RESUMO
Actin is a major structural component of the cytoskeleton in eukaryotic cells, including fungi, plants, and animals, and exists not only in the cytoplasm as cytoskeleton but also in the nucleus. Recently, we developed a novel actin probe, ß-actin-EGFP fusion protein, which exhibited similar monomeric to filamentous ratio as that of endogenous actin, in contrast to the widely used EGFP-ß-actin fusion protein that over-assembles in cells. Unexpectedly, this novel probe visualized an interconnected meshwork of slightly curved beam-like bundles of actin filaments in the nucleus of U2OS cells. These structures were not labeled with rhodamine phalloidin, Lifeact-EGFP or anti-actin antibodies. In addition, immunofluorescence staining and expression of cofilin-EGFP revealed that this nuclear actin structures contained cofilin. We named these actin filaments as phalloidin-negative intranuclear (PHANIN) actin filaments. Since PHANIN actin filaments could not be detected by general detection methods for actin filaments, we propose that PHANIN actin filaments are different from previously reported nuclear actin structures.
Assuntos
Citoesqueleto de Actina , Actinas , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Faloidina/análise , Faloidina/metabolismoRESUMO
Colorectal cancer (CRC) is a heterogenous disease, and patients have differences in therapeutic response. However, the mechanisms underlying interpatient heterogeneity in the response to chemotherapeutic agents remain to be elucidated, and molecular tumor characteristics are required to select patients for specific therapies. Patient-derived organoids (PDOs) established from CRCs recapitulate various biological characteristics of tumor tissues, including cellular heterogeneity and the response to chemotherapy. Patient-derived organoids established from CRCs show various morphologies, but there are no criteria for defining these morphologies, which hampers the analysis of their biological significance. Here, we developed an artificial intelligence (AI)-based classifier to categorize PDOs based on microscopic images according to their similarity in appearance and classified tubular adenocarcinoma-derived PDOs into six types. Transcriptome analysis identified differential expression of genes related to cell adhesion in some of the morphological types. Genes involved in ribosome biogenesis were also differentially expressed and were most highly expressed in morphological types showing CRC stem cell properties. We identified an RNA polymerase I inhibitor, CX-5641, to be an upstream regulator of these type-specific gene sets. Notably, PDO types with increased expression of genes involved in ribosome biogenesis were resistant to CX-5461 treatment. Taken together, these results uncover the biological significance of the morphology of PDOs and provide novel indicators by which to categorize CRCs. Therefore, the AI-based classifier is a useful tool to support PDO-based cancer research.
Assuntos
Adenocarcinoma , Antineoplásicos , Neoplasias Colorretais , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Inteligência Artificial , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Organoides/metabolismoRESUMO
A cryoprotectant known as ice-binding protein (IBP) is thought to facilitate the cold survival of plants, insects, and fungi. Here, we prepared a genetically modified Caenorhabditis elegans strain to synthesize fish-derived IBPs in its body wall muscles and examined whether the antifreeze activity modification of this IBP by point mutation affects the cold tolerance of this worm. We chose a 65-residue IBP identified from notched-fin eelpout, for which the replacement of the 20th alanine residue (A20) modifies its antifreeze activity. These mutant proteins are denoted A20L, A20G, A20T, A20V, and A20I along with the wild-type (WT) protein. We evaluated the survival rate (%) of the transgenic C. elegans that synthesized each IBP mutant following 24 h of preservation at -5, +2, and +5 °C. Significantly, a dramatic improvement in the survival rate was detected for the worms synthesizing the activity-enhanced mutants (A20T and A20I), especially at +2 °C. In contrast, the rate was not improved by the expression of the defective mutants (A20L, A20G, WT and A20V). The survival rate (%) probably correlates with the antifreeze activity of the IBP. These data suggest that IBP protects the cell membrane by employing its ice-binding mechanism, which ultimately improves the cold tolerance of an IBP-containing animal.
Assuntos
Proteínas Anticongelantes , Gelo , Animais , Alanina/genética , Proteínas Anticongelantes/química , Proteínas Anticongelantes/genética , Proteínas Anticongelantes/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Peixes/genética , Congelamento , Proteínas Mutantes/metabolismo , MutaçãoRESUMO
α-Synuclein is a major component of Lewy bodies and Lewy neuritis which are hallmarks of Parkinson's disease, and is known to propagate from cell-to-cell in a prion-like manner. However, the exact mechanism of α-synuclein propagation in cells remains unclear. Despite the increasing number of studies and models of α-synuclein propagation, there is no direct evidence demonstrating whether the propagation is trans-synaptic or synaptic connection-independent, what the direction of propagation is, and what the regulators of α-synuclein propagation are. In this study, we generated a Caenorhabditis elegans model that can help monitoring the neuron-to-neuron propagation of α-synuclein using BiFC system. Using this model, we demonstrated that α-synuclein was propagated into neurons in both anterograde and retrograde manners, with retrograde propagation being dominant. Interestingly, we also found that endophilin, which is a protein required for classical clathrin-mediated endocytic machinery, was not involved in this retrograde propagation. Furthermore, we demonstrated that α-synuclein inhibits neuronal activity through voltage-gated calcium channels. Our findings suggest a possible mechanism for α-synuclein propagation via synapses through a novel uptake pathway.
Assuntos
Aciltransferases/metabolismo , Caenorhabditis elegans/metabolismo , Endocitose , Neurônios/metabolismo , Sinapses/metabolismo , alfa-Sinucleína/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cálcio/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Expressão Gênica , Humanos , Microscopia Confocal , Mutação , Vesículas Sinápticas/metabolismo , Fatores de Tempo , alfa-Sinucleína/genéticaRESUMO
The heterozygous deletion of 15q13.3 is a recurrently observed microdeletion syndrome associated with a relatively mild phenotype including learning disability and language impairment. In contrast, the homozygous deletion of 15q13.3 is extremely rare and is associated with a much severer phenotype that includes epileptic encephalopathy, profound intellectual disability, and hypotonia. Which of the genes within the deleted interval is responsible for the more severe features when biallelically deleted is currently unknown. Here, we report a patient with profound hypotonia, severe intellectual disability, and seizures who had biallelic loss-of-function variants in OTUD7A: a 15q13.3 deletion including the OTUD7A locus, and a frameshift OTUD7A variant c.1125del, p.(Glu375Aspfs*11). Unexpectedly, both aberrations occurred de novo. Our experiment using Caenorhabditis elegans showed that worms carrying a corresponding homozygous variant in the homolog OTUB-2 exhibited weakened muscle contraction suggestive of aberrant neuromuscular transmission. We concluded that the biallelic complete loss of OTUD7A in humans represents a presumably new autosomal recessive disorder characterized by profound hypotonia, severe intellectual disability, and seizures.
Assuntos
Enzimas Desubiquitinantes/genética , Deficiência Intelectual/genética , Hipotonia Muscular/genética , Doenças da Junção Neuromuscular/embriologia , Animais , Caenorhabditis elegans/genética , Pré-Escolar , Mutação da Fase de Leitura/genética , Homozigoto , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/fisiopatologia , Perda de Heterozigosidade/genética , Masculino , Contração Muscular/genética , Contração Muscular/fisiologia , Hipotonia Muscular/fisiopatologia , Doenças da Junção Neuromuscular/complicações , Doenças da Junção Neuromuscular/genética , Doenças da Junção Neuromuscular/fisiopatologia , Convulsões/complicações , Convulsões/genética , Convulsões/fisiopatologia , Tioléster Hidrolases/genéticaRESUMO
To treat malignant glioma, standard fractionated radiotherapy (RT; 60 Gy/30 fractions over 6 weeks) was performed post-surgery in combination with temozolomide to improve overall survival. Malignant glioblastoma recurrence rate is extremely high, and most recurrent tumors originate from the excision cavity in the high-dose irradiation region. In our previous study, protoporphyrin IX physicochemically enhanced reactive oxygen species generation by ionizing radiation and combined treatment with 5-aminolevulinic acid (5-ALA) and ionizing radiation, while radiodynamic therapy (RDT) improved tumor growth suppression in vivo in a melanoma mouse model. We examined the effect of 5-ALA RDT on the standard fractionated RT protocol using U251MG- or U87MG-bearing mice. 5-ALA was orally administered at 60 or 120 mg/kg, 4 h prior to irradiation. In both models, combined treatment with 5-ALA slowed tumor progression and promoted regression compared to treatment with ionizing radiation alone. The standard fractionated RT protocol of 60 Gy in 30 fractions with oral administration of 120 and 240 mg/kg 5-ALA, the human equivalent dose of photodynamic diagnosis, revealed no significant increase in toxicity to normal skin or brain tissue compared to ionizing radiation alone. Thus, RDT is expected to enhance RT treatment of glioblastoma without severe toxicity under clinically feasible conditions.
Assuntos
Ácido Aminolevulínico/farmacologia , Fracionamento da Dose de Radiação , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Radiação Ionizante , Radioterapia , Ácido Aminolevulínico/administração & dosagem , Ácido Aminolevulínico/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Glioblastoma/terapia , Humanos , Camundongos , Fotoquimioterapia/efeitos adversos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/efeitos adversos , Radioterapia/métodos , Raios X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In most animals, avoiding pathogenic bacteria is crucial for better health and a long life span. For this purpose, animals should be able to quickly sense the presence or uptake of pathogens. The intestine could be a candidate organ to induce escape behaviors; however, the intestinal signaling mechanism for acute regulation of neuronal activity is not well understood. Here, we show that adult Caenorhabditis elegans can respond to the pathogenic bacterium Pseudomonas aeruginosa within 30 min of exposure. This behavior was much faster than previously observed avoidance behaviors in response to P. aeruginosa. By genetic screening, we isolated a mutant defective in this quick avoidance behavior and found that the novel F-box protein FBXC-58 is involved. FBXC-58 is expressed in several tissues, but defective avoidance was rescued by expression of the protein in the intestine. Interestingly, we also found that some but not all mutants in the p38-MAPK and insulin-like signaling pathways, which function in the immune response to pathogens in the intestine, were defective in the quick avoidance behavior to P. aeruginosa. These results suggest that a novel signaling pathway in the intestine exists to regulate neuronal activity for a quick behavioral response.
Assuntos
Aprendizagem da Esquiva , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas F-Box/metabolismo , Mucosa Intestinal/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas F-Box/genética , Mucosa Intestinal/microbiologia , Neurônios/metabolismo , Pseudomonas aeruginosa/patogenicidade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Both fluorescent and luminescent observation are widely used to examine real-time gene expression patterns in living organisms. Several fluuorescent and luminescent proteins with specific optical properties have been developed and applied for simultaneous, multi-color observation of more than two gene expression profiles. Compared to fluorescent proteins, however, the application of multi-color luminescent imaging in living organisms is still limited. In this study, we introduced two-color luciferases into the soil nematode C. elegans and performed simultaneous analysis of two gene expression profiles. Using a green-emitting luciferase Eluc (emerald luciferase) and red-emitting luciferase SLR (stable luciferase red), the expression patterns of two genes were simultaneously observed in single animals from embryonic to adult stages over its whole life span. In addition, dual gene activities were observed at the single embryo level, with the simultaneous observation of morphological changes. These are the first application of a two-color luciferase system into a whole animal and suggest that precise relationship of expression patterns of multiple genes of interest can be analyzed over the whole life of the animal, dependent on the changes in genetic and/or environmental conditions.
Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Perfilação da Expressão Gênica , Luciferases/metabolismo , Animais , Animais Geneticamente Modificados , Cor , Corantes Fluorescentes , Regulação da Expressão Gênica , Luminescência , Medições Luminescentes/métodos , Proteínas Luminescentes/genética , Regiões Promotoras GenéticasRESUMO
Recent reports have suggested that 5-aminolevulinic acid (5-ALA), which is a precursor to protoporphyrin IX (PpIX), leads to selective accumulation of PpIX in tumor cells and acts as a radiation sensitizer in vitro and in vivo in mouse models of melanoma, glioma, and colon cancer. In this study, we investigated the effect of PpIX under X-ray irradiation through ROS generation and DNA damage. ROS generation by the interaction between PpIX and X-ray was evaluated by two kinds of probes, 3'-(p-aminophenyl) fluorescein (APF) for hydroxyl radical (â¢OH) detection and dihydroethidium (DHE) for superoxide (O2â¢-). â¢OH showed an increase, regardless of the dissolved oxygen. Meanwhile, the increase in O2â¢- was proportional to the dissolved oxygen. Strand breaks (SBs) of DNA molecule were evaluated by gel electrophoresis, and the enhancement of SBs was observed by PpIX treatment. We also studied the effect of PpIX for DNA damage in cells by X-ray irradiation using a B16 melanoma culture. X-ray irradiation induced γH2AX, DNA double-strand breaks (DSBs) in the context of chromatin, and affected cell survival. Since PpIX can enhance ROS generation even in a hypoxic state and induce DNA damage, combined radiotherapy treatment with 5-ALA is expected to improve therapeutic efficacy for radioresistant tumors.
Assuntos
Quebras de DNA de Cadeia Dupla , Melanoma/metabolismo , Protoporfirinas/metabolismo , Radiossensibilizantes/metabolismo , Ácido Aminolevulínico/metabolismo , Animais , Linhagem Celular Tumoral , Melanoma/genética , Melanoma/radioterapia , Camundongos , Protoporfirinas/efeitos da radiação , Radiossensibilizantes/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Terapia por Raios X/métodos , Raios XRESUMO
Cell migration and axon guidance require proper regulation of the actin cytoskeleton in response to extracellular guidance cues. Rho/Rac small GTPases are essential regulators of actin remodeling. Caenorhabditis elegans CED-10 is a Rac1 homolog that is required for various cellular morphological changes and migration events and is under the control of several guidance signaling pathways. There is still considerable uncertainty regarding events following the activation of guidance receptors by extracellular signals and the regulation of actin dynamics based on spatiotemporally restricted Rac activity. Here we show that the VPS9 domain protein RIN-1 acts as a novel effector for CED-10 in C. elegans. The orthologous mammalian Rin1 protein has previously been identified as an effector for Ras GTPase and is now known to function as a guanine nucleotide exchange factor for Rab5 GTPase. We found that RIN-1 specifically binds to the GTP-bound form of CED-10 and that mutations in rin-1 cause significant defects in migration and axon guidance of restricted neuronal cell types including AVM and HSN neurons, in contrast to the various defects observed in ced-10 mutants. Our analyses place RIN-1 in the Slit-Robo genetic pathway that regulates repulsive signaling for dorsoventral axon guidance. In rin-1 mutants, actin accumulated on both the ventral and dorsal sides of the developing HSN neuron, in contrast to its ventral accumulation in wild type. These results strongly suggest that RIN-1 acts as an effector for CED-10/Rac1 and regulates actin remodeling in response to restricted guidance cues.
Assuntos
Axônios/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Movimento Celular , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Clonagem Molecular , Dados de Sequência Molecular , Mutação , Ligação Proteica , Mapeamento de Interação de Proteínas , Transporte Proteico , Pseudópodes/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/genéticaRESUMO
Stimulating cells by light is an attractive technology to investigate cellular function and deliver innovative cell-based therapy. However, current techniques generally use poorly biopermeable light, which prevents broad applicability. Here, we show that a new type of composite nanomaterial, synthesized from multi-walled carbon nanotubes, magnetic iron nanoparticles, and polyglycerol, enables photothermal and mechanical control of Ca2+ influx into cells overexpressing transient receptor potential vanilloid type-2. The nanohybrid is simply operated by application of highly biotransparent near-infrared light and a magnetic field. The technology may revolutionize remote control of cellular function.
Assuntos
Nanotubos de Carbono , Raios Infravermelhos , Magnetismo , Nanopartículas de Magnetita , NanoestruturasRESUMO
A supramolecular nanohybrid based on carbon nanotubes and liposomes that is highly biocompatible and capable of permeation through cells is described. The nanohybrid can be loaded with a variety of functional molecules and is structurally controlled by near-infrared laser irradiation for the release of molecules from the nanohybrids in a targeted manner via microscopy. We implemented the controlled release of molecules from the nanohybrids and demonstrated remote regulation of the photoinduced nanohybrid functions. As a proof of principle, nanohybrids loaded with amiloride were successfully used in the spatiotemporally targeted blocking of amiloride-sensitive mechanosensory neurons in living Caenorhabditis elegans. Our prototype could inspire new designs for biomimetic parasitism and symbiosis, and biologically active nanorobots for the higher-level manipulation of organisms.
Assuntos
Caenorhabditis elegans/metabolismo , Lipossomos/metabolismo , Nanoestruturas/química , Nanotubos de Carbono/química , Animais , Células HeLa , Humanos , Lipossomos/químicaRESUMO
Recombinant immunoglobulin G (IgG) aggregates are formed during their production. However, the process underlying intracellular/extracellular aggregation in cell culture conditions is not well understood, and no effective method exists to assess IgG aggregates. Here, we establish an approach to detect intracellular aggregates using AF.2A1, a small artificial protein that binds to non-native IgG conformers and aggregates. Fluorescent-labeled AF.2A1 is prepared via conjugation and transfected into antibody-producing Chinese hamster ovary (CHO) cells. Micrographic images show intracellular IgG aggregates in CHO cells. The relative amount of intracellular aggregates (versus total intracellular IgG) differed depending on the type of additives used during cell culture. Interestingly, the relative amount of intracellular aggregates moderately correlates with that of in vitro extracellular IgG aggregates, suggesting they are secreted. This method will allow the investigation of antibody aggregation in cells, and may guide the production of therapeutic antibodies with high yield/quality.
Assuntos
Imunoglobulina G/análise , Animais , Células CHO , Cricetulus , Imunoglobulina G/metabolismo , Agregados Proteicos , Ligação Proteica , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismoRESUMO
Ultraviolet light is quite toxic to all the animals and evoke the avoidance behavior of UV. The soil nematode Caenorhabditis elegans senses UV and is known to avoid UV by using four sensory neurons. However, it is not clear what signaling molecules act for UV avoidance in the neuronal pathway constituted of four sensory neurons. In addition, it is not clear whether this harmful environmental signal can be associated with other benefit signals such as food. In this study, by using newly developed assay system, we found that C. elegans can associate UV and food and changes behavioral strategy against harmful UV signal. This is the first indication that C. elegans shows associate learning with UV and food. Using our assay system, we also found that glutamate is used as a transmitter in both the UV avoidance and UV associate learning neural circuits. However, one sensory neuron showed a significant role for associative learning, compared to a complimentary role in four sensory neurons for direct associative learning, and different sets of glutamate receptors seemed to be acting for UV avoidance and UV associate learning. These findings suggest that a distinct neuronal network is used for UV learning compared to that for direct avoidance behavior of UV.
Assuntos
Aprendizagem , Plasticidade Neuronal , Fototaxia , Células Receptoras Sensoriais/metabolismo , Raios Ultravioleta , Animais , Caenorhabditis elegans , Comportamento Alimentar , Ácido Glutâmico/metabolismo , Receptores de Glutamato/metabolismo , Células Receptoras Sensoriais/fisiologiaRESUMO
The dynamic properties of protein molecules are involved in the relationship between their structure and function. Time-resolved X-ray observation enables capturing the structures of biomolecules with picometre-scale precision. However, this technique has yet to be implemented in living animals. Here, we examined diffracted X-ray blinking (DXB) and diffracted X-ray tracking (DXT) to observe the dynamics of a protein located on intestinal cells in adult Caenorhabditis elegans. This in vivo tissue-specific DXB was examined at temperatures from 20 °C to -10 °C for a recombinant ice-binding protein from Antarctomyces psychrotrophicus (AnpIBP) connected with the cells through a transmembrane CD4 protein equipped with a glycine-serine linker. AnpIBP inhibits ice growth at subzero temperatures by binding to ice crystals. We found that the rotational motion of AnpIBP decreases at -10 °C. In contrast, the motion of the AnpIBP mutant, which has a defective ice-binding ability, did not decrease at -10 °C. The twisting and tilting motional speeds of AnpIBPs measured above 5 °C by DXT were always higher than those of the defective AnpIBP mutant. These results suggest that wild-type AnpIBP is highly mobile in solution, and it is halted at subzero temperatures through ice binding. DXB and DXT allow for exploring protein behaviour in live animals with subnano resolution precision.
RESUMO
We have previously shown that the AEX-1 protein, which is expressed in postsynaptic muscles, retrogradely regulates presynaptic neural activity at the Caenorhabditis elegans neuromuscular junctions. AEX-1 is similar to vertebrate Munc13-4 protein, suggesting a function for vesicle exocytosis from a kind of cells. Compared to emerging evidences of the role of Munc13 proteins in synaptic vesicle release, however, the precise mechanism for vesicle exocytosis by AEX-1 and Munc13-4 is little understood. Here we have identified SYN-1 as a candidate molecule of AEX-1-dependent vesicle exocytosis from non-neuronal cells. The syn-1 gene encodes a C. elegans syntaxin, which is distantly related to the neuronal syntaxin UNC-64. The syn-1 gene is predominantly expressed in non-neuronal tissues and genetically interacts with aex-1 for presynaptic activity. However, the two proteins did not interact physically in our yeast two-hybrid system and mutational SYN-1 did not bypass the requirement of AEX-1 for the behavioral defects in aex-1 mutants, whereas mutant UNC-64 does in unc-13 mutants. These results suggest that a novel molecular interaction between the AEX-1 and syntaxin may regulate vesicle exocytosis for retrograde signal release.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Defecação , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Proteínas Qa-SNARE/metabolismo , Vesículas Sinápticas/metabolismo , Sintaxina 1/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Defecação/genética , Exocitose , Mucosa Intestinal/metabolismo , Intestinos/fisiologia , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Qa-SNARE/genética , Sintaxina 1/genéticaRESUMO
Rab proteins play a critical role in intracellular vesicle trafficking and require post-translational modification by adding lipids at the C-terminus for proper functions. This modification is preceded by the formation of a trimeric protein complex with the Rab escort protein (REP) and the Rab geranylgeranyltransferase (RabGGTase). However, the genetic hierarchy among these proteins and the tissue-specificity of each protein function are not yet clearly understood. Here we identified the Caenorhabditis elegans rep-1 gene and found that a rep-1 mutant showed a mild defect in synaptic transmission and defecation behaviors. Genetic analyses using the exocytic Rab mutants rab-3 or rab-27 suggested that rep-1 functions only in the RAB-27 pathway, and not in the RAB-3 pathway, for synaptic transmission at neuromuscular junctions. However, the disruption of REP-1 did not cause defecation defects compared to severe defects in either RAB-27 or RabGGTase disruption, suggesting that REP-1 is not essential for RAB-27 signaling in defection. Some Rab proteins did not physically interact with REP-1, and localization of these Rab proteins was not severely affected by REP-1 disruption. These findings suggest that REP-1 functions are required in specific Rab pathways and in specific tissues, and that some Rab proteins are functionally prenylated without REP-1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas rab de Ligação ao GTP/análise , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/análise , Regulação da Expressão Gênica , Mutação , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP , Proteínas rab3 de Ligação ao GTP/análise , Proteínas rab3 de Ligação ao GTP/genética , Proteínas rab3 de Ligação ao GTP/metabolismoRESUMO
Na+/K+ ATPase is a plasma membrane-localized sodium pump that maintains the ion gradients between the extracellular and intracellular environments, which in turn controls the cellular resting membrane potential.Recent evidence suggests that the pump is also localized at synapses and regulates synaptic efficacy.However, its precise function at the synapse is unknown. Here we show that two mutations in the alpha subunit of the eat-6 Na+/K+ ATPase in Caenorhabditis elegans dramatically increase the sensitivity to acetylcholine(Ach) agonists and alter the localization of nicotinic Ach receptors at the neuromuscular junction (NMJ).These defects can be rescued by mutated EAT-6 proteins which lack its pump activity, suggesting the presence of a novel function for Ach signaling. The Na+/K+ ATPase accumulates at postsynaptic sites and appears to surround Ach receptors to maintain rigid clusters at the NMJ. Our findings suggest a pump activity-independent, allele-specific role for Na+/K+ ATPase on postsynaptic organization and synaptic efficacy.
Assuntos
Caenorhabditis elegans/enzimologia , Regulação da Expressão Gênica/fisiologia , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , ATPase Trocadora de Sódio-Potássio/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Dados de Sequência Molecular , Mutação , Receptores Colinérgicos/biossínteseRESUMO
Trypanosoma cruzi has three distinct life cycle stages; epimastigote, trypomastigote, and amastigote. Amastigote is the replication stage in host mammalian cells, hence this stage of parasite has clinical significance in drug development research. Presence of extracellular amastigotes (EA) and their infection capability have been known for some decades. Here, we demonstrate that EA can be utilized as an axenic culture to aid in stage-specific study of T. cruzi. Amastigote-like property of axenic amastigote can be sustained in LIT medium at 37°C at least for 1 week, judging from their morphology, amastigote-specific UTR-regulated GFP expression, and stage-specific expression of selected endogenous genes. Inhibitory effect of benznidazole and nifurtimox on axenic amastigotes was comparable to that on intracellular amastigotes. Exogenous nucleic acids can be transfected into EA via conventional electroporation, and selective marker could be utilized for enrichment of transfectants. We also demonstrate that CRISPR/Cas9-mediated gene knockout can be performed in EA. Essentiality of the target gene can be evaluated by the growth capability of the knockout EA, either by continuation of axenic culturing or by host infection and following replication as intracellular amastigotes. By taking advantage of the accessibility and sturdiness of EA, we can potentially expand our experimental freedom in studying amastigote stage of T. cruzi.
Assuntos
Expressão Gênica , Técnicas de Inativação de Genes/métodos , Genética Microbiana/métodos , Biologia Molecular/métodos , Testes de Sensibilidade Parasitária/métodos , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genética , Antiprotozoários/farmacologia , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Eletroporação , Nifurtimox/farmacologia , Nitroimidazóis/farmacologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Automated cell counters that utilize still images of sample cells are widely used. However, they are not well suited to counting slender, aggregate-prone microorganisms such as Trypanosoma cruzi. Here, we developed a motion-based cell-counting system, using an image-recognition method based on a cubic higher-order local auto-correlation feature. The software successfully estimated the cell density of dispersed, aggregated, as well as fluorescent parasites by motion pattern recognition. Loss of parasites activeness due to drug treatment could also be detected as a reduction in apparent cell count, which potentially increases the sensitivity of drug screening assays. Moreover, the motion-based approach enabled estimation of the number of parasites in a co-culture with host mammalian cells, by disregarding the presence of the host cells as a static background.