RESUMO
There is a debate regarding the function of Drp1, a GTPase involved in mitochondrial fission, during the elimination of mitochondria by autophagy. A number of experiments indicate that Drp1 is needed to eliminate mitochondria during mitophagy, either by reducing the mitochondrial size or by providing a noncanonical mitophagy function. Yet, other convincing experimental results support the conclusion that Drp1 is not necessary. Here, we review the possible functions for Drp1 in mitophagy and autophagy, depending on tissues, organisms and stresses, and discuss these apparent discrepancies. In this regard, it appears that the reduction of mitochondria size is often required for mitophagy but not always in a Drp1-dependent manner. Finally, we speculate on Drp1-independent mitochondrial fission mechanism that may take place during mitophagy and on noncanonical roles, which Drp1 may play such as modulating organelle contact sites dynamic during the autophagosome formation.
Assuntos
Dinaminas , Mitofagia , Autofagia , Mitocôndrias , Dinâmica MitocondrialRESUMO
ESCRT (endosomal sorting complex required for transport) machinery has been initially identified for its role during endocytosis, which allows membrane proteins and lipids to be degraded in the lysosome. ESCRT function is required to form intraluminal vesicles permitting internalization of cytosolic components or membrane embedded cargoes and promoting endosome maturation. ESCRT machinery also contributes to multiple key cell mechanisms in which it reshapes membranes. In addition, ESCRT actively participates in different types of autophagy processes for degrading cytosolic components, such as endosomal microautophagy and macroautophagy. During macroautophagy, ESCRT promotes formation of multivesicular bodies, which can fuse with autophagosomes to generate amphisomes. This latter fusion probably brings to autophagosomes key membrane molecules necessary for the subsequent fusion with lysosomes. Interestingly, during macroautophagy, ESCRT proteins could be involved in non-canonical functions such as vesicle tethering or phagophore membrane sealing. Additionally, ESCRT subunits could directly interact with key autophagy related proteins to build a closer connection between endocytosis and autophagy pathways.
Assuntos
Autofagia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Animais , HumanosRESUMO
The sarcoplasmic reticulum is a network of tubules and cisternae localized in close association with the contractile apparatus, and regulates Ca(2+)dynamics within striated muscle cell. The sarcoplasmic reticulum maintains its shape and organization despite repeated muscle cell contractions, through mechanisms which are still under investigation. The ESCRT complexes are essential to organize membrane subdomains and modify membrane topology in multiple cellular processes. Here, we report for the first time that ESCRT-II proteins play a role in the maintenance of sarcoplasmic reticulum integrity inC. elegans ESCRT-II proteins colocalize with the sarcoplasmic reticulum marker ryanodine receptor UNC-68. The localization at the sarcoplasmic reticulum of ESCRT-II and UNC-68 are mutually dependent. Furthermore, the characterization of ESCRT-II mutants revealed a fragmentation of the sarcoplasmic reticulum network, associated with an alteration of Ca(2+)dynamics. Our data provide evidence that ESCRT-II proteins are involved in sarcoplasmic reticulum shaping.
Assuntos
Caenorhabditis elegans/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células Musculares/metabolismo , Contração Muscular/fisiologia , Retículo Sarcoplasmático/metabolismo , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Affinity purifications followed by mass spectrometric analysis are used to identify protein-protein interactions. Because quantitative proteomic data are noisy, it is necessary to develop statistical methods to eliminate false-positives and identify true partners. We present here a novel approach for filtering false interactors, named "SAFER" for mass Spectrometry data Analysis by Filtering of Experimental Replicates, which is based on the reproducibility of the replicates and the fold-change of the protein intensities between bait and control. To identify regulators or targets of autophagy, we characterized the interactors of LGG1, a ubiquitin-like protein involved in autophagosome formation in C. elegans. LGG-1 partners were purified by affinity, analyzed by nanoLC-MS/MS mass spectrometry, and quantified by a label-free proteomic approach based on the mass spectrometric signal intensity of peptide precursor ions. Because the selection of confident interactions depends on the method used for statistical analysis, we compared SAFER with several statistical tests and different scoring algorithms on this set of data. We show that SAFER recovers high-confidence interactors that have been ignored by the other methods and identified new candidates involved in the autophagy process. We further validated our method on a public data set and conclude that SAFER notably improves the identification of protein interactors.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/química , Proteínas Associadas aos Microtúbulos/metabolismo , Proteômica/métodos , Algoritmos , Animais , Autofagia , Proteínas de Caenorhabditis elegans/análise , Interpretação Estatística de Dados , Bases de Dados de Proteínas , Proteínas Associadas aos Microtúbulos/análise , Ligação Proteica , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
For a long time, autophagy has been mainly studied in yeast or mammalian cell lines, and assays for analyzing autophagy in these models have been well described. More recently, the involvement of autophagy in various physiological functions has been investigated in multicellular organisms. Modification of autophagy flux is involved in developmental processes, resistance to stress conditions, aging, cell death and multiple pathologies. So, the use of animal models is essential to understand these processes in the context of different cell types and during the whole life. For ten years, the nematode Caenorhabditis elegans has emerged as a powerful model to analyze autophagy in physiological or pathological contexts. In this article, we present some of the established approaches and the emerging tools available to monitor and manipulate autophagy in C. elegans, and discuss their advantages and limitations.
Assuntos
Envelhecimento/genética , Autofagia/genética , Bioensaio/métodos , Envelhecimento/patologia , Animais , Apoptose/genética , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , HumanosRESUMO
Endosomes and autophagosomes are two vesicular compartments involved in the degradation and recycling of cellular material. They both undergo a maturation process and finally fuse with the lysosome. In mammals, the convergence between endosomes and autophagosomes is a multistep process that can generate intermediate vesicles named amphisomes. Using knockdowns and mutants of the ESCRT machinery (ESCRT-0-ESCRT-III, ATPase VPS-4) and the autophagic pathway (LGG-1, LGG-2, ATG-7, TOR), we analyzed in vivo the functional links between endosomal maturation and autophagy in Caenorhabditis elegans. We report here that, despite a strong heterogeneity of their developmental phenotypes, all ESCRT mutants present an accumulation of abnormal endosomes and autophagosomes. We show that this accumulation of autophagosomes is secondary to the formation of enlarged endosomes and is due to the induction of the autophagic flux and not a blockage of fusion with lysosomes. We demonstrate that the induction of autophagy is not responsible for the lethality of ESCRT mutants but has a protective role on cellular degradation. We also show that increasing the basal level of autophagy reduces the formation of enlarged endosomes in ESCRT mutants. Together, our data indicate that the induction of autophagy is a protective response against the formation of an abnormal vesicular compartment.
Assuntos
Autofagia/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Adaptação Fisiológica , Animais , Animais Geneticamente Modificados , Autofagia/fisiologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Técnicas de Silenciamento de Genes , Genes de Helmintos , Mutação , Fenótipo , Interferência de RNARESUMO
Autophagy is a key lysosomal degradative mechanism allowing a prosurvival response to stresses, especially nutrient starvation. Here we investigate the mechanism of autophagy induction in response to sulfur starvation in Saccharomyces cerevisiae. We found that sulfur deprivation leads to rapid and widespread transcriptional induction of autophagy-related (ATG) genes in ways not seen under nitrogen starvation. This distinctive response depends mainly on the transcription activator of sulfur metabolism Met4. Consistently, Met4 is essential for autophagy under sulfur starvation. Depletion of either cysteine, methionine or SAM induces autophagy flux. However, only SAM depletion can trigger strong transcriptional induction of ATG genes and a fully functional autophagic response. Furthermore, combined inactivation of Met4 and Atg1 causes a dramatic decrease in cell survival under sulfur starvation, highlighting the interplay between sulfur metabolism and autophagy to maintain cell viability. Thus, we describe a pathway of sulfur starvation-induced autophagy depending on Met4 and involving SAM as signaling sulfur metabolite.
Assuntos
Autofagia , S-Adenosilmetionina , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Transdução de Sinais , Enxofre , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Autofagia/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Enxofre/metabolismo , S-Adenosilmetionina/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Metionina/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Quinases , Fatores de Transcrição de Zíper de Leucina BásicaRESUMO
Most of the functions of LC3/GABARAP in macroautophagy/autophagy are considered to depend on their association with the phagophore membrane through a conjugation to a lipid. Using site-directed mutagenesis, we inhibited the conjugation of LGG-1, the single homolog of GABARAP in C. elegans. Mutants that express only cytosolic forms revealed an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy-independent embryonic function.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Autofagia , Proteínas Associadas aos Microtúbulos , AutofagossomosRESUMO
The ubiquitin-like proteins Atg8/LC3/GABARAP are required for multiple steps of autophagy, such as initiation, cargo recognition and engulfment, vesicle closure and degradation. Most of LC3/GABARAP functions are considered dependent on their post-translational modifications and their association with the autophagosome membrane through a conjugation to a lipid, the phosphatidyl-ethanolamine. Contrarily to mammals, C. elegans possesses single homologs of LC3 and GABARAP families, named LGG-2 and LGG-1. Using site-directed mutagenesis, we inhibited the conjugation of LGG-1 to the autophagosome membrane and generated mutants that express only cytosolic forms, either the precursor or the cleaved protein. LGG-1 is an essential gene for autophagy and development in C. elegans, but we discovered that its functions could be fully achieved independently of its localization to the membrane. This study reveals an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy-independent embryonic function. Our data question the use of lipidated GABARAP/LC3 as the main marker of autophagic flux and highlight the high plasticity of autophagy.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Humanos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Autofagia , Autofagossomos/metabolismo , Fagocitose , Mamíferos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismoRESUMO
The ATG8 family of proteins regulates the autophagy process from the autophagosome maturation and cargo recruitment up to degradation. Autophagy dysfunction is involved in the development of multiple diseases. The LC3 interacting region (LIR)-based molecular traps have been designed to isolate endogenous ATG8 proteins and their interactors in order to facilitate the study of selective autophagy events. Here, we summarize protocols describing LC3 traps and sample preparation as well as adaptations for the analysis of ATG8 proteins in different biological models. This protocol was optimized to prepare affinity columns, reduce background, and improve the protein recovery to be analyzed by immunodetection with antibodies recognizing proteins of interest.
Assuntos
Aclimatação , Macroautofagia , Família da Proteína 8 Relacionada à Autofagia/genética , Anticorpos , AutofagiaRESUMO
MARS is an evolutionary conserved supramolecular assembly of aminoacyl-tRNA synthetases found in eukaryotes. This complex was thought to be ubiquitous in the deuterostome and protostome clades of bilaterians because similar complexes were isolated from arthropods and vertebrates. However, several features of the component enzymes suggested that in the nematode Caenorhabditis elegans, a species grouped with arthropods in modern phylogeny, this complex might not exist, or should display a significantly different structural organization. C. elegans was also taken as a model system to study in a multicellular organism amenable to experimental approaches, the reason for existence of these supramolecular entities. Here, using a proteomic approach, we have characterized the components of MARS in C. elegans. We show that this organism evolved a specific structural organization of this complex, which contains several bona fide components of the MARS complexes known so far, but also displays significant variations. These data highlight molecular evolution events that took place after radiation of bilaterians. Remarkably, it shows that expansion of MARS assembly in metazoans is not linear, but is the result of additions but also of subtractions along evolution. We then undertook an experimental approach, using inactivation of the endogenous copy of methionyl-tRNA synthetase by RNAi and expression of transgenic variants, to understand the role in complex assembly and the in vivo functionality, of the eukaryotic-specific domains appended to aminoacyl-tRNA synthetases. We show that rescue of the worms and assembly of transgenic variants into MARS rest on the presence of these appended domains.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Evolução Molecular , Complexos Multienzimáticos/metabolismo , Aminoacil-tRNA Sintetases/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Complexos Multienzimáticos/genética , ProteômicaRESUMO
Autophagy is an essential cellular pathway that ensures degradation of a wide range of substrates including damaged organelles or large protein aggregates. Understanding how this proteolytic pathway is regulated would increase our comprehension on its role in cellular physiology and contribute to identify biomarkers or potential drug targets to develop more specific treatments for disease in which autophagy is dysregulated. Here, we report the development of molecular traps based in the tandem disposition of LC3-interacting regions (LIR). The estimated affinity of LC3-traps for distinct recombinant LC3/GABARAP proteins is in the low nanomolar range and allows the capture of these proteins from distinct mammalian cell lines, S. cerevisiae and C. elegans. LC3-traps show preferences for GABARAP/LGG1 or LC3/LGG2 and pull-down substrates targeted to proteaphagy and mitophagy. Therefore, LC3-traps are versatile tools that can be adapted to multiple applications to monitor selective autophagy events in distinct physiologic and pathologic circumstances.
Assuntos
Caenorhabditis elegans , Macroautofagia , Animais , Autofagia , Caenorhabditis elegans/metabolismo , Mamíferos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Ligação Proteica , Saccharomyces cerevisiae/metabolismoRESUMO
ESCRTs (endosomal sorting complexes required for transport) were first discovered in yeast and are known to be required in the biogenesis of the MVB (multivesicular body). Most ESCRT research has been carried out in vitro using models such as yeast and mammalian cells in culture. The role of the ESCRTs genes in endosome maturation is conserved from yeast to mammals, but little is known about their function during development in multicellular organisms. Since ESCRTs play a leading role in regulating some cell signalling pathways by addressing receptors to the lysosome, it appears important to monitor ESCRT functions in multicellular models. The present review summarizes recent research on the developmental and cellular functions of the ESCRT in Caenorhabditis elegans, Drosophila melanogaster, Mus musculus or Arabidopsis thaliana.
Assuntos
Diferenciação Celular/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Transdução de Sinais/fisiologia , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Endossomos/ultraestrutura , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Lisossomos/ultraestrutura , Modelos Animais , Corpos Multivesiculares/metabolismo , Corpos Multivesiculares/ultraestrutura , Transporte Proteico/fisiologiaRESUMO
Temperature variations induce stressful conditions that challenge the ability of organisms to maintain cell homeostasis. The intensity and duration of heat stress affect cell response very differently, ranging from a beneficial effect - hormesis - to necrotic cell death. There is a strong interplay between the cell response to heat shock and macroautophagy/autophagy, which is induced to cope with stress. Using Caenorhabditis elegans, we developed a new paradigm to study adaptation to acute non-lethal heat-stress (aHS) during development. We found that aHS results in transient fragmentation of mitochondria, decreased cellular respiration, and delayed development. Moreover, an active autophagy flux associated with mitophagy events is triggered in many tissues, enables the rebuilding of the mitochondrial network and modulates the adaptive plasticity of the development, showing that the autophagic response is protective for C. elegans. Using genetic and cellular approaches, we showed that mitochondria are a major site for autophagosome biogenesis in the epidermis, under both standard and heat-stress conditions. We determined that DRP-1 (Dynamin-Related Protein 1) involved in mitochondrial fission, is an important player for the autophagy process and the adaptation to aHS. Our study suggests that DRP-1 is involved in coordinating mitochondrial fission and autophagosome biogenesis during stress.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Adaptação Fisiológica , Animais , Autofagia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Resposta ao Choque Térmico , Mitocôndrias/metabolismoRESUMO
Acute heat stress (aHS) can induce strong developmental defects in Caenorhabditis elegans larva but not lethality or sterility. This stress results in transitory fragmentation of mitochondria, formation of aggregates in the matrix, and decrease of mitochondrial respiration. Moreover, active autophagic flux associated with mitophagy events enables the rebuilding of the mitochondrial network and developmental recovery, showing that the autophagic response is protective. This adaptation to aHS does not require Pink1/Parkin or the mitophagy receptors DCT-1/NIX and FUNDC1. We also find that mitochondria are a major site for autophagosome biogenesis in the epidermis in both standard and heat stress conditions. In addition, we report that the depletion of the dynamin-related protein 1 (DRP-1) affects autophagic processes and the adaptation to aHS. In drp-1 animals, the abnormal mitochondria tend to modify their shape upon aHS but are unable to achieve fragmentation. Autophagy is induced, but autophagosomes are abnormally elongated and clustered on mitochondria. Our data support a role for DRP-1 in coordinating mitochondrial fission and autophagosome biogenesis in stress conditions.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Dinaminas/metabolismo , Resposta ao Choque Térmico , Mitocôndrias/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Dinaminas/genética , MitofagiaRESUMO
The let-413/scribble and dlg-1/discs large genes are key regulators of epithelial cell polarity in C. elegans and other systems but the mechanism how they organize a circumferential junctional belt around the apex of epithelial cells is not well understood. We report here that IP(3)/Ca(2+) signaling is involved in the let-413/dlg-1 pathway for the establishment of epithelial cell polarity during the development in C. elegans. Using RNAi to interfere with let-413 and dlg-1 gene functions during post-embryogenesis, we discovered a requirement for LET-413 and DLG-1 in the polarization of the spermathecal cells. The spermatheca forms an accordion-like organ through which eggs must enter to complete the ovulation process. LET-413- and DLG-1-depleted animals exhibit failure of ovulation. Consistent with this phenotype, the assembly of the apical junction into a continuous belt fails and the PAR-3 protein and microfilaments are no longer localized asymmetrically. All these defects can be suppressed by mutations in IPP-5, an inositol polyphosphate 5-phosphatase and in ITR-1, an inositol triphosphate receptor, which both are supposed to increase the intracellular Ca(2+) level. Analysis of embryogenesis revealed that IP(3)/Ca(2+) signaling is also required during junction assembly in embryonic epithelia.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Sinalização do Cálcio , Guanilato Quinases/fisiologia , Fosfatos de Inositol/metabolismo , Junções Intercelulares/metabolismo , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Polaridade Celular , Desenvolvimento Embrionário , Células Epiteliais , Epitélio , Feminino , Masculino , OvulaçãoRESUMO
BACKGROUND INFORMATION: Within the endocytic pathway, the ESCRT (endosomal sorting complex required for transport) machinery is essential for the biogenesis of MVBs (multivesicular bodies). In yeast, ESCRTs are recruited at the endosomal membrane and are involved in cargo sorting into intralumenal vesicles of the MVBs. RESULTS: In the present study, we characterize the ESCRT-III protein CeVPS-32 (Caenorhabditis elegans vacuolar protein sorting 32) and its interactions with CeVPS-27, CeVPS-23 and CeVPS-4. In contrast with other CevpsE (class E vps) genes, depletion of Cevps-32 is embryonic lethal with severe defects in the remodelling of epithelial cell shape during organogenesis. Furthermore, Cevps-32 animals display an accumulation of enlarged early endosomes in epithelial cells and an accumulation of autophagosomes. The CeVPS-32 protein is enriched in epithelial tissues and in residual bodies during spermatid maturation. We show that CeVPS-32 and CeVPS-27/Hrs (hepatocyte-growth-factor-regulated tyrosine kinase substrate) are enriched in distinct subdomains at the endosomal membrane. CeVPS-27-positive subdomains are also enriched for the ESCRT-I protein CeVPS-23/TSG101 (tumour susceptibility gene 101). The formation of CeVPS-27 subdomains is not affected by the depletion of CeVPS-23, CeVPS-32 or the ATPase CeVPS-4. CONCLUSION: Our results suggest that the formation of membrane subdomains is essential for the maturation of endosomes.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Endossomos/metabolismo , Células Epiteliais/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Autofagia/genética , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/química , Transtornos do Desenvolvimento Sexual , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Desenvolvimento Embrionário , Genes Letais , Genes Reporter , Células Germinativas/metabolismo , Imuno-Histoquímica , Larva/crescimento & desenvolvimento , Larva/metabolismo , Larva/ultraestrutura , Estágios do Ciclo de Vida , Masculino , Membranas/metabolismo , Organogênese , Interferência de RNA , Caracteres Sexuais , Transfecção , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/deficiênciaRESUMO
Mitochondria is a key cellular organelle, which is tightly supervised by multiple oversight cellular mechanisms regulating mitochondrial biogenesis and mitochondria maintenance and/or elimination. Selective autophagy of mitochondria, id est mitophagy, is one of the cellular mechanisms controlling mitochondria homeostasis. The nematode Caenorhabditis elegans has recently emerged as a powerful model organism to study the roles and functions of mitophagy. We present here the current knowledge on cellular and molecular mechanisms underlying the selective elimination of mitochondria by autophagy in C. elegans in the context of developmental processes, aging and adaptive responses to various stresses.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Mitocôndrias/metabolismo , Mitofagia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Mitocôndrias/genéticaRESUMO
Morphogenesis of the Caenorhabditis elegans embryo is driven by actin microfilaments in the epidermis and by sarcomeres in body wall muscles. Both tissues are mechanically coupled, most likely through specialized attachment structures called fibrous organelles (FOs) that connect muscles to the cuticle across the epidermis. Here, we report the identification of new mutations in a gene known as vab-10, which lead to severe morphogenesis defects, and show that vab-10 corresponds to the C. elegans spectraplakin locus. Our analysis of vab-10 reveals novel insights into the role of this plakin subfamily. vab-10 generates isoforms related either to plectin (termed VAB-10A) or to microtubule actin cross-linking factor plakins (termed VAB-10B). Using specific antibodies and mutations, we show that VAB-10A and VAB-10B have distinct distributions and functions in the epidermis. Loss of VAB-10A impairs the integrity of FOs, leading to epidermal detachment from the cuticle and muscles, hence demonstrating that FOs are functionally and molecularly related to hemidesmosomes. We suggest that this isoform protects against forces external to the epidermis. In contrast, lack of VAB-10B leads to increased epidermal thickness during embryonic morphogenesis when epidermal cells change shape. We suggest that this isoform protects cells against tension that builds up within the epidermis.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Epiderme/metabolismo , Citoesqueleto de Actina/metabolismo , Alelos , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/análise , Proteínas de Caenorhabditis elegans/genética , Membrana Celular/metabolismo , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Epiderme/embriologia , Epiderme/ultraestrutura , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Essenciais , Microscopia Eletrônica , Mutação , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
In this chapter, we present a protocol to perform correlative light and electron microscopy (CLEM) on Caenorhabditis elegans embryos. We use a specific fixation method which preserves both the GFP fluorescence and the structural integrity of the samples. Thin sections are first analyzed by light microscopy to detect GFP-tagged proteins, then by transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows to determine the subcellular localization of the fluorescent protein. We have used this method to characterize the roles of autophagy in the phagocytosis of apoptotic cells in C. elegans embryos. We analyzed in apoptotic cell and phagocytic cell the localization of the two homologs of LC3/GABARAP proteins, namely, LGG-1 and LGG-2.