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.

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.

Rohlfing A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "C. elegans Patch-Related Receptors (ptr) Are Involved In The Physiological Responses To Hypertonic Stress"

Miteva Y, Lamitina T, Rohlfing A

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Osmoregulation is invovled in many physiological and pathophysiological processes, including cell growth, apoptosis, immunity and renal function. While the accuracy and sensitivity of this process are well described, the mechanisms by which animal cells and organisms detect deviations in cell size and activate compensatory pathways are poorly understood. In the C. elegans osm mutants physiological responses that are normally only turned on in response to conditions that decrease cell size are constitutive active. The osm-8(n1518) mutants exhibit striking resistance to normally lethal levels of hypertonic stress and have an osmotic stress resistance (Osr) phenotype. The resistance to hyperosmotic stress is at least partially due to a strong constitutive expression of the enzyme gpdh-1 and therefore accumulation of high levels of the osmolyte glycerol even under isotonic conditions. We were able to show, that osm-8(n1518) is encoded by a mucin-like protein. This protein is expressed in the hypodermis and most likely secreted into the extracellular matrix. Genetic studies of osmoregulation in yeast have previously identified mucin-like proteins (Msb2 and Hkr1) as critical regulators of the osmosensitive Hog1 p38 MAP kinases pathway. A genome-wide RNAi screen to identify genes that function downstream of OSM-8 was performed, as p38 MAP kinases function in the Hog1 pathway in yeast. 34 genes, which strongly affect the phenotypes of osm-8 mutants, and 538 genes with mild effects were identified after screening ~18,000 gene knockdowns. P38 MAP kinases pathway genes where not found within these group, but members of the patch-related protein family where identified in the screen as suppressors of the osm-8(n1518) phenotype. The transmembrane protein PTR-23 strongly suppresses the OSM-8 mediated gpdh-1p::GFP expression in the intestine and hypodermis and the Osr phenotype. PTR-23 is also co-expressed with OSM-8 in the hypodermis, which altogether suggests, that PTR-23 functions downstream from OSM-8. The genes ptr-4 and ptr-15 also suppress the Osr Phenotype of osm-8(n1518) mutants but appeared to have a milder effect on the osm-8 mediated gpdh-1p::GFP expression.

Morton E et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Molecular Characterization of the Heat Shock Transcription Factor HSF-1 and Identification of SUMO as a Regulator of HSF-1 in C. elegans"

Brown K, Chaya T, Lamitina T, Morton E

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The heat shock transcription factor HSF-1 activates transcription of target genes that promote protein homeostasis, implying important roles for HSF-1 in stress tolerance, aging, innate immunity, cancer, and protein diseases. Because active HSF-1 mediates resistance to a wide variety of proteotoxic stressors, drugs that upregulate HSF-1 may be therapeutic for a variety of protein misfolding diseases, such as Huntington's disease. Recent studies suggest, however, that HSF-1 activity may also be involved in undesirable phenotypes, including cellular transformation to a cancerous state. While global regulation of HSF-1 might be undesirable, identification of regulators of HSF-1 may allow us to find more nuanced ways to control its activity. Additionally, while many HSF-1-dependent processes have been extensively studied in the model organism Caenorhabditis elegans, the molecular behavior of HSF-1 has not yet been characterized in this system. We have begun to examine molecular characteristics of HSF-1 in C. elegans using a fluorescently tagged HSF-1. Tagged HSF-1 appears to localize to the nucleus under basal conditions and to condense into nuclear granules upon heat shock. To identify HSF-1 regulators, we conducted a high-throughput RNAi screen on an HSF-1-dependent reporter strain of C. elegans. Our screen covered 17,540 genes and identified 44 regulators of the HSF-1-dependent reporter. RNAi inhibition of these genes did not similarly affect an HSF-1-independent reporter, suggesting that they specifically regulate HSF-1-dependent phenotypes. One gene identified by our screen was smo-1, which encodes the sole C. elegans SUMO homolog. SUMO is a small ubiquitin-like molecule used to post-translationally modify proteins. Repression of smo-1 led to hyper-activation of the HSF1-dependent reporter, but only following activation of the reporter by heat shock. Consistent with this phenotype, smo-1(RNAi) also improved whole-animal thermotolerance. Sequence analysis of HSF-1 revealed a consensus site for sumoylation at the N-terminus, and recent studies from other labs confirm that HSF1 is post-translationally sumoylated. These results suggest that SUMO may directly modify HSF-1 in C. elegans in response to specific activating conditions and that such modifications inhibit HSF-1-regulated gene expression and stress resistance in vivo. Our findings have implications for sumoylation as a future target for therapies intended to regulate HSF-1.

Sara Maxwell et al. (2002) European Worm Meeting "Functional analysis of the C. elegans T-box gene family"

Alison Woollard, Roger Pocock, Lizzie Clarke, Sara Maxwell

[European Worm Meeting, 2002]

T-box genes are a group of developmentally important transcription factors united by a common DNA binding domain. T-box genes are present in all metazoan species so far analysed but are absent from yeast. There are 20 T-box genes in C. elegans, more than twice the number found in Drosophila. Many of the C. elegans T-box genes are highly diverged from those found in other species while others have clear orthologues present throughout the metazoan kingdom. One highly conserved T-box gene is mab-9, a member of the tbx20 sub-family1. This was the first C. elegans T-box gene to be identified by mutation and is required for cell fate specification during hindgut and male tail development, and aspects of nervous system function. One other conserved T-box gene has recently been reported to be important for a particular muscle cell fate specification2. We have inactivated the remaining C. elegans T-box genes by RNAi and have found obvious phenotypes only in very few cases. These phenotypes include embryonic lethality, L1 lethality, and a Dpy phenotype with weakly penetrant male tail defects, and will be described in detail. The remaining T-box genes give no obvious phenotype by RNAi. Phylogenetic analysis reveals that several pairs of T-box genes are very similar to eachother and are therefore likely to be the result of recent duplications. This might suggest functional redundancy. Double RNAi experiments have revealed this to be the case with at least two of the T-box gene pairs (see also poster by Pocock et al). Study of the expression patterns of the whole T-box family may suggest other potential redundancy relationships which can be explored by RNAi. Comparison of the C. elegans T-box genes with the set of T-box genes now defined for C. briggsae is being used as a tool for defining potentially important regulatory regions present in orthologous genes.

Hisako Takeshita et al. (2005) International Worm Meeting "Direction of cell polarity is determined by Wnt in C.elegans"

Hisako Takeshita, Hitoshi Sawa, Kota Mizumoto

[International Worm Meeting, 2005]

Asymmetric cell division is a fundamental process that produces cellular diversity during development. In C. elegans, asymmetric divisions of many cells are regulated by Wnt signaling. In particular, asymmetry of the T cell division is regulated by LIN-44/Wnt and the LIN-17/Frizzled receptor. LIN-44/Wnt is expressed in hypodermal cells at the tip of the tail, posterior to the T cell. In lin-44 mutants, the polarity of the T cell division is frequently reversed, while in lin-17 mutants, the division is symmetric. It has been proposed that LIN-44 activates the LIN-17 receptor on the posterior side of the T cell to polarize it. To determine whether LIN-44/Wnt acts as an asymmetric cue that polarizes the T cell, we ectopically expressed LIN-44 in cells anterior to the T cell using the egl-5 promoter (egl-5::LIN-44) to find that polarity reversal phenotype of lin-44 was strongly enhanced. Moreover lin-17 was required for egl-5::LIN-44 to reverse the T-cell polarity. These results suggest that LIN-44 polarize the T cell through the LIN-17 receptor. To further confirm that LIN-44 directly acts on LIN-17 in the T cell, we analyzed the localization of LIN-17::GFP fusion proteins expressed by the seam cell specific promoter (SCM::LIN-17::GFP). This construct rescued defects of lin-17 mutants in the T cell division, indicating that lin-17 functions cell-autonomously in the T cell. We found that LIN-17 localization was localized in the posterior cortex of the T cell in wild type. In lin-44 mutants, the LIN-17 localization was uniform on the cortex. Furthermore, egl-5::LIN-44 reversed the LIN-17 localization to the anterior cortex. These results strongly indicate that LIN-44 directly acts on LIN-17 in the T cell to orient the polarity.

Hryshkevich, Uladzislau et al. (2011) International Worm Meeting "Core promoter T-blocks correlate with gene expression levels in C. elegans."

Yanai, Itai, Hryshkevich, Uladzislau, Hashimshony, Tamar

[International Worm Meeting, 2011]

Core promoters mediate transcription initiation by the integration of diverse regulatory signals encoded in the proximal promoter and enhancers. It has been suggested that genes under simple regulation may have low-complexity permissive promoters. For these genes, the core promoter may serve as the principal regulatory element; however the mechanism by which this occurs is unclear. We report here a periodic poly-thymine motif, we term T-blocks, enriched in occurrences within core promoter forward strands in C. elegans. An increasing number of Tblocks on either strand is associated with increasing nucleosome eviction. Strikingly, only forward strand T-blocks are correlated with expression levels, whereby genes with ³6 T-blocks have five fold higher expression levels than genes with 3 T-blocks. We further demonstrate that differences in T-block numbers between strains predictably affect expression levels of orthologs. Highly expressed genes and genes in operons tend to have a large number of T-blocks, as well as the previously characterized SL1 motif involved in trans-splicing. The presence of T-blocks thus correlates with low nucleosome occupancy and the precision of a trans-splicing motif, suggesting its role at both the DNA and RNA levels. Collectively our results suggest that core promoters may tune gene expression levels through the occurrences of T-blocks, independently of the spatio-temporal regulation mediated by the proximal promoter.