Membrane localization of LGG-1/GABARAP is dispensable for autophagy in C. elegans.

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.

LGG-1/GABARAP lipidation is not required for autophagy and development in Caenorhabditis elegans.

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.

The strange case of Drp1 in autophagy: Jekyll and Hyde?

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.

A DRP-1 dependent autophagy process facilitates rebuilding of the mitochondrial network and modulates adaptation capacity in response to acute heat stress during C. elegans development.

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.

Autophagy facilitates mitochondrial rebuilding after acute heat stress via a DRP-1-dependent process.

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.

Mitophagy during development and stress in C. elegans.

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.

Subcellular Localization of ESCRT-II in the Nematode C. elegans by Correlative Light Electron Microscopy.

In this chapter, we report a protocol to perform correlative light electron microscopy (CLEM) on adult Caenorhabditis elegans. We use a specific fixation protocol, 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 and, subsequently, with transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows determining the subcellular localization of the fluorescent protein.We used CLEM to characterize the subcellular localization of the C. elegans ESCRT-II component VPS-36. VPS-36 protein localization in C. elegans muscle cell is strongly correlated with the sarcoplasmic reticulum network. Together with genetic evidences, the CLEM data support a role for ESCRT-II proteins in sarcoplasmic reticulum membrane shaping.

Correction to: The ESCRT Complexes.

This book was inadvertently published with incorrect affiliations for both the editors. Earlier it was.

ESCRT and autophagies: Endosomal functions and beyond.

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.

The ESCRT-II proteins are involved in shaping the sarcoplasmic reticulum in C. elegans.

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.

Interactions between endosomal maturation and autophagy: analysis of ESCRT machinery during Caenorhabditis elegans development.

Endocytosis and autophagy are key vesicular pathways involved in degradation and recycling of cellular material. Both degradative pathways finally fuse with lysosome but are indeed interconnected at several levels. In particular, the fusion between endosomes and autophagosomes can generate intermediate vesicles named amphisomes. We analyzed the physiological and developmental roles of the ESCRT machinery in a model organism, the nematode Caenorhabditis elegans and showed that the blockage of the endosomal maturation triggers the induction of autophagic activity. This chapter describes several methods for studying endocytosis, autophagy, and their interconnection in C. elegans. A series of genetic, biochemical, and microscopy analyses has been used to study at the cellular and developmental levels, the cross talks between autophagy and endocytosis.

Induction of autophagy in ESCRT mutants is an adaptive response for cell survival in C. elegans.

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.

Allophagy: a macroautophagic process degrading spermatozoid-inherited organelles.

In most animals, during oocyte fertilization the spermatozoon provides DNA and centrioles together with some cytoplasm and organelles, but paternal mitochondria are generally eliminated in the embryo. Using the model animal C. elegans we have shown that paternal organelle degradation is dependent on the formation of autophagosomes a few minutes after fertilization. This macroautophagic process is preceded by an active ubiquitination of some spermatozoon-inherited organelles. Analysis of fertilized mouse embryos suggests that this autophagy event is evolutionarily conserved.

Need an ESCRT for autophagosomal maturation?

Several reports in fly, nematode and mammalian cells have revealed that the inactivation of endosomal sorting complexes required for transport (ESCRT) blocks the endosomal maturation but also leads to the increased number of autophagosomal structures. In this review we compare these data and conclude that the way ESCRT mutations affect the relationships between autophagosomes and endosomes cannot be generalized but depends on the studied species. We propose that the effect of ESCRT mutations on autophagy is directly dependent of the level of interaction between autophagosomes and endosomes. In particular, the formation of amphisomes during autophagosomal maturation could be the key point to explain the differences observed between species. These observations highlight the importance of multiple model organisms to decipher the complexity of relationships between such dynamic vesicles.

Postfertilization autophagy of sperm organelles prevents paternal mitochondrial DNA transmission.

In sexual reproduction of most animals, the spermatozoon provides DNA and centrioles, together with some cytoplasm and organelles, to the oocyte that is being fertilized. Paternal mitochondria and their genomes are generally eliminated in the embryo by an unknown degradation mechanism. We show that, upon fertilization, a Caenorhabditis elegans spermatozoon triggers the recruitment of autophagosomes within minutes and subsequent paternal mitochondria degradation. Whereas the nematode-specific sperm membranous organelles are ubiquitinated before autophagosome formation, the mitochondria are not. The degradation of both paternal structures and mitochondrial DNA requires an LC3-dependent autophagy. Analysis of fertilized mouse embryos shows the localization of autophagy markers, which suggests that this autophagy event is evolutionarily conserved to prevent both the transmission of paternal mitochondrial DNA to the offspring and the establishment of heteroplasmy.

Qri7/OSGEPL, the mitochondrial version of the universal Kae1/YgjD protein, is essential for mitochondrial genome maintenance.

Yeast Qri7 and human OSGEPL are members of the orthologous Kae1(OSGEP)/YgjD protein family, the last class of universally conserved proteins without assigned function. Phylogenetic analyses indicate that the eukaryotic Qri7(OSGEPL) proteins originated from bacterial YgjD proteins. We have recently shown that the archaeal Kae1 protein is a DNA-binding protein that exhibits apurinic endonuclease activity in vitro. We show here that the Qri7/OSGEPL proteins localize in mitochondria and are involved in mitochondrial genome maintenance in two model eukaryotic organisms, Saccharomyces cerevisiae and Caenorhabditis elegans. Furthermore, S. cerevisiae Qri7 complements the loss of the bacterial YgjD protein in Escherichia coli, suggesting that Qri7/OSGEPL and YgjD proteins have retained similar functions in modern organisms. We suggest to name members of the Kae1(OSGEP)/YgjD family UGMP, for Universal Genome Maintenance Proteins.

Differential expression pattern of the four mitochondrial adenine nucleotide transporter ant genes and their roles during the development of Caenorhabditis elegans.

The adenine nucleotide transporter (ANT) mediates exchange of cytosolic ADP and mitochondrial ATP. Although most species contain more than one ANT family member, it is not known whether their roles in developmental processes are redundant or specific. Here, we show that the Caenorhabditis elegans genome encodes four candidate ant genes (ant-1.1, ant-1.2, ant-1.3, and ant-1.4). We have investigated their spatiotemporal expression patterns and discovered that, whereas ANT-1.1 is a ubiquitously expressed mitochondrial protein, the other three ANT proteins show a restricted range of cell type expression. Moreover, only the disruption of ant-1.1 function, through RNA interference (RNAi), gives a mutant phenotype. Most of the ant-1.1(RNAi) mutant embryos arrest before the morphogenesis stage. Furthermore, ant-1.1 is also required postembryonically because RNAi mutants exhibit small size and life-span extension. Our results suggest that ant-1.1 is the only ant gene strictly required for embryonic and postembryonic development in C. elegans.

The Caenorhabditis elegans unc-63 gene encodes a levamisole-sensitive nicotinic acetylcholine receptor alpha subunit.

The anthelmintic drug levamisole causes hypercontraction of body wall muscles and lethality in nematode worms. In the nematode Caenorhabditis elegans, a genetic screen for levamisole resistance has identified 12 genes, three of which (unc-38, unc-29, and lev-1) encode nicotinic acetylcholine receptor (nAChR) subunits. Here we describe the molecular and functional characterization of another levamisole-resistant gene, unc-63, encoding a nAChR alpha subunit with a predicted amino acid sequence most similar to that of UNC-38. Like UNC-38 and UNC-29, UNC-63 is expressed in body wall muscles. In addition, UNC-63 is expressed in vulval muscles and neurons. We also show that LEV-1 is expressed in body wall muscle, thus overlapping the cellular localization of UNC-63, UNC-38, and UNC-29 and suggesting possible association in vivo. This is supported by electrophysiological studies on body wall muscle, which demonstrate that a levamisole-sensitive nAChR present at the C. elegans neuromuscular junction requires both UNC-63 and LEV-1 subunits. Thus, at least four subunits, two alpha types (UNC-38 and UNC-63) and two non-alpha types (UNC-29 and LEV-1), can contribute to levamisole-sensitive muscle nAChRs in nematodes.

Functional genomics of ionotropic acetylcholine receptors in Caenorhabditis elegans and Drosophila melanogaster.

Genetics, genomics and electrophysiology are transforming our understanding of the nicotinic acetylcholine receptors (nAChRs). Caenorhabditis elegans contains the largest known family of nAChR subunit genes (27 members), while Drosophila melanogaster contains an exclusively neuronal nAChR gene family (10 members). In C. elegans, several genetic screens have enabled the identification of nAChR subunits, along with novel proteins that act upstream and downstream of functional nAChRs. The C. elegans genome project has identified many new candidate nAChR subunits and the calculated electrostatic potential energy profiles for the M2 channel-lining regions predict considerable functional diversity. The respective roles of subunits are under investigation using forward and reverse genetics. Electrophysiological and reporter gene studies have demonstrated roles for particular subunits in levamisole-sensitive muscle nAChRs and a role for nAChRs in pharyngeal pumping. Recombinant homomeric and heteromeric C. elegans nAChRs have been expressed in Xenopus laevis oocytes. In D. melanogaster, three new nAChR a subunits have been cloned, one of which shows multiple variant transcripts arising from alternative splicing and A-to-I pre-mRNA editing. Thus, studies on the genetic model organisms C. elegans and D. melanogaster have revealed different routes to generating molecular and functional diversity in the nAChR gene family and are providing new insights into the in vivo functions of individual family members.