Lamitina T (2006) Methods Mol Biol "Functional genomic approaches in C. elegans."

Lamitina T

[Methods Mol Biol, 2006]

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.

Lamitina T et al. (2000) East Coast Worm Meeting "A wee-1 kinase homolog is required for meiosis during spermatogenesis in C. elegans"

L'Hernault SW, Lamitina T

[East Coast Worm Meeting, 2000]

The switch from mitosis to meiosis is a critical decision in the life of all metazoan germ cells. Once this commitment to meiosis is made, the sperm and oocyte must coordinate differentiation with the meiotic cell cycle. While examining spermatogenesis defective (spe) mutants in the nematode C. elegans, we discovered two dominant mutants that show major defects in sperm proliferation. Light and electron microscopic examination reveal that both mutants show similar aspects of cellular differentiation in the absence of either cell division or successful meiosis. Both dominant mutants affect sperm, but do not affect oocytes or any somatic cells. Using these dominant alleles, one can select for a second mutation within spe-37 (gf) that allows production of functional sperm by spe-37 (gf) / wild type heterozygotes. Several of these "second site" spe-37 mutants have been selected and all have recessive phenotypes. Genetic analysis of these recessive mutants uncovers a role for this gene in early embryogenesis and germline proliferation, in addition to its role in sperm as revealed by the dominant mutants). These recessive mutants allow utilization of positional cloning techniques, which reveal that spe-37 encodes the metazoan specific wee-1 homolog Myt1. Many higher eukaryotes, including humans, have a soluble Wee-1p as well as Myt1, which is a single pass integral membrane protein that localizes to the endoplasmic reticulum and Golgi. The kinase region of Myt1 is presumed to face the cytoplasm while the C terminal tail lies within these organelles. The C terminal region is known to be essential for proper Myt1 function, but prior work has not revealed its role. Two independently derived dominant spe-37/Myt1 mutants have an identical missense mutation in this C-terminal region, so they should provide crucial insight into the function of this domain. We have initiate a large scale mutant hunt to recover new dominant and recessive spe-37/Myt1 mutants and to identify other genes in the pathway in which Myt1 functions. This is the first time that it has been possible to combine cell biological and genetic techniques to study this important metazoan kinase. It should be possible to determine the genetic pathway that controls exit from mitosis and entry into meiosis through study of C. elegans Myt1.

Lamitina T et al. (2012) Virulence "To UPR... and beyond! A new role for a BiP/GRP78 protein in the control of antimicrobial peptide expression in C. elegans epidermis."

Lamitina T, Chevet E

[Virulence, 2012]

The fungal pathogen Drechmeria coniospora infects C. elegans and elicits an innate immune response mediated, in part, by the induction of antimicrobial peptides in the epidermis. The signaling pathways controlling this phenomenon remain to be fully characterized. In this issue of Virulence, Couillault and colleagues use both proteomics and genetics to discover an unexpected role for the unfolded protein response (UPR) target chaperone BiP/GRP78/hsp-3 in the control of fungal infection-induced antimicrobial peptide expression in C. elegans. Although the expression of hsp-3 is regulated by the UPR, Couillault and colleagues describe a novel signaling role for this BiP/GRP78 homolog.

Lamitina T et al. (2008) Dev Cell "Dangerous liaisons: the apoptotic engulfment receptor CED-1 links innate immunity to the unfolded protein response."

Cherry S, Lamitina T

[Dev Cell, 2008]

In this issue of Developmental Cell, Haskins et al. describe a molecular link between an apoptotic cell receptor, a branch of the unfolded protein response, and innate immunity. The finding that the plasma membrane phagocytic receptor CED-1 promotes the expression of unfolded response genes suggests a novel regulatory mechanism for the control of this primitive immune system that may couple the presence of pathogen with the production of secreted antimicrobial effectors.

Lamitina T et al. (2006) Proc Natl Acad Sci U S A "Genome-wide RNAi screening identifies protein damage as a regulator of osmoprotective gene expression."

Strange K, Lamitina T, Huang CG

[Proc Natl Acad Sci U S A, 2006]

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 approximately 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.

Samuel T Lamitina et al. (2003) International Worm Meeting "Adaptation of C. elegans to extreme osmotic stress."

K Strange, R Morrison, G Moeckel, K Baman, Samuel T Lamitina

[International Worm Meeting, 2003]

The ability to tightly control solute and water balance is an essential prerequisite for cellular life. Osmotic homeostasis is maintained by the regulated accumulation and loss of inorganic ions and small organic solutes termed organic osmolytes. While cellular osmoregulation has been studied extensively in a variety of cells and tissues, major gaps exist in our molecular understanding of this essential process. Because of its many experimental advantages, C. elegans provides an ideal model system in which to define the genes and genetic pathways required for cellular osmoregulation in animals.To begin defining the genetic basis of cellular osmoregulation in C. elegans, we characterized the ability of worms to survive and adapt to extreme hypertonic stress by adding NaCl to the growth agar. Exposure to high salt agar causes rapid shrinkage, followed by slow recovery of body volume over several hours. Survival is normal on agar containing up to 200 mM NaCl. When grown on 200 mM NaCl for 2 weeks, worms are able to survive and reproduce on agar containing 400 mM NaCl. Worms adapted to hypertonic stress swell and then rapidly return to their initial body volume when returned to low salt agar. We are currently using forward and reverse genetic screens to identify genes required for adaptation to and recovery from hypertonic stress. The functions of these genes are being defined by molecular, biochemical, and physiological approaches.In response to hypertonicity, all organisms accumulate substances called organic osmolytes to counter the damaging effects of hypertonicity. Organic osmolytes are small uncharged or weakly charged compounds that are accumulated to concentrations of 10s to 100s of millimolar in the cytoplasm of hypertonically stressed cells. Importantly, the unique biophysical properties of organic osmolytes do not disturb cellular structure and function. Using HPLC analysis, we have detected a low molecular weight compound (~115 daltons) that increases 15-20 fold in nematodes grown on 200 mM NaCl plates. Accumulation of this compound begins after 6 h of high salt stress and peaks by 24 h. Further tests have shown that this compound is not an amino acid, one of the three major categories of known organic osmolytes. We are attempting to identify the molecular nature of this putative organic osmolyte using mass and NMR spectrometry.

Samuel T Lamitina et al. (2003) International Worm Meeting "Microarray analysis of the hypertonic stress response in C. elegans"

K Strange, Samuel T Lamitina

[International Worm Meeting, 2003]

Osmotic stress alters the expression of genes required for cellular osmoregulation and for the detection and repair of osmotically-induced cellular and molecular damage. The identity and function of many of these genes are unknown. C. elegans provides numerous experimental advantages for identifying osmotically regulated genes and for defining their molecular functions.We have demonstrated recently C. elegans survives and adapts readily to extreme hypertonic stress. Using microarray analysis, we have begun to identify the complement of genes that are transcriptionally regulated during exposure to high NaCl growth agar. When nematodes are adapted to 200 mM NaCl agar for 24 h, approximately 150 genes showed average changes in transcription of >1.7 fold. These included genes that encode proteases, cytoskeletal proteins, extracellular matrix proteins and components of stress response and signaling pathways. Putative metabolic genes accounted for 41% of the genes that were transcriptionally upregulated. Transport of inorganic ions and small organic solutes termed organic osmolytes plays an important role in the adaptation of cells to hypertonic stress. However, upregulation of relatively few channel- or transporter-encoding genes was observed. One putative channel gene that was upregulated (mean change in expression level

Samuel T Lamitina et al. (2002) East Coast Worm Meeting "Cell cycle dependent localization of the C. elegans Myt1 ortholog wee-1.3"

Steven W L'Hernault, Samuel T Lamitina

[East Coast Worm Meeting, 2002]

All eukaryotes control mitotic timing through the activation of MPF, which is composed of a cyclin-dependent kinase and a regulatory cyclin subunit. MPF is negatively regulated by Wee1p kinases and positively regulated by Cdc25p phosphatases. These soluble proteins are actively localized to the nucleus or cytoplasm, respectively, during the cell cycle. Metazoans express another member of the Wee1p kinase family called Myt1, a type 2 transmembrane protein with an N-terminal Wee-1p kinase domain and a C-terminal regulatory domain. We have used a peptide antibody directed towards the C-terminal domain of WEE-1.3 to study its localization during C. elegans development. WEE-1.3 colocalizes with microtubules during sperm meiosis and blastomere mitosis, but is punctate throughout the cytoplasm or associated with the nuclear envelope during interphase and at the G2/M transition point, respectively. The wee-1.3(q89 eb60) mutant is hypomorphic and strongly reduces WEE-1.3 levels in early blastomeres, which frequently contain ectopic microtubule nucleation sites. Missense mutations in a four amino acid region within the WEE-1.3 C-terminal domain cause a dominant spermatogenesis defective (Spe) phenotype that arrests spermatocytes at the G2/M transition. WEE-1.3(gf) localizes to the nuclear envelope in arrested wee-1.3(gf) spermatocytes and this localization is indistinguishable from wild type spermatocytes at a similar stage of development, suggesting that the dominant Spe phenotype is not caused by abnormal protein localization or protein instability. In oocytes, WEE-1.3 is concentrated primarily in the nuclear envelope. wee-1.3 (q89 eb87) mutants delete the transmembrane domain of WEE-1.3 and suppresses the dominant Spe phenotype. Phenotypically, these mutants are viable but recessive sterile, suggesting that the somatic functions that require WEE-1.3 are unaffected by the absence of the WEE-1.3 transmembrane domain. wee-1.3(q89 eb87) mutants result in the localized accumulation of WEE-1.3 within the oocyte nucleus. This suggests that the localization of WEE-1.3 to the oocyte nuclear envelope is important for some aspect of oocyte development.

Samuel T Lamitina et al. (2003) International Worm Meeting "Inhibition of an insulin-like signaling pathway in C. elegans increases resistance to hypertonic stress."

K Strange, L Waters, Samuel T Lamitina

[International Worm Meeting, 2003]

The signaling pathways and molecular mechanisms that protect animal cells from osmotic stress are not well understood. In C. elegans, mutations in genes that comprise the daf-2 signaling pathway increase resistance to thermal, oxidative, UV and hypoxic stress. We have analyzed the effect of these mutations on osmotic stress resistance in nematodes. Animals with reduction-of-function mutations in the insulin-like growth factor receptor gene daf-2 or the PI-3-like kinase gene age-1 exhibit 80-100% survival after 24 h exposure to 400 mM NaCl growth agar whereas wild type worms are all killed. daf-2-induced survival is observed at 20 and 25 degrees C, but not at 16 degrees C consistent with the temperature sensitive nature of the daf-2(e1370) allele. Hypertonic stress resistance in daf-2;daf-16 or age-1;daf-16 double mutants is indistinguishable from wild type worms, suggesting that the effects of daf-2 and age-1 are mediated by the DAF-16 transcription factor. Previous studies have shown that the normal function of DAF-2 and AGE-1 is to prevent the nuclear accumulation of DAF-16, a forkhead-like transcription factor. We therefore tested the hypothesis that the daf signaling pathway is osmotically sensitive by analyzing the subcellular distribution of a DAF-16::GFP fusion protein. Heat shock resulted in a dramatic increase in nuclear localization of DAF-16::GFP within 1 h. DAF-16::GFP worms showed significantly increased resistance to 400 mM NaCl compared to wild type animals, but hypertonic stress had little effect on nuclear localization of DAF-16. These results suggest that hypertonicity itself does not activate the daf/insulin-like signaling pathway. Consistent with this hypothesis, we observed that a daf-16 loss-of-function mutant survives well on 400 mM NaCl agar if it is first grown for 2 weeks on agar containing 200 mM NaCl. A similar adaptive response is seen in wild type animals. Taken together, our results suggest that the transcriptional targets of DAF-16 protect cells from hypertonic stress. However, other transcription factors and signaling pathways likely mediate the adaptive response to hypertonicity. Current studies are aimed at identifying these transcription factors, their transcriptional targets, and the signaling pathways that lead to their activation.

Rudich P et al. (2018) J Genet "Models and mechanisms of repeat expansion disorders: a worm's eye view."

Lamitina T, Rudich P

[J Genet, 2018]

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 nonAUG dependent (RAN) translation' paints a new and unifying picture of how distinct repeat expansion-bearing RNAs might act as substrates for this noncanonical 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 <i>Caenorhabditis elegans</i> 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.