Cuticle Collagen Research Articles

Cuticle Collagen Expression Is Regulated in Response to Environmental Stimuli by the GATA Transcription Factor ELT-3 in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is protected from the environment by the cuticle, an extracellular collagen-based matrix that encloses the animal. Over 170 cuticular collagens are predicted in the C. elegans genome, but the role of each individual collagen is unclear. Stage-specific specialization of the cuticle explains the need for some collagens; however, the large number of collagens suggests that specialization of the cuticle may also occur in response to other environmental triggers. Missense mutations in many collagen genes can disrupt cuticle morphology, producing a helically twisted body causing the animal to move in a stereotypical pattern described as rolling. We find that environmental factors, including diet, early developmental arrest, and population density can differentially influence the penetrance of rolling in these mutants. These effects are in part due to changes in collagen gene expression that are mediated by the GATA family transcription factor ELT-3 We propose a model by which ELT-3 regulates collagen gene expression in response to environmental stimuli to promote the assembly of a cuticle specialized to a given environment.

New Roles for the Heterochronic Transcription Factor LIN-29 in Cuticle Maintenance and Lipid Metabolism at the Larval-to-Adult Transition in Caenorhabditis elegans.

Temporal regulation of gene expression is a crucial aspect of metazoan development. In the roundworm Caenorhabditis elegans, the heterochronic pathway controls multiple developmental events in a time-specific manner. The most downstream effector of this pathway, the zinc-finger transcription factor LIN-29, acts in the last larval stage (L4) to regulate elements of the larval-to-adult switch. Here, we explore new LIN-29 targets and their implications for this developmental transition. We used RNA-sequencing to identify genes differentially expressed between animals misexpressing LIN-29 at an early time point and control animals. Among 230 LIN-29-activated genes, we found that genes encoding cuticle collagens were overrepresented. Interestingly, expression of lin-29 and some of these collagens was increased in adults with cuticle damage, suggesting a previously unknown function for LIN-29 in adult cuticle maintenance. On the other hand, genes involved in fat metabolism were enriched among 350 LIN-29-downregulated targets. We used mass spectrometry to assay lipid content in animals overexpressing LIN-29 and observed reduced fatty acid levels. Many LIN-29-repressed genes are normally expressed in the intestine, suggesting cell-nonautonomous regulation. We identified several LIN-29 upregulated genes encoding signaling molecules that may act as mediators in the regulation of intestinally expressed genes encoding fat metabolic enzymes and vitellogenins. Overall, our results support the model of LIN-29 as a major regulator of adult cuticle synthesis and integrity, and as the trigger for metabolic changes that take place at the important transition from rapid growth during larval life to slower growth and offspring production during adulthood.

Integration of behavioural tests and transcriptome sequencing of C. elegans reveals how the nematode responds to peanut shell biochar amendment

Biochars have drawn wide attention as adsorbents, carbon sequesters and soil re-mediators. However, these substances are ambiguous regarding their effects on the motility, phenotypic changes and potential adaptative mechanisms of soil organisms. This study investigated how peanut shell biochar (PBC) affects the C. elegans model via a one-choice selection test and RNA-seq analysis. The results showed that C. elegans were able to select either PBC or a water control, and a clear preference for PBC was observed after 48 h of exposure, with the chemotaxis index (CI) reaching approximately 1.0. The nematode preferences for PBC vs sterile PBC/graphite were not significant, which demonstrated that initial microorganisms and appearances were not the reasons for the worms' selection, but the selection behaviour was instead determined by volatile odours. The treatments also showed that biochar amendment significantly decreased the body length, brood size and superoxide dismutase (SOD) activity of C. elegans to 960.20 ± 15.23 μm, 173.22 ± 4.56, 165.81 ± 3.82 U/mL SOD, respectively. Then, a possible molecular mechanism of PBC-induced developmental and reproductive effects on C. elegans was explored. Differential gene expression analysis was performed, and 1625 genes (1425 up- and 225 downregulated genes) were regulated in response to PBC treatment. The top 20 regulated genes were col genes (col-129; col-81; col-139; col-71), bli-6, perm-4 and his-24, which indicated that cuticle collagen synthesis, eggshell formation and/or heterochromatin in postembryonic growth may be disrupted following exposure to PBC. Therefore, our study suggested that quality standards be used to test nematode preferences and responses to biochar amendment, with the aim of safe application in soils, seedling substrates or fertilizers.

Label-free quantitative proteomic analysis of molting-related proteins of Trichinella spiralis intestinal infective larvae

Molting is a key step for body-size expansion and environmental adaptation of parasitic nematodes, and it is extremely important for Trichinella spiralis growth and development, but the molting mechanism is not fully understood. In this work, label-free LC–MS/MS was used to determine the proteome differences between T. spiralis muscle larvae (ML) at the encapsulated stage and intestinal infective larvae (IIL) at the molting stage. The results showed that a total of 2885 T. spiralis proteins were identified, 323 of which were differentially expressed. These proteins were involved in cuticle structural elements, regulation of cuticle synthesis, remodeling and degradation, and hormonal regulation of molting. These differential proteins were also involved in diverse intracellular pathways, such as fatty acid biosynthesis, arachidonic acid metabolism, and mucin type O-glycan biosynthesis. qPCR results showed that five T. spiralis genes (cuticle collagen 14, putative DOMON domain-containing protein, glutamine synthetase, cathepsin F and NADP-dependent isocitrate dehydrogenase) had significantly higher transcriptional levels in 10 h IIL than ML (P < 0.05), which were similar to their protein expression levels, suggesting that they might be T. spiralis molting-related genes. Identification and characterization of T. spiralis molting-related proteins will be helpful for developing vaccines and new drugs against the early enteral stage of T. spiralis.

Toxicogenomic responses to zearalenone in Caenorhabditis elegans reveal possible molecular mechanisms of reproductive toxicity

In this study, the possible molecular mechanisms of zearalenone (ZEA)-induced reproductive and developmental toxic effects in Caenorhabditis elegans (C. elegans) were investigated. Differential gene expression profiles were identified, and 171, 245, and 3149 genes were down- or up-regulated (>2.0 fold) in 10, 20, and 40 μg/ml ZEA treated groups, respectively, as compared to untreated controls. Pathway specific mapping showed that the major differentially expressed genes were collagen synthetic pathways regulating genes, col-121 and dpy-17. Real-time PCR reconfirmation of key genes, related to cuticle collagen synthetic pathway, found dramatic changes in the expression of the genes dpy-31, sqt-3, col-121, and dpy-17 following exposure to ZEA (40 μg/ml), which indicated the significance of these genes in ZEA-induced toxicity. Cuticle collagen plays many key roles in the development and reproduction of C. elegans. The hypersensitive responses in transgenic and mutant worms also confirmed the roles of these genes in lethality and reproductive response to ZEA exposure, which indicates that ZEA blocked the normal collagen processing and cuticle formation. Taken together, our results demonstrate that disruption of the collagen biosynthetic pathway might be a key mechanism in ZEA-induced reproductive and developmental toxic effects in C. elegans.

BMP Signaling Determines Body Size via Transcriptional Regulation of Collagen Genes in Caenorhabditis elegans.

Body size is a tightly regulated phenotype in metazoans that depends on both intrinsic and extrinsic factors. While signaling pathways are known to control organ and body size, the downstream effectors that mediate their effects remain poorly understood. In the nematode Caenorhabditis elegans, a Bone Morphogenetic Protein (BMP)-related signaling pathway is the major regulator of growth and body size. We investigated the transcriptional network through which the BMP pathway regulates body size and identified cuticle collagen genes as major effectors of growth control. We demonstrate that cuticle collagens can act as positive regulators (col-41), negative regulators (col-141), or dose-sensitive regulators (rol-6) of body size. Moreover, we find a requirement of BMP signaling for stage-specific expression of cuticle collagen genes. We show that the Smad signal transducers directly bind conserved Smad-binding elements in regulatory regions of col-141 and col-142, but not of col-41 Hence, cuticle collagen genes may be directly and indirectly regulated via the BMP pathway. Our work thus connects a conserved signaling pathway with its critical downstream effectors, advancing insight into how body size is specified. Since collagen mutations and misregulation are implicated in numerous human genetic disorders and injury sequelae, understanding how collagen gene expression is regulated has broad implications.

Three-dimensional behavioural phenotyping of freely moving C. elegans using quantitative light field microscopy.

Behavioural phenotyping of model organisms is widely used to investigate fundamental aspects of organism biology, from the functioning of the nervous system to the effects of genetic mutations, as well as for screening new drug compounds. However, our capacity to observe and quantify the full range and complexity of behavioural responses is limited by the inability of conventional microscopy techniques to capture volumetric image information at sufficient speed. In this article we describe how combining light field microscopy with computational depth estimation provides a new method for fast, quantitative assessment of 3D posture and movement of the model organism Caenorhabditis elegans (C. elegans). We apply this technique to compare the behaviour of cuticle collagen mutants, finding significant differences in 3D posture and locomotion. We demonstrate the ability of quantitative light field microscopy to provide new fundamental insights into C. elegans locomotion by analysing the 3D postural modes of a freely swimming worm. Finally, we consider relative merits of the method and its broader application for phenotypic imaging of other organisms and for other volumetric bioimaging applications.

A Damage Sensor Associated with the Cuticle Coordinates Three Core Environmental Stress Responses in Caenorhabditis elegans.

Extracellular matrix barriers and inducible cytoprotective genes form successive lines of defense against chemical and microbial environmental stressors. The barrier in nematodes is a collagenous extracellular matrix called the cuticle. In Caenorhabditis elegans, disruption of some cuticle collagen genes activates osmolyte and antimicrobial response genes. Physical damage to the epidermis also activates antimicrobial responses. Here, we assayed the effect of knocking down genes required for cuticle and epidermal integrity on diverse cellular stress responses. We found that disruption of specific bands of collagen, called annular furrows, coactivates detoxification, hyperosmotic, and antimicrobial response genes, but not other stress responses. Disruption of other cuticle structures and epidermal integrity does not have the same effect. Several transcription factors act downstream of furrow loss. SKN-1/Nrf and ELT-3/GATA are required for detoxification, SKN-1/Nrf is partially required for the osmolyte response, and STA-2/Stat and ELT-3/GATA for antimicrobial gene expression. Our results are consistent with a cuticle-associated damage sensor that coordinates detoxification, hyperosmotic, and antimicrobial responses through overlapping, but distinct, downstream signaling.

Host Delivered RNAi of Two Cuticle Collagen Genes, Mi-col-1 and Lemmi-5 Hampers Structure and Fecundity in Meloidogyne incognita.

Root-knot nematodes have emerged as devastating parasites causing substantial losses to agricultural economy worldwide. Tomato is the most favored host for major species of root-knot nematodes. Control strategies like use of nematicides have proved to be harmful to the environment. Other control methods like development of resistant cultivars and crop rotation have serious limitations. This study deals with the application of host generated RNA interference toward development of resistance against root-knot nematode Meloidogyne incognita in tomato. Two cuticle collagen genes viz. Mi-col-1 and Lemmi-5 involved in the synthesis and maintenance of the cuticle in M. incognita were targeted through host generated RNA interference. Expression of both Mi-col-1 and Lemmi-5 was found to be higher in adult females followed by egg masses and J2s. Tomato var. Pusa Ruby was transformed with the RNAi constructs of these genes to develop transgenic lines expressing the target dsRNAs. 30.80–35.00% reduction in the number of adult females, 50.06–65.73% reduction in the number of egg mass per plant and 76.47–82.59% reduction in the number of eggs per egg mass were observed for the T1 events expressing Mi-col-1 dsRNA. Similarly, 34.14–38.54% reduction in the number of adult females, 62.34–66.71% reduction in number of egg mass per plant and 67.13–79.76% reduction in the number of eggs per egg mass were observed for the T1 generation expressing Lemmi-5 dsRNA. The multiplication factor of M. incognita reduced significantly in both the cases and the structure of adult females isolated from transgenic plants were heavily distorted. This study demonstrates the role of the cuticle collagen genes Mi-col-1 and Lemmi-5 in the structure and development of M. incognita cuticle inside the host and reinforces the potential of host generated RNA interference for management of plant parasitic nematodes (PPNs).

Human amyloid β peptide and tau co-expression impairs behavior and causes specific gene expression changes in Caenorhabditis elegans

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of extracellular amyloid plaques consisting of Amyloid-β peptide (Aβ) aggregates and neurofibrillary tangles formed by aggregation of hyperphosphorylated microtubule-associated protein tau. We generated a novel invertebrate model of AD by crossing Aβ1–42 (strain CL2355) with either pro-aggregating tau (strain BR5270) or anti-aggregating tau (strain BR5271) pan-neuronal expressing transgenic Caenorhabditis elegans. The lifespan and progeny viability of the double transgenic strains were significantly decreased compared with wild type N2 (P<0.0001). In addition, co-expression of these transgenes interfered with neurotransmitter signaling pathways, caused deficits in chemotaxis associative learning, increased protein aggregation visualized by Congo red staining, and increased neuronal loss. Global transcriptomic RNA-seq analysis revealed 248 up- and 805 down-regulated genes in N2 wild type versus Aβ1–42+pro-aggregating tau animals, compared to 293 up- and 295 down-regulated genes in N2 wild type versus Aβ1–42+anti-aggregating tau animals. Gene set enrichment analysis of Aβ1–42+pro-aggregating tau animals uncovered up-regulated annotation clusters UDP-glucuronosyltransferase (5 genes, P<4.2E−4), protein phosphorylation (5 genes, P<2.60E−02), and aging (5 genes, P<8.1E−2) while the down-regulated clusters included nematode cuticle collagen (36 genes, P<1.5E−21).RNA interference of 13 available top up-regulated genes in Aβ1–42+pro-aggregating tau animals revealed that F-box family genes and nep-4 could enhance life span deficits and chemotaxis deficits while Y39G8C.2 (TTBK2) could suppress these behaviors.Comparing the list of regulated genes from C. elegans to the top 60 genes related to human AD confirmed an overlap of 8 genes: patched homolog 1, PTCH1 (ptc-3), the Rab GTPase activating protein, TBC1D16 (tbc-16), the WD repeat and FYVE domain-containing protein 3, WDFY3 (wdfy-3), ADP-ribosylation factor guanine nucleotide exchange factor 2, ARFGEF2 (agef-1), Early B-cell Factor, EBF1 (unc-3), d-amino-acid oxidase, DAO (daao-1), glutamate receptor, metabotropic 1, GRM1 (mgl-2), prolyl 4-hydroxylase subunit alpha 2, P4HA2 (dpy-18 and phy-2). Taken together, our C. elegans double transgenic model provides insight on the fundamental neurobiologic processes underlying human AD and recapitulates selected transcriptomic changes observed in human AD brains.

Isolation, cloning, and characterization of a cuticle collagen gene, Mi-col-5, in Meloidogyne incognita.

Cuticle collagens form a major part of the nematode cuticle and are responsible for maintaining the overall shape of the animal and its protection from the external environment. Although substantial research on cuticle collagen genes has been carried out in Caenorhabditis elegans, their isolation and characterization in plant parasitic nematodes have been limited to a few genes only. In this study, a cuticle collagen gene, Mi-col-5, was isolated from root-knot nematode, Meloidogyne incognita. A partial segment of 402bp was first cloned and analyzed on Gbrowse followed by subsequent cloning of the 1047bp long full cDNA specifying the open reading frame. The deduced amino acid sequence showed 92% sequence identity with that of Mj-col-5. However, a transmembrane helix was predicted in Mi-col-5 which was not present in Mj-col-5. The conserved pattern of cysteine residues in Mi-col-5 suggested that it belonged to group 2 of nematode cuticle collagens but with a longer carboxy terminal region as was the case with Mj-col-5. Domain prediction revealed the presence of a nematode cuticle collagen N terminal domain and a pfam collagen domain along with collagen triple helix repeats. A phylogenetic tree based on the amino acid sequences showed evolutionary relationship of Mi-col-5 with cuticle collagens genes of other nematodes. 3D models for Mi-col-5 were predicted with the best confidence score of -2.78. Expression of Mi-col-5 transcript was found to be maximum in egg masses followed by adult females and J2s suggesting its role in the early stages of the development of the nematode during its life cycle.

In-vivo high resolution AFM topographic imaging of Caenorhabditis elegans reveals previously unreported surface structures of cuticle mutants

Atomic force microscopy (AFM) is a powerful method for topographic imaging of surfaces with nanometer resolution. AFM offers significant advantages over scanning electron microscopy (SEM) including the acquisition of quantitative 3D-images and biomechanical information. More importantly, for in-vivo biological imaging, AFM does not require sample dehydration/labeling. We show for the first time high-resolution topographical images of the cuticle of the model organism C. elegans under physiological conditions using AFM. C. elegans is used extensively for drug screening and to study pathogen adherence in innate immunity; both applications highly depend on the integrity of the nematode's cuticle. Mutations affecting both drug adsorption and pathogen clearance have been proposed to relate to changes in the cuticle structure, but never visually examined in high resolution. In this study we use AFM to visualize the topography of wild-type adult C. elegans as well as several cuticle collagen mutants and describe previously unseen anatomical differences.

Fluid dynamics alter Caenorhabditis elegans body length via TGF-β/DBL-1 neuromuscular signaling.

Skeletal muscle wasting is a major obstacle for long-term space exploration. Similar to astronauts, the nematode Caenorhabditis elegans displays negative muscular and physical effects when in microgravity in space. It remains unclear what signaling molecules and behavior(s) cause these negative alterations. Here we studied key signaling molecules involved in alterations of C. elegans physique in response to fluid dynamics in ground-based experiments. Placing worms in space on a 1G accelerator increased a myosin heavy chain, myo-3, and a transforming growth factor-β (TGF-β), dbl-1, gene expression. These changes also occurred when the fluid dynamic parameters viscosity/drag resistance or depth of liquid culture were increased on the ground. In addition, body length increased in wild type and body wall cuticle collagen mutants, rol-6 and dpy-5, grown in liquid culture. In contrast, body length did not increase in TGF-β, dbl-1, or downstream signaling pathway, sma-4/Smad, mutants. Similarly, a D1-like dopamine receptor, DOP-4, and a mechanosensory channel, UNC-8, were required for increased dbl-1 expression and altered physique in liquid culture. As C. elegans contraction rates are much higher when swimming in liquid than when crawling on an agar surface, we also examined the relationship between body length enhancement and rate of contraction. Mutants with significantly reduced contraction rates were typically smaller. However, in dop-4, dbl-1, and sma-4 mutants, contraction rates still increased in liquid. These results suggest that neuromuscular signaling via TGF-β/DBL-1 acts to alter body physique in response to environmental conditions including fluid dynamics.

The Effect of In Vitro Cultivation on the Transcriptome of Adult Brugia malayi.

BackgroundFilarial nematodes cause serious and debilitating infections in human populations of tropical countries, contributing to an entrenched cycle of poverty. Only one human filarial parasite, Brugia malayi, can be maintained in rodents in the laboratory setting. It has been a widely used model organism in experiments that employ culture systems, the impact of which on the worms is unknown.Methodology/Principal FindingsUsing Illumina RNA sequencing, we characterized changes in gene expression upon in vitro maintenance of adult B. malayi female worms at four time points: immediately upon removal from the host, immediately after receipt following shipment, and after 48 h and 5 days in liquid culture media. The dramatic environmental change and the 24 h time lapse between removal from the host and establishment in culture caused a globally dysregulated gene expression profile. We found a maximum of 562 differentially expressed genes based on pairwise comparison between time points. After an initial shock upon removal from the host and shipping, a few stress fingerprints remained after 48 h in culture and until the experiment was stopped. This was best illustrated by a strong and persistent up-regulation of several genes encoding cuticle collagens, as well as serpins.Conclusions/SignificanceThese findings suggest that B. malayi can be maintained in culture as a valid system for pharmacological and biological studies, at least for several days after removal from the host and adaptation to the new environment. However, genes encoding several stress indicators remained dysregulated until the experiment was stopped.

A highly conserved, inhibitable astacin metalloprotease from Teladorsagia circumcincta is required for cuticle formation and nematode development

Parasitic nematodes cause chronic, debilitating infections in both livestock and humans worldwide, and many have developed multiple resistance to the currently available anthelmintics. The protective collagenous cuticle of these parasites is required for nematode survival and its synthesis has been studied extensively in the free-living nematode, Caenorhabditis elegans. The collagen synthesis pathway is a complex, multi-step process involving numerous key enzymes, including the astacin metalloproteases. Nematode astacinsare crucial for C. elegans development, having specific roles in hatching, moulting and cuticle synthesis. NAS-35 (also called DPY-31) is a homologue of a vertebrate procollagen C-proteinase and performs a central role in cuticle formation of C. elegans as its mutation causes temperature-sensitive lethality and cuticle defects. The characterisation of DPY-31 from the ovine gastrointestinal nematode Teladorsagia circumcincta and its ability to rescue the C. elegans mutant is described. Compounds with a hydroxamate functional group have previously been shown to be potent inhibitors of procollagen C-proteinases and were therefore examined for inhibitory activity against the T. circumcincta enzyme. Phenotypic screening against T. circumcincta, Haemonchus contortus and C. elegans larval stages identified compounds that caused body morphology phenotypes consistent with the inhibition of proteases involved in cuticle collagen synthesis. These compounds correspondingly inhibited the activity of recombinant T. circumcincta DPY-31, supporting the hypothesis that this enzyme may represent a potentially novel anthelmintic drug target.

A Deep Sequencing Approach to Comparatively Analyze the Transcriptome of Lifecycle Stages of the Filarial Worm, Brugia malayi

BackgroundDeveloping intervention strategies for the control of parasitic nematodes continues to be a significant challenge. Genomic and post-genomic approaches play an increasingly important role for providing fundamental molecular information about these parasites, thus enhancing basic as well as translational research. Here we report a comprehensive genome-wide survey of the developmental transcriptome of the human filarial parasite Brugia malayi.Methodology/Principal FindingsUsing deep sequencing, we profiled the transcriptome of eggs and embryos, immature (≤3 days of age) and mature microfilariae (MF), third- and fourth-stage larvae (L3 and L4), and adult male and female worms. Comparative analysis across these stages provided a detailed overview of the molecular repertoires that define and differentiate distinct lifecycle stages of the parasite. Genome-wide assessment of the overall transcriptional variability indicated that the cuticle collagen family and those implicated in molting exhibit noticeably dynamic stage-dependent patterns. Of particular interest was the identification of genes displaying sex-biased or germline-enriched profiles due to their potential involvement in reproductive processes. The study also revealed discrete transcriptional changes during larval development, namely those accompanying the maturation of MF and the L3 to L4 transition that are vital in establishing successful infection in mosquito vectors and vertebrate hosts, respectively.Conclusions/SignificanceCharacterization of the transcriptional program of the parasite's lifecycle is an important step toward understanding the developmental processes required for the infectious cycle. We find that the transcriptional program has a number of stage-specific pathways activated during worm development. In addition to advancing our understanding of transcriptome dynamics, these data will aid in the study of genome structure and organization by facilitating the identification of novel transcribed elements and splice variants.

Regulation of Fertility, Survival, and Cuticle Collagen Function by the Caenorhabditis elegans eaf-1 and ell-1 Genes

EAF2, an androgen-regulated protein, interacts with members of the ELL (eleven-nineteen lysine-rich leukemia) transcription factor family and also acts as a tumor suppressor. Although these proteins control transcriptional elongation and perhaps modulate the effects of other transcription factors, the mechanisms of their actions remain largely unknown. To gain new insights into the biology of the EAF2 and ELL family proteins, we used Caenorhabditis elegans as a model to explore the in vivo roles of their worm orthologs. Through the use of transgenic worms, RNAi, and an eaf-1 mutant, we found that both genes are expressed in multiple cell types throughout the worm life cycle and that they play important roles in fertility, survival, and body size regulation. ELL-1 and EAF-1 likely contribute to these activities in part through modulating cuticle synthesis, given that we observed a disrupted cuticle structure in ell-1 RNAi-treated or eaf-1 mutant worms. Consistent with disruption of cuticle structure, loss of either ELL-1 or EAF-1 suppressed the rol phenotype of specific collagen mutants, possibly through the control of dpy-3, dpy-13, and sqt-3 collagen gene expression. Furthermore, we also noted the regulation of collagen expression by ELL overexpression in PC3 human prostate cancer cells. Together, these results reveal important roles for the eaf-1 and ell-1 genes in the regulation of extracellular matrix components.

C. elegans ADAMTS ADT-2 regulates body size by modulating TGFβ signaling and cuticle collagen organization

Organismal growth and body size are influenced by both genetic and environmental factors. We have utilized the strong molecular genetic techniques available in the nematode Caenorhabditis elegans to identify genetic determinants of body size. In C. elegans, DBL-1, a member of the conserved family of secreted growth factors known as the Transforming Growth Factor β superfamily, is known to play a major role in growth control. The mechanisms by which other determinants of body size function, however, is less well understood. To identify additional genes involved in body size regulation, a genetic screen for small mutants was previously performed. One of the genes identified in that screen was sma-21. We now demonstrate that sma-21 encodes ADT-2, a member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted metalloproteases. ADAMTS proteins are believed to remodel the extracellular matrix and may modulate the activity of extracellular signals. Genetic interactions suggest that ADT-2 acts in parallel with or in multiple size regulatory pathways. We demonstrate that ADT-2 is required for normal levels of expression of a DBL-1-responsive transcriptional reporter. We further demonstrate that adt-2 regulatory sequences drive expression in glial-like and vulval cells, and that ADT-2 activity is required for normal cuticle collagen fibril organization. We therefore propose that ADT-2 regulates body size both by modulating TGFβ signaling activity and by maintaining normal cuticle structure.

Tyrosylprotein sulfotransferase regulates collagen secretion in Caenorhabditis elegans.

The sulfation of tyrosine residues is an important post-translational modification involved in the regulation of protein function. We examined the activity of worm tyrosylprotein sulfotransferase (TPST-1) on a typical cuticle collagen, ROL-6, in C. elegans. We verified that TPST-1 sulfates three tyrosine residues of ROL-6 in vitro. We found that these tyrosine residues are important for the secretion of ROL-6::GFP. Mutant ROL-6::GFP proteins that contain more than two substitutions of the target tyrosine residues are severely deficient in cuticle localization. Consistently, knock down of tpst-1 blocked the cuticle localization of ROL-6::GFP. Therefore, the sulfation of ROL-6 by TPST-1 is critical for the proper localization of ROL-6. We also confirmed that worm TPST-1 is localized to the trans-Golgi network (TGN). Our results indicate that TPST-1 regulates cuticle organization by promoting the transport of ROL-6 from the TGN to the cuticle.

Collagen processing and cuticle formation is catalysed by the astacin metalloprotease DPY-31 in free-living and parasitic nematodes

The exoskeleton or cuticle performs many key roles in the development and survival of all nematodes. This structure is predominantly collagenous in nature and requires numerous enzymes to properly fold, modify, process and cross-link these essential structural proteins. The cuticle structure and its collagen components are conserved throughout the nematode phylum but differ from the collagenous matrices found in vertebrates. This structure, its formation and the enzymology of nematode cuticle collagen biogenesis have been elucidated in the free-living nematode Caenorhabditis elegans. The dpy-31 gene in C. elegans encodes a procollagen C-terminal processing enzyme of the astacin metalloprotease or bone morphogenetic protein class that, when mutated, results in a temperature-sensitive lethal phenotype associated with cuticle defects. In this study, orthologues of this essential gene have been identified in the phylogenetically diverse parasitic nematodes Haemonchus contortus and Brugia malayi. The DPY-31 protein is expressed in the gut and secretory system of C. elegans, a location also confirmed when a B. malayi transcriptional dpy-31 promoter–reporter gene fusion was expressed in C. elegans. Functional conservation between the nematode enzymes was supported by the fact that heterologous expression of the H. contortus dpy-31 orthologue in a C. elegans dpy-31 mutant resulted in the full rescue of the mutant body form. This interspecies conservation was further established when the recombinant nematode enzymes were found to have a similar range of inhibitable protease activities. In addition, the recombinant DPY-31 enzymes from both H. contortus and B. malayi were shown to efficiently process the C. elegans cuticle collagen SQT-3 at the correct C-terminal procollagen processing site.