CNS, we have electrophysiologically and morphologically examined the process of acquisition of auditory responsiveness by zebrafish macular HCs and the Mauthner cells (M-cells) in vivo. The M-cells are a paired large reticulospinal neurons in the hindbrain; they receive direct inputs from the VIIIth nerve afferents and initiate an acoustic startle response. Whole-cell recordings from the M-cells showed that sound-evoked postsynaptic currents were first observed around 40 h postfertilization (hpf); during subsequent development, onset latency decreased and amplitude increased. The appearance and development of microphonic potentials in the inner ear coincided with those of the acoustic responses of the M-cell, whereas the functional auditory circuits from the macular HCs to the M-cell were already formed at 27 hpf. These results suggest that the functional maturation of inner ear after formation of the auditory pathway is a critical process in the acquisition of auditory inputs by CNS neurons.