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1.
Nat Commun ; 12(1): 4912, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389721

RESUMO

Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between these multimodular megaenzymes. In the canal-associated neurons (CANs) of Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides biosynthesized by animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional enzymes expressed in the CANs that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes and discover a mechanism for trafficking intermediates between a PKS and an NRPS. Specifically, the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking mechanism provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.


Assuntos
Vias Biossintéticas/genética , Proteínas de Caenorhabditis elegans/genética , Peptídeo Sintases/genética , Peptídeos/metabolismo , Policetídeo Sintases/genética , Policetídeos/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografia Líquida/métodos , Enzimas/genética , Enzimas/metabolismo , Expressão Gênica , Espectrometria de Massas/métodos , Estrutura Molecular , Mutação , Neurônios/metabolismo , Peptídeo Sintases/metabolismo , Peptídeos/química , Policetídeo Sintases/metabolismo , Policetídeos/química
2.
Nature ; 596(7871): 257-261, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34349261

RESUMO

An animal's nervous system changes as its body grows from birth to adulthood and its behaviours mature1-8. The form and extent of circuit remodelling across the connectome is unknown3,9-15. Here we used serial-section electron microscopy to reconstruct the full brain of eight isogenic Caenorhabditis elegans individuals across postnatal stages to investigate how it changes with age. The overall geometry of the brain is preserved from birth to adulthood, but substantial changes in chemical synaptic connectivity emerge on this consistent scaffold. Comparing connectomes between individuals reveals substantial differences in connectivity that make each brain partly unique. Comparing connectomes across maturation reveals consistent wiring changes between different neurons. These changes alter the strength of existing connections and create new connections. Collective changes in the network alter information processing. During development, the central decision-making circuitry is maintained, whereas sensory and motor pathways substantially remodel. With age, the brain becomes progressively more feedforward and discernibly modular. Thus developmental connectomics reveals principles that underlie brain maturation.


Assuntos
Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Caenorhabditis elegans/citologia , Conectoma , Modelos Neurológicos , Vias Neurais , Sinapses/fisiologia , Envelhecimento/metabolismo , Animais , Encéfalo/anatomia & histologia , Encéfalo/ultraestrutura , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/ultraestrutura , Individualidade , Interneurônios/citologia , Microscopia Eletrônica , Neurônios/citologia , Comportamento Estereotipado
3.
Nature ; 596(7871): 285-290, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34321666

RESUMO

Ageing is driven by a loss of cellular integrity1. Given the major role of ubiquitin modifications in cell function2, here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome3, a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament4 and the EPS-8 modulator of RAC signalling5. While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.


Assuntos
Envelhecimento/metabolismo , Caenorhabditis elegans/metabolismo , Proteoma/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Citoesqueleto de Actina/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas do Citoesqueleto/metabolismo , Intestinos/microbiologia , Longevidade , Músculos/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteoma/química , Proteínas rac de Ligação ao GTP/metabolismo
4.
Methods Mol Biol ; 2276: 397-407, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060057

RESUMO

Caenorhabditis elegans is a highly versatile model system, intensively used for functional, genetic, cytometric, and integrative studies. Due to its simplicity and large muscle cell number, C. elegans has frequently been used to study mitochondrial deficiencies caused by disease or drug toxicity. Here we describe a robust and efficient method to visualize and quantify mitochondrial morphology in vivo. This method has many practical and technical advantages above traditional (manual) methods and provides a comprehensive analysis of mitochondrial morphology.


Assuntos
Caenorhabditis elegans/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia Intravital/métodos , Microscopia Confocal/métodos , Mitocôndrias/ultraestrutura , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Mitocôndrias/metabolismo
5.
Aging (Albany NY) ; 13(10): 13474-13495, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34091442

RESUMO

The medicinal fungus Ganoderma lucidum is used as a dietary supplement and health tonic, but whether it affects longevity remains unclear. We show here that a water extract of G. lucidum mycelium extends lifespan of the nematode Caenorhabditis elegans. The G. lucidum extract reduces the level of fibrillarin (FIB-1), a nucleolar protein that correlates inversely with longevity in various organisms. Furthermore, G. lucidum treatment increases expression of the autophagosomal protein marker LGG-1, and lifespan extension is abrogated in mutant C. elegans strains that lack atg-18, daf-16, or sir-2.1, indicating that autophagy and stress resistance pathways are required to extend lifespan. In cultured human cells, G. lucidum increases concentrations of the LGG-1 ortholog LC3 and reduces levels of phosphorylated mTOR, a known inhibitor of autophagy. Notably, low molecular weight compounds (<10 kDa) isolated from the G. lucidum water extract prolong lifespan of C. elegans and the same compounds induce autophagy in human cells. These results suggest that G. lucidum can increase longevity by inducing autophagy and stress resistance.


Assuntos
Autofagia , Caenorhabditis elegans/citologia , Caenorhabditis elegans/fisiologia , Longevidade/fisiologia , Reishi/química , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Humanos , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
6.
Methods Mol Biol ; 2326: 3-18, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34097257

RESUMO

Crude oil disasters, such as the Deepwater Horizon accident, have caused severe environmental contamination and damage, affecting the health of marine and terrestrial organisms. Some previous studies have demonstrated cleanup efforts using chemical dispersant induced more potent toxicities than oil alone due to an increase in bioavailability of crude oil components, such as PAHs. However, there still lacks a systematic procedure that provides methods to determine genotypic and phenotypic changes following exposure to environmental toxicants or toxicant mixture, such as dispersed crude oil. Here, we describe methods for identifying a mechanism of dispersed crude oil-induced reproductive toxicity in the model organisms, Caenorhabditis elegans (C. elegans). Due to the genetic malleability of C. elegans, two mutant strains outlined in this chapter were used to identify a pathway responsible for inducing apoptosis: MD701 bcIs39 [lim-7p::ced-1::GFP + lin-15(+)], a mutant strain that allows visualization of apoptotic bodies via a green fluorescent protein fused to CED-1; and TJ1 (cep-1(gk138) I.), a p53/CEP-1 defective strain that is unable to activate apoptosis via the p53/CEP-1 pathway. In addition, qRT-PCR was utilized to demonstrate the aberrant expression of apoptosis (ced-13, ced-3, ced-4, ced-9, cep-1, dpl-1, efl-1, efl-2, egl-1, egl-38, lin-35, pax-2, and sir-2.1) and cytochrome P450 (cyp14a3, cyp35a1, cyp35a2, cyp35a5, and cyp35c1) protein-coding genes following exposure to dispersed crude oil. The procedure outlined here can be applicable to determine whether environmental contaminants, most of time contaminant mixture, cause reproductive toxicity by activation of the proapoptotic, p53/CEP-1 pathway.


Assuntos
Apoptose/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Exposição Ambiental/efeitos adversos , Poluentes Ambientais/efeitos adversos , Células Germinativas/efeitos dos fármacos , Petróleo/efeitos adversos , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Exposição Ambiental/análise , Poluentes Ambientais/análise , Poluentes Ambientais/toxicidade , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células Germinativas/citologia , Células Germinativas/metabolismo , Petróleo/análise , Petróleo/toxicidade
8.
PLoS Genet ; 17(6): e1009607, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34081703

RESUMO

Early endosomes are the sorting hub on the endocytic pathway, wherein sorting nexins (SNXs) play important roles for formation of the distinct membranous microdomains with different sorting functions. Tubular endosomes mediate the recycling of clathrin-independent endocytic (CIE) cargoes back toward the plasma membrane. However, the molecular mechanism underlying the tubule formation is still poorly understood. Here we screened the effect on the ARF-6-associated CIE recycling endosomal tubules for all the SNX members in Caenorhabditis elegans (C. elegans). We identified SNX-3 as an essential factor for generation of the recycling tubules. The loss of SNX-3 abolishes the interconnected tubules in the intestine of C. elegans. Consequently, the surface and total protein levels of the recycling CIE protein hTAC are strongly decreased. Unexpectedly, depletion of the retromer components VPS-26/-29/-35 has no similar effect, implying that the retromer trimer is dispensable in this process. We determined that hTAC is captured by the ESCRT complex and transported into the lysosome for rapid degradation in snx-3 mutants. Interestingly, EEA-1 is increasingly recruited on early endosomes and localized to the hTAC-containing structures in snx-3 mutant intestines. We also showed that SNX3 and EEA1 compete with each other for binding to phosphatidylinositol-3-phosphate enriching early endosomes in Hela cells. Our data demonstrate for the first time that PX domain-only C. elegans SNX-3 organizes the tubular endosomes for efficient recycling and retrieves the CIE cargo away from the maturing sorting endosomes by competing with EEA-1 for binding to the early endosomes. However, our results call into question how SNX-3 couples the cargo capture and membrane remodeling in the absence of the retromer trimer complex.


Assuntos
Fatores de Ribosilação do ADP/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Endossomos/metabolismo , Células Epiteliais/metabolismo , Nexinas de Classificação/genética , Proteínas de Transporte Vesicular/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Transporte Biológico , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células Epiteliais/citologia , Regulação da Expressão Gênica , Células HeLa , Humanos , Intestinos/citologia , Lisossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise , Transdução de Sinais , Nexinas de Classificação/deficiência , Proteínas de Transporte Vesicular/metabolismo
9.
PLoS Genet ; 17(6): e1009618, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34115759

RESUMO

Coordination of neurite extension with surrounding glia development is critical for neuronal function, but the underlying molecular mechanisms remain poorly understood. Through a genome-wide mutagenesis screen in C. elegans, we identified dyf-4 and daf-6 as two mutants sharing similar defects in dendrite extension. DAF-6 encodes a glia-specific patched-related membrane protein that plays vital roles in glial morphogenesis. We cloned dyf-4 and found that DYF-4 encodes a glia-secreted protein. Further investigations revealed that DYF-4 interacts with DAF-6 and functions in a same pathway as DAF-6 to regulate sensory compartment formation. Furthermore, we demonstrated that reported glial suppressors of daf-6 could also restore dendrite elongation and ciliogenesis in both dyf-4 and daf-6 mutants. Collectively, our data reveal that DYF-4 is a regulator for DAF-6 which promotes the proper formation of the glial channel and indirectly affects neurite extension and ciliogenesis.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Genoma Helmíntico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Comunicação Celular , Cílios/genética , Cílios/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutagênese , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
PLoS Comput Biol ; 17(6): e1009034, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34061834

RESUMO

Increasing interest has emerged in new mathematical approaches that simplify the study of complex differentiation processes by formalizing Waddington's landscape metaphor. However, a rational method to build these landscape models remains an open problem. Here we study vulval development in C. elegans by developing a framework based on Catastrophe Theory (CT) and approximate Bayesian computation (ABC) to build data-fitted landscape models. We first identify the candidate qualitative landscapes, and then use CT to build the simplest model consistent with the data, which we quantitatively fit using ABC. The resulting model suggests that the underlying mechanism is a quantifiable two-step decision controlled by EGF and Notch-Delta signals, where a non-vulval/vulval decision is followed by a bistable transition to the two vulval states. This new model fits a broad set of data and makes several novel predictions.


Assuntos
Caenorhabditis elegans/citologia , Modelos Biológicos , Animais , Teorema de Bayes , Diferenciação Celular , Fator de Crescimento Epidérmico/metabolismo , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Projetos de Pesquisa , Vulva/crescimento & desenvolvimento
11.
Nucleic Acids Res ; 49(W1): W80-W85, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-33956141

RESUMO

Recent innovations in genetics and imaging are providing the means to reconstruct cell lineages, either by tracking cell divisions using live microscopy, or by deducing the history of cells using molecular recorders. A cell lineage on its own, however, is simply a description of cell divisions as branching events. A major goal of current research is to integrate this description of cell relationships with information about the spatial distribution and identities of the cells those divisions produce. Visualizing, interpreting and exploring these complex data in an intuitive manner requires the development of new tools. Here we present CeLaVi, a web-based visualization tool that allows users to navigate and interact with a representation of cell lineages, whilst simultaneously visualizing the spatial distribution, identities and properties of cells. CeLaVi's principal functions include the ability to explore and manipulate the cell lineage tree; to visualise the spatial distribution of cell clones at different depths of the tree; to colour cells in the 3D viewer based on lineage relationships; to visualise various cell qualities on the 3D viewer (e.g. gene expression, cell type) and to annotate selected cells/clones. All these capabilities are demonstrated with four different example data sets. CeLaVi is available at http://www.celavi.pro.


Assuntos
Linhagem da Célula , Software , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/crescimento & desenvolvimento , Ciona intestinalis/citologia , Ciona intestinalis/embriologia , Crustáceos/citologia , Crustáceos/embriologia , Gástrula/citologia , Expressão Gênica , Larva/citologia
12.
Biochem Biophys Res Commun ; 562: 36-42, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34034091

RESUMO

There are six different longevity models in Caenorhabditis elegans. Previous studies have identified several convergence points, such as hlh-30, daf-16, and klf-3, required for lifespan extension in these longevity models. However, it is not clear whether there other such convergence points. In this study, based on analysis of transcriptome data, we found that the expression of klo-1/klotho was elevated in several longevity models. klo-1 was required for lifespan extension in the glp-1(e2141) and isp-1(qm150) mutants. klo-1 extended the lifespan of glp-1(e2141) and isp-1(qm150) worms by activating extracellular-signal-regulated kinase (ERK). In addition, klo-1 and mpk-1 (the homologous gene encoding ERK) regulated autophagy in glp-1(e2141) mutants, suggesting that klo-1 regulates lifespan by activating autophagy.


Assuntos
Autofagia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/fisiologia , Longevidade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Animais , Caenorhabditis elegans/genética , Sistema de Sinalização das MAP Quinases , Mutação/genética
13.
PLoS Genet ; 17(5): e1009567, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34014923

RESUMO

The widely conserved kinase Aurora B regulates important events during cell division. Surprisingly, recent work has uncovered a few functions of Aurora-family kinases that do not require kinase activity. Thus, understanding this important class of cell cycle regulators will require strategies to distinguish kinase-dependent from independent functions. Here, we address this need in C. elegans by combining germline-specific, auxin-induced Aurora B (AIR-2) degradation with the transgenic expression of kinase-inactive AIR-2. Through this approach, we find that kinase activity is essential for AIR-2's major meiotic functions and also for mitotic chromosome segregation. Moreover, our analysis revealed insight into the assembly of the ring complex (RC), a structure that is essential for chromosome congression in C. elegans oocytes. AIR-2 localizes to chromosomes and recruits other components to form the RC. However, we found that while kinase-dead AIR-2 could load onto chromosomes, other components were not recruited. This failure in RC assembly appeared to be due to a loss of RC SUMOylation, suggesting that there is crosstalk between SUMOylation and phosphorylation in building the RC and implicating AIR-2 in regulating the SUMO pathway in oocytes. Similar conditional depletion approaches may reveal new insights into other cell cycle regulators.


Assuntos
Aurora Quinase B/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/enzimologia , Segregação de Cromossomos , Oócitos/enzimologia , Animais , Caenorhabditis elegans/genética , Cromossomos/metabolismo , Meiose/genética , Mitose/genética , Oócitos/citologia , Fosforilação , Reprodutibilidade dos Testes , Fuso Acromático/enzimologia , Sumoilação
14.
Nat Commun ; 12(1): 2583, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972516

RESUMO

Quantitative micromechanical characterization of single cells and multicellular tissues or organisms is of fundamental importance to the study of cellular growth, morphogenesis, and cell-cell interactions. However, due to limited manipulation capabilities at the microscale, systems used for mechanical characterizations struggle to provide complete three-dimensional coverage of individual specimens. Here, we combine an acoustically driven manipulation device with a micro-force sensor to freely rotate biological samples and quantify mechanical properties at multiple regions of interest within a specimen. The versatility of this tool is demonstrated through the analysis of single Lilium longiflorum pollen grains, in combination with numerical simulations, and individual Caenorhabditis elegans nematodes. It reveals local variations in apparent stiffness for single specimens, providing previously inaccessible information and datasets on mechanical properties that serve as the basis for biophysical modelling and allow deeper insights into the biomechanics of these living systems.


Assuntos
Imageamento Tridimensional/métodos , Micromanipulação/instrumentação , Micromanipulação/métodos , Microscopia de Força Atômica/métodos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Acústica , Animais , Fenômenos Biomecânicos , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/citologia , Parede Celular/ultraestrutura , Lilium/citologia , Microscopia Eletrônica de Varredura , Morfogênese , Células Vegetais , Pólen/citologia , Pólen/ultraestrutura
15.
Genes Cells ; 26(6): 411-425, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33817914

RESUMO

Interneurons, innervated by multiple sensory neurons, need to integrate information from these sensory neurons and respond to sensory stimuli adequately. Mechanisms how sensory information is integrated to form responses of interneurons are not fully understood. In Caenorhabditis elegans, loss-of-function mutations of egl-4, which encodes a cGMP-dependent protein kinase (PKG), cause a defect in chemotaxis to odorants. Our genetic and imaging analyses revealed that the response property of AIY interneuron to an odorant is reversed in the egl-4 mutant, while the responses of two upstream olfactory neurons, AWA and AWC, are largely unchanged. Cell- ablation experiments show that AIY in the egl-4 mutant functions to suppress chemotaxis. Furthermore, the reversal of AIY response occurs only in the presence of sensory signals from both AWA and AWC. These results suggest that sensory signals are inadequately integrated in the egl-4 mutant. We also show that egl-4 expression in AWA and another sensory neuron prevents the reversed AIY response and restores chemotaxis in the egl-4 mutants. We propose that EGL-4/PKG, by suppressing aberrant integration of signals from olfactory neurons, converts the response property of an interneuron to olfactory stimuli and maintains the role of the interneuron in the circuit to execute chemotactic behavior.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Quimiotaxia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Interneurônios/metabolismo , Sensação , Animais , Proteínas de Caenorhabditis elegans/genética , Cálcio/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/genética , Interneurônios/citologia , Mutação/genética , Neurônios Receptores Olfatórios/metabolismo , Pentanóis/farmacologia , Células Receptoras Sensoriais/metabolismo
16.
Adv Genet ; 107: 33-87, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33641748

RESUMO

Developmental programs are under strict genetic control that favors robustness of the process. In order to guarantee the same outcome in different environmental situations, development is modulated by input pathways, which inform about external conditions. In the nematode Caenorhabditis elegans, the process of postembryonic development involves a series of stereotypic cell divisions, the progression of which is controlled by the nutritional status of the animal. C. elegans can arrest development at different larval stages, leading to cell arrest of the relevant divisions of the stage. This means that studying the nutritional control of development in C. elegans we can learn about the mechanisms controlling cell division in an in vivo model. In this work, we reviewed the current knowledge about the nutrient sensing pathways that control the progression or arrest of development in response to nutrient availability, with a special focus on the arrest at the L1 stage.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crescimento & desenvolvimento , Larva/fisiologia , Envelhecimento/fisiologia , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Insulina/metabolismo
17.
Elife ; 102021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33781383

RESUMO

Despite recent improvements in microscope technologies, segmenting and tracking cells in three-dimensional time-lapse images (3D + T images) to extract their dynamic positions and activities remains a considerable bottleneck in the field. We developed a deep learning-based software pipeline, 3DeeCellTracker, by integrating multiple existing and new techniques including deep learning for tracking. With only one volume of training data, one initial correction, and a few parameter changes, 3DeeCellTracker successfully segmented and tracked ~100 cells in both semi-immobilized and 'straightened' freely moving worm's brain, in a naturally beating zebrafish heart, and ~1000 cells in a 3D cultured tumor spheroid. While these datasets were imaged with highly divergent optical systems, our method tracked 90-100% of the cells in most cases, which is comparable or superior to previous results. These results suggest that 3DeeCellTracker could pave the way for revealing dynamic cell activities in image datasets that have been difficult to analyze.


Assuntos
Rastreamento de Células/métodos , Aprendizado Profundo , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Imagem com Lapso de Tempo/métodos , Animais , Encéfalo/diagnóstico por imagem , Caenorhabditis elegans/citologia , Rastreamento de Células/instrumentação , Coração/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/instrumentação , Imageamento Tridimensional/instrumentação , Esferoides Celulares , Imagem com Lapso de Tempo/instrumentação , Células Tumorais Cultivadas , Peixe-Zebra
18.
Genes (Basel) ; 12(2)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578809

RESUMO

Infiltration of the endothelial layer of the blood-brain barrier by leukocytes plays a critical role in health and disease. When passing through the endothelial layer during the diapedesis process lymphocytes can either follow a paracellular route or a transcellular one. There is a debate whether these two processes constitute one mechanism, or they form two evolutionary distinct migration pathways. We used artificial intelligence, phylogenetic analysis, HH search, ancestor sequence reconstruction to investigate further this intriguing question. We found that the two systems share several ancient components, such as RhoA protein that plays a critical role in controlling actin movement in both mechanisms. However, some of the key components differ between these two transmigration processes. CAV1 genes emerged during Trichoplax adhaerens, and it was only reported in transcellular process. Paracellular process is dependent on PECAM1. PECAM1 emerged from FASL5 during Zebrafish divergence. Lastly, both systems employ late divergent genes such as ICAM1 and VECAM1. Taken together, our results suggest that these two systems constitute two different mechanical sensing mechanisms of immune cell infiltrations of the brain, yet these two systems are connected. We postulate that the mechanical properties of the cellular polarity is the main driving force determining the migration pathway. Our analysis indicates that both systems coevolved with immune cells, evolving to a higher level of complexity in association with the evolution of the immune system.


Assuntos
Células Endoteliais/metabolismo , Leucócitos/metabolismo , Proteínas/genética , Migração Transcelular de Célula/genética , Transcriptoma , Migração Transendotelial e Transepitelial/genética , Animais , Evolução Biológica , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Caenorhabditis elegans/classificação , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Galinhas/classificação , Galinhas/genética , Galinhas/metabolismo , Ciona intestinalis/classificação , Ciona intestinalis/citologia , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Drosophila melanogaster/classificação , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Endoteliais/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Leucócitos/citologia , Camundongos , Pan troglodytes/classificação , Pan troglodytes/genética , Pan troglodytes/metabolismo , Petromyzon/classificação , Petromyzon/genética , Petromyzon/metabolismo , Filogenia , Placozoa/classificação , Placozoa/citologia , Placozoa/genética , Placozoa/metabolismo , Proteínas/classificação , Proteínas/metabolismo , Anêmonas-do-Mar/classificação , Anêmonas-do-Mar/citologia , Anêmonas-do-Mar/genética , Anêmonas-do-Mar/metabolismo , Tubarões/classificação , Tubarões/genética , Tubarões/metabolismo , Peixe-Zebra/classificação , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
19.
Nature ; 591(7848): 99-104, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33627875

RESUMO

Neuropil is a fundamental form of tissue organization within the brain1, in which densely packed neurons synaptically interconnect into precise circuit architecture2,3. However, the structural and developmental principles that govern this nanoscale precision remain largely unknown4,5. Here we use an iterative data coarse-graining algorithm termed 'diffusion condensation'6 to identify nested circuit structures within the Caenorhabditis elegans neuropil, which is known as the nerve ring. We show that the nerve ring neuropil is largely organized into four strata that are composed of related behavioural circuits. The stratified architecture of the neuropil is a geometrical representation of the functional segregation of sensory information and motor outputs, with specific sensory organs and muscle quadrants mapping onto particular neuropil strata. We identify groups of neurons with unique morphologies that integrate information across strata and that create neural structures that cage the strata within the nerve ring. We use high resolution light-sheet microscopy7,8 coupled with lineage-tracing and cell-tracking algorithms9,10 to resolve the developmental sequence and reveal principles of cell position, migration and outgrowth that guide stratified neuropil organization. Our results uncover conserved structural design principles that underlie the architecture and function of the nerve ring neuropil, and reveal a temporal progression of outgrowth-based on pioneer neurons-that guides the hierarchical development of the layered neuropil. Our findings provide a systematic blueprint for using structural and developmental approaches to understand neuropil organization within the brain.


Assuntos
Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Neurópilo/química , Neurópilo/metabolismo , Algoritmos , Animais , Encéfalo/citologia , Encéfalo/embriologia , Caenorhabditis elegans/química , Caenorhabditis elegans/citologia , Movimento Celular , Difusão , Interneurônios/metabolismo , Neurônios Motores/metabolismo , Neuritos/metabolismo , Neurópilo/citologia , Células Receptoras Sensoriais/metabolismo
20.
J Vis Exp ; (167)2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33554967

RESUMO

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::GαS(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.


Assuntos
Caenorhabditis elegans/citologia , Imageamento Tridimensional , Degeneração Neural/diagnóstico por imagem , Degeneração Neural/patologia , Neurotoxinas/toxicidade , Envelhecimento/patologia , Criação de Animais Domésticos , Animais , Apoptose , Tampões (Química) , Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Ácido Glutâmico/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Necrose , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neuroproteção/efeitos dos fármacos , RNA/isolamento & purificação , Fatores de Risco , Transcriptoma/genética
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