RESUMO
In the nervous system, the specific identity of a neuron is established and maintained by terminal selector transcription factors that directly activate large batteries of terminal differentiation genes and positively regulate their own expression via feedback loops. However, how this is achieved in a reliable manner despite noise in gene expression, genetic variability or environmental perturbations remains poorly understood. We addressed this question using the AIY cholinergic interneurons of C. elegans, whose specification and differentiation network is well characterized. Via a genetic screen, we found that a loss of function of PRC1 chromatin factors induces a stochastic loss of AIY differentiated state in a small proportion of the population. PRC1 factors act directly in the AIY neuron and independently of PRC2 factors. By quantifying mRNA and protein levels of terminal selector transcription factors in single neurons, using smFISH and CRISPR tagging, we observed that, in PRC1 mutants, terminal selector expression is still initiated during embryonic development but the level is reduced, and expression is subsequently lost in a stochastic manner during maintenance phase in part of the population. We also observed variability in the level of expression of terminal selectors in wild type animals and, using correlation analysis, established that this noise comes from both intrinsic and extrinsic sources. Finally, we found that PRC1 factors increase the resistance of AIY neuron fate to environmental stress, and also secure the terminal differentiation of other neuron types. We propose that PRC1 factors contribute to the consistency of neuronal cell fate specification and maintenance by protecting neurons against noise and perturbations in their differentiation program.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Neural bHLH transcription factors play a key role in the early steps of neuronal specification in many animals. We have previously observed that the Achaete-Scute HLH-3, the Olig HLH-16 and their binding partner the E-protein HLH-2 activate the terminal differentiation program of a specific class of cholinergic neurons, AIY, in Caenorhabditis elegans. Here we identify a role for a fourth bHLH, the Neurogenin NGN-1, in this process, raising the question of why so many neural bHLHs are required for a single neuronal specification event. Using quantitative imaging we show that the combined action of different bHLHs is needed to activate the correct level of expression of the terminal selector transcription factors TTX-3 and CEH-10 that subsequently initiate and maintain the expression of a large battery of terminal differentiation genes. Surprisingly, the different bHLHs have an antagonistic effect on another target, the proapoptotic BH3-only factor EGL-1, normally not expressed in AIY and otherwise detrimental for its specification. We propose that the use of multiple neural bHLHs allows robust neuronal specification while, at the same time, preventing spurious activation of deleterious genes.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sistema Nervoso/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismoRESUMO
To investigate the early stages of cell-cell interactions occurring between living biological samples, imaging methods with appropriate spatiotemporal resolution are required. Among the techniques currently available, those based on optical trapping are promising. Methods to image trapped objects, however, in general suffer from a lack of three-dimensional resolution, due to technical constraints. Here, we have developed an original setup comprising two independent modules: holographic optical tweezers, which offer a versatile and precise way to move multiple objects simultaneously but independently, and a confocal microscope that provides fast three-dimensional image acquisition. The optical decoupling of these two modules through the same objective gives users the possibility to easily investigate very early steps in biological interactions. We illustrate the potential of this setup with an analysis of infection by the fungus Drechmeria coniospora of different developmental stages of Caenorhabditis elegans. This has allowed us to identify specific areas on the nematode's surface where fungal spores adhere preferentially. We also quantified this adhesion process for different mutant nematode strains, and thereby derive insights into the host factors that mediate fungal spore adhesion.
Assuntos
Caenorhabditis elegans/microbiologia , Comunicação Celular , Hypocreales/citologia , Hypocreales/fisiologia , Microscopia Confocal/métodos , Pinças Ópticas , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/crescimento & desenvolvimento , Lentes , Microscopia Confocal/instrumentação , Movimento , Esporos Fúngicos/citologia , Esporos Fúngicos/fisiologiaRESUMO
Metazoan transcription factors control distinct networks of genes in specific tissues, yet understanding how these networks are integrated into physiology, development, and homeostasis remains challenging. Inactivation of the nuclear hormone receptor nhr-25 ameliorates developmental and metabolic phenotypes associated with loss of function of an acyl-CoA synthetase gene, acs-3. ACS-3 activity prevents aberrantly high NHR-25 activity. Here, we investigated this relationship further by examining gene expression patterns following acs-3 and nhr-25 inactivation. Unexpectedly, we found that the acs-3 mutation or nhr-25 RNAi resulted in similar transcriptomes with enrichment in innate immunity and stress response gene expression. Mutants of either gene exhibited distinct sensitivities to pathogens and environmental stresses. Only nhr-25 was required for wild-type levels of resistance to the bacterial pathogen P. aeruginosa and only acs-3 was required for wild-type levels of resistance to osmotic stress and the oxidative stress generator, juglone. Inactivation of either acs-3 or nhr-25 compromised lifespan and resistance to the fungal pathogen D. coniospora. Double mutants exhibited more severe defects in the lifespan and P. aeruginosa assays, but were similar to the single mutants in other assays. Finally, acs-3 mutants displayed defects in their epidermal surface barrier, potentially accounting for the observed sensitivities. Together, these data indicate that inactivation of either acs-3 or nhr-25 causes stress sensitivity and increased expression of innate immunity/stress genes, most likely by different mechanisms. Elevated expression of these immune/stress genes appears to abrogate the transcriptional signatures relevant to metabolism and development.
Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Coenzima A Ligases/deficiência , Proteínas de Ligação a DNA/deficiência , Estresse Fisiológico , Fatores de Transcrição/deficiência , Animais , Animais Geneticamente Modificados , Peptídeos Catiônicos Antimicrobianos/genética , Caenorhabditis elegans/imunologia , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Estudos de Associação Genética , Longevidade/genética , Mutação , Fenótipo , Interferência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
The nematode C. elegans responds to infection by the fungus Drechmeria coniospora with a rapid increase in the expression of antimicrobial peptide genes. To investigate further the molecular basis of this innate immune response, we took a two-dimensional difference in-gel electrophoresis (2D-DIGE) approach to characterize the changes in host protein that accompany infection. We identified a total of 68 proteins from differentially represented spots and their corresponding genes. Through class testing, we identified functional categories that were enriched in our proteomic data set. One of these was "protein processing in endoplasmic reticulum," pointing to a potential link between innate immunity and endoplasmic reticulum function. This class included HSP-3, a chaperone of the BiP/GRP78 family known to act coordinately in the endoplasmic reticulum with its paralog HSP-4 to regulate the unfolded protein response (UPR). Other studies have shown that infection of C. elegans can provoke a UPR. We observed, however, that in adult C. elegans infection with D. coniospora did not induce a UPR, and conversely, triggering a UPR did not lead to an increase in expression of the well-characterized antimicrobial peptide gene nlp-29. On the other hand, we demonstrated a specific role for hsp-3 in the regulation of nlp-29 after infection that is not shared with hsp-4. Epistasis analysis allowed us to place hsp-3 genetically between the Tribbles-like kinase gene nipi-3 and the protein kinase C delta gene tpa-1. The precise function of hsp-3 has yet to be determined, but these results uncover a hitherto unsuspected link between a BiP/GRP78 family protein and innate immune signaling.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/química , Caenorhabditis elegans/imunologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/análise , Eletroforese em Gel Bidimensional , Epiderme/química , Epiderme/imunologia , Perfilação da Expressão GênicaRESUMO
Like other multicellular organisms, the model nematode C. elegans responds to infection by inducing the expression of defense genes. Among the genes upregulated in response to a natural fungal pathogen is nlp-29, encoding an antimicrobial peptide. In a screen for mutants that fail to express nlp-29 following fungal infection, we isolated alleles of tpa-1, homologous to the mammalian protein kinase C (PKC) delta. Through epistasis analyses, we demonstrate that C. elegans PKC acts through the p38 MAPK pathway to regulate nlp-29. This involves G protein signaling and specific C-type phospholipases acting upstream of PKCdelta. Unexpectedly and unlike in mammals, tpa-1 does not act via D-type protein kinases, but another C. elegans PKC gene, pkc-3, functions nonredundantly with tpa-1 to control nlp-29 expression. Finally, the tribbles-like kinase nipi-3 acts upstream of PKCdelta in this antifungal immune signaling cascade. These findings greatly expand our understanding of the pathways involved in C. elegans innate immunity.
Assuntos
Proteínas de Caenorhabditis elegans/imunologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/imunologia , Fungos/imunologia , Imunidade Inata , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Animais , Proteínas de Ligação ao GTP/metabolismo , Regulação Fúngica da Expressão Gênica , Modelos Biológicos , Proteína Quinase C/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Encounters with pathogens provoke changes in gene transcription that are an integral part of host innate immune responses. In recent years, studies with invertebrate model organisms have given insights into the origin, function, and evolution of innate immunity. Here, we use genome-wide transcriptome analysis to characterize the consequence of natural fungal infection in Caenorhabditis elegans. We identify several families of genes encoding putative antimicrobial peptides (AMPs) and proteins that are transcriptionally up-regulated upon infection. Many are located in small genomic clusters. We focus on the nlp-29 cluster of six AMP genes and show that it enhances pathogen resistance in vivo. The same cluster has a different structure in two other Caenorhabditis species. A phylogenetic analysis indicates that the evolutionary diversification of this cluster, especially in cases of intra-genomic gene duplications, is driven by natural selection. We further show that upon osmotic stress, two genes of the nlp-29 cluster are strongly induced. In contrast to fungus-induced nlp expression, this response is independent of the p38 MAP kinase cascade. At the same time, both involve the epidermal GATA factor ELT-3. Our results suggest that selective pressure from pathogens influences intra-genomic diversification of AMPs and reveal an unexpected complexity in AMP regulation as part of the invertebrate innate immune response.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/imunologia , Interações Hospedeiro-Patógeno , Imunidade Inata/fisiologia , Fungos Mitospóricos/efeitos dos fármacos , Animais , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Expressão Gênica , Genoma , Análise de Sequência com Séries de Oligonucleotídeos , Organismos Geneticamente Modificados , Filogenia , Seleção Genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Understanding the mechanisms of Salmonella virulence is an important challenge. The capacity of this intracellular bacterial pathogen to cause diseases depends on the expression of virulence factors including the second type III secretion system (TTSS-2), which is used to translocate into the eukaryotic cytosol a set of effector proteins that divert the biology of the host cell and shape the bacterial replicative niche. Yet little is known about the eukaryotic functions affected by individual Salmonella effectors. Here we report that the TTSS-2 effector PipB2 interacts with the kinesin light chain, a subunit of the kinesin-1 motor complex that drives anterograde transport along microtubules. Translocation of PipB2 is both necessary and sufficient for the recruitment of kinesin-1 to the membrane of the Salmonella-containing vacuole. In vivo, PipB2 contributes to the attenuation of Salmonella mutant strains in mice. Taken together, our data indicate that the TTSS-2-mediated fine-tuning of kinesin-1 activity associated with the bacterial vacuole is crucial for the virulence of Salmonella.
Assuntos
Proteínas de Bactérias/metabolismo , Cinesinas/metabolismo , Salmonella/metabolismo , Salmonella/patogenicidade , Animais , Proteínas de Bactérias/genética , Células da Medula Óssea/citologia , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Feminino , Fêmur/citologia , Deleção de Genes , Células HeLa , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Salmonella/classificação , Salmonella/genética , Salmonella/crescimento & desenvolvimento , Salmonelose Animal/microbiologia , Vacúolos/microbiologia , VirulênciaRESUMO
Mutations in the XNP/ATR-X gene cause several X-linked mental retardation syndromes in humans. The XNP/ATR-X gene encodes a DNA-helicase belonging to the SNF2 family. It has been proposed that XNP/ATR-X might be involved in chromatin remodelling. The lack of a mouse model for the ATR-X syndrome has, however, hampered functional studies of XNP/ATR-X. C. elegans possesses one homolog of the XNP/ATR-X gene, named xnp-1. By analysing a deletion mutant, we show that xnp-1 is required for the development of the embryo and the somatic gonad. Moreover, we show that abrogation of xnp-1 function in combination with inactivation of genes of the NuRD complex, as well as lin-35/Rb and hpl-2/HP1 leads to a stereotyped block of larval development with a cessation of growth but not of cell division. We also demonstrate a specific function for xnp-1 together with lin-35 or hpl-2 in the control of transgene expression, a process known to be dependent on chromatin remodelling. This study thus demonstrates that in vivo XNP-1 acts in association with RB, HP1 and the NuRD complex during development.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/fisiologia , Proteínas de Helminto/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , DNA Helicases/genética , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Genes de Helmintos , Gônadas/crescimento & desenvolvimento , Proteínas de Helminto/genética , Fator 1 Nuclear de Hepatócito , Fator 1-alfa Nuclear de Hepatócito , Histona Desacetilases/genética , Histona Desacetilases/fisiologia , Humanos , Larva/crescimento & desenvolvimento , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Interferência de RNA , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
Both plants and animals respond to infection by synthesizing compounds that directly inhibit or kill invading pathogens. We report here the identification of infection-inducible antimicrobial peptides in Caenorhabditis elegans. Expression of two of these peptides, NLP-29 and NLP-31, was differentially regulated by fungal and bacterial infection and was controlled in part by tir-1, which encodes an ortholog of SARM, a Toll-interleukin 1 receptor (TIR) domain protein. Inactivation of tir-1 by RNA interference caused increased susceptibility to infection. We identify protein partners for TIR-1 and show that the small GTPase Rab1 and the f subunit of ATP synthase participate specifically in the control of antimicrobial peptide gene expression. As the activity of tir-1 was independent of the single nematode Toll-like receptor, TIR-1 may represent a component of a previously uncharacterized, but conserved, innate immune signaling pathway.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/imunologia , Proteínas do Citoesqueleto/metabolismo , Imunidade Inata/imunologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas do Domínio Armadillo , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas do Citoesqueleto/genética , Imunidade Inata/genética , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Micoses/imunologia , Micoses/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Receptores Toll-LikeRESUMO
The term innate immunity refers to a number of evolutionary ancient mechanisms that serve to defend animals and plants against infection. Genetically tractable model organisms, especially Drosophila, have contributed greatly to advances in our understanding of mammalian innate immunity. Essentially, nothing is known about immune responses in the nematode Caenorhabditis elegans. Using high-density cDNA microarrays, we show here that infection of C. elegans by the Gram-negative bacterium Serratia marcescens provokes a marked upregulation of the expression of many genes. Among the most robustly induced are genes encoding lectins and lysozymes, known to be involved in immune responses in other organisms. Certain infection-inducible genes are under the control of the DBL-1/TGFbeta pathway. We found that dbl-1 mutants exhibit increased susceptibility to infection. Conversely, overexpression of the lysozyme gene lys-1 augments the resistance of C. elegans to S. marcescens. These results constitute the first demonstration of inducible antibacterial defenses in C. elegans and open new avenues for the investigation of evolutionary conserved mechanisms of innate immunity.
Assuntos
Caenorhabditis elegans/imunologia , Serratia marcescens/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/microbiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão GênicaRESUMO
Practically and ethically attractive as model systems, invertebrate organisms are increasingly recognized as relevant for the study of bacterial pathogenesis. We show here that the nematode Caenorhabditis elegans is susceptible to a surprisingly broad range of bacteria and may constitute a useful model for the study of both pathogens and symbionts.
Assuntos
Caenorhabditis elegans/microbiologia , Aeromonas hydrophila/patogenicidade , Agrobacterium tumefaciens/patogenicidade , Animais , Bactérias/patogenicidade , Brucella/patogenicidade , Caenorhabditis elegans/crescimento & desenvolvimento , Dickeya chrysanthemi/patogenicidade , Escherichia coli/patogenicidade , Modelos Animais , Mycobacterium fortuitum/patogenicidade , Mycobacterium marinum/patogenicidade , Pectobacterium carotovorum/patogenicidade , Photorhabdus/patogenicidade , Shewanella/patogenicidade , Shewanella putrefaciens/patogenicidade , Xenorhabdus/patogenicidadeRESUMO
We recently reported the identification of EFA6 (exchange factor for ARF6), a brain-specific Sec7-domain-containing guanine nucleotide exchange factor that works specifically on ARF6. Here, we have characterized the product of a broadly expressed gene encoding a novel 1056 amino-acid protein that we have named EFA6B. We show that EFA6B, which contains a Sec7 domain that is highly homologous to EFA6, works as an ARF6-specific guanine exchange factor in vitro. Like EFA6, which will be referred to as EFA6A from now on, EFA6B is involved in membrane recycling and colocalizes with ARF6 in actin-rich membrane ruffles and microvilli-like protrusions on the dorsal cell surface in transfected baby hamster kidney cells. Strikingly, homology between EFA6A and EFA6B is not limited to the Sec7 domain but extends to an adjacent pleckstrin homology (PH) domain and a approximately 150 amino-acid C-terminal region containing a predicted coiled coil motif. Association of EFA6A with membrane ruffles and microvilli-like structures depends on the PH domain, which probably interacts with phosphatidylinositol 4,5-biphosphate. Moreover, we show that overexpression of the PH domain/C-terminal region of EFA6A or EFA6B in the absence of the Sec7 domain promotes lengthening of dorsal microvillar protrusions. This morphological change requires the integrity of the coiled-coil motif. Lastly, database analysis reveals that the EFA6-family comprises at least four members in humans and is conserved in multicellular organisms throughout evolution. Our results suggest that EFA6 family guanine exchange factors are modular proteins that work through the coordinated action of the catalytic Sec7 domain to promote ARF6 activation, through the PH domain to regulate association with specific subdomains of the plasma membrane and through the C-terminal region to control actin cytoskeletal reorganization.