Replication stress promotes cell elimination by extrusion.

Cell extrusion is a mechanism of cell elimination that is used by organisms as diverse as sponges, nematodes, insects and mammals1-3. During extrusion, a cell detaches from a layer of surrounding cells while maintaining the continuity of that layer4. Vertebrate epithelial tissues primarily eliminate cells by extrusion, and the dysregulation of cell extrusion has been linked to epithelial diseases, including cancer1,5. The mechanisms that drive cell extrusion remain incompletely understood. Here, to analyse cell extrusion by Caenorhabditis elegans embryos3, we conducted a genome-wide RNA interference screen, identified multiple cell-cycle genes with S-phase-specific function, and performed live-imaging experiments to establish how those genes control extrusion. Extruding cells experience replication stress during S phase and activate a replication-stress response via homologues of ATR and CHK1. Preventing S-phase entry, inhibiting the replication-stress response, or allowing completion of the cell cycle blocked cell extrusion. Hydroxyurea-induced replication stress6,7 triggered ATR-CHK1- and p53-dependent cell extrusion from a mammalian epithelial monolayer. We conclude that cell extrusion induced by replication stress is conserved among animals and propose that this extrusion process is a primordial mechanism of cell elimination with a tumour-suppressive function in mammals.

Deletion of VPS50 protein in mouse brain impairs synaptic function and behavior.

BACKGROUND: The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans. Patients with mutations in VPS50 show severe developmental delay and intellectual disability, characteristics that have been associated with autism spectrum disorders (ASDs). The mechanisms that link VPS50 mutations to ASD are unknown. RESULTS: To examine the role of VPS50 in mammalian brain function and behavior, we used the CRISPR/Cas9 system to generate knockouts of VPS50 in both cultured murine cortical neurons and living mice. In cultured neurons, KO of VPS50 did not affect the number of synaptic vesicles but did cause mislocalization of the V-ATPase V1 domain pump and impaired synaptic activity, likely as a consequence of defects in vesicle acidification and vesicle content. In mice, mosaic KO of VPS50 in the hippocampus altered synaptic transmission and plasticity and generated robust cognitive impairments. CONCLUSIONS: We propose that VPS50 functions as an accessory protein to aid the recruitment of the V-ATPase V1 domain to synaptic vesicles and in that way plays a crucial role in controlling synaptic vesicle acidification. Understanding the mechanisms controlling behaviors and synaptic function in ASD-associated mutations is pivotal for the development of targeted interventions, which may open new avenues for therapeutic strategies aimed at ASD and related conditions.

A Caenorhabditis elegans protein with a PRDM9-like SET domain localizes to chromatin-associated foci and promotes spermatocyte gene expression, sperm production and fertility.

To better understand the tissue-specific regulation of chromatin state in cell-fate determination and animal development, we defined the tissue-specific expression of all 36 C. elegans presumptive lysine methyltransferase (KMT) genes using single-molecule fluorescence in situ hybridization (smFISH). Most KMTs were expressed in only one or two tissues. The germline was the tissue with the broadest KMT expression. We found that the germline-expressed C. elegans protein SET-17, which has a SET domain similar to that of the PRDM9 and PRDM7 SET-domain proteins, promotes fertility by regulating gene expression in primary spermatocytes. SET-17 drives the transcription of spermatocyte-specific genes from four genomic clusters to promote spermatid development. SET-17 is concentrated in stable chromatin-associated nuclear foci at actively transcribed msp (major sperm protein) gene clusters, which we term msp locus bodies. Our results reveal the function of a PRDM9/7-family SET-domain protein in spermatocyte transcription. We propose that the spatial intranuclear organization of chromatin factors might be a conserved mechanism in tissue-specific control of transcription.

Presumptive TRP channel CED-11 promotes cell volume decrease and facilitates degradation of apoptotic cells in Caenorhabditis elegans.

Apoptotic cells undergo a series of morphological changes. These changes are dependent on caspase cleavage of downstream targets, but which targets are significant and how they facilitate the death process are not well understood. In Caenorhabditis elegans an increase in the refractility of the dying cell is a hallmark morphological change that is caspase dependent. We identify a presumptive transient receptor potential (TRP) cation channel, CED-11, that acts in the dying cell to promote the increase in apoptotic cell refractility. CED-11 is required for multiple other morphological changes during apoptosis, including an increase in electron density as visualized by electron microscopy and a decrease in cell volume. In ced-11 mutants, the degradation of apoptotic cells is delayed. Mutation of ced-11 does not cause an increase in cell survival but can enhance cell survival in other cell-death mutants, indicating that ced-11 facilitates the death process. In short, ced-11 acts downstream of caspase activation to promote the shrinkage, death, and degradation of apoptotic cells.

Mutations in Nonessential eIF3k and eIF3l Genes Confer Lifespan Extension and Enhanced Resistance to ER Stress in Caenorhabditis elegans.

The translation initiation factor eIF3 is a multi-subunit protein complex that coordinates the assembly of the 43S pre-initiation complex in eukaryotes. Prior studies have demonstrated that not all subunits of eIF3 are essential for the initiation of translation, suggesting that some subunits may serve regulatory roles. Here, we show that loss-of-function mutations in the genes encoding the conserved eIF3k and eIF3l subunits of the translation initiation complex eIF3 result in a 40% extension in lifespan and enhanced resistance to endoplasmic reticulum (ER) stress in Caenorhabditis elegans. In contrast to previously described mutations in genes encoding translation initiation components that confer lifespan extension in C. elegans, loss-of-function mutations in eif-3.K or eif-3.L are viable, and mutants show normal rates of growth and development, and have wild-type levels of bulk protein synthesis. Lifespan extension resulting from EIF-3.K or EIF-3.L deficiency is suppressed by a mutation in the Forkhead family transcription factor DAF-16. Mutations in eif-3.K or eif-3.L also confer enhanced resistance to ER stress, independent of IRE-1-XBP-1, ATF-6, and PEK-1, and independent of DAF-16. Our data suggest a pivotal functional role for conserved eIF3k and eIF3l accessory subunits of eIF3 in the regulation of cellular and organismal responses to ER stress and aging.

Transcriptional landscape of a hypoxia response identifies cell-specific pathways for adaptation.

How the HIF-1 (Hypoxia-Inducible) transcription factor drives and coordinates distinct responses to low oxygen across diverse cell types is poorly understood. We present a multi-tissue single-cell gene-expression atlas of the hypoxia response of the nematode Caenorhabditis elegans . This atlas highlights how cell-type-specific HIF-1 responses overlap and diverge among and within neuronal, intestinal, and muscle tissues. Using the atlas to guide functional analyses of candidate muscle-specific HIF-1 effectors, we discovered that HIF-1 activation drives downregulation of the tspo-1 ( TSPO, Translocator Protein) gene in vulval muscle cells to modulate a hypoxia-driven change in locomotion caused by contraction of body-wall muscle cells. We further showed that in human cardiomyocytes HIF-1 activation decreases levels of TSPO and thereby alters intracellular cholesterol transport and the mitochondrial network. We suggest that TSPO-1 is an evolutionarily conserved mediator of HIF-1-dependent modulation of muscle and conclude that our gene-expression atlas can help reveal how HIF-1 drives cell-specific adaptations to hypoxia.

A DEAD-box helicase drives the partitioning of a pro-differentiation NAB protein into nuclear foci.

How cells regulate gene expression in a precise spatiotemporal manner during organismal development is a fundamental question in biology. Although the role of transcriptional condensates in gene regulation has been established, little is known about the function and regulation of these molecular assemblies in the context of animal development and physiology. Here we show that the evolutionarily conserved DEAD-box helicase DDX-23 controls cell fate in Caenorhabditis elegans by binding to and facilitating the condensation of MAB-10, the C. elegans homolog of mammalian NGFI-A-binding (NAB) protein. MAB-10 is a transcriptional cofactor that functions with the early growth response (EGR) protein LIN-29 to regulate the transcription of genes required for exiting the cell cycle, terminal differentiation, and the larval-to-adult transition. We suggest that DEAD-box helicase proteins function more generally during animal development to control the condensation of NAB proteins important in cell identity and that this mechanism is evolutionarily conserved. In mammals, such a mechanism might underlie terminal cell differentiation and when dysregulated might promote cancerous growth.

Deletion of VPS50 protein in mice brain impairs synaptic function and behavior.

VPS50, is an accessory protein, involved in the synaptic and dense core vesicle acidification and its alterations produce behavioral changes in C.elegans. Here, we produce the mosaic knock out (mKO) of VPS50 using CRISPR/Cas9 system in both cortical cultured neurons and whole animals to evaluate the effect of VPS50 in regulating mammalian brain function and behavior. While mKO of VPS50 does not change the number of synaptic vesicles, it produces a mislocalization of the V-ATPase pump that likely impact in vesicle acidification and vesicle content to impair synaptic and neuronal activity in cultured neurons. In mice, mKO of VPS50 in the hippocampus, alter synaptic transmission and plasticity, and generated robust cognitive impairments associate to memory formation. We propose that VPS50 is an accessory protein that aids the correct recruitment of the V-ATPase pump to synaptic vesicles, thus having a crucial role controlling synaptic vesicle acidification and hence synaptic transmission.

Endoplasmic Reticulum Homeostasis Is Modulated by the Forkhead Transcription Factor FKH-9 During Infection of Caenorhabditis elegans.

Animals have evolved critical mechanisms to maintain cellular and organismal proteostasis during development, disease, and exposure to environmental stressors. The Unfolded Protein Response (UPR) is a conserved pathway that senses and responds to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) lumen. We have previously demonstrated that the IRE-1-XBP-1 branch of the UPR is required to maintain Caenorhabditis elegans ER homeostasis during larval development in the presence of pathogenic Pseudomonas aeruginosa In this study, we identify loss-of-function mutations in four conserved transcriptional regulators that suppress the larval lethality of xbp-1 mutant animals caused by immune activation in response to infection by pathogenic bacteria: FKH-9, a forkhead family transcription factor; ARID-1, an ARID/Bright domain-containing transcription factor; HCF-1, a transcriptional regulator that associates with histone modifying enzymes; and SIN-3, a subunit of a histone deacetylase complex. Further characterization of FKH-9 suggests that loss of FKH-9 enhances resistance to the ER toxin tunicamycin and results in enhanced ER-associated degradation (ERAD). Increased ERAD activity of fkh-9 loss-of-function mutants is accompanied by a diminished capacity to degrade cytosolic proteasomal substrates and a corresponding increased sensitivity to the proteasomal inhibitor bortezomib. Our data underscore how the balance between ER and cytosolic proteostasis can be influenced by compensatory activation of ERAD during the physiological ER stress of infection and immune activation.

Distinct Neural Circuits Control Rhythm Inhibition and Spitting by the Myogenic Pharynx of C. elegans.

Neural circuits have long been known to modulate myogenic muscles such as the heart, yet a mechanistic understanding at the cellular and molecular levels remains limited. We studied how light inhibits pumping of the Caenorhabditis elegans pharynx, a myogenic muscular pump for feeding, and found three neural circuits that alter pumping. First, light inhibits pumping via the I2 neuron monosynaptic circuit. Our electron microscopic reconstruction of the anterior pharynx revealed evidence for synapses from I2 onto muscle that were missing from the published connectome, and we show that these "missed synapses" are likely functional. Second, light inhibits pumping through the RIP-I1-MC neuron polysynaptic circuit, in which an inhibitory signal is likely transmitted from outside the pharynx into the pharynx in a manner analogous to how the mammalian autonomic nervous system controls the heart. Third, light causes a novel pharyngeal behavior, reversal of flow or "spitting," which is induced by the M1 neuron. These three neural circuits show that neurons can control a myogenic muscle organ not only by changing the contraction rate but also by altering the functional consequences of the contraction itself, transforming swallowing into spitting. Our observations also illustrate why connectome builders and users should be cognizant that functional synaptic connections might exist despite the absence of a declared synapse in the connectome.

Interstitial ciliates: benthic microaerophiles or planktonic anaerobes?

We observed marine benthic interstitial ciliates Geleia sp. and Tracheloraphis sp. inhabiting the water column of a chemically stratified salt pond. This habitat is uncharacteristic for interstitial ciliates, yet they displayed active and abundant planktonic populations (up to 800 and 250 cells/liter, respectively) and a well-defined pattern of vertical distribution. Completely absent from the oxygenated epilimnion, they first appeared at the oxic/anoxic interface and were present throughout the anoxic hypolimnion. The data could not be explained by a passive removal (e.g. by currents) of these ciliates from their conventional habitat (soft sediments) to water column. The results suggest that 1) these ciliates favored an anoxic environment, and 2) they switched to a planktonic lifestyle as appropriate conditions (seasonal anoxia) developed in the water column. This sharply contrasts the classic view of these ciliates as specifically benthic and aerobic (albeit microaerophilic) organisms. We hypothesize that Geleia sp. and Tracheloraphis sp. can readily grow in either water column or benthos, but are typically found in sediments simply because they contain their preferred (anoxic) niche.