Schultz, Jessica L. et al. (2013) International Worm Meeting "A Complex Issue: Understanding vMSP Receptor Heteromeric Complexing Behavior."

Miller, Michael, Lee, Se-Jin, Schultz, Jessica L., Han, Sung Min

[International Worm Meeting, 2013]

Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease with an unknown pathogenesis and limited therapeutic treatments. A major limitation hindering the development of therapeutic treatments is a lack of understanding of the disease's molecular pathways. In humans, a P56S point mutation in the VAPB/ALS8 MSP domain is associated with ALS and late-onset spinal muscular atrophy (SMA) (Funke et al., 2010; Millecamps et al., 2010; Nishimura et al., 2004). The N-terminal MSP domain is cleaved from the C-terminus of the VAPB protein, and is secreted in a cell-type specific manner (Tsuda et al., 2008). However, the P56S mutation inhibits secretion of the MSP domain. Genetic and biochemical evidence support the hypothesis that the MSP domain interacts with the VAB-1 Eph receptor, ROBO/SAX-3 receptor, and CLR-1 Lar-like protein tyrosine phosphatase receptor, which are collectively called growth cone guidance receptors (Miller et al, 2001; Miller et al., 2003; Tsuda et al., 2008; Han et al., 2012). In C. elegans, secreted vMSP acts on CLR-1 and ROBO/SAX-3 receptors expressed in striated muscle, promoting Arp2/3-dependent actin remodeling. This remodeling is critical for proper placement of mitochondria to actin-rich myofilament I-bands (Han et al., 2012). We hypothesize that the vMSP receptors form heteromeric complexes to promote signaling critical for actin remodeling and correct mitochondria placement. To begin testing this hypothesis, I am expressing combinations of VAB-1, SAX-3, and CLR-1 in cultured cells and investigating putative complex formation via co-immunoprecipitation. My preliminary data suggest that SAX-3 complexes with both VAB-1 and CLR-1. Data will also be presented on the role of C. elegans VAPB/VPR-1 in regulating mitochondria in motor neurons. The results could provide insight into growth cone guidance receptor interactions and pathways involved in ALS.

Schultz, Robbie D. et al. (2013) International Worm Meeting "DBL-1 TGF-b localization and the physiological basis of body size regulation in C. elegans."

Gumienny, Tina L., Ellis, E. Ann, Schultz, Robbie D.

[International Worm Meeting, 2013]

Transforming Growth Factor-b (TGF-b) is a family of secreted cell signaling ligands. DBL-1, a C. elegans TGF-b superfamily member, is secreted from nervous tissue, but must be trafficked to the epidermis where it binds to receptors. Alteration of the DBL-1 pathway generates distinct, dose-dependent phenotypes, where animals with increased or decreased signaling are long or small respectively. However, how body size is altered in dbl-1 pathway variants is still poorly understood. The goals of this project are to determine subcellular localization of secreted DBL-1, analyze its regulation between secreting and receiving cells, and elucidate the molecular and physiological underpinnings of body length regulation by DBL-1 pathway signaling. We found that that secreted GFP-tagged DBL-1 localizes in a discrete punctate pattern along the dorsal and ventral nerve cords. Using a whole-mount microwave-based immunofluorescence method we developed, we found that mouse BMP4, a DBL-1 homolog, rescues body size defects in C. elegans caused by loss of DBL-1 pathway signaling, but also co-localizes with GFP-tagged DBL-1. Immunocytochemical studies also show that these punctae co-localize with cell-cell contact sites and caveolar bodies in the ventral nerve cord. These studies are the first to decipher the subcellular localization of DBL-1, expanding the knowledge of TGF-b trafficking in C. elegans. Microarray analyses have revealed DBL-1 signaling regulates transcription of cuticular components, which may together affect body size. Our genetic and pharmacological studies reveal that DBL-1 signaling affects cuticular permeability, altering sensitivity to soluble anesthetics based on the dose of DBL-1. Using transmission electron microscopy, we found that ultrastructural composition of the cuticle is affected in a dose-dependent manner in dbl-1 variants. These results show that DBL-1-mediated body size differences directly correlate with changes in cuticular organization. These studies expand our understanding of the molecular mechanisms involved in regulation of body size by the DBL-1 pathway.

Schultz, Robbie D. et al. (2011) International Worm Meeting "The role of extracellular ZP-domain protein DYF-7 in body size development."

Gumienny, Tina L., Schultz, Robbie D.

[International Worm Meeting, 2011]

Body size development is strictly regulated in C. elegans through genetic and environmental components. The most well studied of these genetic regulators is the TGF-b DBL-1 pathway. Loss of DBL-1 signaling leads to decreased body size as well as abnormal development of male-specific structures. Mutants lacking DYF-7 display a small body size and do not properly sense their environment. DYF-7 is a transmembrane zona pellucida (ZP) domain-containing protein expressed in head and tail neurons as well as seam cells. Previous studies have shown that DYF-7 is involved in neural tip anchoring, which explains its sensing defect, but its role in body size development has remained unexplored. To determine if DYF-7 regulates body size though DBL-1 signaling, we performed epistasis and expression studies. Our epistasis analyses between dyf-7 and dbl-1 pathway members reveal that double mutants are significantly smaller than either single mutant, indicating DYF-7 regulates body length independent from the TGF-b DBL-1 pathway. We also found GFP-tagged DBL-1 is properly expressed and localized in animals lacking DYF-7 signaling. Further, male-specific structures appear normal in dyf-7 mutants. An additional mode of body size regulation is through organization of the cuticle, which surrounds the exterior of the animal. The cuticle in dyf-7 mutants has morphological defects that could contribute to the small body size phenotype. These results indicate that DYF-7 acts through cuticle organization, independent of the TGF-b pathway, to regulate body size.

Guo, Yiqing et al. (2013) International Worm Meeting "Differential expression of germline genes in the presence/absence of H3K9me2."

Maine, Eleanor, Guo, Yiqing

[International Worm Meeting, 2013]

In C. elegans, indirect immunofluorescence experiments indicate a high level of histone H3 lysine 9 dimethylation (H3K9me2) on any unpaired/unsynapsed chromosomes during first meiotic prophase, particularly in pachytene stage nuclei (Kelly et al 2002; Bean et al 2004). In contrast, H3K9me2 marks are relatively low on paired chromosomes at this time. H3K9me2 marks are typically associated with transcriptional repression. Thus, the elevated level of H3K9me2 on unpaired chromosomes may play a role in meiotic silencing of unpaired chromosomes (MSUC). Germline H3K9me2 marks require activity of MET-2 (Bessler et al 2010), a SET domain protein that is the only C. elegans ortholog of human SETDB1 H3K9 methyltransferase (Schultz et al 2002). To begin to investigate the importance of H3K9me2 marks on unpaired chromosomes, we performed RNA-seq on mRNA isolated from him-8 vs met-2;him-8 adult hermaphrodite gonads. Our data identify genes that are expressed in the gonads and that differentially expressed in met-2(0) and met 2(+). In most cases, the differentially expressed transcripts are elevated in met-2(0), consistent with a role for H3K9me2 in limiting transcription. Although elevated transcripts are not preferentially X-linked, we observe a greater average fold-change for X-linked transcripts than for autosomal transcripts. To evaluate whether the unpaired status of the X contributes to this pattern, we are evaluating additional genotypes. As a complementary approach, we are also investigating the distribution of H3K9me2 on paired vs unpaired chromosomes using a ChIP-seq approach.

Song, Myung Chul et al. (2015) International Worm Meeting "C09F5.1, the BRICHOS containing protein functions in stress resistance."

Han, Ching Tack, Song, Kyung Hee, Song, Myung Chul, Kim, Sung Hee

[International Worm Meeting, 2015]

C09F5.1, one of several genes expressed differentially in heat sensitive C. elegans mutants was isolated. To confirm whether C09F5.1 is involved in heat stress response, the expression pattern of C09F5.1 in heat stress condition was analyzed, showing the induction of expression of mRNA and protein by 33degC heat treatment in wild type N2. The induction of C09F5.1 protein by heat treatment delayed in heat shock transcription factor-1(HSF-1) mutants (sy441) compared to N2 implying that the HSF regulates the transcription of C09F5.1 as HSPs. C09F5.1 RNAi-treated worms became more sensitive to heat stress than control worms. These results indicate that C09F5.1 transcribed by HSF-1 may function in heat stress response. To find the putative functional domain of C09F5.1, the computational analysis of C09F5.1 protein was done. The results showed that C09F5.1 consists of an N-terminal region, a type II transmembrane domain and a BRICHOS domain. The various BRICHOS proteins are known related with familial dementia (BRI2), chondrosarcoma (Chondromodulin-1) and interstitial lung disease (SP-C). BRICHOS domain containing proteins function as molecular chaperone(Sanchez-Pulido, Devos et al. 2002). Furthermore recent studies revealed that BRICHOS domain directly interacts with Abeta42 peptide and delays amyloid-beta fibril formation(Willander, Presto et al. 2012, Hermansson, Schultz et al. 2014). When the C09F5.1 was expressed in animal cells (293T), the C09F5.1 protein directly interacted with Abeta42. In order to identify the chaperone activity of C09F5.1 on abnormal protein aggregation, we generated transgenic worms expressing each of the C09F5.1 protein with full length, BRICHOS deletion and N-terminus deletion in Abeta42 expressing worm. The transgenic worms expressing the BRICHOS domain deleted mutant showed the increased survival rate compared to worms expressing Abeta42 solely. These results suggest C09F5.1 BRICHOS containing protein has chaperonin functions under stress but not through the BRICHOS domain only. Currently we are studying the mechanism of protection from amyloid toxicity by the N-terminus of C09F5.1.

Mongan NP et al. (1997) International C. elegans Meeting "AN EXTENSIVE NICOTINIC ACETYLCHOLINE RECEPTOR GENE FAMILY IN CAENORHABDITIS ELEGANS."

Baylis HA, Mongan NP, Sattelle DB

[International C. elegans Meeting, 1997]

Although the nicotinic acetylcholine receptors (nAChRs) are the best characterized ionotropic neurotransmitter receptors, the extent of the molecular and functional diversity of the nAChR gene family is not known for a single organism. A number of C. elegans nAChR subunit genes have been identified previously using genetic approaches [lev-1(non-a),unc-38(a),unc-29(non-a),deg-3(a)] and cross-species probing of C. elegans cDNA libraries [acr-2(non-a),acr-3(non-a),and Ce-21(a)]. nAChR a-subunits may be distinguished from all other ionotropic receptor subunits by the presence of adjacent cysteines at positions equivalent to 192,193 in the Torpedo a-subunit. We have applied an RT-PCR strategy to confirm the expression of 8 novel putative nAChR a-subunits predicted by GENEFINDER. Analysis of the regions of the a-subunit considered to (a) contribute to the acetylcholine binding site and (b) the channel lining, has revealed a diversity which may underlie distinct functional roles for members of this multi-gene family.

C Huang et al. (1999) International C. elegans Meeting "Each C. elegans Laminin a Subunit Mediates Distinct Aspects of Morphogenesis"

WG Wadsworth, C Huang, PD Yurchenco

[International C. elegans Meeting, 1999]

Laminin is a heterotrimeric glycoprotein composed of a , b , and g subunits. The genome sequencing project reveals two a , one b and one g laminin subunit genes. Based on sequence analyses, major features and known binding sites are conserved. The predicted a chains, laminin a A and laminin a B, are most similar to the vertebrate a 1/ a 2 and a 5 chains respectively, while the b and g resemble the b 1/ b 2 and g 1 chains. Thus, there are two predicted laminin trimers, a A bg and a B bg . Mutations in epi-1 , which encodes laminin a B, have been isolated and characterized (abstract ECWM 98). They cause defects that are consistent with the developmental roles for basement membranes (BMs). We report the expression patterns of laminin a A and a B. By immunostaining, both first appear at gastrulation between germ layers. However, laminin a A is heavily deposited anteriorly, surrounding pharygeal precursors, whereas laminin a B is more posteriorly localized. Laminin a B becomes localized to the BMs associated with pharynx, intestine, gonad, body wall, body wall muscle, and muscles throughout development. In contrast, laminin a A accumulates in pharyngeal BM, intestinal BM and body wall muscle BM during elongation and its level in intestinal BM and body wall muscle BM gradually decreases. In larvae and adults, laminin a A is only sometimes detected weakly in pharyngeal BM and body wall muscle BM. It is primarily localized in the spermatheca. Injection of dsRNA directed to epi-1 produces phenotypes similar to those observed in epi-1 alleles and the injection of dsRNA directed to lam3 , which encodes laminin aB, causes L1 larval arrest with severe pharyngeal defects. Double dsRNA-mediated interference directed to both genes produces embryos that arrest at morphogenesis with phenotypes more severe than those caused by the RNAi of either epi-1 or lam3 alone, indicating that both genes contribute to morphogenesis. Together, our RNAi and immunostaining results indicate that laminin a B has a broad role in maintaining the structural integrity of BMs and for regulating many aspects of morphogenesis, while laminin a A is restricted to specialized membranes and is required for the morphogenesis of specific tissues.

Yanjue Wu et al. (2005) International Worm Meeting "EGb 761 INHIBITS AMYLOID BETA OLIGOMERIZATION AND TOXICITY IN TRANSGENIC CAENORHABDITIS ELEGANS"

Christopher D Link, Yves Christen, Astrid G Zepeda, Yanjue Wu, Zhixin Wu, Yuan Luo

[International Worm Meeting, 2005]

Amyloid peptide aggregation has been postulated to link oxidative stress and neurodegeneration seen in Alzheimer's diesease (AD). We have previously demonstrated that the standardized Ginkgo Biloba extract, EGb 761, inhibits A aggregation in vitro and attenuates the expression of a stress response gene small heat shock protein (hsp 16-2) in Caenorhabditis elegans. In this study, we use EGb 761 as a pharmacological modulator to associate A species with the levels of oxidative free radicals and with A-induced toxicity in an A-expressing transgenic C.elegans model, which can express A in muscle cells after temperature up-shift. We observed that EGb 761 alleviated A-induced toxicity which correlated with its ability to specifically inhibit A oligomerization. Furthermore, EGb 761 reduces intracellular levels of hydrogen peroxide in the transgenic C.elegans. These findings suggest that A oligomers and A-induced oxidative strss are crucial for A toxicity, and EGb 761 has a clear therapeutic potential for prevention and treatment of AD.

Lyashenko, Alexey et al. (2015) International Worm Meeting "Line scanning fluorescent confocal microscope for a 3D multi-neuron imaging of C. elegans."

Polanco, Edward, Lyashenko, Alexey, Arisaka, Katsushi

[International Worm Meeting, 2015]

Conventional confocal microscope uses a physical aperture to reduce the amount of out of focus light to the image sensor. We developed a line scanning confocal microscope that utilizes a software controlled rolling shutter on a CMOS camera for a high-speed 3D volume imaging of dozens of active neurons. The microscope setup allows for a real time worm tracking for freely navigating C. elegans under a localized external stimulation for phototaxis and thermotaxis. An external photo stimulation for optogenetics was also realized.

Paul Maddox et al. (2007) International Worm Meeting "Functional genomics identifies a Myb domain-containing protein family required for assembly of CENP-A chromatin."

Joost Monen, Arshad Desai, Francie Hyndman, Karen Oegema, Paul Maddox

[International Worm Meeting, 2007]

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2-associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain-containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.