Soto, Rony et al. (2020) MicroPubl Biol

Van Buskirk, Cheryl, Soto, Rony

[MicroPubl Biol, 2020]

Across species, sleep is increased following exposure to damaging conditions, a phenomenon known as stress-induced sleep (SIS) (Hill et al. 2014; Lenz et al. 2015; Zada et al. 2019). In C. elegans, SIS is dependent on the ALA interneuron (Hill et al. 2014; Nelson et al. 2014), which promotes a coordinated quiescent state through the collective action of several neuropeptides (Nelson et al. 2014; Nath et al. 2016). Recently it has been shown that ALA ablation can suppress phenotypes associated with loss of the cephalic sensilla sheath (CEPsh) glia, such as prolonged larval development and locomotor pausing in adults (Katz et al. 2018), indicating that these glia attenuate certain aspects of ALA function. The CEPsh glia form a tubular structure surrounding the sensory endings of the CEP neurons and also extend thin processes that sheath the nerve ring, including the synapse between ALA and the postsynaptic AVE, a major locomotor interneuron. While the ALA is likely to promote SIS via peptidergic rather than synaptic signaling (Nelson et al. 2014; Nath et al. 2016), we wished to determine whether CEPsh glia ablation may impact the SIS-promoting function of ALA. To this end, we examined three independent lines that genetically ablate the four CEPsh glial cells (Katz et al. 2018), and compared the SIS responses of these strains to wild type and to ALA specification-defective ceh-17(np1) mutant animals. We examined three conditions known to trigger ALA-dependent sleep: noxious heat, pore-forming Cry5B toxin, and ultraviolet light exposure (Figure 1). We found that while the glia-ablated (GA) lines displayed a trend toward enhanced heat-SIS at the 15min time point, there were no significant differences in heat-SIS between the GA lines and wild-type. The GA lines also showed wild-type Cry5B-SIS. Surprisingly, two of the GA lines showed reduced UV-SIS, rather than the enhanced sleep that would be predicted if the CEPsh glia attenuate ALAs peptidergic function. Our data indicate that CEPsh glia are largely dispensable for stress-induced sleep, but may modulate the UVSIS response. As UV exposure elicits a delayed sleep response relative to other SIS triggers, we speculate that UV damage may activate ALA in a manner that is distinct, and partially supported by glial function.

Soto, Rony et al. (2015) International Worm Meeting "Disruption of proteostasis induces sleep following heat shock."

Soto, Rony, Hill, Andrew, Van Buskirk, Cheryl, Sarian, Adrig, Mansfield, Richard

[International Worm Meeting, 2015]

Recent work from our lab has shown that Caenorhabditis elegans sleeps after exposure to stressful stimuli, including heat shock. We hypothesize that disruption of proteostasis is the underlying trigger of this behavioral quiescence. Therefore, we expect animals with constitutive chaperone activity to require less sleep after heat shock compared to wild type. To test our hypothesis, we examined the behavioral responses of daf-21 mutants after heat shock. DAF-21/HSP90 is a negative regulator of the heat shock response, and these mutants are known to display heightened chaperone activity. We find that these mutants show a reduced sleep response relative to wild type following heat shock. This result supports our hypothesis that a core function of sleep may be related to mitigating disruptions of proteostasis. We aim to further test our model by examining the effects of sleep on proteostasis, by examining metastable protein function in wild type and sleep-defective mutants. Our results on these temperature sensitive mutants will be presented.

Soto , Rony et al. (2017) International Worm Meeting "UNC-103/ERG potassium channel contributes to locomotor quiescence during sleep."

Soto , Rony, Van Buskirk, Cheryl, Sternberg, Paul W.

[International Worm Meeting, 2017]

In recent years the field of sleep research has been bolstered by studies in non-mammalian model organisms like C. elegans, promising to shed light on the long-standing mystery of the core function of sleep. C. elegans has been found to experience two distinct types of behavioral quiescence that each fulfill all of the behavioral criteria for sleep. The first to be discovered was developmentally-timed sleep (DTS), which occurs at the end of each of the four larval stages (Raizen et al., 2008). The other sleep state can be triggered at any time by exposure to damaging conditions such as noxious heat, tissue damage, and UV exposure, and is referred to as stress-induced sleep (SIS) or recovery sleep (RS) (Hill et al., 2014; Iannacone et al., 2016). We have shown that recovery sleep in C. elegans is dependent on EGF signaling, and that robust sleep can be induced at any time, in an EGFR-dependent manner, via forced expression of the EGF ligand LIN-3 (Van Buskirk and Sternberg, 2007). We have taken advantage of this 'forced-sleep' assay to uncover genes required for sleep regulation. We are particularly interested in determining which potassium channel genes, if any, contribute to C. elegans sleep, as K+ channel mutations produce short sleepers in Drosophila and zebrafish. An RNAi screen of K+ channel genes and their regulators identified UNC-103, an ERG-type potassium channel, as required for EGF-induced sleep. Here we show that UNC-103 is required for locomotor quiescence during recovery sleep (RS) and contributes to locomotor quiescence during lethargus (DTS) as well, indicating that these two types of sleep share common downstream effectors. We present evidence for developmental compensation by other K+ channels in the unc-103 null mutant, as short exposure unc-103 RNAi produces a greater sleep disruption than long exposure. We find that the application of ERG-blockers disrupts sleep in an UNC-103-dependent manner. Last, we present the results of our current site of action analyses aimed at determining in which neurons the widely expressed UNC-103 is required during sleep.

Goetting, Desiree et al. (2017) International Worm Meeting "Food deprivation suppresses sleep via AMP kinase and circumvents the lethal effect of sleep loss in C. elegans."

Soto, Rony, Van Buskirk, Cheryl, Goetting, Desiree

[International Worm Meeting, 2017]

A major challenge in understanding the function and evolution of sleep lies in identifying the mechanisms that offset the vulnerabilities that come with it, such as the inability to forage or escape. The nematode C. elegans has recently been identified as a useful model for the dissection of sleep function and evolution, as animals experience a primitive sleep-like state that is triggered by conditions that cause cellular damage. In response to noxious environmental conditions such as extreme heat, animals enter a state of behavioral quiescence characterized by a reduction in sensory responsiveness and a cessation of feeding and locomotion. This recovery sleep, or RS, appears to be beneficial under certain conditions, as sleepless mutant animals are impaired for survival following noxious heat exposure. We wished to investigate how the decision to enter into RS may be influenced by additional environmental inputs that could potentially alter the physiological benefit to be gained from sleep. Here we show that food deprivation suppresses RS, and that this effect is exacerbated as population density increases. In addition to suppressing sleep drive, food deprivation protects against the lethality associated with sleep loss, suggesting that food-deprived animals have a reduced need for sleep. We show that suppression of sleep drive during periods of food deprivation requires AMP kinase. Additionally, we show that competence to engage in RS is dependent on the neuroendocrine signal DAF-7/TGF- beta , activating a previously identified neural circuit that shifts several aspects of development and metabolism from conservation to utilization of energetic resources. These data suggest that recovery sleep in C. elegans is an energetically costly activity that can be suppressed when environmental conditions are unfavorable and animals are required to compete for resources.

Katz, D.J. et al. (2019) International Worm Meeting "SPR-5;MET-2 maternal reprogramming antagonizes H3K36me3 in the control of germline versus soma."

Carpenter, B.S., Katz, D.J.

[International Worm Meeting, 2019]

In C. elegans, the H3K4me2 demethylase, SPR-5, and the H3K9 methyltransferase, MET-2, are maternally deposited into the oocyte where they cooperate to reestablish the epigenetic ground state of the newly formed zygote. Here, we show that the progeny of spr-5;met-2 mutants display a severe developmental delay that is associated with the ectopic expression of MES-4 germline genes. Previous work from the Strome and Kelly Labs demonstrates that maternally deposited MES-4 transgenerationally maintains H3K36me2/3 at ~200 germline genes in a transcription-independent manner, and this is required to reactivate germline genes in the subsequent generation. We hypothesized that SPR-5;MET-2 reprogramming may prevent MES-4 from ectopically maintaining H3K36me2/3 at germline genes in somatic tissues. By performing ChIP-seq on L1 progeny from spr-5;met-2 mutants, we find that MES-4 germline genes ectopically accumulate H3K36me3 in somatic tissues. Additionally, knocking down MES-4 suppresses the ectopic expression of MES-4 germline genes and rescues the developmental delay. These data suggest a model where SPR-5;MET-2 maternal reprogramming antagonizes H3K36me2/3 to enable the proper transgenerational control of germline versus somatic cell fates. Without SPR-5;MET-2 reprogramming, somatic cells struggle to specify their proper cell fate amongst the noise of inappropriate germline gene transcription, leading to developmental delay. A similar mechanism may underlie the developmental delay of Soto and Kabuki Syndrome patients, who have mutations in histone modifying enzymes.

He XY et al. (1998) J Biol Chem "A human brain L-3-hydroxyacyl-coenzyme A dehydrogenase is identical to an amyloid beta-peptide-binding protein involved in Alzheimer's disease."

He XY, Schulz H, Yang SY

[J Biol Chem, 1998]

A novel L-3-hydroxyacyl-CoA dehydrogenase from human brain has been cloned, expressed, purified, and characterized. This enzyme is a homotetramer with a molecular mass of 108 kDa. Its subunit consists of 261 amino acid residues and has structural features characteristic of short chain dehydrogenases. It was found that the amino acid sequence of this human brain enzyme is identical to that of an endoplasmic reticulum amyloid beta-peptide-binding protein (ERAB), which mediates neurotoxicity in Alzheimer's disease (Yan, S. D., Fu, J., Soto, C., Chen, X., Zhu, H., Al-Mohanna, F., Collison, K., Zhu, A., Stern, E., Saido, T., Tohyama, M., Ogawa, S., Roher, A., and Stern, D. (1997) Nature 389, 689-695). The purification of human brain short chain L-3-hydroxyacyl-CoA dehydrogenase made it possible to characterize the structural and catalytic properties of ERAB. This NAD+-dependent dehydrogenase catalyzes the reversible oxidation of L-3-hydroxyacyl-CoAs to form 3-ketoacyl-CoAs, but it does not act on the D-isomers. The catalytic rate constant of the purified enzyme was estimated to be 37 s-1 with apparent Km values of 89 and 20 microM for acetoacetyl-CoA and NADH, respectively. The activity ratio of this enzyme for substrates with chain lengths of C4, C8, and C16 was approximately 1:2:2. The human short chain L-3-hydroxyacyl-CoA dehydrogenase gene is organized into six exons and five introns and maps to chromosome Xp11.2. The amino-terminal NAD-binding region of the dehydrogenase is encoded by the first three exons, whereas the other exons code for the carboxyl-terminal substrate-binding region harboring putative catalytic residues. The results of this study lead to the conclusion that ERAB involved in neuronal dysfunction is encoded by the human short chain L-3-hydroxyacyl-CoA dehydrogenase gene.

K Kasuya et al. (1999) International C. elegans Meeting "C. elegans homologue of Sra-1, a target of Rac GTPase, is involved in hypodermal morphogenesis"

C Mello, M Soto, K Kaibuchi, K Kasuya, H Qadota

[International C. elegans Meeting, 1999]

The Rho family small GTPases (Rho, Rac, Cdc42) regulate cell motility and cell polarity through the rearrangement of actin cytoskeleton and through cell adhesion. We have previously identified Sra-1 as an effector for Rac1 GTPase in mammalian cells. Sra-1 directly interacts with GTP-bound Rac1 in vitro , but little is known about the physiological functions of Sra-1. Here, we examined the physiological functions of Sra-1 using C. elegans . We identified CeSra-1 as a C. elegans homologue of Sra-1 (51% identical to human Sra-1). To study CeSra-1 functions, we used RNA-mediated interference. We found that CeSra-1(RNAi) shows about 100% embryonic lethality with failure of the hypodermis to enclose embryos. In CeSra-1(RNAi) embryos visualized by MH27 immunostaining, differentiation of the hypodermal cells seems to be normal, but the dorsal intercalation and ventral enclosure did not occur. To investigate further the function of CeSra-1, we observed the RNAi phenotype of C. elegans homologues of the possible Sra-1 associated proteins previously identified in mammalian cells. Among them, only CeHEM-2(RNAi) shows a similar phenotype to that of CeSra-1(RNAi). Since the mutant allele of CeHEM-2, inx-1 , was isolated by Soto et al . (this meeting), we named CeSra-1 inx-2 . We raised antibodies to both proteins. Based on immunostaining results, both CeSra-1/INX-2 and CeHEM-2/INX-1 are expressed at a minimum in the hypodermis during the embryogenesis. Furthermore, we found that CeSra-1/INX-2 interacts with CeHEM-2/INX-1 in the two-hybrid system. Taken together, these results suggest that CeSra-1/CeHEM-2 complexes are involved in hypodermal morphogenesis.

Yanxia Bei et al. (2001) International C. elegans Meeting "Phosphotyrosine signaling acts in parallel with Wnt signaling to specify endoderm and to control cleavage orientation in early C. elegans embryos"

John Collins, Jennifer Hogan, Craig C Mello, Yanxia Bei, Ka Ming Pang, Martha Soto

[International C. elegans Meeting, 2001]

At the 4-cell stage of embryogenesis in C. elegans a cell contact mediated induction specifies both the endoderm fate and the anterior-posterior (A/P) orientation of the endoderm precursor called EMS. In vitro manipulations of the 4-cell stage blastomeres indicate that the signaling cell in this induction is the posterior-most cell called P2 and we refer to this signaling process as P2/EMS signaling. Wnt signaling components have been implicated in P2/EMS signaling for cell fate determination. Some of the components in the pathway are also important for the A/P orientation of the EMS division. However, with the exception of an intriguing allele of cdk-1 (See abstract by Soto et al.), no mutants identified to date prevent the A/P cleavage orientation of EMS cell in the intact embryo. Here we show that SRC-1, a C elegans homolog of pp60 c-src , and MES-1, a protein distantly related to receptor tyrosine kinases, are responsible for the more intense accumulation of phosphotyrosine at the junction between P2 and EMS. The accumulation of the intense phosphotyrosine at this junction correlates precisely with the localization of the MES-1 protein at the P2/EMS contact site. The src-1 and mes-1 mutants synergize with mutants in mom-2/wnt, mom-3, mom-5/FZ, and sgk-1/GSK-3ß to cause a complete loss of the endoderm fate and a left-right (L/R) rather than A/P division of EMS. These results suggest that phosphotyrosine and Wnt signaling act in parallel in the intact embryo to specify endoderm and to orient the division axis of the endoderm precursor cell EMS. In mammalian cells cell adhesion components are substrates for the SRC tyrosine kinase and results from Drosophila have shown that cell adhesion plays a role in cell polarity. Previous work in C.elegans has shown that classic components of cell adhesion junctions including E-cadherin (HMR-1), a -catenin (HMP-1) and ß-catenin (HMP-2) are cortically localized at all cell-cell contact sites in the early embryo, but are not required for P2/EMS signaling. Genetic analysis of L/R EMS divisions in cdk-1(ne236) have suggested a model that EMS has a groundstate potential to divide A/P and that misregulation of another ß-catenin (WRM-1) in the early embryo may lead to masking of this groundstate potential (see abstract by Soto et al.). Consistent with this model, we find that inhibition of WRM-1 or of HMR-1 by RNAi can restore the EMS A/P division orientation (but not endoderm) in our double mutants deficient in P2/EMS signaling. These results suggest that SRC-1 and MES-1 function in parallel with Wnt pathway components to unmask the potential of EMS to orient its division axis. Although the in-vivo localization of WRM-1 is still not known it is tempting to speculate that as in other systems this ß-catenin homolog is localized at cell-contact sites and that local signaling at the EMS/P2 junction leads to the release of WRM-1. This release may accomplish two things, unmasking of a cortical site that directs the A/P division of EMS and activation of WRM-1 for signaling downstream to specify the endoderm fate. We are currently testing predictions of this model by looking for changes in the localization of adhesion components such as HMR-1, HMP-1, and HMP-2 in cells undergoing P2/EMS signaling. We are also examining the genetic requirements for EMS spindle orientation in blastomere isolation assays where cell contacts are broken and then restored. Our preliminary results have shown that the intense phosphotyrosine at the EMS-P2 boundary can only rarely be restored in such experiments. Similar experiments have shown that SRC-1 appears to function in both cells for the phosphotyrosine accumulation at cell-cell contact sites.

Carpenter, Brandon et al. (2021) International Worm Meeting "C. elegans establishes germline versus soma by balancing histone methylation"

Carpenter, Brandon, Katz, David

[International Worm Meeting, 2021]

Embryos undergo extensive reprogramming at fertilization to prevent the inappropriate inheritance of histone methylation. In C. elegans, the H3K4me2 demethylase, SPR-5, and the H3K9 methyltransferase, MET-2, are maternally deposited into the oocyte where they cooperate to reestablish the epigenetic ground state of the newly formed zygote. Previous work from the Strome and Kelly Labs demonstrates that maternally deposited MES-4 maintains H3K36me2/3 at germline genes between generations to help re-establish the germline. To determine whether the MES-4 germline inheritance system antagonizes spr-5; met-2 maternal reprogramming, we examined the interaction between these two systems. Here, we show that the progeny of spr-5; met-2 mutants display a severe developmental delay that is associated with the ectopic expression of MES-4 germline genes in somatic tissues. By performing ChIP-seq on L1 progeny from spr-5; met-2 mutants, we find that MES-4 germline genes ectopically accumulate H3K36me3 in somatic tissues. Additionally, knocking down MES-4 suppresses the ectopic expression of MES-4 germline genes and rescues the developmental delay. We also show that the developmental delay is dependent upon the H3K4 methyltransferase, SET-2. More recent data from our lab uncovered a synergistic interaction between SPR-5; MET-2 maternal reprogramming and MEC Complex member, MEP-1. This interaction links maternal reprogramming of histone methylation to the somatic mechanisms that suppress germline gene expression in somatic tissues. Together, these data suggest a model where SPR-5; MET-2 maternal reprogramming antagonizes H3K36me2/3 to enable the proper transgenerational control of germline versus somatic cell fates. Without SPR-5; MET-2 reprogramming, somatic cells struggle to specify their proper cell fate amongst the noise of inappropriate germline gene transcription, leading to developmental delay. A similar mechanism may underlie the developmental delay of Soto and Kabuki Syndrome patients who have mutations in histone modifying enzymes.

Kuniaki Nakamura et al. (2002) East Coast Worm Meeting "Isolation and characterization of the cks-1 mutant in C. elegans"

Kuniaki Nakamura, Craig C Mello, Soyoung Kim

[East Coast Worm Meeting, 2002]

In wild-type embryos the 4-cell stage blastomere, EMS, undergoes an inductive interaction that specifies the endoderm and orients the division axis of the EMS cell onto the anterior/posterior (a/p) axis of the embryo. In our temperature-sensitive mutant screen, we isolated one recessive mutant, ne549, with a penetrant EMS division orientation defect and ectopic endoderm produced by the C blastomere. A mutant with a very similar phenotype, cdk-1(ne236) was identified in a previous screen by Martha Soto (See ECWM abstract). We mapped ne549 to the right of unc-24 on LGIV, into a region that contains a C. elegans homolog of the highly conserved CDK-1 interacting protein, CKS-1, which encodes a conserved 13 kD protein required for cyclin-dependent kinase activity. We obtained rescue of ne549 with a single gene PCR product that contains the cks-1(+) allele, and we found a single point mutation in a highly conserved residue within the open reading of cks-1. Thus we conclude that ne549 is an allele of cks-1. RNAi of cks-1 causes a drastic cell division defect during early embryogenesis, therefore ne549 is clearly a special allele that alters but does not abolish the function of CKS-1. Given the similarity between cks-1(ne549) and cdk-1(ne236) phenotypes, we hypothesize that the two proteins function together in an activity that is required for endoderm specification and the control of division orientation. Although each single mutant exhibits wild-type cell-cycles we found that double mutants between cks-1(ne549) and cdk-1(ne236), resemble the null (RNAi) phenotypes for the individual mutants. This finding suggests that both mutants impair a function(s) of the kinase complex that are required for the cell-cycle. How do cell-cycle molecule(s) function to control both the orientation of the cell division and cell fate? This is a very interesting question but has yet to be answered in any organism. As one potential avenue to address the targets or interactors of CKS-1, we have started a suppressor screen for cks-1 mutants. So far we have identified 50 dominant suppressors, which arise at knock-out frequency and define at least three loci. We will report on our progress toward mapping, and characterizing these suppressors, as well as our further analysis of the cks-1(ne549) mutant.