Endogenous transcriptional reporters as new tools for the study of the osmotic stress response.

monitoring of gene expression using standard transcriptional reporters is a powerful and commonly used tool for genetic dissection of the osmotic stress response in . Like all transgene reporters, these reporters have important limitations that restrict their utility. To overcome these limitations, we created three different reporters using CRISPR/Cas9 methods to insert several variants of GFP into the endogenous locus.

Regulation of the hypertonic stress response by the 3' mRNA cleavage and polyadenylation complex.

Maintenance of osmotic homeostasis is one of the most aggressively defended homeostatic set points in physiology. One major mechanism of osmotic homeostasis involves the upregulation of proteins that catalyze the accumulation of solutes called organic osmolytes. To better understand how osmolyte accumulation proteins are regulated, we conducted a forward genetic screen in Caenorhabditis elegans for mutants with no induction of osmolyte biosynthesis gene expression (Nio mutants).

Length-dependent RNA foci formation and Repeat Associated non-AUG dependent translation in a G C model.

GC-rich repeat expansion mutations are implicated in several neurodegenerative diseases and can lead to repeat associated non-AUG-dependent (RAN) translation and concentrations of nuclear RNA foci. To model ALS/FTD, we engineered to express pure GGGGCC (G C ) repeats of varying lengths and observed RAN translation and nuclear RNA foci. RNA foci were observed in animals expressing ≥20 G C repeats while RAN translation occured in animals expressing ≥33 G C repeats.

The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity.

A hexanucleotide repeat expansion in the gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs PR50 and GR50 are highly toxic when expressed in , and this toxicity depends on nuclear localization of the DPR.

The C. elegans Hypertonic Stress Response: Big Insights from Shrinking Worms.

Cell volume is one of the most aggressively defended physiological set points in biology. Changes in intracellular ion and water concentrations, which are induced by changes in metabolism or environmental exposures, disrupt protein folding, enzymatic activity, and macromolecular assemblies. To counter these challenges, cells and organisms have evolved multifaceted, evolutionarily conserved molecular mechanisms to restore cell volume and repair stress induced damage.

The O-GlcNAc transferase OGT is a conserved and essential regulator of the cellular and organismal response to hypertonic stress.

The conserved O-GlcNAc transferase OGT O-GlcNAcylates serine and threonine residues of intracellular proteins to regulate their function. OGT is required for viability in mammalian cells, but its specific roles in cellular physiology are poorly understood. Here we describe a conserved requirement for OGT in an essential aspect of cell physiology: the hypertonic stress response.

Measuring RAN Peptide Toxicity in C. elegans.

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington's disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation.

PolyQ-independent toxicity associated with novel translational products from CAG repeat expansions.

Expanded CAG nucleotide repeats are the underlying genetic cause of at least 14 incurable diseases, including Huntington's disease (HD). The toxicity associated with many CAG repeat expansions is thought to be due to the translation of the CAG repeat to create a polyQ protein, which forms toxic oligomers and aggregates. However, recent studies show that HD CAG repeats undergo a non-canonical form of translation called Repeat-associated non-AUG dependent (RAN) translation.

Models and mechanisms of repeat expansion disorders: a worm's eye view.

The inappropriate genetic expansion of various repetitive DNA sequences underlies over 20 distinct inherited diseases. The genetic context of these repeats in exons, introns and untranslated regions has played a major role in thinking about the mechanisms by which various repeat expansions might cause disease. Repeat expansions in exons are thought to give rise to expanded toxic protein repeats (i.

Nuclear localized C9orf72-associated arginine-containing dipeptides exhibit age-dependent toxicity in C. elegans.

A hexanucleotide repeat expansion mutation in the C9orf72 gene represents a prevalent genetic cause of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Non-canonical translation of this repeat gives rise to several distinct dipeptide protein species that could play pathological roles in disease. Here, we show in the model system Caenorhabditis elegans that expression of the arginine-containing dipeptides, but not alanine-containing dipeptides, produces toxic phenotypes in multiple cellular contexts, including motor neurons.

The homeodomain-interacting protein kinase HPK-1 preserves protein homeostasis and longevity through master regulatory control of the HSF-1 chaperone network and TORC1-restricted autophagy in Caenorhabditis elegans.

An extensive proteostatic network comprised of molecular chaperones and protein clearance mechanisms functions collectively to preserve the integrity and resiliency of the proteome. The efficacy of this network deteriorates during aging, coinciding with many clinical manifestations, including protein aggregation diseases of the nervous system. A decline in proteostasis can be delayed through the activation of cytoprotective transcriptional responses, which are sensitive to environmental stress and internal metabolic and physiological cues.

Loss of RAD-23 Protects Against Models of Motor Neuron Disease by Enhancing Mutant Protein Clearance.

Unlabelled: Misfolded proteins accumulate and aggregate in neurodegenerative disease. The existence of these deposits reflects a derangement in the protein homeostasis machinery. Using a candidate gene screen, we report that loss of RAD-23 protects against the toxicity of proteins known to aggregate in amyotrophic lateral sclerosis.

Stress signaling: serotonin spreads systemic stress.

Cells respond to elevated temperatures through a well-characterized heat-shock response that enables short-term survival, long-term adaptation and mitigation of macromolecular damage. New work reveals a cell non-autonomous layer of stress-response regulation between neurons and the gonad involving serotonin.

FER-1/Dysferlin promotes cholinergic signaling at the neuromuscular junction in C. elegans and mice.

Dysferlin is a member of the evolutionarily conserved ferlin gene family. Mutations in Dysferlin lead to Limb Girdle Muscular Dystrophy 2B (LGMD2B), an inherited, progressive and incurable muscle disorder. However, the molecular mechanisms underlying disease pathogenesis are not fully understood.

Caenorhabditis elegans HSF-1 is an essential nuclear protein that forms stress granule-like structures following heat shock.

The heat shock transcription factor (HSF) is a conserved regulator of heat shock-inducible gene expression. Organismal roles for HSF in physiological processes such as development, aging, and immunity have been defined largely through studies of the single Caenorhabditis elegans HSF homolog, hsf-1. However, the molecular and cell biological properties of hsf-1 in C.

Global analysis of RNA secondary structure in two metazoans.

The secondary structure of RNA is necessary for its maturation, regulation, processing, and function. However, the global influence of RNA folding in eukaryotes is still unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach to identify the paired (double-stranded RNA [dsRNA]) and unpaired (single-stranded RNA [ssRNA]) components of the Drosophila melanogaster and Caenorhabditis elegans transcriptomes, which allows us to identify conserved features of RNA secondary structure in metazoans.

To UPR… and beyond! A new role for a BiP/GRP78 protein in the control of antimicrobial peptide expression in C. elegans epidermis.

The fungal pathogen Drechmeria coniospora infects C. elegans and elicits an innate immune response mediated, in part, by the induction of antimicrobial peptides in the epidermis. The signaling pathways controlling this phenomenon remain to be fully characterized.

Stress and aging induce distinct polyQ protein aggregation states.

Many age-related diseases are known to elicit protein misfolding and aggregation. Whereas environmental stressors, such as temperature, oxidative stress, and osmotic stress, can also damage proteins, it is not known whether aging and the environment impact protein folding in the same or different ways. Using polyQ reporters of protein folding in both Caenorhabditis elegans and mammalian cell culture, we show that osmotic stress, but not other proteotoxic stressors, induces rapid (minutes) cytoplasmic polyQ aggregation.

The cystic-fibrosis-associated ΔF508 mutation confers post-transcriptional destabilization on the C. elegans ABC transporter PGP-3.

Membrane proteins make up ∼30% of the proteome. During the early stages of maturation, this class of proteins can experience localized misfolding in distinct cellular compartments, such as the cytoplasm, endoplasmic reticulum (ER) lumen and ER membrane. ER quality control (ERQC) mechanisms monitor folding and determine whether a membrane protein is appropriately folded or is misfolded and warrants degradation.

Biomechanical profiling of Caenorhabditis elegans motility.

Caenorhabditis elegans locomotion is a stereotyped behavior that is ideal for genetic analysis. We integrated video microscopy, image analysis algorithms, and fluid mechanics principles to describe the C. elegans swim gait.

Daf-2 signaling modifies mutant SOD1 toxicity in C. elegans.

The DAF-2 Insulin/IGF-1 signaling (IIS) pathway is a strong modifier of Caenorhabditis elegans longevity and healthspan. As aging is the greatest risk factor for developing neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS), we were interested in determining if DAF-2 signaling modifies disease pathology in mutant superoxide dismutase 1 (SOD1) expressing C. elegans.

The Caenorhabditis elegans mucin-like protein OSM-8 negatively regulates osmosensitive physiology via the transmembrane protein PTR-23.

The molecular mechanisms of animal cell osmoregulation are poorly understood. Genetic studies of osmoregulation in yeast have identified mucin-like proteins as critical regulators of osmosensitive signaling and gene expression. Whether mucins play similar roles in higher organisms is not known.

TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress: organic osmolyte-dependent and -independent pathways.

TonEBP (tonicity-responsive enhancer binding protein) is a transcription factor that promotes cellular accumulation of organic osmolytes in the hypertonic renal medulla by stimulating expression of its target genes. Genetically modified animals with deficient TonEBP activity in the kidney suffer from severe medullary atrophy in association with cell death, demonstrating that TonEBP is essential for the survival of the renal medullary cells. Using both TonEBP knockout cells and RNA interference of TonEBP, we found that TonEBP promoted cellular adaptation to hypertonic stress.