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

The C. elegans PUM1, 2-like RNA binding protein PUF-8 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 PUF-8's ability to interact with ced-3caspase mRNA thereby repressing the activity of the pro-apoptotic ced-3caspase 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 PUF-8's ability to repress the expression of the anti-apoptotic ced-9Bcl-2 gene. 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-8PUM1, 2 acts at the post-transcriptional level to level out these differences, thereby ensuring proper cell number homeostasis

Eukaryotic Translation Initiation Factor 5B Activity Regulates Larval Growth Rate and Germline Development in Caenorhabditis elegans

In C. elegans, a population of proliferating germ cells is maintained via GLP-1/Notch signaling; in the absence of GLP-1 signaling, germ cells prematurely enter meiosis and differentiate. We previously identified ego (enhancer of glp-1) genes that promote germline proliferation and interact genetically with the GLP-1 signaling pathway. Here, we report that iffb-1 (initiation factor five B) is an ego gene. iffb-1 encodes the sole C. elegans isoform of eukaryotic translation initiation factor 5B, a protein essential for translation. We have used RNA interference and a deletion mutation to determine the developmental consequences of reduced iffb-1 activity. Our data indicate that maternal iffb-1 gene expression is sufficient for embryogenesis, and zygotic iffb-1 expression is required for development beyond late L1/ early L2 stage. Partial reduction in iffb-1 expression delays larval development and can severely disrupt proliferation and differentiation of germ cells. We hypothesize that germline development is particularly sensitive to iffb-1 expression level

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

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.publishedVersio

Engulfment pathways promote programmed cell death by enhancing the unequal segregation of apoptotic potential

Components of the conserved engulfment pathways promote programmed cell death in Caenorhabditis elegans (C. elegans) through an unknown mechanism. Here we report that the phagocytic receptor CED-1 mEGF10 is required for the formation of a dorsal-ventral gradient of CED-3 caspase activity within the mother of a cell programmed to die and an increase in the level of CED-3 protein within its dying daughter. Furthermore, CED-1 becomes enriched on plasma membrane regions of neighbouring cells that appose the dorsal side of the mother, which later forms the dying daughter. Therefore, we propose that components of the engulfment pathways promote programmed cell death by enhancing the polar localization of apoptotic factors in mothers of cells programmed to die and the unequal segregation of apoptotic potential into dying and surviving daughters. Our findings reveal a novel function of the engulfment pathways and provide a better understanding of how apoptosis is initiated during C. elegans development

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\u27 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

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 theC.elegansorthologue of MELK, PIG-1, promotes apoptosis by partitioning an anti-apoptotic factor. TheC.elegansNSM 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 is required for partitioning of CES-1 Snail, a transcriptional repressor of the pro-apoptotic geneegl-1BH3-only.pig-1MELK is controlled by both aces-1Snail- andpar-4LKB1-dependent pathway, and may act through phosphorylation and cortical enrichment of nonmuscle myosin II prior to neuroblast division. We propose thatpig-1MELK-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 whichces-1Snail controls its own activity through the formation of a gradient of CES-1 Snail protein. Author summary Apoptosis is critical for the elimination of 'unwanted' cells. What distinguishes wanted from unwanted cells in developing animals is poorly understood. We report that in theC.elegansNSM neuroblast lineage, the level of CES-1, a Snail-family member and transcriptional repressor of the pro-apoptotic geneegl-1, contributes to this process. In addition, we demonstrate thatC.elegansPIG-1, the orthologue of mammalian proto-oncoprotein MELK, plays a critical role in controlling CES-1(Snail)levels. Specifically, during NSM neuroblast division, PIG-1(MELK)controls partitioning of CES-1(Snail)into one but not the other daughter cell thereby promoting the making of one wanted and one unwanted cell. Furthermore, we present evidence that PIG-1(MELK)acts prior to NSM neuroblast division by locally activating the actomyosin network

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

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