PUF-8, a C. elegans ortholog of the RNA-binding proteins PUM1 and PUM2, is required for robustness of the cell death fate.

During C. elegans development, 1090 somatic cells are generated, of which 959 survive and 131 die, many through apoptosis. We present evidence that PUF-8, a C. elegans ortholog of the mammalian RNA-binding proteins PUM1 and PUM2, is required for the robustness of this 'survival and death' pattern. We found that PUF-8 prevents the inappropriate death of cells that normally survive, and we present evidence that this anti-apoptotic activity of PUF-8 is dependent on the ability of PUF-8 to interact with ced-3 (a C. elegans ortholog of caspase) mRNA, thereby repressing the activity of the pro-apoptotic ced-3 gene. PUF-8 also promotes the death of cells that are programmed to die, and we propose that this pro-apoptotic activity of PUF-8 may depend on the ability of PUF-8 to repress the expression of the anti-apoptotic ced-9 gene (a C. elegans ortholog of Bcl2). Our results suggest that stochastic differences in the expression of genes within the apoptosis pathway can disrupt the highly reproducible and robust survival and death pattern during C. elegans development, and that PUF-8 acts at the post-transcriptional level to level out these differences, thereby ensuring proper cell number homeostasis.

A caspase-RhoGEF axis contributes to the cell size threshold for apoptotic death in developing Caenorhabditis elegans.

A cell's size affects the likelihood that it will die. But how is cell size controlled in this context and how does cell size impact commitment to the cell death fate? We present evidence that the caspase CED-3 interacts with the RhoGEF ECT-2 in Caenorhabditis elegans neuroblasts that generate "unwanted" cells. We propose that this interaction promotes polar actomyosin contractility, which leads to unequal neuroblast division and the generation of a daughter cell that is below the critical "lethal" size threshold. Furthermore, we find that hyperactivation of ECT-2 RhoGEF reduces the sizes of unwanted cells. Importantly, this suppresses the "cell death abnormal" phenotype caused by the partial loss of ced-3 caspase and therefore increases the likelihood that unwanted cells die. A putative null mutation of ced-3 caspase, however, is not suppressed, which indicates that cell size affects CED-3 caspase activation and/or activity. Therefore, we have uncovered novel sequential and reciprocal interactions between the apoptosis pathway and cell size that impact a cell's commitment to the cell death fate.

miRNAs cooperate in apoptosis regulation during C. elegans development.

Programmed cell death occurs in a highly reproducible manner during Caenorhabditis elegans development. We demonstrate that, during embryogenesis, miR-35 and miR-58 bantam family microRNAs (miRNAs) cooperate to prevent the precocious death of mothers of cells programmed to die by repressing the gene egl-1, which encodes a proapoptotic BH3-only protein. In addition, we present evidence that repression of egl-1 is dependent on binding sites for miR-35 and miR-58 family miRNAs within the egl-1 3' untranslated region (UTR), which affect both mRNA copy number and translation. Furthermore, using single-molecule RNA fluorescent in situ hybridization (smRNA FISH), we show that egl-1 is transcribed in the mother of a cell programmed to die and that miR-35 and miR-58 family miRNAs prevent this mother from dying by keeping the copy number of egl-1 mRNA below a critical threshold. Finally, miR-35 and miR-58 family miRNAs can also dampen the transcriptional boost of egl-1 that occurs specifically in a daughter cell that is programmed to die. We propose that miRNAs compensate for lineage-specific differences in egl-1 transcriptional activation, thus ensuring that EGL-1 activity reaches the threshold necessary to trigger death only in daughter cells that are programmed to die.

The permanently chaperone-active small heat shock protein Hsp17 from Caenorhabditis elegans exhibits topological separation of its N-terminal regions.

Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind nonnative proteins in an ATP-independent manner. Caenorhabditis elegans encodes 16 different sHsps, among them Hsp17, which is evolutionarily distinct from other sHsps in the nematode. The structure and mechanism of Hsp17 and how these may differ from other sHsps remain unclear. Here, we find that Hsp17 has a distinct expression pattern, structural organization, and chaperone function. Consistent with its presence under nonstress conditions, and in contrast to many other sHsps, we determined that Hsp17 is a mono-disperse, permanently active chaperone in vitro, which interacts with hundreds of different C. elegans proteins under physiological conditions. Additionally, our cryo-EM structure of Hsp17 reveals that in the 24-mer complex, 12 N-terminal regions are involved in its chaperone function. These flexible regions are located on the outside of the spherical oligomer, whereas the other 12 N-terminal regions are engaged in stabilizing interactions in its interior. This allows the same region in Hsp17 to perform different functions depending on the topological context. Taken together, our results reveal structural and functional features that further define the structural basis of permanently active sHsps.

PIG-1 MELK-dependent phosphorylation of nonmuscle myosin II promotes apoptosis through CES-1 Snail partitioning.

The mechanism(s) through which mammalian kinase MELK promotes tumorigenesis is not understood. We find that the C. elegans orthologue of MELK, PIG-1, promotes apoptosis by partitioning an anti-apoptotic factor. The C. elegans NSM neuroblast divides to produce a larger cell that differentiates into a neuron and a smaller cell that dies. We find that in this context, PIG-1 MELK is required for partitioning of CES-1 Snail, a transcriptional repressor of the pro-apoptotic gene egl-1 BH3-only. pig-1 MELK is controlled by both a ces-1 Snail- and par-4 LKB1-dependent pathway, and may act through phosphorylation and cortical enrichment of nonmuscle myosin II prior to neuroblast division. We propose that pig-1 MELK-induced local contractility of the actomyosin network plays a conserved role in the acquisition of the apoptotic fate. Our work also uncovers an auto-regulatory loop through which ces-1 Snail controls its own activity through the formation of a gradient of CES-1 Snail protein.

Autophagy compensates for defects in mitochondrial dynamics.

Compromising mitochondrial fusion or fission disrupts cellular homeostasis; however, the underlying mechanism(s) are not fully understood. The loss of C. elegans fzo-1MFN results in mitochondrial fragmentation, decreased mitochondrial membrane potential and the induction of the mitochondrial unfolded protein response (UPRmt). We performed a genome-wide RNAi screen for genes that when knocked-down suppress fzo-1MFN(lf)-induced UPRmt. Of the 299 genes identified, 143 encode negative regulators of autophagy, many of which have previously not been implicated in this cellular quality control mechanism. We present evidence that increased autophagic flux suppresses fzo-1MFN(lf)-induced UPRmt by increasing mitochondrial membrane potential rather than restoring mitochondrial morphology. Furthermore, we demonstrate that increased autophagic flux also suppresses UPRmt induction in response to a block in mitochondrial fission, but not in response to the loss of spg-7AFG3L2, which encodes a mitochondrial metalloprotease. Finally, we found that blocking mitochondrial fusion or fission leads to increased levels of certain types of triacylglycerols and that this is at least partially reverted by the induction of autophagy. We propose that the breakdown of these triacylglycerols through autophagy leads to elevated metabolic activity, thereby increasing mitochondrial membrane potential and restoring mitochondrial and cellular homeostasis.

Mitochondrial Alkbh1 localizes to mtRNA granules and its knockdown induces the mitochondrial UPR in humans and C. elegans.

The Fe(II) and 2-oxoglutarate-dependent oxygenase Alkb homologue 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA and histones. Thereby different enzymatic activities have been identified including, among others, demethylation of N3-methylcytosine (m3C) in RNA- and single-stranded DNA oligonucleotides, demethylation of N1-methyladenosine (m1A) in tRNA or formation of 5-formyl cytosine (f5C) in tRNA. In accordance with the different substrates, Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria. Here, we describe further evidence for a role of human Alkbh1 in regulation of mitochondrial protein biogenesis, including visualizing localization of Alkbh1 into mitochondrial RNA granules with super-resolution 3D SIM microscopy. Electron microscopy and high-resolution respirometry analyses revealed an impact of Alkbh1 level on mitochondrial respiration, but not on mitochondrial structure. Downregulation of Alkbh1 impacts cell growth in HeLa cells and delays development in Caenorhabditis elegans, where the mitochondrial role of Alkbh1 seems to be conserved. Alkbh1 knockdown, but not Alkbh7 knockdown, triggers the mitochondrial unfolded protein response (UPRmt) in C. elegans.

Twenty million years of evolution: The embryogenesis of four Caenorhabditis species are indistinguishable despite extensive genome divergence.

The four Caenorhabditis species C. elegans, C. briggsae, C. remanei and C. brenneri show more divergence at the genomic level than humans compared to mice (Stein et al., 2003; Cutter et al., 2006, 2008). However, the behavior and anatomy of these nematodes are very similar. We present a detailed analysis of the embryonic development of these species using 4D-microscopic analyses of embryos including lineage analysis, terminal differentiation patterns and bioinformatical quantifications of cell behavior. Further functional experiments support the notion that the early development of all four species depends on identical induction patterns. Based on our results, the embryonic development of all four Caenorhabditis species are nearly identical, suggesting that an apparently optimal program to construct the body plan of nematodes has been conserved for at least 20 million years. This contrasts the levels of divergence between the genomes and the protein orthologs of the Caenorhabditis species, which is comparable to the level of divergence between mouse and human. This indicates an intricate relationship between the structure of genomes and the morphology of animals.

Genetic control of programmed cell death during animal development.

The elimination of unwanted cells by programmed cell death is a common feature of animal development. Genetic studies in the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the mouse have not only revealed the molecular machineries that cause the programmed demise of specific cells, but have also allowed us to get a glimpse of the types of pathways that regulate these machineries during development. Rather than serving as a broad overview of programmed cell death during development, this review focuses on recent advances in our understanding of the regulation of specific programmed cell death events during nematode, fly, and mouse development. Recent studies have revealed that many of the regulatory pathways involved play additional important roles in development, which confirms that the programmed cell death fate is an integral aspect of animal development.

Phagocytic receptor signaling regulates clathrin and epsin-mediated cytoskeletal remodeling during apoptotic cell engulfment in C. elegans.

The engulfment and subsequent degradation of apoptotic cells by phagocytes is an evolutionarily conserved process that efficiently removes dying cells from animal bodies during development. Here, we report that clathrin heavy chain (CHC-1), a membrane coat protein well known for its role in receptor-mediated endocytosis, and its adaptor epsin (EPN-1) play crucial roles in removing apoptotic cells in Caenorhabditis elegans. Inactivating epn-1 or chc-1 disrupts engulfment by impairing actin polymerization. This defect is partially suppressed by inactivating UNC-60, a cofilin ortholog and actin server/depolymerization protein, further indicating that EPN-1 and CHC-1 regulate actin assembly during pseudopod extension. CHC-1 is enriched on extending pseudopods together with EPN-1, in an EPN-1-dependent manner. Epistasis analysis places epn-1 and chc-1 in the same cell-corpse engulfment pathway as ced-1, ced-6 and dyn-1. CED-1 signaling is necessary for the pseudopod enrichment of EPN-1 and CHC-1. CED-1, CED-6 and DYN-1, like EPN-1 and CHC-1, are essential for the assembly and stability of F-actin underneath pseudopods. We propose that in response to CED-1 signaling, CHC-1 is recruited to the phagocytic cup through EPN-1 and acts as a scaffold protein to organize actin remodeling. Our work reveals novel roles of clathrin and epsin in apoptotic-cell internalization, suggests a Hip1/R-independent mechanism linking clathrin to actin assembly, and ties the CED-1 pathway to cytoskeleton remodeling.

Coordination of cell proliferation and cell fate determination by CES-1 snail.

The coordination of cell proliferation and cell fate determination is critical during development but the mechanisms through which this is accomplished are unclear. We present evidence that the Snail-related transcription factor CES-1 of Caenorhabditis elegans coordinates these processes in a specific cell lineage. CES-1 can cause loss of cell polarity in the NSM neuroblast. By repressing the transcription of the BH3-only gene egl-1, CES-1 can also suppress apoptosis in the daughters of the NSM neuroblasts. We now demonstrate that CES-1 also affects cell cycle progression in this lineage. Specifically, we found that CES-1 can repress the transcription of the cdc-25.2 gene, which encodes a Cdc25-like phosphatase, thereby enhancing the block in NSM neuroblast division caused by the partial loss of cya-1, which encodes Cyclin A. Our results indicate that CDC-25.2 and CYA-1 control specific cell divisions and that the over-expression of the ces-1 gene leads to incorrect regulation of this functional 'module'. Finally, we provide evidence that dnj-11 MIDA1 not only regulate CES-1 activity in the context of cell polarity and apoptosis but also in the context of cell cycle progression. In mammals, the over-expression of Snail-related genes has been implicated in tumorigenesis. Our findings support the notion that the oncogenic potential of Snail-related transcription factors lies in their capability to, simultaneously, affect cell cycle progression, cell polarity and apoptosis and, hence, the coordination of cell proliferation and cell fate determination.

Impaired complex IV activity in response to loss of LRPPRC function can be compensated by mitochondrial hyperfusion.

Mitochondrial morphology changes in response to various stimuli but the significance of this is unclear. In a screen for mutants with abnormal mitochondrial morphology, we identified MMA-1, the Caenorhabditis elegans homolog of the French Canadian Leigh Syndrome protein LRPPRC (leucine-rich pentatricopeptide repeat containing). We demonstrate that reducing mma-1 or LRPPRC function causes mitochondrial hyperfusion. Reducing mma-1/LRPPRC function also decreases the activity of complex IV of the electron transport chain, however without affecting cellular ATP levels. Preventing mitochondrial hyperfusion in mma-1 animals causes larval arrest and embryonic lethality. Furthermore, prolonged LRPPRC knock-down in mammalian cells leads to mitochondrial fragmentation and decreased levels of ATP. These findings indicate that in a mma-1/LRPPRC-deficient background, hyperfusion allows mitochondria to maintain their functions despite a reduction in complex IV activity. Our data reveal an evolutionary conserved mechanism that is triggered by reduced complex IV function and that induces mitochondrial hyperfusion to transiently compensate for a drop in the activity of the electron transport chain.

A molecular switch that governs mitochondrial fusion and fission mediated by the BCL2-like protein CED-9 of Caenorhabditis elegans.

Depending on the cellular context, BCL2-like proteins promote mitochondrial fusion or fission. What determines which of these two opposing processes they promote has so far been unknown. Furthermore, the mechanisms through which BCL2-like proteins affect mitochondrial dynamics remain to be fully understood. The BCL2-like protein CED-9 of Caenorhabditis elegans has previously been shown to promote mitochondrial fusion by physically interacting with the mitochondrial fusion protein FZO-1. Here, we report that CED-9 also physically interacts with the mitochondrial fission protein DRP-1 and that this interaction can be enhanced when CED-9 is associated with the BH3-only protein EGL-1. In addition, we show that the EGL-1-CED-9 complex promotes mitochondrial fission by recruiting DRP-1 to mitochondria and that the egl-1 gene is required for CED-9-dependent mitochondrial fission in vivo. Based on these results, we propose that EGL-1 converts CED-9 into a mitochondrial receptor for DRP-1, thereby shifting its activity from profusion to profission. We hypothesize that BCL2-like proteins act as mitochondrial receptors for DRP-1-like proteins in higher organisms as well and that BH3-only proteins play a general role as modifiers of the function in mitochondrial dynamics of BCL2-like proteins. We speculate that this function of BCL2-like proteins may be as couplers of mitochondrial fusion and fission.

A genetic screen identifies C. elegans eif-3.H and hrpr-1 as pro-apoptotic genes and potential activators of egl-1 expression.

During C. elegans development, 1090 somatic cells are generated of which 131 reproducibly die, many through apoptosis. The C. elegans BH3-only gene egl-1 is the key activator of apoptosis in somatic tissues, and it is predominantly expressed in 'cell death' lineages i.e. lineages in which apoptotic cell death occurs. egl-1 expression is regulated at the transcriptional and post-transcriptional level. For example, we previously showed that the miR-35 and miR-58 families of miRNAs repress egl-1 expression in mothers of 'unwanted' cells by binding to the 3' UTR of egl-1 mRNA, thereby increasing egl-1 mRNA turnover. In a screen for RNA-binding proteins with a role in the post-transcriptional control of egl-1 expression, we identified EIF-3.H (ortholog of human eIF3H) and HRPR-1 (ortholog human hnRNP R/Q) as potential activators of egl-1 expression. In addition, we demonstrate that the knockdown of the eif-3.H or hrpr-1 gene by RNA-mediated interference (RNAi) results in the inappropriate survival of unwanted cells during C. elegans development. Our study provides novel insight into how egl-1 expression is controlled to cause the reproducible pattern of cell death observed during C. elegans development.

ULP-2 SUMO protease regulates UPRmt and mitochondrial homeostasis in Caenorhabditis elegans.

Mitochondria are the powerhouses of cells, responsible for energy production and regulation of cellular homeostasis. When mitochondrial function is impaired, a stress response termed mitochondrial unfolded protein response (UPRmt) is initiated to restore mitochondrial function. Since mitochondria and UPRmt are implicated in many diseases, it is important to understand UPRmt regulation. In this study, we show that the SUMO protease ULP-2 has a key role in regulating mitochondrial function and UPRmt. Specifically, down-regulation of ulp-2 suppresses UPRmt and reduces mitochondrial membrane potential without significantly affecting cellular ROS. Mitochondrial networks are expanded in ulp-2 null mutants with larger mitochondrial area and increased branching. Moreover, the amount of mitochondrial DNA is increased in ulp-2 mutants. Downregulation of ULP-2 also leads to alterations in expression levels of mitochondrial genes involved in protein import and mtDNA replication, however, mitophagy remains unaltered. In summary, this study demonstrates that ULP-2 is required for mitochondrial homeostasis and the UPRmt.

The story behind the emergence of different forms of cell death.

Various types of cell death program are needed for cells to respond to changes in physiological conditions. In this collection of Voices, we asked scientists to tell the story behind their contributions to the identification and mechanistic dissection of cell death pathways and to discuss future directions for such research.

Recombinant expression, biophysical characterization, and cardiolipin-induced changes of two Caenorhabditis elegans cytochrome c proteins.

Cytochrome c (cyt c) is one of the most widely studied biomolecules, but not much is known about this protein from nematodes. Recombinant expression of Caenorhabditis elegans CYC-2.1 and CYC-2.2 allowed for detailed characterization of their structural features, redox properties, stabilities, and interactions with cardiolipin (CL)-containing liposomes. Using a variety of spectroscopic tools, we show that CYC-2.1 and CYC-2.2 adopt a globular α-helical fold with His/Met heme ligation. The longer CYC-2.2 has a lower thermodynamic stability than CYC-2.1 and lacks His residues to misligate to the heme in the protein's denatured state. Both C. elegans proteins bind to CL-containing liposomes, and these interactions promote the proteins' peroxidase activity but to a much greater degree for CYC-2.2. Dye-to-heme distance distributions from time-resolved fluorescence resonance energy transfer in bimane-labeled CYC-2.1 and CYC-2.2 revealed similar populations of extended and compact conformers for CL-bound proteins, suggesting that their distinct peroxidase activities in the presence of CL arise from differences in the local heme environments for the two polypeptide ensembles. Without inhibition from His misligation, a less stable and more prone to unfolding CYC-2.2 allows for better access of substrates to the heme and thus exhibits higher peroxidase activity. Similar features of the conformational ensembles of CYC-2.1 and CYC-2.2 to those of mammalian cyt c suggest that C. elegans proteins, particularly the former, could serve as useful models for examining the mechanism of cyt c-CL interactions in live organisms.

Mitochondrial involvement in cell death of non-mammalian eukaryotes.

Although mitochondria are essential organelles for long-term survival of eukaryotic cells, recent discoveries in biochemistry and genetics have advanced our understanding of the requirements for mitochondria in cell death. Much of what we understand about cell death is based on the identification of conserved cell death genes in Drosophila melanogaster and Caenorhabditis elegans. However, the role of mitochondria in cell death in these models has been much less clear. Considering the active role that mitochondria play in apoptosis in mammalian cells, the mitochondrial contribution to cell death in non-mammalian systems has been an area of active investigation. In this article, we review the current research on this topic in three non-mammalian models, C. elegans, Drosophila, and Saccharomyces cerevisiae. In addition, we discuss how non-mammalian models have provided important insight into the mechanisms of human disease as they relate to the mitochondrial pathway of cell death. The unique perspective derived from each of these model systems provides a more complete understanding of mitochondria in programmed cell death. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.

Methods to Study the Mitochondrial Unfolded Protein Response (UPRmt) in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is a powerful model to study cellular stress responses. Due to its transparency and ease of genetic manipulation, C. elegans is especially suitable for fluorescence microscopy. As a result, studies of C. elegans using different fluorescent reporters have led to the discovery of key players of cellular stress response pathways, including the mitochondrial unfolded protein response (UPRmt). UPRmt is a protective retrograde signaling pathway that ensures mitochondrial homeostasis. The nuclear genes hsp-6 and hsp-60 encode mitochondrial chaperones and are highly expressed upon UPRmt induction. The transcriptional reporters of these genes, hsp-6::gfp and hsp-60::gfp, have been instrumental for monitoring this pathway in live animals. Additional tools for studying UPRmt include fusion proteins of ATFS-1 and DVE-1, ATFS-1::GFP and DVE-1::GFP, key players of the UPRmt pathway. In this protocol, we discuss advantages and limitations of currently available methods and reporters, and we provide detailed instructions on how to image and quantify reporter expression.

Genetic screen identifies non-mitochondrial proteins involved in the maintenance of mitochondrial homeostasis.

The mitochondrial unfolded protein response (UPR mt ) is an important stress response that ensures the maintenance of mitochondrial homeostasis in response to various types of cellular stress. We previously described a genetic screen for Caenorhabditis elegans genes, which when inactivated cause UPR mt activation, and reported genes identified that encode mitochondrial proteins. We now report additional genes identified in the screen. Importantly, these include genes that encode non-mitochondrial proteins involved in processes such as the control of gene expression, post-translational modifications, cell signaling and cellular trafficking. Interestingly, we identified several genes that have been proposed to participate in the transfer of lipids between peroxisomes, ER and mitochondria, suggesting that lipid transfer between these organelles is essential for mitochondrial homeostasis. In conclusion, this study shows that the maintenance of mitochondrial homeostasis is not only dependent on mitochondrial processes but also relies on non-mitochondrial processes and pathways. Our results reinforce the notion that mitochondrial function and cellular function are intimately connected.