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.

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.

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.

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.

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.

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.

In vivo labeling of endogenous genomic loci in C. elegans using CRISPR/dCas9.

Visualization of genomic loci with open chromatin state has been reported in mammalian tissue culture cells using a CRISPR/Cas9-based system that utilizes an EGFP-tagged endonuclease-deficient Cas9 protein (dCas9::EGFP) (Chen et al. 2013). Here, we adapted this approach for use in Caenorhabditis elegans . We generated a C. elegans strain that expresses the dCas9 protein fused to two nuclear-localized EGFP molecules (dCas9::NLS::2xEGFP::NLS) in an inducible manner. Using this strain, we report the visualization in live C. elegans embryos of two endogenous repetitive loci, rrn-4 and rrn-1 , from which 5S and 18S ribosomal RNAs are constitutively generated.

Genome-wide RNAi screen for regulators of UPRmt in Caenorhabditis elegans mutants with defects in mitochondrial fusion.

Mitochondrial dynamics plays an important role in mitochondrial quality control and the adaptation of metabolic activity in response to environmental changes. The disruption of mitochondrial dynamics has detrimental consequences for mitochondrial and cellular homeostasis and leads to the activation of the mitochondrial unfolded protein response (UPRmt), a quality control mechanism that adjusts cellular metabolism and restores homeostasis. To identify genes involved in the induction of UPRmt in response to a block in mitochondrial fusion, we performed a genome-wide RNAi screen in Caenorhabditis elegans mutants lacking the gene fzo-1, which encodes the ortholog of mammalian Mitofusin, and identified 299 suppressors and 86 enhancers. Approximately 90% of these 385 genes are conserved in humans, and one-third of the conserved genes have been implicated in human disease. Furthermore, many have roles in developmental processes, which suggests that mitochondrial function and their response to stress are defined during development and maintained throughout life. Our dataset primarily contains mitochondrial enhancers and non-mitochondrial suppressors of UPRmt, indicating that the maintenance of mitochondrial homeostasis has evolved as a critical cellular function, which, when disrupted, can be compensated for by many different cellular processes. Analysis of the subsets "non-mitochondrial enhancers" and "mitochondrial suppressors" suggests that organellar contact sites, especially between the ER and mitochondria, are of importance for mitochondrial homeostasis. In addition, we identified several genes involved in IP3 signaling that modulate UPRmt in fzo-1 mutants and found a potential link between pre-mRNA splicing and UPRmt activation.

MitoSegNet: Easy-to-use Deep Learning Segmentation for Analyzing Mitochondrial Morphology.

While the analysis of mitochondrial morphology has emerged as a key tool in the study of mitochondrial function, efficient quantification of mitochondrial microscopy images presents a challenging task and bottleneck for statistically robust conclusions. Here, we present Mitochondrial Segmentation Network (MitoSegNet), a pretrained deep learning segmentation model that enables researchers to easily exploit the power of deep learning for the quantification of mitochondrial morphology. We tested the performance of MitoSegNet against three feature-based segmentation algorithms and the machine-learning segmentation tool Ilastik. MitoSegNet outperformed all other methods in both pixelwise and morphological segmentation accuracy. We successfully applied MitoSegNet to unseen fluorescence microscopy images of mitoGFP expressing mitochondria in wild-type and catp-6 ATP13A2 mutant C. elegans adults. Additionally, MitoSegNet was capable of accurately segmenting mitochondria in HeLa cells treated with fragmentation inducing reagents. We provide MitoSegNet in a toolbox for Windows and Linux operating systems that combines segmentation with morphological analysis.

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.

Msp1 cooperates with the proteasome for extraction of arrested mitochondrial import intermediates.

The mitochondrial AAA ATPase Msp1 is well known for extraction of mislocalized tail-anchored ER proteins from the mitochondrial outer membrane. Here, we analyzed the extraction of precursors blocking the import pore in the outer membrane. We demonstrate strong genetic interactions of Msp1 and the proteasome with components of the TOM complex, the main translocase in the outer membrane. Msp1 and the proteasome both contribute to the removal of arrested precursor proteins that specifically accumulate in these mutants. The proteasome activity is essential for the removal as proteasome inhibitors block extraction. Furthermore, the proteasomal subunit Rpn10 copurified with Msp1. The human Msp1 homologue has been implicated in neurodegenerative diseases, and we show that the lack of the Caenorhabditis elegans Msp1 homologue triggers an import stress response in the worm, which indicates a conserved role in metazoa. In summary, our results suggest a role of Msp1 as an adaptor for the proteasome that drives the extraction of arrested and mislocalized proteins at the mitochondrial outer membrane.

Tunable light and drug induced depletion of target proteins.

Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to investigate the role of CED-3 in apoptosis during Caenorhabditis elegans development. These independent tools can be combined for spatial and temporal depletion of different sets of proteins, can help to distinguish immediate cellular responses from long-term adaptation effects and can facilitate the exploration of complex networks.

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.

Compromised Mitochondrial Protein Import Acts as a Signal for UPRmt.

The induction of the mitochondrial unfolded protein response (UPRmt) results in increased transcription of the gene encoding the mitochondrial chaperone HSP70. We systematically screened the C. elegans genome and identified 171 genes that, when knocked down, induce the expression of an hsp-6 HSP70 reporter and encode mitochondrial proteins. These genes represent many, but not all, mitochondrial processes (e.g., mitochondrial calcium homeostasis and mitophagy are not represented). Knockdown of these genes leads to reduced mitochondrial membrane potential and, hence, decreased protein import into mitochondria. In addition, it induces UPRmt in a manner that is dependent on ATFS-1 but that is not antagonized by the kinase GCN-2. We propose that compromised mitochondrial protein import signals the induction of UPRmt and that the mitochondrial targeting sequence of ATFS-1 functions as a sensor for this signal.

PCMD-1 Organizes Centrosome Matrix Assembly in C. elegans.

Centrosomes, the major microtubule-organizing centers of animal cells, are essential for the assembly of a bipolar spindle during mitosis. Spindle defective-5 (SPD-5), the main scaffold protein of the centrosome matrix in Caenorhabditis elegans, forms a thin core around non-mitotic centrioles. Upon mitotic entry, the SPD-5-containing centrosome matrix expands in a Polo-like-kinase 1 (PLK-1)-dependent manner and this enables an enhanced microtubule nucleation activity during mitosis. How the non-mitotic centrosome core is formed and how this core facilitates robust SPD-5 expansion at mitotic entry remains unknown. Here, we present evidence that the coiled-coil protein pericentriolar matrix deficient-1 (PCMD-1) is necessary for the efficient loading of SPD-5, SPD-2, and PLK-1 to the non-mitotic centrosome core. Furthermore, we demonstrate that the absence of PCMD-1 disrupts pericentriolar material (PCM) recruitment and integrity. The expansion of centrosomes into spherical structures at the mitotic entry is compromised. We propose that PCMD-1 acts as a molecular platform for mitotic regulators and for components of the PCM, thereby allowing functional interactions between them, which in turn is necessary for the organization of the mitotic centrosome and, hence, spindle bipolarity.

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.

Caenorhabditis elegans ced-3 Caspase Is Required for Asymmetric Divisions That Generate Cells Programmed To Die.

Caspases have functions other than in apoptosis. Here, we report that Caenorhabditis elegans CED-3 caspase regulates asymmetric cell division. Many of the 131 cells that are "programmed" to die during C. elegans development are the smaller daughter of a neuroblast that divides asymmetrically by size and fate. We have previously shown that CED-3 caspase is activated in such neuroblasts, and that before neuroblast division, a gradient of CED-3 caspase activity is formed in a ced-1 MEGF10 ( m ultiple EGF -like domains 10 )-dependent manner. This results in the nonrandom segregation of active CED-3 caspase or "apoptotic potential" into the smaller daughter. We now show that CED-3 caspase is necessary for the ability of neuroblasts to divide asymmetrically by size. In addition, we provide evidence that a pig-1 MELK (maternal embryonic leucine zipper kinase)-dependent reciprocal gradient of "mitotic potential" is formed in the QL.p neuroblast, and that CED-3 caspase antagonizes this mitotic potential. Based on these findings, we propose that CED-3 caspase plays a critical role in the asymmetric division by size and fate of neuroblasts, and that this contributes to the reproducibility and robustness with which the smaller daughter cell is produced and adopts the apoptotic fate. Finally, the function of CED-3 caspase in this context is dependent on its activation through the conserved egl-1 BH3-only, ced-9 Bcl-2, and ced-4 Apaf-1 pathway. In mammals, caspases affect various aspects of stem cell lineages. We speculate that the new nonapoptotic function of C. elegans CED-3 caspase in asymmetric neuroblast division is relevant to the function(s) of mammalian caspases in stem cells.

Overlapping expression patterns and functions of three paralogous P5B ATPases in Caenorhabditis elegans.

P5B ATPases are present in the genomes of diverse unicellular and multicellular eukaryotes, indicating that they have an ancient origin, and that they are important for cellular fitness. Inactivation of ATP13A2, one of the four human P5B ATPases, leads to early-onset Parkinson's disease (Kufor-Rakeb Syndrome). The presence of an invariant PPALP motif within the putative substrate interaction pocket of transmembrane segment M4 suggests that all P5B ATPases might have similar transport specificity; however, the identity of the transport substrate(s) remains unknown. Nematodes of the genus Caenorhabditis possess three paralogous P5B ATPase genes, catp-5, catp-6 and catp-7, which probably originated from a single ancestral gene around the time of origin of the Caenorhabditid clade. By using CRISPR/Cas9, we have systematically investigated the expression patterns, subcellular localization and biological functions of each of the P5B ATPases of C. elegans. We find that each gene has a unique expression pattern, and that some tissues express more than one P5B. In some tissues where their expression patterns overlap, different P5Bs are targeted to different subcellular compartments (e.g., early endosomes vs. plasma membrane), whereas in other tissues they localize to the same compartment (plasma membrane). We observed lysosomal co-localization between CATP-6::GFP and LMP-1::RFP in transgenic animals; however, this was an artifact of the tagged LMP-1 protein, since anti-LMP-1 antibody staining of native protein revealed that LMP-1 and CATP-6::GFP occupy different compartments. The nematode P5Bs are at least partially redundant, since we observed synthetic sterility in catp-5(0); catp-6(0) and catp-6(0) catp-7(0) double mutants. The double mutants exhibit defects in distal tip cell migration that resemble those of ina-1 (alpha integrin ortholog) and vab-3 (Pax6 ortholog) mutants, suggesting that the nematode P5Bs are required for ina-1and/or vab-3 function. This is potentially a conserved regulatory interaction, since mammalian ATP13A2, alpha integrin and Pax6 are all required for proper dopaminergic neuron function.