James F Morley et al. (1999) International C. elegans Meeting "The Stressed Worm Project"

Richard I Morimoto, Sanjeev Satyal, James F Morley, Josh Segal

[International C. elegans Meeting, 1999]

Genes encoding molecular chaperones are activated in response to a variety of stresses and developmental events. The study of chaperones and co-chaperones has traditionally focused on experiments in bacteria, yeast and mammalian tissue culture cells. We have begun to investigate the regulation of the stress response and the function of molecular chaperones in the nematode, C. elegans . These studies will help us to understand the relationship of the stress response to processes such as cell death and development. We have performed a molecular evolutionary analysis of chaperone genes by reciprocal BLAST searches of the C. elegans and S. cerevisiae genomes, focusing on the HSP40, HSP70, and HSP90 families. Dendrogram analysis of predicted protein sequences reveals: a) 13 HSP70s, consisting of orthologs of the soluble cytoplasmic SSAs, two orthologs of the ER KAR2/BiP, and orthologs of mitochondrial HSP70 b) a single ortholog of the co-chaperone BAG-1 c) three HSP90s, comprised of one direct ortholog of the cytoplasmic HSC82 and HSP82 chaperones, an ortholog of the ER protein GRP94, and a ~70kD protein which is not directly related to any yeast HSP90 d) seven HSP40 orthologs. This analysis has served as a basis for our rationale in selecting genes for deletion. The transcriptional regulation of chaperone genes involves an interplay between a heat shock transcription factor and various positive and negative regulators. We have generated a deletion mutation in heat shock factor binding protein ( hsb-1 ), a negative regulator of the heat shock response. A deletion in hsb-1 gives rise to no gross morphological abnormalities, however, hsb-1 mutant animals have a significantly shorter lifespan than N2 animals. To study the in vivo regulation of chaperone activity, we have generated a deletion mutation in the co-chaperone bag-1 . BAG-1 is a negative regulator of Hsp70 and interacts with important signaling molecules including BCL-2, RAF-1, and steroid receptors. Transgenic strains expressing a bag-1 promoter::LacZ/GFP fusion exhibit X-gal staining in the intestine, head and tail neurons. Deletion of bag-1 does not affect timing of developmental stages, embryonic and larval viability, or lifespan. However, bag-1 mutant animals exhibit a significant increase in brood size compared to the N2 strain.

James F Morley et al. (2001) International C. elegans Meeting "HEAT SHOCK TRANSCRIPTION FACTOR (HSF) ACTIVITY REGULATES THE APPEARANCE OF POLYGLUTAMINE PROTEIN AGGREGATES"

Li-Jung Tai, Richard I Morimoto, James F Morley

[International C. elegans Meeting, 2001]

Disruption of protein folding homeostasis resulting in protein misfolding and aggregation is associated with many human diseases including Huntington's disease where expansion of a polyglutamine (polyQ) tract to greater than 37-40 residues leads to the formation of protein aggregates, neuronal dysfunction and premature cell death. The expression of expanded chimeric polyglutamine (polyQ) -YFP proteins as extrachromosomal arrays in body wall muscle cells results in the formation of visible protein aggregates associated with a10-fold reduction in motility. Immunohistochemical studies of Q82-expressing animals reveal displacement of actin and myosin filaments. In contrast, neither visible aggregates nor altered motility were detected in animals expressing Q19 or Q29-YFP. Animals expressing an intermediate polyQ tract (Q40) exhibit striking polymorphism, as different muscle cells in the same animal contain soluble or aggregated polyQ. The severity of motility defect in Q40 animals corresponds to the proportion of cells with aggregates. Our laboratory and others have previously identified individual molecular chaperones as modulators of the aggregation phenotype. As chaperones are co-regulated during the heat shock response by HSF, we have examined whether altering the levels of HSF by RNAi and tissue-specific overexpression would impact the biochemical events that influence protein aggregation. Expression of a full length Ce-hsf cDNA from the unc-54 promoter results in more rapid activation of an hsp-16::lacZ reporter in body wall muscle cells, confirming overexpression of functional HSF. In unc-54::Q82-yfp; myo-2::Q82-gfp; unc-54::hsf animals, we observe the appearance of soluble patches of fluorescence in body wall muscle, but not in the pharynx. However, expression of a C-terminal HSF fragment that lacks DNA binding and trimerization domains has no effect on activation of the heat shock response or distribution of Q82-YFP. Injection of dsRNA transcribed from a full-length hsf cDNA inhibits activation of the heat shock response in heat shocked hsp-16::lacZ animals, confirming inactivation of HSF by RNAi. Inhibition of HSF in Q19 or Q29 animals leads to sporadic formation of aggregates similar to those observed in Q82 strains. Consistent with the transient nature of RNAi phenotypes, F1 progeny of injected animals were affected, but F2 animals exhibit normal activation of the heat shock response and distribution of Q19. Normal activation of the heat shock response and distribution of Q19 in animals injected with unc-22 dsRNA demonstrated the specificity of hsf RNAi effects. These data suggest that Q19 and Q29 solubility is maintained by an active process that requires HSF activity. The heat shock response is considered an acute response to changes in environmental conditions, and the requirement for HSF activity to maintain Q19 and Q29 solubility in the absence of stress reveals a novel role for HSF. We are currently testing whether manipulations of HSF levels that influence aggregate formation are associated with altered motility.

Yoko Honda et al. (2005) International Worm Meeting "Study of space-flight effect on the aging of C. elegans in the International C. elegans Experiment(ICE)-First"

Shuji Honda, Noriaki Ishioka, Yoko Honda

[International Worm Meeting, 2005]

As exposures to the space environment become longer, information regarding the effects on biological aging will become important. We have not yet fully clarified aging processes in space environments. The aging process and lifespan are affected by various kinds of environmental factors including oxygen concentration (1), temperature and radiation. The aging phenomena that we usually see occur under certain conditions on the ground. Space environments differ from ground environments especially with regard to the radiation spectrum and gravity. We participated in the International C. elegans Experiment(ICE)First that examined the effects of a 10-day space flight on the nematode C. elegans. C. elegans has frequently been used for study of aging because of its short lifespan. Recently, Morley et al. reported that Huntington's-like polyglutamine (polyQ)-repeat proteins expressed in the muscle of C. elegans form aggregates as the animals age, and that this aggregation is delayed in long-lived mutants(2). We measured the polyQ aggregates in these nematodes after space flight as an aging marker. Herndon et al. showed that the sarcomere orientation in the body-wall muscle becomes disorderly as the animals age(3). We also observed the sarcomere orientation in the body-wall muscle of these nematodes after space flight as another aging marker. Acknowledgement: We thank Dr. R. L. Morimoto (Northwestern University) for providing us polyQ-YFP C. elegans strains. We also thank CGC for other strains. ICE-First was mainly conducted by the French Space Agency (CNES), with support of the European Space Agency and the Space Research Organization of the Netherlands. We are grateful to Dr. Michel Viso (CNES), Dr. K. Kuriyama (JAXA) and Dr. A. Higashitani (Tohoku University) for their support and suggestion for our experiment. References: 1) Honda S., Ishii N, Suzuki K, Matsuo M. J Gerontol. 48:B57-61. 1993 2) James F. Morley, Heather R. Brignull, Jill J. Weyers, and Richard I. Morimoto. Proc. Natl. Acad. Sci. USA, 99: 10417-10422. 2002 3) Herndon LA, Schmeissner PJ, Dudaronek JM, Brown PA, Listner KM, Sakano Y, Paupard MC, Hall DH, Driscoll M. Nature. 419:808-814. 2002

Wei-meng Woo et al. (2004) West Coast Worm Meeting "The extracellular matrix protein F-spondin is essential for muscle attachment and epidermal elongation"

Chris Suh, Yishi Jin, Andrew D Chisholm, Mei Ding, Christie Emigh, Wei-Meng Woo, Jian Cao

[West Coast Worm Meeting, 2004]

In C. elegans epidermal intermediate filaments (IFs) and their associated structures, the trans-epidermal attachments, are essential for embryonic epidermal elongation (Woo et al 2004). The formation of muscle contractile units and trans-epidermal attachments are mutually dependent during epidermal elongation. To understand how the connection between epidermis and muscle is established and how the two tissues communicate during organogenesis, we performed a screen for epidermal elongation-defective mutants. One locus identified in this screen was defined by three lethal alleles and mapped to the cluster of LG II. Subsequent analysis showed that these mutations were allelic to vab-13 and ven-3 . By genetic mapping and allele sequencing we showed that all these mutations affect F10E7.4, which encodes the C. elegans member of the F-spondin family of secreted proteins. F-spondin has been shown to play roles in axon guidance, cell migration, and angiogenesis. Our genetic analysis shows that in C. elegans F-spondin is required for epidermal elongation and muscle attachment, as well as for proper positioning of neuronal processes. Using GFP reporters, we found that F-spondin is expressed in body muscle cells and is a secreted protein. Thus, F-spondin may function in embryogenesis in communication between muscle and epidermis. Immunostaining of F-spondin mutants suggest that F-spondin may indirectly affect the organization of epidermal actin microfilaments and trans-epidermal attachments . We are examining the expression patterns of muscle and basement membrane components in F-spondin mutants. To study the signaling pathways regulated by F-spondin, we are testing mutations in candidate receptor genes for genetic interactions with F-spondin mutations.

Bonnie Kaas et al. (2008) C.elegans Neuronal Development Meeting "Regulation of Levels of UNC-13 at Synapses"

Rebecca Lawson, Rebecca Kohn, Thomas Limouze, Ryan Wennell, Bonnie Kaas

[C.elegans Neuronal Development Meeting, 2008]

Release of neurotransmitters from neurons is highly regulated. Several proteins play roles in this process, including UNC-13, and decreased release of neurotransmitters in unc-13 mutants results in paralysis. We identified an F-box protein that interacts with UNC-13. F-box proteins participate in ubiquitin ligase complexes and in Drosophila, DUNC-13 is degraded via the ubiquitin proteasome pathway. This UNC-13/F-box interaction may therefore indicate that UNC-13 is tagged for proteasomal degradation with ubiquitin by the ligase complex in C. elegans. The C. elegans knockout consortium isolated a strain with a large deletion in the coding region of the gene that codes for the F-box protein. If the F-box protein is indeed involved in the degradation of UNC-13, this strain would be expected to have higher levels of UNC-13, which could result in changes in phenotypes. We characterized the F-box deletion mutant by assaying brood size, developmental rate, and body bends per minute. Aldicarb assays were used to determine whether a deletion in the gene coding for the F-box protein alters the response to inhibitors of acetylcholinesterase. We found that the deletion resulted in some changes in developmental rate and in aldicarb sensitivity. We are continuing to study strains with mutations in both the gene coding for the F-box protein and in unc-13.

Yoko Honda et al. (2006) East Asia C. elegans Meeting "Study of space-flight effect on the aging of C. elegans in the International C. elegans Experiment (ICE)-First."

Akira Higashibata, Noriaki Ishioka, Masashi Tanaka, Shuji Honda, Yoko Honda

[East Asia C. elegans Meeting, 2006]

As exposures to the space environment become longer, information regarding the effects on biological aging will become important. We have not yet fully clarified aging processes in space environments. The aging process and lifespan are affected by various kinds of environmental factors including oxygen concentration<SUP>1)</SUP>, temperature and radiation. The aging phenomena that we usually see occur under certain conditions on the ground. Space environments differ from ground environments especially with regard to the radiation spectrum and gravity. We participated in the International Caenorhabditis elegans Experiment (ICE)-First that examined the effects of a 10-day space flight on the nematode C. elegans. C. elegans has frequently been used for study of aging because of its short lifespan and powerful genetic system. Recently, Morley et al. reported that Huntington's-like polyglutamine (polyQ)-repeat proteins expressed in the muscle of C. elegans form aggregates as the animals age, and that this aggregation is delayed in long-lived mutants<SUP>2)</SUP>. We measured the polyQ aggregates in these nematodes after space flight as an aging marker. We also examined the effects of space flight on the gene expression by DNA microarray analysis and real time PCR. The expression of several genes specifically for nervous systems was suppressed in space-flown animals, suggesting inactivation of nervous functions in space. The relationship between aging and changes in the gene expression in space will be discussed. 1) Honda S et al. Oxygen-dependent perturbation of life span and aging rate in the nematode. J Gerontol. 48:B57-61, 1993. 2) James F et al. The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA, 99: 10417-10422, 2002.

Nathalie Velarde et al. (2005) International Worm Meeting "F-actin dynamics in early C. elegans development"

Nathalie Velarde, Fabio Piano

[International Worm Meeting, 2005]

Actin has been shown to play key roles during early development in the C. elegans embryo (Schneider and Bowerman, 2003). However, it has been difficult to faithfully reproduce F-actin dynamics in vivo during early embryogenesis. To visualize F-actin we have generated a transgenic line that uses the F-actin binding domain of Drosophila moesin to decorate endogenous actin filaments with GFP (GFP::Moe). The GFP::Moe fusion line appears to be specific for filamentous actin and allows the visualization of F-actin dynamics in C. elegans in embryos. Preliminary evidence shows that F-actin is very dynamic in many cellular processes from prior to fertilization through the first mitotic cellular division. During fertilization a posterior actin cap forms and then dissipates. Prior to the first cell division F-actin becomes enriched in the anterior, similar to the anterior PAR proteins. As seen in par-3 mutants (Kirby et. al., 1990), depleting the embryo of PAR-6 with RNAi prevents this enrichment, suggesting that PAR-6 and the other anterior PARs may be required for F-actin to accumulate in the anterior. We have also observed the presence of highly dynamic actin comets in the early embryo. Preliminary evidence suggests that these comets may be involved with endocytic processes. Using results from large-scale RNAi surveys, we are employing the GFP::Moe line to perform secondary screening of genes associated with pseudocleavage defects to further characterize this process. We will present our progress in using this GFP::Moe line to study genetic interactions involving actin dynamics in early development.

Yoko Honda et al. (2007) International Worm Meeting "Study of space-flight effect on the aging of C. elegans in the International C. elegans Experiment (ICE)-First."

Yoko Honda, Masashi Tanaka, Noriaki Ishioka, Akira Higashibata, Shuji Honda

[International Worm Meeting, 2007]

. Yoko Honda1, Akira Higashibata2, Noriaki Ishioka2, Masashi Tanaka1, Shuji Honda1. 1) Genomics for Longevity & Health, Tokyo Metropolitan Institute of Gerontology(TMIG), Tokyo, Japan; 2) Japan Aerospace Exploration Agency(JAXA), Tsukuba, Japan. As exposures to the space environment become longer, information regarding the effects on biological aging will become important. We have not yet fully clarified aging processes in space environments. The aging process and lifespan are affected by various kinds of environmental factors including oxygen concentration (1), temperature and radiation. The aging phenomena that we usually see occur under certain conditions on the ground. Space environments differ from ground environments especially with regard to the radiation spectrum and gravity. We participated in the International Caenorhabditis elegans Experiment (ICE)-First that examined the effects of a 10-day space flight on the nematode C. elegans. C. elegans has frequently been used for study of aging because of its short lifespan and powerful genetic system. Recently, Morley et al. reported that Huntington''s-like polyglutamine (polyQ)-repeat proteins expressed in the muscle of C. elegans form aggregates as the animals age, and that this aggregation is delayed in long-lived mutants (2). We measured the polyQ aggregates in these nematodes after space flight as an aging marker. We also examined the effects of space flight on the gene expression by DNA microarray analysis and real time PCR. The expression of more than ten genes specifically for nervous systems was suppressed in space-flown animals, suggesting inactivation of nervous functions in space. Next, effects of inactivation of these genes on lifespan were tested by RNAi in the laboratory. 1) Honda S et al.Oxygen-dependent perturbation of life span and aging rate in the nematode. J Gerontol. 48:B57-61. 1993 2) James F et al. The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA, 99: 10417-10422. 2002.

Atsushi Yamanaka et al. (2001) International C. elegans Meeting "Biochemical and functional analysis of SKP1 family in Caenorhabditis elegans"

Yasumi Ohshima, Kei-ichi Nakayama, Masayoshi Yada, Atsushi Yamanaka, Hiroyuki Imaki

[International C. elegans Meeting, 2001]

The ubiuquitin-proteasome pathway is a key mechanism for substrate-specific degradation to control the abundance of a number of proteins. SCF complex, one of ubiquitin-protein ligases (E3s), regulates cell cycle progression, signal transduction, and many other biological systems. The SCF complex consists of invariable components, such as Skp1, Cul-1 and Rbx1, and variable components called F-box proteins that bind to Skp1 through the F-box motif. F-box proteins are substrate-specific adaptor subunits that recruit substrates to the SCF complex. Surprisingly, we found that the genome of Caenorhabditis elegans ( C. elegans ) contains at least 20 Skp1-like sequenses, whereas one or a few Skp1 is present in humans. Therefore, we studied C. elegans Skp1-like proteins (CeSkp1) that are likely to be variable components of SCF complex in addition to F-box proteins. At least, seven CeSkp1s were associated with C. elegans Cul-1 (CeCul-1) in yeast two-hybrid system as well as co-immunoprecipitation assay in mammalian cells, and these expression patterns were different in C. elegans . By RNA interference (RNAi), two of these CeSkp1s showed embyonic lethality and four showed the phenotype of slow growth. There were differences among CeSkp1s in ability to interact with F-box proteins. These results suggest that CeSkp1s, like F-box proteins, act as variable components of SCF complex in C. elegans .

Soto, Martha et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Arp2/3-based Regulation of Junctional Dynamics During Morphogenetic Cell Movements and Polarization."

Bernadskaya, Yelena, Patel, Falshruti, Soto, Martha

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Morphogenesis requires dynamic interaction between the adherens junctions and the actin cytoskeleton. This interaction is mediated by ?-catenin, which was proposed to be a stable bridge between the junctions and F-actin at the apical regions of polarized epithelial cells. However, ?-catenin cannot bind to F-actin and its adherens junction partner, ?-catenin, simultaneously, suggesting the bridge is dynamic and complex. ?-catenin was thought to inhibit Arp2/3-based branched actin at the junctions for normal maturation of adherens junctions. Our research suggests a new positive role for Arp2/3-dependent branched actin in junctional maturation and during embryonic cell movements of C. elegans . In this study we investigate how progressive WAVE/Scar and Arp2/3-depen dent accumulation of ?-catenin at the adherens junctions contributes to the accumulation of F-actin at apical regions of epithelial cells. We find that removing Arp2/3-dependent actin nucleation disrupts junctional maturation and results in altered levels of adherens junctional proteins in epithelial tissues. In particular, there is a drop in both ?-catenin and F-actin levels at the apical intestine, and altered organization of the apical intestine. Subcellular fractionation reveals that loss of Arp2/3-dependent actin nucleation reduces the amount of ?-catenin in the membrane-associated pool. These data demonstrate an essential role for Arp2/3 to dynamically remodel F-actin to support adherens junctions and polarized F-actin during cell migration and tissue morphogenesis.