60 results on '"Todd Lamitina"'
Search Results
2. The O-GlcNAc transferase OGT is a conserved and essential regulator of the cellular and organismal response to hypertonic stress.
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Sarel J Urso, Marcella Comly, John A Hanover, and Todd Lamitina
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Genetics ,QH426-470 - Abstract
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. Through a forward genetic screen in Caenorhabditis elegans, we discovered OGT is acutely required for osmoprotective protein expression and adaptation to hypertonic stress. Gene expression analysis shows that ogt-1 functions through a post-transcriptional mechanism. Human OGT partially rescues the C. elegans phenotypes, suggesting that the osmoregulatory functions of OGT are ancient. Intriguingly, expression of O-GlcNAcylation-deficient forms of human or worm OGT rescue the hypertonic stress response phenotype. However, expression of an OGT protein lacking the tetracopeptide repeat (TPR) domain does not rescue. Our findings are among the first to demonstrate a specific physiological role for OGT at the organismal level and demonstrate that OGT engages in important molecular functions outside of its well described roles in post-translational O-GlcNAcylation of intracellular proteins.
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- 2020
- Full Text
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3. PolyQ-independent toxicity associated with novel translational products from CAG repeat expansions.
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Paige Rudich, Simon Watkins, and Todd Lamitina
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Medicine ,Science - Abstract
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. RAN translation of the CAG sense and CUG anti-sense RNAs produces six distinct repeat peptides: polyalanine (polyAla, from both CAG and CUG repeats), polyserine (polySer), polyleucine (polyLeu), polycysteine (polyCys), and polyglutamine (polyGln). The toxic potential of individual CAG-derived RAN polypeptides is not well understood. We developed pure C. elegans protein models for each CAG RAN polypeptide using codon-varied expression constructs that preserve RAN protein sequence but eliminate repetitive CAG/CUG RNA. While all RAN polypeptides formed aggregates, only polyLeu was consistently toxic across multiple cell types. In GABAergic neurons, which exhibit significant neurodegeneration in HD patients, codon-varied (Leu)38, but not (Gln)38, caused substantial neurodegeneration and motility defects. Our studies provide the first in vivo evaluation of CAG-derived RAN polypeptides in a multicellular model organism and suggest that polyQ-independent mechanisms, such as RAN-translated polyLeu peptides, may have a significant pathological role in CAG repeat expansion disorders.
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- 2020
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4. 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.
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Ritika Das, Justine A Melo, Manjunatha Thondamal, Elizabeth A Morton, Adam B Cornwell, Beresford Crick, Joung Heon Kim, Elliot W Swartz, Todd Lamitina, Peter M Douglas, and Andrew V Samuelson
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Genetics ,QH426-470 - Abstract
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. The homeodomain-interacting protein kinase (hipk) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole Caenorhabditis elegans Hipk homolog, hpk-1, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of hpk-1 is deleterious to these phenotypes. We show that HPK-1 preserves proteostasis and extends longevity through distinct but complementary genetic pathways defined by the heat shock transcription factor (HSF-1), and the target of rapamycin complex 1 (TORC1). We demonstrate that HPK-1 antagonizes sumoylation of HSF-1, a post-translational modification associated with reduced transcriptional activity in mammals. We show that inhibition of sumoylation by RNAi enhances HSF-1-dependent transcriptional induction of chaperones in response to heat shock. We find that hpk-1 is required for HSF-1 to induce molecular chaperones after thermal stress and enhances hormetic extension of longevity. We also show that HPK-1 is required in conjunction with HSF-1 for maintenance of proteostasis in the absence of thermal stress, protecting against the formation of polyglutamine (Q35::YFP) protein aggregates and associated locomotory toxicity. These functions of HPK-1/HSF-1 undergo rapid down-regulation once animals reach reproductive maturity. We show that HPK-1 fortifies proteostasis and extends longevity by an additional independent mechanism: induction of autophagy. HPK-1 is necessary for induction of autophagosome formation and autophagy gene expression in response to dietary restriction (DR) or inactivation of TORC1. The autophagy-stimulating transcription factors pha-4/FoxA and mxl-2/Mlx, but not hlh-30/TFEB or the nuclear hormone receptor nhr-62, are necessary for extended longevity resulting from HPK-1 overexpression. HPK-1 expression is itself induced by transcriptional mechanisms after nutritional stress, and post-transcriptional mechanisms in response to thermal stress. Collectively our results position HPK-1 at a central regulatory node upstream of the greater proteostatic network, acting at the transcriptional level by promoting protein folding via chaperone expression, and protein turnover via expression of autophagy genes. HPK-1 therefore provides a promising intervention point for pharmacological agents targeting the protein homeostasis system as a means of preserving robust longevity.
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- 2017
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5. The cystic-fibrosis-associated ΔF508 mutation confers post-transcriptional destabilization on the C. elegans ABC transporter PGP-3
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Liping He, Jennifer Skirkanich, Lorenza Moronetti, Rosemary Lewis, and Todd Lamitina
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Medicine ,Pathology ,RB1-214 - Abstract
SUMMARY 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. ERQC plays crucial roles in human diseases, such as cystic fibrosis, in which deletion of a single amino acid (F508) results in the misfolding and degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel. We introduced the ΔF508 mutation into Caenorhabditis elegans PGP-3, a 12-transmembrane ABC transporter with 15% identity to CFTR. When expressed in intestinal epithelial cells, PGP-3wt was stable and efficiently trafficked to the apical plasma membrane through a COPII-dependent mechanism. However, PGP-3ΔF508 was post-transcriptionally destabilized, resulting in reduced total and apical membrane protein levels. Genetic or physiological activation of the osmotic stress response pathway, which causes accumulation of the chemical chaperone glycerol, stabilized PGP-3ΔF508. Efficient degradation of PGP-3ΔF508 required the function of several C. elegans ER-associated degradation (ERAD) homologs, suggesting that destabilization occurs through an ERAD-type mechanism. Our studies show that the ΔF508 mutation causes post-transcriptional destabilization and degradation of PGP-3 in C. elegans epithelial cells. This model, combined with the power of C. elegans genetics, provides a new opportunity to genetically dissect metazoan ERQC.
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- 2012
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6. Global Analysis of RNA Secondary Structure in Two Metazoans
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Fan Li, Qi Zheng, Paul Ryvkin, Isabelle Dragomir, Yaanik Desai, Subhadra Aiyer, Otto Valladares, Jamie Yang, Shelly Bambina, Leah R. Sabin, John I. Murray, Todd Lamitina, Arjun Raj, Sara Cherry, Li-San Wang, and Brian D. Gregory
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Biology (General) ,QH301-705.5 - Abstract
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. From this analysis, we find that ssRNAs and dsRNAs are significantly correlated with specific epigenetic modifications. Additionally, we find key structural patterns across protein-coding transcripts that indicate that RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in animals. Finally, we identify and characterize 546 mRNAs whose folding pattern is significantly correlated between these metazoans, suggesting that their structure has some function. Overall, our findings provide a global assessment of RNA folding in animals.
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- 2012
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7. Daf-2 signaling modifies mutant SOD1 toxicity in C. elegans.
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Marco Boccitto, Todd Lamitina, and Robert G Kalb
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Medicine ,Science - Abstract
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. Worms with pan-neuronal G85R SOD1 expression demonstrate significantly impaired locomotion as compared to WT SOD1 expressing controls and they develop insoluble SOD1 aggregates. Reductions in DAF-2 signaling, either through a hypomorphic allele or neuronally targeted RNAi, decreases the abundance of aggregated SOD1 and results in improved locomotion in a DAF-16 dependant manner. These results suggest that manipulation of the DAF-2 Insulin/IGF-1 signaling pathway may have therapeutic potential for the treatment of ALS.
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- 2012
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8. The Caenorhabditis elegans mucin-like protein OSM-8 negatively regulates osmosensitive physiology via the transmembrane protein PTR-23.
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Anne-Katrin Rohlfing, Yana Miteva, Lorenza Moronetti, Liping He, and Todd Lamitina
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Genetics ,QH426-470 - Abstract
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. Here, we show that mutations in the Caenorhabditis elegans mucin-like gene osm-8 specifically disrupt osmoregulatory physiological processes. In osm-8 mutants, normal physiological responses to hypertonic stress, such as the accumulation of organic osmolytes and activation of osmoresponsive gene expression, are constitutively activated. As a result, osm-8 mutants exhibit resistance to normally lethal levels of hypertonic stress and have an osmotic stress resistance (Osr) phenotype. To identify genes required for Osm-8 phenotypes, we performed a genome-wide RNAi osm-8 suppressor screen. After screening ~18,000 gene knockdowns, we identified 27 suppressors that specifically affect the constitutive osmosensitive gene expression and Osr phenotypes of osm-8 mutants. We found that one suppressor, the transmembrane protein PTR-23, is co-expressed with osm-8 in the hypodermis and strongly suppresses several Osm-8 phenotypes, including the transcriptional activation of many osmosensitive mRNAs, constitutive glycerol accumulation, and osmotic stress resistance. Our studies are the first to show that an extracellular mucin-like protein plays an important role in animal osmoregulation in a manner that requires the activity of a novel transmembrane protein. Given that mucins and transmembrane proteins play similar roles in yeast osmoregulation, our findings suggest a possible evolutionarily conserved role for the mucin-plasma membrane interface in eukaryotic osmoregulation.
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- 2011
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9. A suite of MATLAB-based computational tools for automated analysis of COPAS Biosort data
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Elizabeth Morton and Todd Lamitina
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C. elegans ,computational method ,high-throughput screening ,flow cytometry ,Wormsorter ,Biology (General) ,QH301-705.5 - Abstract
Complex Object Parametric Analyzer and Sorter (COPAS) devices are large-object, fluorescence-capable flow cytometers used for high-throughput analysis of live model organisms, including Drosophila melanogaster, Caenorhabditis elegans, and zebrafish. The COPAS is especially useful in C. elegans high-throughput genome-wide RNA interference (RNAi) screens that utilize fluorescent reporters. However, analysis of data from such screens is relatively labor-intensive and time-consuming. Currently, there are no computational tools available to facilitate high-throughput analysis of COPAS data. We used MATLAB to develop algorithms (COPAquant, COPAmulti, and COPAcompare) to analyze different types of COPAS data. COPAquant reads single-sample files, filters and extracts values and value ratios for each file, and then returns a summary of the data. COPAmulti reads 96-well autosampling files generated with the ReFLX adapter, performs sample filtering, graphs features across both wells and plates, performs some common statistical measures for hit identification, and outputs results in graphical formats. COPAcompare performs a correlation analysis between replicate 96-well plates. For many parameters, thresholds may be defined through a simple graphical user interface (GUI), allowing our algorithms to meet a variety of screening applications. In a screen for regulators of stress-inducible GFP expression, COPAquant dramatically accelerated data analysis and allowed us to rapidly move from raw data to hit identification. Because the COPAS file structure is standardized and our MATLAB code is freely available, our algorithms should be extremely useful for analysis of COPAS data from multiple platforms and organisms. The MATLAB code is freely available at our web site (www.med.upenn.edu/lamitinalab/downloads.shtml).
- Published
- 2010
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10. Genetic and physiological activation of osmosensitive gene expression mimics transcriptional signatures of pathogen infection in C. elegans.
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Anne-Katrin Rohlfing, Yana Miteva, Sridhar Hannenhalli, and Todd Lamitina
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Medicine ,Science - Abstract
The soil-dwelling nematode C. elegans is a powerful system for comparative molecular analyses of environmental stress response mechanisms. Infection of worms with bacterial and fungal pathogens causes the activation of well-characterized innate immune transcriptional programs in pathogen-exposed hypodermal and intestinal tissues. However, the pathophysiological events that drive such transcriptional responses are not understood. Here, we show that infection-activated transcriptional responses are, in large part, recapitulated by either physiological or genetic activation of the osmotic stress response. Microarray profiling of wild type worms exposed to non-lethal hypertonicity identified a suite of genes that were also regulated by infection. Expression profiles of five different osmotic stress resistant (osr) mutants under isotonic conditions reiterated the wild type transcriptional response to osmotic stress and also showed substantial similarity to infection-induced gene expression under isotonic conditions. Computational, transgenic, and functional approaches revealed that two GATA transcription factors previously implicated in infection-induced transcriptional responses, elt-2 and elt-3, are also essential for coordinated tissue-specific activation of osmosensitive gene expression and promote survival under osmotically stressful conditions. Together, our data suggest infection and osmotic adaptation share previously unappreciated transcriptional similarities which might be controlled via regulation of tissue-specific GATA transcription factors.
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- 2010
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11. Regulation of the hypertonic stress response by the 3′ mRNA cleavage and polyadenylation complex
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Sarel J. Urso, Anson Sathaseevan, W. Brent Derry, and Todd Lamitina
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Genetics - Abstract
Maintenance of osmotic homeostasis is one of the most aggressively defended homeostatic setpoints 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 forward genetic screen inC. elegansfor mutants with no induction of osmolyte biosynthesis gene expression (Nio mutants).nio-3mutants encoded a missense mutation incpf-2/CstF64 whilenio-7mutants encoded a missense mutation insymk-1/Symplekin. Bothcpf-2andsymk-1are nuclear components of the highly conserved 3’ mRNA cleavage and polyadenylation complex.cpf-2andsymk-1block the hypertonic induction ofgpdh-1and other osmotically induced mRNAs, suggesting they act at the transcriptional level. We generated a functional auxin-inducible degron (AID) allele forsymk-1and found that acute, post-developmental degradation in the intestine and hypodermis was sufficient to cause the Nio phenotype.symk-1andcpf-2exhibit genetic interactions that strongly suggest they function through alterations in 3’ mRNA cleavage and/or alternative polyadenylation. Consistent with this hypothesis, we find that inhibition of several other components of the mRNA cleavage complex also cause a Nio phenotype.cpf-2andsymk-1specifically affect the osmotic stress response since heat shock-induced upregulation of ahsp-16.2::GFPreporter is normal in these mutants. Our data suggest a model in which alternative polyadenylation of one or more mRNAs is essential to regulate the hypertonic stress response.
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- 2023
12. Length-dependent RNA foci formation and Repeat Associated non-AUG dependent translation in a
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Todd, Lamitina
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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
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- 2022
13. The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity
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Carley Snoznik, Paige Rudich, Todd Lamitina, Jelena Mojsilovic-Petrovic, James Oosten, Valentina Medvedeva, and Robert G. Kalb
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SPOP ,Ligases ,Ubiquitin ,RNA interference ,C9orf72 ,Animals ,Caenorhabditis elegans ,Gene ,Cells, Cultured ,Cell Nucleus ,Motor Neurons ,Gene knockdown ,Multidisciplinary ,DNA Repeat Expansion ,biology ,C9orf72 Protein ,Amyotrophic Lateral Sclerosis ,Nuclear Proteins ,Dipeptides ,Biological Sciences ,biology.organism_classification ,Cell biology ,Ubiquitin ligase ,Bromodomain ,Rats ,Repressor Proteins ,Proteasome ,Spinal Cord ,Frontotemporal Dementia ,biology.protein ,Trinucleotide repeat expansion ,Nuclear localization sequence ,Genetic screen - Abstract
A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs (PR)50 and (GR)50 are highly toxic when expressed in C. elegans and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNAi screen for suppressors of (PR)50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by (PR)50 expression. All of these genes have vertebrate homologs and 7/12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. SPOP is also required for (GR)50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP. Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. Finally, we find that genetic inhibition of bet-1, the C. elegans homolog of the known SPOP ubiquitination targets BRD2/3/4, suppresses the protective effect of SPOP mutations. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent a novel entry point for therapeutic intervention to treat C9 FTD/ALS.Significance statementThe G4C2 repeat expansion in the C9orf72 gene is a major cause of Fronto-Temporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS). Unusual translation of the repeat sequence produces two highly toxic dipeptide repeat proteins, PRX and GRX, which accumulate in the brain tissue of individuals with these diseases. Here, we show that PR and GR toxicity in both C. elegans and mammalian neurons depends on the E3 ubiquitin ligase adaptor SPOP. SPOP acts through the bromodomain protein BET-1 to mediate dipeptide toxicity. SPOP inhibitors, which are currently being developed to treat SPOP-dependent renal cancer, also protect neurons against DPR toxicity. Our findings identify a highly conserved and ‘druggable’ pathway that may represent a new strategy for treating these currently incurable diseases.
- Published
- 2021
14. The O-GlcNAc transferase OGT is a conserved and essential regulator of the cellular and organismal response to hypertonic stress
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Todd Lamitina, Sarel J. Urso, Marcella E. Comly, and John A. Hanover
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Threonine ,Cancer Research ,Genetic Screens ,Nematoda ,Regulator ,Gene Identification and Analysis ,QH426-470 ,Synthetic Genome Editing ,Biochemistry ,Genome Engineering ,0302 clinical medicine ,Fluorescence Microscopy ,RNA interference ,Gene expression ,Serine ,Genetics (clinical) ,Caenorhabditis elegans ,Hypertonic ,Regulation of gene expression ,0303 health sciences ,Microscopy ,biology ,Physics ,Crispr ,Classical Mechanics ,Eukaryota ,Light Microscopy ,Animal Models ,Phenotype ,Cell biology ,Nucleic acids ,Experimental Organism Systems ,Genetic interference ,Caenorhabditis Elegans ,Physical Sciences ,Engineering and Technology ,Synthetic Biology ,Epigenetics ,Research Article ,Cell physiology ,Osmotic shock ,Bioengineering ,Research and Analysis Methods ,N-Acetylglucosaminyltransferases ,Gene Expression Regulation, Enzymologic ,03 medical and health sciences ,Model Organisms ,Protein Domains ,Osmotic Pressure ,Osmotic Shock ,Pressure ,Genetics ,Tonicity ,Animals ,Humans ,Caenorhabditis elegans Proteins ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Alleles ,030304 developmental biology ,Organisms ,Biology and Life Sciences ,Cell Biology ,Synthetic Genomics ,biology.organism_classification ,Invertebrates ,Genetic Loci ,Animal Studies ,Caenorhabditis ,RNA ,Zoology ,Protein Processing, Post-Translational ,030217 neurology & neurosurgery ,Function (biology) ,Genetic screen - Abstract
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. Through a forward genetic screen in Caenorhabditis elegans, we discovered OGT is acutely required for osmoprotective protein expression and adaptation to hypertonic stress. Gene expression analysis shows that ogt-1 functions through a post-transcriptional mechanism. Human OGT partially rescues the C. elegans phenotypes, suggesting that the osmoregulatory functions of OGT are ancient. Intriguingly, expression of O-GlcNAcylation-deficient forms of human or worm OGT rescue the hypertonic stress response phenotype. However, expression of an OGT protein lacking the tetracopeptide repeat (TPR) domain does not rescue. Our findings are among the first to demonstrate a specific physiological role for OGT at the organismal level and demonstrate that OGT engages in important molecular functions outside of its well described roles in post-translational O-GlcNAcylation of intracellular proteins., Author summary The ability to sense and adapt to changes in the environment is an essential feature of cellular life. Changes in environmental salt and water concentrations can rapidly cause cell volume swelling or shrinkage and, if left unchecked, will lead to cell and organismal death. All organisms have developed similar physiological strategies for maintaining cell volume. However, the molecular mechanisms that control these physiological outputs are not well understood in animals. Using unbiased genetic screening in C. elegans, we discovered that a highly conserved enzyme called O-GlcNAc transferase (OGT) is essential for regulating physiological responses to increased environmental solute levels. A human form of OGT can functionally substitute for worm OGT, showing that this role is conserved across evolution. Surprisingly, the only known enzymatic activity of OGT was not required for this role, suggesting this enzyme has important undescribed molecular functions. Our studies reveal a new animal-specific role for OGT in the response to osmotic stress and show that C. elegans is an important model for defining the conserved molecular mechanisms that respond to alterations in cell volume.
- Published
- 2020
15. Measuring RAN Peptide Toxicity in C. elegans
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Paige, Rudich, Carley, Snoznik, Noah, Puleo, and Todd, Lamitina
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Neurons ,DNA Repeat Expansion ,Animals ,RNA, Antisense ,Caenorhabditis elegans ,Peptide Chain Initiation, Translational ,Peptide Fragments ,Article - Abstract
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. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.
- Published
- 2020
16. Measuring RAN Peptide Toxicity in C. elegans
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Noah Puleo, Todd Lamitina, Carley Snoznik, and Paige Rudich
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General Immunology and Microbiology ,General Chemical Engineering ,General Neuroscience ,Neurodegeneration ,Reading frame ,Translation (biology) ,Computational biology ,Biology ,medicine.disease ,General Biochemistry, Genetics and Molecular Biology ,Antisense RNA ,Start codon ,Proteotoxicity ,Ran ,medicine ,Trinucleotide repeat expansion - Abstract
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. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.
- Published
- 2020
17. PolyQ-independent toxicity associated with novel translational products from CAG repeat expansions
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Simon C. Watkins, Paige Rudich, and Todd Lamitina
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0301 basic medicine ,Nematoda ,ved/biology.organism_classification_rank.species ,Toxicology ,Pathology and Laboratory Medicine ,Biochemistry ,0302 clinical medicine ,Animal Cells ,Sense (molecular biology) ,Medicine and Health Sciences ,Caenorhabditis elegans ,Neurons ,Motor Neurons ,Genetics ,Microscopy ,0303 health sciences ,Multidisciplinary ,biology ,Neurodegeneration ,Eukaryota ,Light Microscopy ,Neurodegenerative Diseases ,Translation (biology) ,Animal Models ,3. Good health ,Huntington Disease ,Experimental Organism Systems ,Neurology ,Caenorhabditis Elegans ,Genetic Diseases ,Medicine ,Cellular Types ,Anatomy ,Research Article ,Repetitive Sequences, Amino Acid ,congenital, hereditary, and neonatal diseases and abnormalities ,Cell type ,Fluorescence Recovery after Photobleaching ,Science ,Muscle Tissue ,Research and Analysis Methods ,Protein Aggregates ,03 medical and health sciences ,Model Organisms ,mental disorders ,medicine ,Animals ,Humans ,RNA, Antisense ,Caenorhabditis elegans Proteins ,Model organism ,030304 developmental biology ,Sequence (medicine) ,Clinical Genetics ,Muscle Cells ,Toxicity ,ved/biology ,Autosomal Dominant Diseases ,Organisms ,Biology and Life Sciences ,Polypeptides ,RNA ,Cell Biology ,medicine.disease ,biology.organism_classification ,Invertebrates ,nervous system diseases ,Disease Models, Animal ,Biological Tissue ,030104 developmental biology ,Cellular Neuroscience ,Protein Biosynthesis ,Ran ,Animal Studies ,Caenorhabditis ,Peptides ,Trinucleotide Repeat Expansion ,Trinucleotide repeat expansion ,030217 neurology & neurosurgery ,Neuroscience - Abstract
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 calledRepeat-associatednon-AUG dependent (RAN) translation. RAN translation of the CAG sense and CUG anti-sense RNAs produces six distinct repeat peptides: polyalanine (polyAla, from both CAG and CUG repeats), polyserine (polySer), polyleucine (polyLeu), polycysteine (polyCys), and polyglutamine (polyGln). The toxic potential of individual CAG-derived RAN polypeptides is not well understood. We developed pureC. elegansprotein models for each CAG RAN polypeptide using codon-varied expression constructs that preserve RAN protein sequence but eliminate repetitive CAG/CUG RNA. While all RAN polypeptides formed aggregates, only polyLeu was consistently toxic across multiple cell types. In GABAergic neurons, which exhibit significant neurodegeneration in HD patients, codon-varied (Leu)38, but not (Gln)38, caused substantial neurodegeneration and motility defects. Our studies provide the firstin vivoevaluation of CAG-derived RAN polypeptides and suggest that polyQ-independent mechanisms, such as RAN-translated polyLeu peptides, may have a significant pathological role in CAG repeat expansion disorders.
- Published
- 2020
18. Models and mechanisms of repeat expansion disorders: a worm's eye view
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Paige, Rudich and Todd, Lamitina
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Disease Models, Animal ,Fragile X Syndrome ,Tremor ,Animals ,Humans ,Ataxia ,Caenorhabditis elegans ,Trinucleotide Repeat Expansion ,Article - Abstract
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.e. polyQ). Repeat expansions in introns and UTRs (i.e. FXTAS) are thought to produce aberrant repeat bearing RNAs that interact with and sequester a wide variety of essential proteins, resulting in cellular toxicity. However, a new phenomenon termed ‘Repeat-associated non-AUG dependent (RAN) translation’ paints a new and unifying picture of how distinct repeat expansion bearing RNAs might act as substrates for this non-canonical form of translation, leading to the production of a wide range of repeat sequence specific-encoded toxic proteins. Here, we review how the model system C. elegans has been utilized to model many repeat disorders and discuss how RAN translation could be a previously unappreciated contributor to the toxicity associated with these different models.
- Published
- 2018
19. 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
- Author
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Justine A. Melo, Elizabeth A. Morton, Peter M. Douglas, Manjunatha Thondamal, Todd Lamitina, Andrew V. Samuelson, Adam Cornwell, Elliot W. Swartz, Joung Heon Kim, Beresford Crick, and Ritika Das
- Subjects
0301 basic medicine ,Cancer Research ,Aging ,Nematoda ,SUMO protein ,mTORC1 ,QH426-470 ,Biochemistry ,RNA interference ,Animal Cells ,Homeostasis ,Genetics (clinical) ,Genetics ,Regulation of gene expression ,Neurons ,Cell Death ,Physics ,TOR Serine-Threonine Kinases ,Classical Mechanics ,Eukaryota ,Animal Models ,SUMOylation ,Cell biology ,Nucleic acids ,Genetic interference ,Experimental Organism Systems ,Cell Processes ,Caenorhabditis Elegans ,Physical Sciences ,Mechanical Stress ,Epigenetics ,Post-translational modification ,Cellular Types ,Research Article ,Signal Transduction ,Autophagic Cell Death ,Longevity ,Biology ,Mechanistic Target of Rapamycin Complex 1 ,Protein Serine-Threonine Kinases ,Research and Analysis Methods ,03 medical and health sciences ,Model Organisms ,Stress, Physiological ,DNA-binding proteins ,Autophagy ,Animals ,Gene Regulation ,Caenorhabditis elegans Proteins ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Biology and life sciences ,Organisms ,Proteins ,Cell Biology ,Invertebrates ,Regulatory Proteins ,Heat shock factor ,030104 developmental biology ,Proteostasis ,Thermal Stresses ,Gene Expression Regulation ,Chaperone (protein) ,Cellular Neuroscience ,Multiprotein Complexes ,biology.protein ,Caenorhabditis ,TFEB ,RNA ,Gene expression ,Protein Processing, Post-Translational ,Transcription Factors ,Neuroscience ,Molecular Chaperones - Abstract
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. The homeodomain-interacting protein kinase (hipk) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole Caenorhabditis elegans Hipk homolog, hpk-1, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of hpk-1 is deleterious to these phenotypes. We show that HPK-1 preserves proteostasis and extends longevity through distinct but complementary genetic pathways defined by the heat shock transcription factor (HSF-1), and the target of rapamycin complex 1 (TORC1). We demonstrate that HPK-1 antagonizes sumoylation of HSF-1, a post-translational modification associated with reduced transcriptional activity in mammals. We show that inhibition of sumoylation by RNAi enhances HSF-1-dependent transcriptional induction of chaperones in response to heat shock. We find that hpk-1 is required for HSF-1 to induce molecular chaperones after thermal stress and enhances hormetic extension of longevity. We also show that HPK-1 is required in conjunction with HSF-1 for maintenance of proteostasis in the absence of thermal stress, protecting against the formation of polyglutamine (Q35::YFP) protein aggregates and associated locomotory toxicity. These functions of HPK-1/HSF-1 undergo rapid down-regulation once animals reach reproductive maturity. We show that HPK-1 fortifies proteostasis and extends longevity by an additional independent mechanism: induction of autophagy. HPK-1 is necessary for induction of autophagosome formation and autophagy gene expression in response to dietary restriction (DR) or inactivation of TORC1. The autophagy-stimulating transcription factors pha-4/FoxA and mxl-2/Mlx, but not hlh-30/TFEB or the nuclear hormone receptor nhr-62, are necessary for extended longevity resulting from HPK-1 overexpression. HPK-1 expression is itself induced by transcriptional mechanisms after nutritional stress, and post-transcriptional mechanisms in response to thermal stress. Collectively our results position HPK-1 at a central regulatory node upstream of the greater proteostatic network, acting at the transcriptional level by promoting protein folding via chaperone expression, and protein turnover via expression of autophagy genes. HPK-1 therefore provides a promising intervention point for pharmacological agents targeting the protein homeostasis system as a means of preserving robust longevity., Author summary Aging is the gradual and progressive decline of vitality. A hallmark of aging is the decay of protective mechanisms that normally preserve the robustness and resiliency of cells and tissues. Proteostasis is the term that applies specifically to those mechanisms that promote stability of the proteome, the collection of polypeptides that cells produce, by a combination of chaperone-assisted folding and degradation of misfolded or extraneous proteins. We have identified hpk-1 (encoding a homeodomain-interacting protein kinase) in the nematode C. elegans as an important transcriptional regulatory component of the proteostasis machinery. HPK-1 promotes proteostasis by linking two distinct mechanisms: first by stimulating chaperone gene expression via the heat shock transcription factor (HSF-1), and second by stimulating autophagy gene expression in opposition to the target of rapamycin (TOR) kinase signaling pathway. HPK-1 therefore provides an attractive target for interventions to preserve physiological resiliency during aging by preserving the overall health of the proteome.
- Published
- 2017
20. Calcium Homeostasis Modulator (CALHM) Ion Channels: Structure, Functions and Physiological Roles
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J. Kevin Foskett, Akiyuki Taruno, E Tanis Jessica, Adam P. Siebert, Zhongming Ma, Philippe Marambaud, and Todd Lamitina
- Subjects
Calcium metabolism ,Taste ,Structure function ,Connexin ,Pharmacology (medical) ,Biology ,Ion channel ,Cell biology - Published
- 2014
21. Stress Signaling: Serotonin Spreads Systemic Stress
- Author
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Todd Lamitina and Arjumand Ghazi
- Subjects
Serotonin ,medicine.medical_specialty ,Gonad ,Cell ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Internal medicine ,medicine ,Animals ,Heat shock ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Regulation of gene expression ,Agricultural and Biological Sciences(all) ,Biochemistry, Genetics and Molecular Biology(all) ,Stress signaling ,Serotonin metabolism ,Cell biology ,Endocrinology ,medicine.anatomical_structure ,Gene Expression Regulation ,Systemic stress ,General Agricultural and Biological Sciences ,Heat-Shock Response ,Transcription Factors - Abstract
SummaryCells 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.
- Published
- 2015
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22. FER-1/Dysferlin promotes cholinergic signaling at the neuromuscular junction in C. elegans and mice
- Author
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S. Todd Lamitina, Jessica E. Tanis, Predrag Krajacic, Emidio E. Pistilli, and Tejvir S. Khurana
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medicine.medical_specialty ,QH301-705.5 ,Science ,Muscle disorder ,General Biochemistry, Genetics and Molecular Biology ,Neuromuscular junction ,Dysferlin ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,Limb-girdle ,medicine ,Synaptic transmission ,Biology (General) ,Muscular dystrophy ,030304 developmental biology ,0303 health sciences ,biology ,Skeletal muscle ,medicine.disease ,Cell biology ,medicine.anatomical_structure ,Endocrinology ,biology.protein ,Cholinergic ,Synaptic signaling ,General Agricultural and Biological Sciences ,030217 neurology & neurosurgery ,Limb-girdle muscular dystrophy ,Research Article ,LGMD2B - Abstract
Summary 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. We found that both loss-of-function mutations and muscle-specific overexpression of C. elegans fer-1, the founding member of the Dysferlin gene family, caused defects in muscle cholinergic signaling. To determine if Dysferlin-dependent regulation of cholinergic signaling is evolutionarily conserved, we examined the in vivo physiological properties of skeletal muscle synaptic signaling in a mouse model of Dysferlin-deficiency. In addition to a loss in muscle strength, Dysferlin −/− mice also exhibited a cholinergic deficit manifested by a progressive, frequency-dependent decrement in their compound muscle action potentials following repetitive nerve stimulation, which was observed in another Dysferlin mouse model but not in a Dysferlin-independent mouse model of muscular dystrophy. Oral administration of Pyridostigmine bromide, a clinically used acetylcholinesterase inhibitor (AchE.I) known to increase synaptic efficacy, reversed the action potential defect and restored in vivo muscle strength to Dysferlin −/− mice without altering muscle pathophysiology. Our data demonstrate a previously unappreciated role for Dysferlin in the regulation of cholinergic signaling and suggest that such regulation may play a significant pathophysiological role in LGMD2B disease.
- Published
- 2013
23. CLHM-1 is a Functionally Conserved and Conditionally Toxic Ca2+-Permeable Ion Channel in Caenorhabditis elegans
- Author
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Jessica E. Tanis, Todd Lamitina, Predrag Krajacic, J. K. Foskett, Liping He, and Zhongming Ma
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Sensory Receptor Cells ,Motility ,Biology ,Membrane Potentials ,Cell membrane ,Xenopus laevis ,Species Specificity ,Extracellular ,medicine ,Animals ,Humans ,Transgenes ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Ion channel ,Membrane potential ,Membrane Glycoproteins ,Voltage-dependent calcium channel ,General Neuroscience ,Cell Membrane ,Articles ,Calcium Channel Blockers ,biology.organism_classification ,Electric Stimulation ,Cell biology ,medicine.anatomical_structure ,Touch ,Nerve Degeneration ,Oocytes ,Calcium ,CALHM1 ,Calcium Channels ,Locomotion - Abstract
Disruption of neuronal Ca(2+) homeostasis contributes to neurodegenerative diseases through mechanisms that are not fully understood. A polymorphism in CALHM1, a recently described ion channel that regulates intracellular Ca(2+) levels, is a possible risk factor for late-onset Alzheimer's disease. Since there are six potentially redundant CALHM family members in humans, the physiological and pathophysiological consequences of CALHM1 function in vivo remain unclear. The nematode Caenorhabditis elegans expresses a single CALHM1 homolog, CLHM-1. Here we find that CLHM-1 is expressed at the plasma membrane of sensory neurons and muscles. Like human CALHM1, C. elegans CLHM-1 is a Ca(2+)-permeable ion channel regulated by voltage and extracellular Ca(2+). Loss of clhm-1 in the body-wall muscles disrupts locomotory kinematics and biomechanics, demonstrating that CLHM-1 has a physiologically significant role in vivo. The motility defects observed in clhm-1 mutant animals can be rescued by muscle-specific expression of either C. elegans CLHM-1 or human CALHM1, suggesting that the function of these proteins is conserved in vivo. Overexpression of either C. elegans CLHM-1 or human CALHM1 in neurons is toxic, causing degeneration through a necrotic-like mechanism that is partially Ca(2+) dependent. Our data show that CLHM-1 is a functionally conserved ion channel that plays an important but potentially toxic role in excitable cell function.
- Published
- 2013
24. Caenorhabditis elegansHSF-1 is an essential nuclear protein that forms stress granule-like structures following heat shock
- Author
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Todd Lamitina and Elizabeth A. Morton
- Subjects
endocrine system ,Aging ,biology ,Mutant ,Cell Biology ,biology.organism_classification ,Cell biology ,Green fluorescent protein ,Heat shock factor ,Stress granule ,Nuclear protein ,Heat shock ,Transcription factor ,Caenorhabditis elegans - Abstract
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. elegans are incompletely understood. We generated animals expressing physiological levels of an HSF-1::GFP fusion protein and examined its function, localization, and regulation in vivo. HSF-1::GFP was functional, as measured by its ability to rescue phenotypes associated with two hsf-1 mutant alleles. Rescue of hsf-1 development phenotypes was abolished in a DNA-binding-deficient mutant, demonstrating that the transcriptional targets of hsf-1 are critical to its function even in the absence of stress. Under nonstress conditions, HSF-1::GFP was found primarily in the nucleus. Following heat shock, HSF-1::GFP rapidly and reversibly redistributed into dynamic, subnuclear structures that share many properties with human nuclear stress granules, including colocalization with markers of active transcription. Rapid formation of HSF-1 stress granules required HSF-1 DNA-binding activity, and the threshold for stress granule formation was altered by growth temperature. HSF-1 stress granule formation was not induced by inhibition of IGF signaling, a pathway previously suggested to function upstream of hsf-1. Our findings suggest that development, stress, and aging pathways may regulate HSF-1 function in distinct ways, and that HSF-1 nuclear stress granule formation is an evolutionarily conserved aspect of HSF-1 regulation in vivo.
- Published
- 2012
25. Biomechanical Profiling of Caenorhabditis elegans Motility
- Author
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Xiaoning Shen, Paulo E. Arratia, Todd Lamitina, Prashant K. Purohit, and Predrag Krajacic
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Genetics ,Microscopy, Video ,Temperature ,Biomechanics ,Motility ,Video microscopy ,Computational biology ,Biology ,Bioinformatics ,biology.organism_classification ,Biomechanical Phenomena ,Notes ,Hydrodynamics ,Image Processing, Computer-Assisted ,Animals ,Caenorhabditis elegans ,Gait ,Algorithms ,Locomotion ,Software ,Swimming ,Mechanical Phenomena - Abstract
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. Quantification of body shapes and external hydrodynamics and model-based estimates of biomechanics reveal that mutants affecting similar biological processes exhibit related patterns of biomechanical differences. Therefore, biomechanical profiling could be useful for predicting the function of previously unstudied motility genes.
- Published
- 2012
26. Stress and aging induce distinct polyQ protein aggregation states
- Author
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Robert G. Kalb, Lorenza E. Moronetti Mazzeo, Devin Dersh, Marco Boccitto, and Todd Lamitina
- Subjects
Aging ,Multidisciplinary ,Osmotic shock ,Biological Sciences ,Biology ,Protein aggregation ,biology.organism_classification ,medicine.disease_cause ,Cell biology ,Mice ,Oxidative Stress ,Osmotic Pressure ,Stress, Physiological ,Cytoplasm ,medicine ,Osmoregulation ,Animals ,Osmotic pressure ,Protein folding ,Peptides ,Caenorhabditis elegans ,Oxidative stress - Abstract
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. Osmotic stress-induced polyQ aggregates could be distinguished from aging-induced polyQ aggregates based on morphological, biophysical, cell biological, and biochemical criteria, suggesting that they are a unique misfolded-protein species. The insulin-like growth factor signaling mutant daf-2 , which inhibits age-induced polyQ aggregation and protects C. elegans from stress, did not prevent the formation of stress-induced polyQ aggregates. However, osmotic stress resistance mutants, which genetically activate the osmotic stress response, strongly inhibited the formation of osmotic polyQ aggregates. Our findings show that in vivo, the same protein can adopt distinct aggregation states depending on the initiating stressor and that stress and aging impact the proteome in related but distinct ways.
- Published
- 2012
27. Global Analysis of RNA Secondary Structure in Two Metazoans
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John I. Murray, Subhadra Aiyer, Paul Ryvkin, Sara Cherry, Leah R. Sabin, Otto Valladares, Isabelle Dragomir, Qi Zheng, Yaanik Desai, Fan Li, Li-San Wang, Shelly Bambina, Brian D. Gregory, Jamie Yang, Todd Lamitina, and Arjun Raj
- Subjects
Molecular Sequence Data ,Computational biology ,Biology ,Chromosomes ,General Biochemistry, Genetics and Molecular Biology ,Epigenesis, Genetic ,Nucleic acid secondary structure ,7SK RNA ,Animals ,RNA, Messenger ,Caenorhabditis elegans ,Base Pairing ,lcsh:QH301-705.5 ,Conserved Sequence ,RNA, Double-Stranded ,Genetics ,Genome ,Base Sequence ,Intron ,RNA ,Non-coding RNA ,MicroRNAs ,RNA silencing ,Drosophila melanogaster ,lcsh:Biology (General) ,RNA editing ,Protein Biosynthesis ,Nucleic Acid Conformation ,RNA, Helminth ,Transcriptome ,Small nuclear RNA - Abstract
SummaryThe 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. From this analysis, we find that ssRNAs and dsRNAs are significantly correlated with specific epigenetic modifications. Additionally, we find key structural patterns across protein-coding transcripts that indicate that RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in animals. Finally, we identify and characterize 546 mRNAs whose folding pattern is significantly correlated between these metazoans, suggesting that their structure has some function. Overall, our findings provide a global assessment of RNA folding in animals.
- Published
- 2012
28. TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress: organic osmolyte-dependent and -independent pathways
- Author
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Steffan N. Ho, William Y. Go, Soo Youn Choi, Sun Woo Lim, Sang Do Lee, S. Todd Lamitina, and H. Moo Kwon
- Subjects
Mice, Knockout ,Osmosis ,Cellular adaptation ,Physiology ,Hypertonic Solutions ,Articles ,Fibroblasts ,Biology ,Adaptation, Physiological ,Mice ,Biochemistry ,Stress, Physiological ,RNA interference ,Osmolyte ,Hypertonic Stress ,Heat shock protein ,Enhancer binding ,Models, Animal ,Animals ,RNA Interference ,Signal transduction ,Transcription factor ,Cells, Cultured ,Signal Transduction ,Transcription Factors - Abstract
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. Microarray analyses revealed that the genetic response to hypertonicity was dominated by TonEBP in that expression of totally different sets of genes was increased by hypertonicity in those cells with TonEBP vs. those without TonEBP activity. Of over 100 potentially new TonEBP-regulated genes, we selected seven for further analyses and found that their expressions were all dependent on TonEBP. RNA interference experiments showed that some of these genes, asporin, insulin-like growth factor-binding protein-5 and -7, and an extracellular lysophospholipase D, plus heat shock protein 70, a known TonEBP target gene, contributed to the adaptation to hypertonicity without promoting organic osmolyte accumulation. We conclude that TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress in addition to organic osmolyte accumulation.
- Published
- 2011
29. C. elegans dysferlin homologfer-1is expressed in muscle, andfer-1mutations initiate altered gene expression of muscle enriched genes
- Author
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Olga Lozynska, Todd Lamitina, Predrag Krajacic, Jane Hermanowski, and Tejvir S. Khurana
- Subjects
Transcription, Genetic ,Physiology ,Muscle Proteins ,Polymerase Chain Reaction ,Dysferlin ,Gene expression ,Genetics ,medicine ,Animals ,Cluster Analysis ,Humans ,Myocyte ,RNA, Messenger ,Muscular dystrophy ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Gene ,Cells, Cultured ,Research Articles ,Oligonucleotide Array Sequence Analysis ,Regulation of gene expression ,Muscle Cells ,Sequence Homology, Amino Acid ,biology ,Muscles ,Membrane Proteins ,Reproducibility of Results ,biology.organism_classification ,medicine.disease ,Molecular biology ,Gene Expression Regulation ,Organ Specificity ,Mutation ,biology.protein ,Limb-girdle muscular dystrophy - Abstract
Mutations in the human dysferlin gene cause Limb Girdle Muscular Dystrophy 2B (LGMD2B). The Caenorhabditis elegans dysferlin homolog, fer-1, affects sperms development but is not known to be expressed in or have a functional roles outside of the male germline. Using several approaches, we show that fer-1 mRNA is present in C. elegans muscle cells but is absent from neurons. In mammals, loss of muscle-expressed dysferlin causes transcriptional deregulation of muscle expressed genes. To determine if similar alterations in gene expression are initiated in C. elegans due to loss of muscle-expressed fer-1, we performed whole genome Affymetrix microarray analysis of two loss-of-function fer-1 mutants. Both mutants gave rise to highly similar changes in gene expression and altered the expression of 337 genes. Using multiple analysis methods, we show that this gene set is enriched for genes known to regulate the structure and function of muscle. However, these transcriptional changes do not appear to be in response to gross sarcomeric damage, since genetically sensitized fer-1 mutants exhibit normal thin filament organization. Our data suggest that processes other than sarcomere stability may be affected by loss of fer-1 in C. elegans muscle. Therefore, C. elegans may be an attractive model system in which to explore new muscle-specific functions of the dysferlin protein and gain insights into the molecular pathogenesis of LGMD2B.
- Published
- 2009
30. Loss of RAD-23 Protects Against Models of Motor Neuron Disease by Enhancing Mutant Protein Clearance
- Author
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Jiou Wang, Todd Lamitina, Preetika Gupta, Lei Zhang, Jiayin Lu, Chia-Yen Wu, Angela M. Jablonski, Shachee Doshi, Brian C. Kraemer, Robert G. Kalb, Jelena Mojsilovic-Petrovic, Hannes Lans, Lyle W. Ostrow, Mariangela Sabatella, Nicole F. Liachko, and Molecular Genetics
- Subjects
Male ,Genotype ,SOD1 ,Green Fluorescent Proteins ,Biology ,Motor Activity ,DNA-binding protein ,Neuroprotection ,Animals, Genetically Modified ,Rats, Sprague-Dawley ,Mice ,Ubiquitin ,Mutant protein ,medicine ,Animals ,Humans ,Motor Neuron Disease ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Cells, Cultured ,Photobleaching ,General Neuroscience ,Neurodegeneration ,Articles ,Motor neuron ,medicine.disease ,Cell biology ,Rats ,DNA-Binding Proteins ,Disease Models, Animal ,medicine.anatomical_structure ,Proteotoxicity ,Biochemistry ,Animals, Newborn ,Gene Expression Regulation ,Mutation ,biology.protein ,RNA Interference - Abstract
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. Loss of RAD-23 suppresses the locomotor deficit ofCaenorhabditis elegansengineered to express mutTDP-43 or mutSOD1 and also protects against aging and proteotoxic insults. Knockdown of RAD-23 is further neuroprotective against the toxicity of SOD1 and TDP-43 expression in mammalian neurons. Biochemical investigation indicates that RAD-23 modifies mutTDP-43 and mutSOD1 abundance, solubility, and turnover in association with altering the ubiquitination status of these substrates. In human amyotrophic lateral sclerosis spinal cord, we find that RAD-23 abundance is increased and RAD-23 is mislocalized within motor neurons. We propose a novel pathophysiological function for RAD-23 in the stabilization of mutated proteins that cause neurodegeneration.SIGNIFICANCE STATEMENTIn this work, we identify RAD-23, a component of the protein homeostasis network and nucleotide excision repair pathway, as a modifier of the toxicity of two disease-causing, misfolding-prone proteins, SOD1 and TDP-43. Reducing the abundance of RAD-23 accelerates the degradation of mutant SOD1 and TDP-43 and reduces the cellular content of the toxic species. The existence of endogenous proteins that act as “anti-chaperones” uncovers new and general targets for therapeutic intervention.
- Published
- 2015
31. Isolation of C. elegans Deletion Mutants Following ENU Mutagenesis and Thermostable Restriction Enzyme PCR Screening
- Author
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Kevin Strange, Peter Agre, Chunyi George Huang, and Todd Lamitina
- Subjects
Genetics ,Mutant ,Mutagenesis (molecular biology technique) ,Bioengineering ,Helminth genetics ,Biology ,Applied Microbiology and Biotechnology ,Biochemistry ,Molecular biology ,law.invention ,Restriction enzyme ,chemistry.chemical_compound ,chemistry ,law ,Molecular Biology ,Gene ,Gene knockout ,DNA ,Polymerase chain reaction ,Biotechnology - Abstract
The ability to generate null mutants is essential for studying gene function. Gene knockouts in Caenorhabditis elegans can be generated in a high throughput manner using chemical mutagenesis followed by polymerase chain reaction (PCR) assays to detect deletions in a gene of interest. However, current methods for identifying deletions are time and labor intensive and are unable to efficiently detect small deletions. In this study, we expanded the method pioneered by Wei et al., which used the thermostable restriction enzyme PspGI and tested the usefulness of other thermostable restriction enzymes including BstUI, Tsp45I, ApeKI, and TfiI. We designed primers to flank one or multiple thermostable restriction enzymes sites in the genes of interest. The use of multiple enzymes and the optimization of PCR primer design enabled us to isolate deletion in 66.7% of the genes screened. The size of the deletions varied from 330 bp to 1 kb. This method should make it possible for small academic laboratories to rapidly isolate deletions in their genes of interest.
- Published
- 2006
32. Transcriptional targets of DAF-16 insulin signaling pathway protectC.elegansfrom extreme hypertonic stress
- Author
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S. Todd Lamitina and Kevin Strange
- Subjects
Transcription, Genetic ,Physiology ,medicine.medical_treatment ,Hypertonic Solutions ,Biology ,Osmotic Pressure ,Somatomedins ,Transcription (biology) ,RNA interference ,medicine ,Daf-16 ,Animals ,Insulin ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Genes, Helminth ,Oligonucleotide Array Sequence Analysis ,Trehalose ,Forkhead Transcription Factors ,Cell Biology ,Biochemistry ,Osmolyte ,Hypertonic Stress ,Daf-2 ,Signal transduction ,Signal Transduction ,Transcription Factors - Abstract
All cells adapt to hypertonic stress by regulating their volume after shrinkage, by accumulating organic osmolytes, and by activating mechanisms that protect against and repair hypertonicity-induced damage. In mammals and nematodes, inhibition of signaling from the DAF-2/IGF-1 insulin receptor activates the DAF-16/FOXO transcription factor, resulting in increased life span and resistance to some types of stress. We tested the hypothesis that inhibition of insulin signaling in Caenorhabditis elegans also increases hypertonic stress resistance. Genetic inhibition of DAF-2 or its downstream target, the AGE-1 phosphatidylinositol 3-kinase, confers striking resistance to a normally lethal hypertonic shock in a DAF-16-dependent manner. However, insulin signaling is not inhibited by or required for adaptation to hypertonic conditions. Microarray studies have identified 263 genes that are transcriptionally upregulated by DAF-16 activation. We identified 14 DAF-16-upregulated genes by RNA interference screening that are required for age- 1 hypertonic stress resistance. These genes encode heat shock proteins, proteins of unknown function, and trehalose synthesis enzymes. Trehalose levels were elevated approximately twofold in age- 1 mutants, but this increase was insufficient to prevent rapid hypertonic shrinkage. However, age- 1 animals unable to synthesize trehalose survive poorly under hypertonic conditions. We conclude that increased expression of proteins that protect eukaryotic cells against environmental stress and/or repair stress-induced molecular damage confers hypertonic stress resistance in C. elegans daf- 2/ age- 1 mutants. Elevated levels of solutes such as trehalose may also function in a cytoprotective manner. Our studies provide novel insights into stress resistance in animal cells and a foundation for new studies aimed at defining molecular mechanisms underlying these essential processes.
- Published
- 2005
33. Adaptation of the nematodeCaenorhabditis elegansto extreme osmotic stress
- Author
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Kevin Strange, Rebecca Morrison, Gilbert W. Moeckel, and S. Todd Lamitina
- Subjects
Glycerol ,Saline Solution, Hypertonic ,Glycerol-3-Phosphate Dehydrogenase (NAD+) ,Osmotic shock ,Physiology ,Glycerolphosphate Dehydrogenase ,Cell Biology ,Water-Electrolyte Balance ,Inorganic ions ,Biology ,Adaptation, Physiological ,Gene Expression Regulation, Enzymologic ,Yeast ,chemistry.chemical_compound ,Biochemistry ,chemistry ,Osmotic Pressure ,Hypertonic Stress ,Osmolyte ,Osmoregulation ,Animals ,Osmotic pressure ,Caenorhabditis elegans - Abstract
The ability to control osmotic balance is essential for cellular life. Cellular osmotic homeostasis is maintained by accumulation and loss of inorganic ions and organic osmolytes. Although osmoregulation has been studied extensively in many cell types, major gaps exist in our molecular understanding of this essential process. Because of its numerous experimental advantages, the nematode Caenorhabditis elegans provides a powerful model system to characterize the genetic basis of animal cell osmoregulation. We therefore characterized the ability of worms to adapt to extreme osmotic stress. Exposure of worms to high-salt growth agar causes rapid shrinkage. Survival is normal on agar containing up to 200 mM NaCl. When grown on 200 mM NaCl for 2 wk, worms are able to survive well on agar containing up to 500 mM NaCl. HPLC analysis demonstrated that levels of the organic osmolyte glycerol increase 15- to 20-fold in nematodes grown on 200 mM NaCl agar. Accumulation of glycerol begins 3 h after exposure to hypertonic stress and peaks by 24 h. Glycerol accumulation is mediated primarily by synthesis from metabolic precursors. Consistent with this finding, hypertonicity increases transcriptional expression of glycerol 3-phosphate dehydrogenase, an enzyme that is rate limiting for hypertonicity-induced glycerol synthesis in yeast. Worms adapted to high salt swell and then return to their initial body volume when exposed to low-salt agar. During recovery from hypertonic stress, glycerol levels fall rapidly and glycerol excretion increases approximately fivefold. Our studies provide the first description of osmotic adaptation in C. elegans and provide the foundation for genetic and functional genomic analysis of animal cell osmoregulation.
- Published
- 2004
34. To UPR… and beyond!
- Author
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Eric Chevet and Todd Lamitina
- Subjects
Microbiology (medical) ,Editor's Corner ,Immunology ,Antimicrobial peptides ,Microbiology ,Heat shock protein ,Animals ,Electrophoresis, Gel, Two-Dimensional ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Heat-Shock Proteins ,Regulation of gene expression ,Innate immune system ,biology ,Gene Expression Profiling ,biology.organism_classification ,Cell biology ,Infectious Diseases ,Gene Expression Regulation ,Chaperone (protein) ,biology.protein ,Unfolded protein response ,Parasitology ,Epidermis ,Signal transduction ,Antimicrobial Cationic Peptides - Abstract
The nematode C. elegans responds to infection by the fungus Drechmeria coniospora with a rapid increase in the expression of antimicrobial peptide genes. To investigate further the molecular basis of this innate immune response, we took a two-dimensional difference in-gel electrophoresis (2D-DIGE) approach to characterize the changes in host protein that accompany infection. We identified a total of 68 proteins from differentially represented spots and their corresponding genes. Through class testing, we identified functional categories that were enriched in our proteomic data set. One of these was "protein processing in endoplasmic reticulum," pointing to a potential link between innate immunity and endoplasmic reticulum function. This class included HSP-3, a chaperone of the BiP/GRP78 family known to act coordinately in the endoplasmic reticulum with its paralog HSP-4 to regulate the unfolded protein response (UPR). Other studies have shown that infection of C. elegans can provoke a UPR. We observed, however, that in adult C. elegans infection with D. coniospora did not induce a UPR, and conversely, triggering a UPR did not lead to an increase in expression of the well-characterized antimicrobial peptide gene nlp-29. On the other hand, we demonstrated a specific role for hsp-3 in the regulation of nlp-29 after infection that is not shared with hsp-4. Epistasis analysis allowed us to place hsp-3 genetically between the Tribbles-like kinase gene nipi-3 and the protein kinase C delta gene tpa-1. The precise function of hsp-3 has yet to be determined, but these results uncover a hitherto unsuspected link between a BiP/GRP78 family protein and innate immune signaling.
- Published
- 2012
35. The cystic fibrosis-associated ΔF508 mutation confers post-transcriptional destabilization on the C. elegans ABC transporter PGP-3
- Author
-
Todd Lamitina, Liping He, Rosemary Lewis, Jennifer Skirkanich, and Lorenza Moronetti
- Subjects
Research Report ,ATP Binding Cassette Transporter, Subfamily B ,Cystic Fibrosis ,Transcription, Genetic ,Neuroscience (miscellaneous) ,Cystic Fibrosis Transmembrane Conductance Regulator ,lcsh:Medicine ,Medicine (miscellaneous) ,ATP-binding cassette transporter ,Endoplasmic-reticulum-associated protein degradation ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Immunology and Microbiology (miscellaneous) ,Osmotic Pressure ,Stress, Physiological ,lcsh:Pathology ,Animals ,Humans ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,ΔF508 ,030304 developmental biology ,0303 health sciences ,biology ,Protein Stability ,Endoplasmic reticulum ,Cell Membrane ,lcsh:R ,Cell Polarity ,Endoplasmic Reticulum-Associated Degradation ,Apical membrane ,Cystic fibrosis transmembrane conductance regulator ,Cell biology ,Membrane protein ,Gene Knockdown Techniques ,Mutation ,biology.protein ,ATP-Binding Cassette Transporters ,Chemical chaperone ,030217 neurology & neurosurgery ,lcsh:RB1-214 - Abstract
Summary Membrane proteins comprise ~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. ERQC plays critical roles in human diseases, such as cystic fibrosis, where deletion of a single amino acid (F508) results in the misfolding and degradation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel. We introduced the ΔF508 mutation into C. elegans PGP-3, a 12 transmembrane ABC transporter with 15% identity to CFTR. When expressed in intestinal epithelial cells, PGP-3wt was stable and efficiently trafficked to the apical plasma membrane through a COPII-dependent mechanism. However, PGP-3ΔF508 was post-transcriptionally destabilized, resulting in reduced total and apical membrane protein levels. Genetic or physiological activation of the osmotic stress response pathway, which causes accumulation of the chemical chaperone glycerol, stabilized PGP-3ΔF508. Efficient degradation of PGP-3ΔF508 required the function of several C. elegans endoplasmic reticulum-associated degradation (ERAD) homologs, suggesting that destabilization occurs through an ERAD-type mechanism. Our studies show that the ΔF508 mutation causes post-transcriptional destabilization and degradation of PGP-3 in C. elegans epithelial cells. This model, combined with the power of C. elegans genetics, provides a new opportunity to genetically dissect metazoan ERQC.
- Published
- 2012
36. A suite of MATLAB-based computational tools for automated analysis of COPAS Biosort data
- Author
-
Todd Lamitina and Elizabeth A. Morton
- Subjects
Computer science ,Gene Expression ,computer.software_genre ,Bioinformatics ,General Biochemistry, Genetics and Molecular Biology ,Article ,Software ,Animals ,Fluorometry ,MATLAB ,Caenorhabditis elegans ,Heat-Shock Proteins ,Zebrafish ,computer.programming_language ,Graphical user interface ,Adapter (computing) ,business.industry ,Computational Biology ,Object (computer science) ,File format ,Flow Cytometry ,High-Throughput Screening Assays ,Identification (information) ,Drosophila melanogaster ,Data analysis ,RNA Interference ,Data mining ,business ,computer ,Algorithms ,Biotechnology - Abstract
Complex Object Parametric Analyzer and Sorter (COPAS) devices are large-object, fluorescence-capable flow cytometers used for high-throughput analysis of live model organisms, including Drosophila melanogaster, Caenorhabditis elegans, and zebrafish. The COPAS is especially useful in C. elegans high-throughput genome-wide RNA interference (RNAi) screens that utilize fluorescent reporters. However, analysis of data from such screens is relatively labor-intensive and time-consuming. Currently, there are no computational tools available to facilitate high-throughput analysis of COPAS data. We used MATLAB to develop algorithms (COPAquant, COPAmulti, and COPAcompare) to analyze different types of COPAS data. COPAquant reads single-sample files, filters and extracts values and value ratios for each file, and then returns a summary of the data. COPAmulti reads 96-well autosampling files generated with the ReFLX adapter, performs sample filtering, graphs features across both wells and plates, performs some common statistical measures for hit identification, and outputs results in graphical formats. COPAcompare performs a correlation analysis between replicate 96-well plates. For many parameters, thresholds may be defined through a simple graphical user interface (GUI), allowing our algorithms to meet a variety of screening applications. In a screen for regulators of stress-inducible GFP expression, COPAquant dramatically accelerated data analysis and allowed us to rapidly move from raw data to hit identification. Because the COPAS file structure is standardized and our MATLAB code is freely available, our algorithms should be extremely useful for analysis of COPAS data from multiple platforms and organisms. The MATLAB code is freely available at our web site ( www.med.upenn.edu/lamitinalab/downloads.shtml ).
- Published
- 2010
37. Material properties of Caenorhabditis elegans swimming at low Reynolds number
- Author
-
Paulo E. Arratia, Todd Lamitina, Predrag Krajacic, Prashant K. Purohit, and Josué Sznitman
- Subjects
Nematode caenorhabditis elegans ,Muscle, Motility, and Motor Proteins ,Biophysics ,Motility ,FOS: Physical sciences ,Biology ,Models, Biological ,Quantitative Biology::Cell Behavior ,symbols.namesake ,Viscosity ,Gait (human) ,Undulatory locomotion ,Elastic Modulus ,Animals ,Physics - Biological Physics ,Caenorhabditis elegans ,Swimming ,Physics::Biological Physics ,Quantitative Biology::Neurons and Cognition ,Ecology ,Fluid Dynamics (physics.flu-dyn) ,Reynolds number ,Physics - Fluid Dynamics ,Muscular Dystrophy, Animal ,biology.organism_classification ,Quantitative Biology::Genomics ,Biomechanical Phenomena ,Biological Physics (physics.bio-ph) ,Mutation ,symbols ,Material properties - Abstract
Undulatory locomotion, as seen in the nematode \emph{Caenorhabditis elegans}, is a common swimming gait of organisms in the low Reynolds number regime, where viscous forces are dominant. While the nematode's motility is expected to be a strong function of its material properties, measurements remain scarce. Here, the swimming behavior of \emph{C.} \emph{elegans} are investigated in experiments and in a simple model. Experiments reveal that nematodes swim in a periodic fashion and generate traveling waves which decay from head to tail. The model is able to capture the experiments' main features and is used to estimate the nematode's Young's modulus $E$ and tissue viscosity $\eta$. For wild-type \emph{C. elegans}, we find $E\approx 3.77$ kPa and $\eta \approx-860$ Pa$\cdot$s; values of $\eta$ for live \emph{C. elegans} are negative because the tissue is generating rather than dissipating energy. Results show that material properties are sensitive to changes in muscle functional properties, and are useful quantitative tools with which to more accurately describe new and existing muscle mutants., Comment: To appear in Biophysical Journal
- Published
- 2009
38. Strongyloides stercoralis: cell- and tissue-specific transgene expression and co-transformation with vector constructs incorporating a common multifunctional 3' UTR
- Author
-
Meera V. Sundaram, Ariel B. Junio, Xinshe Li, Holman C. Massey, Thomas J. Nolan, S. Todd Lamitina, and James B. Lok
- Subjects
Untranslated region ,Microinjections ,Transgene ,Immunology ,Genetic Vectors ,Green Fluorescent Proteins ,Molecular Sequence Data ,Biology ,Article ,Green fluorescent protein ,Dogs ,Transformation, Genetic ,Genes, Reporter ,Gene expression ,Animals ,Transgenes ,Serial Passage ,Caenorhabditis elegans ,Promoter Regions, Genetic ,Gene ,3' Untranslated Regions ,Regulation of gene expression ,Promoter ,General Medicine ,DNA, Helminth ,Molecular biology ,Transformation (genetics) ,Infectious Diseases ,Gene Expression Regulation ,Parasitology ,Female ,Gerbillinae ,Strongyloides stercoralis - Abstract
Transgenesis is a valuable methodology for studying gene expression patterns and gene function. It has recently become available for research on some parasitic nematodes, including Strongyloides stercoralis. Previously, we described a vector construct, comprising the promoter and 3′ UTR of the S. stercoralis gene Ss era-1 that gives expression of GFP in intestinal cells of developing F1 progeny. In the present study, we identified three new S. stercoralis promoters, which, in combination with the Ss era-1 3′ UTR, can drive expression of GFP or the red fluorescent protein, mRFPmars, in tissue-specific fashion. These include Ss act-2, which drives expression in body wall muscle cells, Ss gpa-3, which drives expression in amphidial and phasmidial neurons and Ss rps-21, which drives ubiquitous expression in F1 transformants and in the gonads of microinjected P0 female worms. Concomitant microinjection of vectors containing GFP and mRFPmars gave dually transformed F1 progeny, suggesting that these constructs could be used as co-injection markers for other transgenes of interest. We have developed a vector “toolkit” for S. stercoralis including constructs with the Ss era-1 3′ UTR and each of the promoters described above.
- Published
- 2007
39. Functional analysis of the aquaporin gene family in Caenorhabditis elegans
- Author
-
Todd Lamitina, Kevin Strange, Chunyi George Huang, and Peter Agre
- Subjects
Glycerol ,Cell Membrane Permeability ,Genotype ,Microinjections ,Physiology ,Xenopus ,Mutant ,Green Fluorescent Proteins ,Molecular Sequence Data ,Aquaporin ,Gene Expression ,Sodium Chloride ,Aquaporins ,Genes, Reporter ,Gene expression ,Gene family ,Animals ,Amino Acid Sequence ,Cloning, Molecular ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Promoter Regions, Genetic ,Phylogeny ,Water transport ,biology ,Water ,Cell Biology ,Water-Electrolyte Balance ,biology.organism_classification ,Cell biology ,Aquaporin 4 ,Phenotype ,Mutation ,Osmoregulation ,Oocytes - Abstract
Aquaporin channels facilitate the transport of water, glycerol, and other small solutes across cell membranes. The physiological roles of many aquaporins remain unclear. To better understand aquaporin function, we characterized the aquaporin gene family in the nematode Caenorhabditis elegans. Eight canonical aquaporin-encoding genes ( aqp) are present in the worm genome. Expression of aqp-2, aqp-3, aqp-4, aqp-6, or aqp-7 in Xenopus oocytes increased water permeability five- to sevenfold. Glycerol permeability was increased three to sevenfold by expression of aqp-1, aqp-3, or aqp-7. Green fluorescent protein transcriptional and translational reporters demonstrated that aqp genes are expressed in numerous C. elegans cell types, including the intestine, excretory cell, and hypodermis, which play important roles in whole animal osmoregulation. To define the role of C. elegans aquaporins in osmotic homeostasis, we isolated deletion alleles for four aqp genes, aqp-2, aqp-3, aqp-4, and aqp-8, which are expressed in osmoregulatory tissues and mediate water transport. Single, double, triple, and quadruple aqp mutant animals exhibited normal survival, development, growth, fertility, and movement under normal and hypertonic culture conditions. aqp-2; aqp-3; aqp-4; aqp-8 quadruple mutants exhibited a slight defect in recovery from hypotonic stress but survived hypotonic stress as well as wild-type animals. These results suggest that C. elegans aquaporins are not essential for whole animal osmoregulation and/or that deletion of aquaporin genes activates mechanisms that compensate for loss of water channel function.
- Published
- 2007
40. Functional genomic approaches in C. elegans
- Author
-
Todd, Lamitina
- Subjects
Phenotype ,Gene Transfer Techniques ,Animals ,RNA Interference ,Genomics ,Caenorhabditis elegans ,Genes, Helminth ,RNA, Double-Stranded - Abstract
The nematode Caenorhabditis elegans is an extraordinarily powerful model organism for the application of functional genomic approaches. Two such approaches, whole genome microarray analysis and genome-wide RNA interference (RNAi)-mediated phenotypic screening, are highly advanced and can be used by virtually any laboratory to study biological processes of interest. Using studies of the osmotic stress response in C. elegans as an example, this chapter describes methods for conducting whole genome microarray experiments and for carrying out genome-wide reverse-genetic screens using a commercially available C. elegans bacterial RNAi feeding library. Both approaches are complimentary and can be used to rapidly gain genome-wide insights into the genes and gene networks controlling specific physiological processes.
- Published
- 2006
41. Functional Genomic Approaches in C. elegans
- Author
-
Todd Lamitina
- Subjects
Osmotic stress response ,Nematode caenorhabditis elegans ,Whole genome microarray ,ved/biology ,RNA interference ,Phenotypic screening ,ved/biology.organism_classification_rank.species ,Gene regulatory network ,Computational biology ,Biology ,Model organism ,Gene - Abstract
The nematode Caenorhabditis elegans is an extraordinarily powerful model organism for the application of functional genomic approaches. Two such approaches, whole genome microarray analysis and genome-wide RNA interference (RNAi)-mediated phenotypic screening, are highly advanced and can be used by virtually any laboratory to study biological processes of interest. Using studies of the osmotic stress response in C. elegans as an example, this chapter describes methods for conducting whole genome microarray experiments and for carrying out genome-wide reverse-genetic screens using a commercially available C. elegans bacterial RNAi feeding library. Both approaches are complimentary and can be used to rapidly gain genome-wide insights into the genes and gene networks controlling specific physiological processes.
- Published
- 2006
42. Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis
- Author
-
Keith Nehrke, Catherine Lorin-Nebel, Todd Lamitina, Kevin Strange, Juan Xing, Xiaohui Yan, and Ana Y. Estevez
- Subjects
Ovulation ,Physiology ,Molecular Sequence Data ,Biology ,Endoplasmic Reticulum ,Article ,Animals, Genetically Modified ,03 medical and health sciences ,0302 clinical medicine ,Intestinal mucosa ,RNA interference ,Animals ,Homeostasis ,Inositol 1,4,5-Trisphosphate Receptors ,Amino Acid Sequence ,Calcium Signaling ,Stromal Interaction Molecule 1 ,Cloning, Molecular ,Intestinal Mucosa ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Defecation ,030304 developmental biology ,Calcium signaling ,SOC channels ,0303 health sciences ,Sequence Homology, Amino Acid ,Endoplasmic reticulum ,Membrane Proteins ,STIM1 ,Articles ,biology.organism_classification ,Cell biology ,Electrophysiology ,Fertility ,Mutation ,Calcium ,Female ,RNA Interference ,Calcium Channels ,Signal transduction ,030217 neurology & neurosurgery ,Muscle Contraction - Abstract
1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.
- Published
- 2006
43. Genome-wide RNAi screening identifies protein damage as a regulator of osmoprotective gene expression
- Author
-
Todd Lamitina, Kevin Strange, and Chunyi George Huang
- Subjects
Glycerol ,Osmosis ,Green Fluorescent Proteins ,RNA interference ,Gene expression ,Protein biosynthesis ,Animals ,Caenorhabditis elegans ,Promoter Regions, Genetic ,Gene ,Regulation of gene expression ,Multidisciplinary ,biology ,Models, Genetic ,Genomics ,Biological Sciences ,biology.organism_classification ,Cell biology ,Gene Expression Regulation ,Genetic Techniques ,Osmolyte ,Protein Biosynthesis ,RNA Interference ,Chemical chaperone ,Plasmids - Abstract
The detection, stabilization, and repair of stress-induced damage are essential requirements for cellular life. All cells respond to osmotic stress-induced water loss with increased expression of genes that mediate accumulation of organic osmolytes, solutes that function as chemical chaperones and restore osmotic homeostasis. The signals and signaling mechanisms that regulate osmoprotective gene expression in animal cells are poorly understood. Here, we show that gpdh-1 and gpdh-2 , genes that mediate the accumulation of the organic osmolyte glycerol, are essential for survival of the nematode Caenorhabditis elegans during osmotic stress. Expression of GFP driven by the gpdh-1 promoter ( P gpdh-1 :: GFP ) is detected only during hypertonic stress but is not induced by other stressors. Using P gpdh-1 :: GFP expression as a phenotype, we screened ≈16,000 genes by RNAi feeding and identified 122 that cause constitutive activation of gpdh-1 expression and glycerol accumulation. Many of these genes function to regulate protein translation and cotranslational protein folding and to target and degrade denatured proteins, suggesting that the accumulation of misfolded proteins functions as a signal to activate osmoprotective gene expression and organic osmolyte accumulation in animal cells. Consistent with this hypothesis, 73% of these protein-homeostasis genes have been shown to slow age-dependent protein aggregation in C. elegans . Because diverse environmental stressors and numerous disease states result in protein misfolding, mechanisms must exist that discriminate between osmotically induced and other forms of stress-induced protein damage. Our findings provide a foundation for understanding how these damage-selectivity mechanisms function.
- Published
- 2006
44. GCK‐3, a Caenorhabditis elegans homologue of PASK, is essential for whole‐animal osmotic homeostasis
- Author
-
Samuel Todd Lamitina, Kevin Strange, and Keith P. Choe
- Subjects
biology ,Kinase ,Genetics ,SUPERFAMILY ,WHOLE ANIMAL ,biology.organism_classification ,Molecular Biology ,Biochemistry ,Caenorhabditis elegans ,Homeostasis ,Biotechnology ,Cell biology - Abstract
We recently identified a new member of the Ste20 kinase superfamily in C. elegans, GCK-3, which is a homologue of mammalian PASK. PASK is expressed in transporting epithelia including the kidney, a...
- Published
- 2006
45. osm‐8 mutations constitutively activate osmosensitive gene expression in C. elegans
- Author
-
Samuel Todd Lamitina, Kevin Strange, and Yiliu Chen
- Subjects
Gene expression ,Genetics ,Biology ,Molecular Biology ,Biochemistry ,Biotechnology ,Cell biology - Published
- 2006
46. Isolation of C. elegans deletion mutants following ENU mutagenesis and thermostable restriction enzyme PCR screening
- Author
-
Chunyi George, Huang, Peter, Agre, Kevin, Strange, and Todd, Lamitina
- Subjects
Alkylating Agents ,Mutagenesis ,Ethylnitrosourea ,Mutation ,Animals ,DNA, Helminth ,Caenorhabditis elegans ,Deoxyribonucleases, Type II Site-Specific ,Polymerase Chain Reaction ,Gene Deletion ,Genes, Helminth - Abstract
The ability to generate null mutants is essential for studying gene function. Gene knockouts in Caenorhabditis elegans can be generated in a high throughput manner using chemical mutagenesis followed by polymerase chain reaction (PCR) assays to detect deletions in a gene of interest. However, current methods for identifying deletions are time and labor intensive and are unable to efficiently detect small deletions. In this study, we expanded the method pioneered by Wei et al., which used the thermostable restriction enzyme PspGI and tested the usefulness of other thermostable restriction enzymes including BstUI, Tsp45I, ApeKI, and TfiI. We designed primers to flank one or multiple thermostable restriction enzymes sites in the genes of interest. The use of multiple enzymes and the optimization of PCR primer design enabled us to isolate deletion in 66.7% of the genes screened. The size of the deletions varied from 330 bp to 1 kb. This method should make it possible for small academic laboratories to rapidly isolate deletions in their genes of interest.
- Published
- 2005
47. Dominant mutations in the Caenorhabditis elegans Myt1 ortholog wee-1.3 reveal a novel domain that controls M-phase entry during spermatogenesis
- Author
-
Steven W. L'Hernault and S. Todd Lamitina
- Subjects
Male ,Mutant ,Molecular Sequence Data ,Mitosis ,Helminth genetics ,Cell Cycle Proteins ,Protein Serine-Threonine Kinases ,medicine.disease_cause ,Germline ,Suppression, Genetic ,Meiosis ,medicine ,Animals ,Amino Acid Sequence ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Spermatogenesis ,Molecular Biology ,Gene ,Alleles ,Genes, Helminth ,Genes, Dominant ,Genetics ,Mutation ,biology ,Base Sequence ,Sequence Homology, Amino Acid ,Gene Expression Regulation, Developmental ,Nuclear Proteins ,DNA, Helminth ,Protein-Tyrosine Kinases ,biology.organism_classification ,Phenotype ,Female ,Gene Deletion ,Developmental Biology - Abstract
Regulatory phosphorylation of the Cdc2p kinase by Wee1p-type kinases prevents eukaryotic cells from entering mitosis or meiosis at an inappropriate time. The canonical Wee1p kinase is a soluble protein that functions in the eukaryotic nucleus. All metazoa also have a membrane-associated Wee1p-like kinase named Myt1, and we describe the first genetic characterization of this less well-studied kinase. The Caenorhabditis elegans Myt1 ortholog is encoded by the wee-1.3 gene, and six dominant missense mutants prevent primary spermatocytes from entering M phase but do not affect either oocyte meiosis or any mitotic division. These six dominantwee-1.3(gf) mutations are located in a four amino acid region near the C terminus and they cause self-sterility of hermaphrodites. Second-site intragenic suppressor mutations in wee-1.3(gf) restore self-fertility to these dominant sterile hermaphrodites, permitting genetic dissection of this kinase. Ten intragenic wee-1.3 suppressor mutations were recovered and they form an allelic series that includes semi-dominant,hypomorphic and null mutations. These mutants reveal that WEE-1.3 protein is required for embryonic development, germline proliferation and initiation of meiosis during spermatogenesis. This suggests that a novel, sperm-specific pathway negatively regulates WEE-1.3 to allow the G2/M transition of male meiosis I, and that dominant wee-1.3 mutants prevent this negative regulation.
- Published
- 2002
48. The mechanism of ran import into the nucleus by nuclear transport factor 2
- Author
-
Anita H. Corbett, Steven W. L'Hernault, Todd Lamitina, and B. Booth Quimby
- Subjects
Nucleocytoplasmic Transport Proteins ,Saccharomyces cerevisiae ,Molecular Sequence Data ,Karyopherins ,Biochemistry ,medicine ,Small GTPase ,Amino Acid Sequence ,Molecular Biology ,Cell Nucleus ,Nucleoplasm ,biology ,Nuclear Proteins ,Biological Transport ,Cell Biology ,biology.organism_classification ,Cell biology ,medicine.anatomical_structure ,ran GTP-Binding Protein ,Cytoplasm ,Nucleocytoplasmic Transport ,Ran ,Nuclear transport ,Carrier Proteins ,Nucleus - Abstract
The small GTPase Ran is essential for virtually all nucleocytoplasmic transport events. It is hypothesized that Ran drives vectorial transport of macromolecules into and out of the nucleus via the establishment of a Ran gradient between the cytoplasm and nucleoplasm. Although Ran shuttles between the nucleus and cytoplasm, it is concentrated in the nucleus at steady state. We show that nuclear transport factor 2 (NTF2) is required to concentrate Ran in the nucleus in the budding yeast, Saccharomyces cerevisiae. To analyze the mechanism of Ran import into the nucleus by NTF2, we use mutants in a variety of nuclear transport factors along with biochemical analyses of NTF2 complexes. We find that Ran remains concentrated in the nucleus when importin-mediated protein import is disrupted and demonstrate that NTF2 does not form a stable complex with the transport receptor, importin-beta. Consistent with a critical role for NTF2 in establishing and maintaining the Ran gradient, we show that NTF2 is required for early embryogenesis in Caenorhabditis elegans. Our data distinguish between two possible mechanisms for Ran import by NTF2 and demonstrate that Ran import is independent from importin-beta-mediated protein import.
- Published
- 2000
49. Dangerous Liaisons: The Apoptotic Engulfment Receptor CED-1 Links Innate Immunity to the Unfolded Protein Response
- Author
-
Todd Lamitina and Sara Cherry
- Subjects
Protein Folding ,Apoptosis ,Models, Biological ,Article ,General Biochemistry, Genetics and Molecular Biology ,Immune system ,Immunity ,Escherichia coli ,Animals ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Receptor ,Molecular Biology ,Innate immune system ,biology ,Effector ,fungi ,Membrane Proteins ,Salmonella enterica ,Cell Biology ,biology.organism_classification ,Immunity, Innate ,Cell biology ,Genes ,Membrane protein ,Mutation ,Unfolded protein response ,Developmental Biology - Abstract
The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), has been implicated in the normal physiology of the immune defense and in several disorders including diabetes, cancer, and neurodegenerative disease. Here we show that the apoptotic receptor CED-1 and a network of PQN/ABU proteins involved in a non-canonical UPR response are required for proper defense to pathogen infection in Caenorhabditis elegans. A full-genome microarray analysis indicates that CED-1 functions to activate the expression of pqn/abu genes. We also show that ced-1 and pqn/abu genes are required for survival of C. elegans exposed to live S. enterica and that overexpression of pqn/abu genes confers protection to pathogen-mediated killing. The results indicate that unfolded protein response genes, regulated in a CED-1-dependent manner, are involved in the C. elegans immune response to live bacteria.
- Published
- 2008
50. Genetic and Physiological Activation of Osmosensitive Gene Expression Mimics Transcriptional Signatures of Pathogen Infection in C. elegans
- Author
-
Sridhar Hannenhalli, Anne-Katrin Rohlfing, S. Todd Lamitina, and Yana Miteva
- Subjects
Osmosis ,Immunology/Innate Immunity ,lcsh:Medicine ,Sodium Chloride ,Biology ,GATA Transcription Factors ,03 medical and health sciences ,0302 clinical medicine ,RNA interference ,Actinomycetales ,Gene expression ,Transcriptional regulation ,Animals ,Cluster Analysis ,lcsh:Science ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Gene ,Transcription factor ,Oligonucleotide Array Sequence Analysis ,030304 developmental biology ,Genetics ,Regulation of gene expression ,0303 health sciences ,Multidisciplinary ,Reverse Transcriptase Polymerase Chain Reaction ,Gene Expression Profiling ,lcsh:R ,Genetics and Genomics/Gene Expression ,Salt Tolerance ,Gene expression profiling ,Gene Expression Regulation ,Host-Pathogen Interactions ,Physiology/Integrative Physiology ,GATA transcription factor ,lcsh:Q ,RNA Interference ,Physiology/Immune Response ,030217 neurology & neurosurgery ,Research Article - Abstract
The soil-dwelling nematode C. elegans is a powerful system for comparative molecular analyses of environmental stress response mechanisms. Infection of worms with bacterial and fungal pathogens causes the activation of well-characterized innate immune transcriptional programs in pathogen-exposed hypodermal and intestinal tissues. However, the pathophysiological events that drive such transcriptional responses are not understood. Here, we show that infection-activated transcriptional responses are, in large part, recapitulated by either physiological or genetic activation of the osmotic stress response. Microarray profiling of wild type worms exposed to non-lethal hypertonicity identified a suite of genes that were also regulated by infection. Expression profiles of five different osmotic stress resistant (osr) mutants under isotonic conditions reiterated the wild type transcriptional response to osmotic stress and also showed substantial similarity to infection-induced gene expression under isotonic conditions. Computational, transgenic, and functional approaches revealed that two GATA transcription factors previously implicated in infection-induced transcriptional responses, elt-2 and elt-3, are also essential for coordinated tissue-specific activation of osmosensitive gene expression and promote survival under osmotically stressful conditions. Together, our data suggest infection and osmotic adaptation share previously unappreciated transcriptional similarities which might be controlled via regulation of tissue-specific GATA transcription factors.
- Published
- 2010
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