MC Hresko et al. (1999) International C. elegans Meeting "Myotactin, a novel adhesion molecule involved in muscle-hypodermal signaling."

RH Waterston, MC Hresko, P Shrimankar, LA Schriefer

[International C. elegans Meeting, 1999]

A physical link between the bodywall muscle and the hypodermis is required for locomotion in C. elegans . This linkage is mediated in part by hemidesmosome-like structures, called fibrous organelles (FO; Francis and Waterston, 91), found in regions of the hypodermis adjacent to muscle. We are interested in understanding the signals required to establish and maintain the correct spatial relationship between muscle and the FO. The localization of a protein we have named myotactin suggests it may participate in muscle-hypodermal communication. Myotactin is expressed by dorsal and ventral hypodermal cells and in adults localizes in regions of the hypodermis known to contain high concentrations of FO. However, during part of embryogenesis, the position of myotactin mirrors that of the contractile apparatus in the forming muscle. Myotactin is a novel transmembrane protein with at least 32 fibronectin type III repeats extracellularly and a unique cytoplasmic tail, consistent with a role in muscle-hypodermal linkage. Analysis of let-805 mutants st456 and s2706 , which we believe to be myotactin loss-of-function mutants, suggests myotactin is involved in muscle cell adhesion and in maintaining the connection between muscle and FO. Like known muscle ( pat class) mutants, the myotactin mutants arrest elongation at the 2-fold stage, display little or no movement, and die before hatching. Further examination of the mutants shows the muscle forms as in wild type, but the muscle cells detach from the hypodermis when contractions begin, consistent with a role in muscle cell adhesion. A second aspect of the myotactin mutant phenotype is delocalization of FO. Initially, the FO appear to form and localize as in wild type. However, after the muscle cells detach, FO are seen all through the dorsal and ventral hypodermis. Confocal and immunoelectron microscopy results demonstrate a close spatial relationship between myotactin and FO. However, a molecular link between myotactin and FO has not been shown, and therefore it's not clear how myotactin is involved in maintaining the connection between muscle and FO. To investigate in more detail the role myotactin plays in FO development, we are performing a 2-hybrid screen to identify potential myotactin-interacting proteins.

Zhang, Huimin et al. (2009) International Worm Meeting "A Tension-dependent Pathway Promoting Fibrous Organelle Maturation During Embryonic Morphogenesis."

Zhang, Huimin, Labouesse, Michel, Landmann, Frederic, Zahreddine, Hala, Nagamatsu, Yasuko, Diogon, Marie

[International Worm Meeting, 2009]

C. elegans body-wall muscles are required for embryonic development. Loss of muscle tension results in embryonic elongation arrest at the 2-fold stage. However how muscle contraction promotes hypodermal extension still remains unknown. To address this issue, we focused on the attachment complex fibrous organelles(FO), the only structure connecting muscles to the hypodermis. We present evidence that a mechanotransduction pathway might exist between muscles and FOs, contributing to hypodermal elongation. C. elegans FOs are composed of two hemidesmosome-like complexes at both sides of the hypodermis, connected by intermediate filaments(IF) in between. Intact FOs are very important for transmitting muscle tension to the cuticle. Consistent with previous reports, our results showed that the FO organization changes when muscles start to contract, and their integrity requires muscle tension. To analyze FO functions in C. elegans, we performed a genome-wide RNAi screen, searching for genes that decrease the viability of a weak VAB-10/plakin mutant, a key component of the FO complex. We identified 14 candidate genes. In particular, we showed that the p21-activated kinase homologue PAK-1 is a novel FO component. Strong mutations in this gene combined with the viable vab-10 allele destabilize FOs, resulting in severe epidermal defects. By 2-dimensional electrophoresis and antibody staining, we found that PAK-1 controls phosphorylation of IFs and their organization. More interestingly, hypodermal localized PAK-1 activity requires tension exerted by muscles, suggesting that PAK-1 is downstream of a novel mechanotransduction pathway. Combining biochemical and genetic approaches, we propose that this mechano-sensing machinery does not involve PAK-1 protein delocalization or phosphorylation. Instead, preliminary results showed that constitutively active CDC-42 can rescue the loss of IF phosphorylation observed in muscle mutants, indicating that muscle tension more likely activates PAK-1 through Rac/Cdc-42 signaling. Altogether, we suggest that C.elegans muscle contraction generates a mechanical response in the hypodermis through FOs. Possibly by turning on CED-10/CDC-42 signalling, two classic upstream activators of PAK-1, the muscle tension triggers a chemical signal that finally results in phosphorylation of IFs. This process enables FO maturation into a junction able to resist mechanical stress. Our finding suggests that the C.elegans fibrous organelles are not only an attachment structure, but also a mechanosensor transducing physical tension into chemical signals.

Jonathan M Rhine et al. (2005) International Worm Meeting "PAT-12, a Novel Member of the C. elegans Fibrous Organelle"

Jonathan M Rhine, Benjamin D Williams

[International Worm Meeting, 2005]

In C. elegans, contractions of the body-wall muscles are transmitted across the hypodermis to the cuticular exoskeleton through pairs of hemidesmosome-like structures called fibrous organelles (FO). Though several key FO components have been identified, much remains unknown. We have found that pat-12, a gene identified in a screen for Pat mutants (Paralyzed, Arrested elongation at Two-fold), encodes a novel member of the FO complex. In our model, the PAT-12 protein functions within the FO as an adaptor molecule providing necessary linkages between FO proteins. Without PAT-12, the FOs fail as muscle cell contractions begin during embryogenesis, causing the muscle cells to detach from the body wall. We confirmed that T17H7.4 corresponds to pat-12 by standard transgenic rescue of pat-12(st430) mutants with three overlapping cosmids (C24A1, F20B8, and F42G9) that together include the entire predicted gene T17H7.4. Each cosmid includes part of T17H7.4, and when injected singly fails to give phenotypic rescue. We then performed sequence analysis on DNA isolated by PCR from wild-type and arrested pat-12(st430) embryos. The pat-12(st430) sequence had a single base pair change relative to wild-type that disrupts a splice acceptor site of the 12th exon of predicted isoform T17H7.4j. Alternative splicing is predicted to produce at least 11 different isoforms, but only four of them include exon 12 and thus are likely to be affected by the st430 mutation. The predicted PAT-12 protein sequence has no homology to any vertebrate proteins, but does share significant homology to several proteins with unknown function identified in two other nematode species. We generated an antibody against PAT-12 by immunizing mice against a peptide [N-CRRNLAGDFPSEHSKSKIKLDPLEQR-C] present in all four of predicted isoforms containing exon 12 (A, C, J, and K) and a fifth isoform (B) that does not. This antiserum specifically stains the hypodermal FOs in wild-type adults, and the FOs and pharynx in wild-type ~430 minute embryos. Interestingly, in pat-12(st430) arrested embryos the FO staining is not detectable, but the pharyngeal staining pattern is unaffected. We conclude that the pat-12(st430) allele blocks the synthesis of one or more PAT-12 isoforms that contribute to FOs. By examining worms containing a minigene in which an isoform-specific promoter drives GFP expression, we determined that isoforms A, C, and K are expressed in hypodermal cells. Currently, we are constructing minigenes of these isoforms to test for rescue of st430 and to serve as GFP reporters. To date we have rescued st430 mutants with isoform K fused to GFP, which localizes to the fibrous organelles, confirming earlier antibody staining experiments.

Hetherington , Suzannah et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The nematode-specific protein GEI-16/PAT-12 organizes and/or maintains fibrous organelle components at the apical membrane."

Behm, Carolyn, Gally, Christelle, Hetherington , Suzannah, Fritz, Julie-Anne, Labouesse, Michel

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

Morphogenesis of the Caenorhabditis elegans embryo is driven by the epidermis and requires input from the body wall muscles. Both tissues are mechanically coupled through trans-epithelial attachment structures called fibrous organelles (FOs). Here, we report the identification of a new FO component called GEI-16. We initially identified this gene through a RNA interference (RNAi) screen for potential anthelminthic drug targets. GEI-16 proved necessary for maintaining the tight link between muscles and the cuticle, since RNAi against gei-16 caused muscle and cuticle detachment. In parallel, w e demonstrated an interaction between the core FO component VAB-10A and GEI-16 by yeast two-hybrid analysis. We confirmed by RT-PCRs that gei-16 encodes at least 8 different transcripts, and identified two mutations in the gene, gei-16(tm2298) and pat-12(st430) that display embryonic elongation defects typical of FO-defective mutants and fail to complement. Hence we suggest that pat-12 and gei - 16 correspond to the same gene. We developed GFP-tagged fusion proteins for the main classes of transcripts and observed that one of them, which could rescue the embryonic lethality of both gei-16/pat-12 alleles, was specifically localized to FOs. Its absence specifically affected FO organization, particularly at the apical membrane where intermediate filaments, MUP-4 and VAB-10 stripes were lost. Consistent with this view, knockdown of IFs or VAB-10 induced GEI-16::GFP disorganization mostly at the apical side showing an interdependent localization of these FOs components. In contrast, basal markers like LET-805 or UNC-52 did not display major disorganization problem when gei-16/pat-12 was absent. Altogether our data suggest that GEI-16 could be an epidermal scaffolding protein responsible for the organization and/or maintenance of FOs mainly at the apical side during embryonic development.

Williams, Kyle C. et al. (2011) International Worm Meeting "Essential Functions of IFA-2 Domains in C. elegans Fibrous Organelles."

Plenefisch, John, Williams, Kyle C.

[International Worm Meeting, 2011]

The C. elegans fibrous organelle (FO) complex consists of apical and basal hemidesmosomes linked by cytoplasmic intermediate filaments (IFs). FOs are found most prominently in the epidermis where they overlie body wall muscle and serve to transmit force from the muscle to the cuticle. IFA-2, one of four epidermally expressed IFs whose loss results in epidermal fragility and failure of muscle-cuticle force transmission, localizes to FOs. Unlike IFB-1 and IFA-3 that are required embryonically, IFA-2, although expressed in the embryo, is not essential for FO function until postembryonic stages. This distinctive phenotype allows us to specifically explore the functions of IFA-2 domains, and map their interactions with other FO associated proteins. All IFs contain three domains: a central rod domain and globular head and tail domains. The rod is essential for assembly of IFs into mature filaments while less is known about the functions of the head and tail domains. Roles of the head and tail domains of IFA-2 were examined by expressing GFP-tagged IFA-2 variants in transgenic animals and examining their phenotype and localization in wild-type and null backgrounds. Mutant variants included IFA-2 deleted for the head domain (DH), deleted for the tail domain (DT), deleted for both (RO), and variants that contained only the head domain (HO) or tail domain (TO). The expression and function of DH is virtually indistinguishable from full-length IFA-2. DT results in a lowered incorporation of IFA-2 into FOs and is unable to rescue a null allele of ifa-2, suggesting the tail domain is essential to IFA-2 function. Though both HO and TO are expressed well in the hypodermis at all stages, incorporation into FOs is variable between individual animals. High levels of early-stage HO incorporation into FOs correlate with a dominant-negative muscle detachment phenotype, while low-level incorporation results in healthy animals. This suggests the head domain interacts with FO components essential to tissue integrity. To determine the protein components of the FOs that interact with the head and tail domains, yeast two-hybrid and co-localization experiments are in progress. This work should help to elucidate the role of IFA-2 in FO function and reveal the underlying defects leading to tissue fragility.

James CL et al. (1996) European Worm Meeting "An Orphan Neurotransmitter Transporter Gene from C.elegans"

Lynch AS, MacGregor D, Coates D, James CL, Isaac RE

[European Worm Meeting, 1996]

We have utilised conserved regions in neurotransmitter transporter proteins (NTPs) to clone a novel nematode NTP gene through TBLASTN searches of data from the C.elegans genome sequencing project. Cosmid T25B6 was found to contain a putative gene for a protein showing homology fo mammalian NTPs. This predicted gene was cloned and a segment of the DNA, including the promoter was subcloned into LacZ translational fusion vector. This construct was transformed into C.elegans and gave rise to beta-galactosidase expression in neurons located in the head, in the lateral nerve processes and in the vulva and tail regions, the expression pattern in the male is similar with the addition of expression in the copulatory apparatus. The promoter is being characterised through a series of deletions of the 5'flanking region of the T25b6.2::LacZ construct.

Michel Labouesse et al. (2008) Development & Evolution Meeting "Cytoskeleton and Junction Remodelling during C. elegans Embryonic Elongation"

Hala Zahreddine, Michel Labouesse, Christelle GALLY, Huimin Zhang, Frederic Wissler, Marie Diogon

[Development & Evolution Meeting, 2008]

Elongation of C. elegans embryos provides an invaluable system to investigate several cell biological processes occurring in all epithelia, such as establishment of cell polarity, junction assembly, cell shape changes, cytoskeleton dynamics. Embryonic elongation is a rather complex morphogenetic process since it involves distinct epidermal cells and muscles. While many important players controlling this process have been identified, their integration in time and space remain poorly defined. I will outline our recent progress in two areas that contribute to elongation, the spatiotemporal regulation of myosin II activity in the epidermis, and the mechanisms involved in remodelling fibrous organelles (FOs) in response to muscle tension. To define how myosin II activity is controlled we generated several GFP probes to visualize the actomyosin cytoskeleton in vivo. We dissected the mechanism of MLC-4/rMLC activation through phosphorylation at two conserved Ser and Thr residues. We showed that MLC-4 is primarily required in lateral seam cells, and that its activity is repressed in dorsal/ventral cells through a Rho-specific RhoGAP (RGA-2). In addition, we identified the two protein kinases that phosphorylate MLC-4 during elongation and completed the characterisation of myosin II structure by identifying its essential light chain. FOs are the adhesion complexes that attach dorsal/ventral epidermal cells to the extracellular matrix and thereby connect muscles to the cuticle. They are functionally and molecularly related to hemidesmosomes, since they contain a plakin (VAB-10) and intermediate filaments. To define how FOs are assembled and how they contribute to elongation, we performed a genome-wide RNAi enhancer screen looking for clones that decrease the viability of a weak vab-10 mutant. We identified 14 genes that have human homologues with predicted roles in transcription, RNA processing, protein stability or cytoskeleton remodelling. Most of these genes interact together and thus form a functional network. Among them, PAK-1/p21-activated kinase (a novel FO component) controls intermediate filament polymerization and/or anchoring, CRT-1/calreticulin the folding of the ECM component UNC-52/perlecan, and the HECT-domain E3-ligase EEL-1 the abundance of the UNC-52 receptor myotactin. We conclude that this functional network helps maintain FO structure when submitted to external tension.

Vahdati Nia, Behrad et al. (2015) International Worm Meeting "Comparative Analysis of Human Alzheimer's Risk Factor Genes from GWAS and Linkage Analyses.."

Lee, Deborah, Vahdati Nia, Behrad, Ta, Phong, Munassi, Steven, Murakami, Shin

[International Worm Meeting, 2015]

Alzheimer's disease (AD) is the major cause of dementia. The vast majority of AD patients have late onset AD (LOAD) that has been becoming more problematic as people continue to age and the average lifespan of the population increases. Genome-wide association studies (GWAS) and linkage analyses have identified genes with possible risk susceptibility to LOAD and other neurological diseases. How these genes are involved in the molecular mechanism of Alzheimer's disease is still not well understood. Using meta-analysis, a new list of genes was created by comparing with multiple databases of human and C.elegnas ortholog genes. Matching registry test was performed on these genes to find their ortholog counterparts. Using meta-analysis, we narrowed down human genes associated with Alzheimer's disease to 320 (referred to as AD genes). Of them, 93 genes were found to have ortholog counterparts in C. elegans. Interestingly, each ontology search program has some fructuations fo the gene number identified. In addition, there were databases with no update, which were excluded from the study. Of the genes identified, 94% were identified in one ontology database, 85% were identified in two databases, 75% were identified in three databases, and 55% were matched by all four search engines. Importantly, the human counterparts share risk susceptibility with other neurological disorders, including Parkinson's disease, and Amyotrophic lateral sclerosis. The genes identified with high confidence levels should provide reasonable insights to those neurological conditions..

Gramstrup Petersen, Jakob et al. (2011) International Worm Meeting "EGL-13, a SOX transcription factor, regulates neuronal cell fate determination of the BAG neurons."

Pocock, Roger, Gramstrup Petersen, Jakob

[International Worm Meeting, 2011]

Responses to changes in the oxygen environment are crucial for maintaining homeostasis and survival. In C. elegans these responses are orchestrated by soluble guanylate cyclases acting primarily in the two sets of sensory neurons - BAG and URX. The molecular factors that determine BAG cell fate are unknown. Therefore, we have conducted an EMS mutagenesis screen to isolate mutants that have lost BAG cell fate. From the screen we isolated three mutants, rp13, rp14 and rp15. rp14 has a strong Egl phenotype due to defective connection of the gonad (Cog phenotype). Due to the phenotype we hypothesized that rp14 may be an allele of egl-13. We find that egl-13::GFP is expressed in the BAG neurons and that the egl-13(ku194) allele causes 100% loss of BAG expression using the pflp-19::GFP reporter strain. egl-13 is a member of the SOX transcription factor family and has previously been described to play an essential role for the proper development of the utse1. Vertebrate homologs of EGL-13 are SOX5 and SOX6 which have been shown to be involved in chondrogenesis and cell cycle progression of neural progenitors in the chick spinal cord2,3. The discovery that egl-13 plays a role in neuronal cell fate specification in the worm might reveal that SOX5 and SOX6 also plays specific roles in the development of the human nervous system. 1Cinar, H. N., Richards, K. L., Oommen, K. S. & Newman, A. P. The EGL-13 SOX domain transcription factor affects the uterine pi cell lineages in Caenorhabditis elegans. Genetics 165, 1623-1628 (2003). 2Smits, P. et al. The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell 1, 277-290, doi:S1534-5807(01)00003-X [pii] (2001). 3Martinez-Morales, P. L., Quiroga, A. C., Barbas, J. A. & Morales, A. V. SOX5 controls cell cycle progression in neural progenitors by interfering with the WNT-beta-catenin pathway. EMBO Rep 11, 466-472, doi:embor201061 [pii] 10.1038/embor.2010.61 (2010).

SB. Hetherington et al. (2006) European Worm Meeting "GEI-16, a Putative Component of the Hemidesmosome, is Necessary for Tissue Stability and Epithelial Morphogenesis"

FI. Pellerone, SB. Hetherington, CA. Behm

[European Worm Meeting, 2006]

The Caenorhabditis elegans hemidesmosome or fibrous organelle (FO) is a trans-epidermal attachment structure that spans the hypodermis, linking the basal hypodermal membrane to the cuticle, thereby transducing the force of muscle contraction to the exoskeleton and propagating movement. The hemidesmosome has a similar structure and the same localisation and function as the vertebrate hemidesmosome, but there is little direct homology between the components except for the intermediate filaments (IFs), and the plectin VAB-10A (Boscher, 2003; Hahn, 2001). . gei-16 was isolated in a RNA interference (RNAi) screen for potential anthelminthic drug targets because it fulfilled the selection criteria of being nematode specific, and when knocked down results in lethality at many stages of development, sterility and paralysis. Specifically, RNAi of gei-16 in C.elegans results in slow development, and muscle weakness before paralysis, arrest and death in the later larval and adult stages. It appears that gei-16 is necessary for maintaining adhesion of the muscles and cuticle to the hypodermis as depletion of gei-16 message causes 100% penetrance of a muscle detachment phenotype, and a 47% penetrance of a cuticular detachment phenotype. Epithelial morphogenesis is also affected with larvae and adults showing uneven excretory channels and abnormal vulval morphogenesis. RNAi affected worms often arrest or die before the maturation of the reproductive system. Worms that survive to adulthood produce few eggs, which are retained within the uterus and often fail to hatch. This appears to be due to the combined failure of vulval morphogenesis, detachment of the vulval muscles and a failure of the embryo to elongate. Localisation of gei-16 mRNA to all muscle-apposed epithelia is consistent with a role in epithelial tissue stability and morphogenesisan expression pattern which mirrors that of hemidesmosome components. Furthermore, RNAi in GFP reporter strains of C. elegans shows that components of the hemidesmosome are disorganised in gei-16 RNAi worms. Results from the interactome data set show that gei-16 binds to mua-6, an IF in the hemidesmosome (Li et al, 2000). . References:. BOSHER, et al. (2003) The Caenorhabditis elegans vab-10 spectraplakin isoforms protect the epidermis against internal and external forces. J Cell Biol, 161, 757-68.. HAHN, B. S. & LABOUESSE, M. (2001) Tissue integrity: hemidesmosomes and resistance to stress. Curr Biol, 11, R858-61.. LI, S, et al. (2004) A map of the interactome network of the metazoan C. elegans. Science, 303, 540-3.