The cell signaling adaptor protein EPS-8 is essential for C. elegans epidermal elongation and interacts with the ankyrin repeat protein VAB-19

Background

The epidermal cells of the C. elegans embryo undergo coordinated cell shape changes that result in the morphogenetic process of elongation. The cytoskeletal ankyrin repeat protein VAB-19 is required for cell shape changes and localizes to cell-matrix attachment structures. The molecular functions of VAB-19 in this process are obscure, as no previous interactors for VAB-19 have been described.

Methodology/Principal Findings

In screens for VAB-19 binding proteins we identified the signaling adaptor EPS-8. Within C. elegans epidermal cells, EPS-8 and VAB-19 colocalize at cell-matrix attachment structures. The central domain of EPS-8 is necessary and sufficient for its interaction with VAB-19. eps-8 null mutants, like vab-19 mutants, are defective in epidermal elongation and in epidermal-muscle attachment. The eps-8 locus encodes two isoforms, EPS-8A and EPS-8B, that appear to act redundantly in epidermal elongation. The function of EPS-8 in epidermal development involves its N-terminal PTB and central domains, and is independent of its C-terminal SH3 and actin-binding domains. VAB-19 appears to act earlier in the biogenesis of attachment structures and may recruit EPS-8 to these structures.

Conclusions/Significance

EPS-8 and VAB-19 define a novel pathway acting at cell-matrix attachments to regulate epithelial cell shape. This is the first report of a role for EPS-8 proteins in cell-matrix attachments. The existence of EPS-8B-like isoforms in Drosophila suggests this function of EPS-8 proteins could be conserved among other organisms.     

The Caenorhabditis elegans vab-10 spectraplakin isoforms protect the epidermis against internal and external forces

Morphogenesis of the Caenorhabditis elegans embryo is driven by actin microfilaments in the epidermis and by sarcomeres in body wall muscles. Both tissues are mechanically coupled, most likely through specialized attachment structures called fibrous organelles (FOs) that connect muscles to the cuticle across the epidermis. Here, we report the identification of new mutations in a gene known as vab-10, which lead to severe morphogenesis defects, and show that vab-10 corresponds to the C. elegans spectraplakin locus. Our analysis of vab-10 reveals novel insights into the role of this plakin subfamily. vab-10 generates isoforms related either to plectin (termed VAB-10A) or to microtubule actin cross-linking factor plakins (termed VAB-10B). Using specific antibodies and mutations, we show that VAB-10A and VAB-10B have distinct distributions and functions in the epidermis. Loss of VAB-10A impairs the integrity of FOs, leading to epidermal detachment from the cuticle and muscles, hence demonstrating that FOs are functionally and molecularly related to hemidesmosomes. We suggest that this isoform protects against forces external to the epidermis. In contrast, lack of VAB-10B leads to increased epidermal thickness during embryonic morphogenesis when epidermal cells change shape. We suggest that this isoform protects cells against tension that builds up within the epidermis.     

The ephrin VAB-2/EFN-1 functions in neuronal signaling to regulate epidermal morphogenesis in C. elegans

The Eph receptor VAB-1 is required in neurons for epidermal morphogenesis during C. elegans embryogenesis. Two models were proposed for the non-autonomous role of VAB-1: neuronal VAB-1 might signal directly to epidermis, or VAB-1 signaling between neurons might be required for epidermal development. We show that the ephrin VAB-2 (also known as EFN-1) is a ligand for VAB-1 and can function in neurons to regulate epidermal morphogenesis. In the absence of VAB-1 signaling, ephrin-expressing neurons are disorganized. vab-2/efn-1 mutations synergize with vab-1 kinase alleles, suggesting that VAB-2/EFN-1 may partly function in a kinase-independent VAB-1 pathway. Our data indicate that ephrin signaling between neurons is required nonautonomously for epidermal morphogenesis in C. elegans.     

The VAB-1 Eph receptor tyrosine kinase and SAX-3/Robo neuronal receptors function together during C. elegans embryonic morphogenesis

Mutations that affect the single C. elegans Eph receptor tyrosine kinase VAB-1 cause defects in cell movements during embryogenesis. Here, we provide genetic and molecular evidence that the VAB-1 Eph receptor functions with another neuronal receptor, SAX-3/Robo, for proper embryogenesis. Our analysis of sax-3 mutants shows that SAX-3/Robo functions with the VAB-1 Eph receptor for gastrulation cleft closure and ventral epidermal enclosure. In addition, SAX-3 functions autonomously for epidermal morphogenesis independently of VAB-1. A double-mutant combination between vab-1 and slt-1 unmasks a role for the SLT-1 ligand in embryogenesis. We provide evidence for a physical interaction between the VAB-1 tyrosine kinase domain and the juxtamembrane and CC1 region of the SAX-3/Robo receptor. Gene dosage, non-allelic non-complementation experiments and co-localization of the two receptors are consistent with a model in which these two receptors form a complex and function together during embryogenesis.     

The cytoskeleton and epidermal morphogenesis in C. elegans

During Caenorhabditis elegans development, the process of epidermal elongation converts the bean-shaped embryo into the long thin shape of the larval worm. Epidermal elongation results from changes in the shape of epidermal cells, which in turn result from changes in the epidermal cytoskeleton, the extracellular matrix, and in cell-matrix adhesion junctions. Here, we review the roles of cytoskeletal filament systems in epidermal cell shape change during elongation. Genetic and cell biological analyses have established that all three major cytoskeletal filament systems (actin microfilaments, microtubules, and intermediate filaments (IFs)) play distinct and essential roles in epidermal cell shape change. Recent work has also highlighted the importance of communication between these systems for their integrated function in epidermal elongation. Epidermal cells undergo reciprocal interactions with underlying muscle cells, which regulate the position and function of IF-containing cell-matrix adhesion structures within the epidermis. Elongation thus exemplifies the reciprocal tissue interactions of organogenesis.     

The cell junction protein VAB-9 regulates adhesion and epidermal morphology in C. elegans

Epithelial cell junctions are essential for cell polarity, adhesion and morphogenesis. We have analysed VAB-9, a cell junction protein in Caenorhabditis elegans. VAB-9 is a predicted four-pass integral membrane protein that has greatest similarity to BCMP1 (brain cell membrane protein 1, a member of the PMP22/EMP/Claudin family of cell junction proteins) and localizes to the adherens junction domain of C. elegans apical junctions. Here, we show that VAB-9 requires HMR-1/cadherin for localization to the cell membrane, and both HMP-1/alpha-catenin and HMP-2/beta-catenin for maintaining its distribution at the cell junction. In vab-9 mutants, morphological defects correlate with disorganization of F-actin at the adherens junction; however, localization of the cadherin-catenin complex and epithelial polarity is normal. These results suggest that VAB-9 regulates interactions between the cytoskeleton and the adherens junction downstream of or parallel to alpha-catenin and/or beta-catenin. Mutations in vab-9 enhance adhesion defects through functional loss of the cell junction genes apical junction molecule 1 (ajm-1) and discs large 1 (dlg-1), suggesting that VAB-9 is involved in cell adhesion. Thus, VAB-9 represents the first characterized tetraspan adherens junction protein in C. elegans and defines a new family of such proteins in higher eukaryotes.     

Alpha spectrin is essential for morphogenesis and body wall muscle formation in Caenorhabditis elegans

A common feature of multicellular animals is the ubiquitous presence of the spectrin cytoskeleton. Although discovered over 30 yr ago, the function of spectrin in non-erythrocytes has remained elusive. We have found that the spc-1 gene encodes the only alpha spectrin gene in the Caenorhabditis elegans genome. During embryogenesis, alpha spectrin localizes to the cell membrane in most if not all cells, starting at the first cell stage. Interestingly, this localization is dependent on beta spectrin but not beta(Heavy) spectrin. Furthermore, analysis of spc-1 mutants indicates that beta spectrin requires alpha spectrin to be stably recruited to the cell membrane. Animals lacking functional alpha spectrin fail to complete embryonic elongation and die just after hatching. These mutant animals have defects in the organization of the hypodermal apical actin cytoskeleton that is required for elongation. In addition, we find that the process of elongation is required for the proper differentiation of the body wall muscle. Specifically, when compared with myofilaments in wild-type animals the myofilaments of the body wall muscle in mutant animals are abnormally oriented relative to the longitudinal axis of the embryo, and the body wall muscle cells do not undergo normal cell shape changes.     

Kank Is an EB1 Interacting Protein that Localises to Muscle-Tendon Attachment Sites in Drosophila

Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. We report the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. We found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. We found that the mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells.     

Intermediate filaments are required for C. elegans epidermal elongation

Cytoplasmic intermediate filaments (cIFs) are thought to provide mechanical strength to vertebrate cells; however, their function in invertebrates has been largely unexplored. The Caenorhabditis elegans genome encodes multiple cIFs. The C. elegans ifb-1 locus encodes two cIF isoforms, IFB-1A and IFB-1B, that differ in their head domains. We show that both IFB-1 isoforms are expressed in epidermal cells, within which they are localized to muscle-epidermal attachment structures. Reduction in IFB-1A function by mutation or RNA interference (RNAi) causes epidermal fragility, abnormal epidermal morphogenesis, and muscle detachment, consistent with IFB-1A providing mechanical strength to epidermal attachment structures. Reduction in IFB-1B function causes morphogenetic defects and defective outgrowth of the excretory cell. Reduction in function of both IFB-1 isoforms results in embryonic arrest due to muscle detachment and failure in epidermal cell elongation at the 2-fold stage. Two other cIFs, IFA-2 and IFA-3, are expressed in epidermal cells. We show that loss of function in IFA-3 results in defects in morphogenesis indistinguishable from those of embryos lacking ifb-1. In contrast, IFA-2 is not required for embryonic morphogenesis. Our data indicate that IFB-1 and IFA-3 are likely the major cIF isoforms in embryonic epidermal attachment structures.     

VAB-10 spectraplakin acts in cell and nuclear migration in Caenorhabditis elegans

Cytoskeletal regulation is important in cell migration. The Caenorhabditis elegans gonadal distal tip cells (DTCs) offer a simple model with which to investigate the mechanism of cell migration in organogenesis. Here, we report that one of the spectraplakin isoforms, VAB-10B1, plays an essential role in cell and nuclear migration of DTCs by regulating the actin and microtubule (MT) cytoskeleton. In the vab-10(tk27) mutant, which lacks VAB-10B1, alignment of filamentous (F)-actin and MTs was weakly and severely disorganized, respectively, which resulted in a failure to translocate the DTC nucleus and a premature termination of DTC migration. An MT growing-tip marker, EBP-2-GFP, revealed that polarized outgrowth of MTs towards the nuclei of migrating DTCs was strikingly impaired in tk27 animals. A vab-10 mini-gene encoding only the actin- and MT-binding domains significantly rescued the gonadal defects, suggesting that VAB-10B1 has a role in linking actin and MT filaments. These results suggest that VAB-10B1/spectraplakin regulates the polarized alignment of MTs, possibly by linking F-actin and MTs, which enables normal nuclear translocation and cell migration of DTCs.     

The C. elegans LAR-like receptor tyrosine phosphatase PTP-3 and the VAB-1 Eph receptor tyrosine kinase have partly redundant functions in morphogenesis

Receptor-like protein-tyrosine phosphatases (RPTPs) form a diverse family of cell surface molecules whose functions remain poorly understood. The LAR subfamily of RPTPs has been implicated in axon guidance and neural development. Here we report the molecular and genetic analysis of the C. elegans LAR subfamily member PTP-3. PTP-3 isoforms are expressed in many tissues in early embryogenesis, and later become localized to neuronal processes and to epithelial adherens junctions. Loss of function in ptp-3 causes low-penetrance defects in gastrulation and epidermal development similar to those of VAB-1 Eph receptor tyrosine kinase mutants. Loss of function in ptp-3 synergistically enhances phenotypes of mutations in the C. elegans Eph receptor VAB-1 and a subset of its ephrin ligands, but does not show specific interactions with several other RTKs or morphogenetic mutants. The genetic interaction of vab-1 and ptp-3 suggests that LAR-like RPTPs and Eph receptors have related and partly redundant functions in C. elegans morphogenesis.     

Caenorhabditis elegans beta-G spectrin is dispensable for establishment of epithelial polarity, but essential for muscular and neuronal function

The Caenorhabditis elegans genome encodes one alpha spectrin subunit, a beta spectrin subunit (beta-G), and a beta-H spectrin subunit. Our experiments show that the phenotype resulting from the loss of the C. elegans alpha spectrin is reproduced by tandem depletion of both beta-G and beta-H spectrins. We propose that alpha spectrin combines with the beta-G and beta-H subunits to form alpha/beta-G and alpha/beta-H heteromers that perform the entire repertoire of spectrin function in the nematode. The expression patterns of nematode beta-G spectrin and vertebrate beta spectrins exhibit three striking parallels including: (1) beta spectrins are associated with the sites of cell-cell contact in epithelial tissues; (2) the highest levels of beta-G spectrin occur in the nervous system; and (3) beta spectrin-G in striated muscle is associated with points of attachment of the myofilament apparatus to adjacent cells. Nematode beta-G spectrin associates with plasma membranes at sites of cell-cell contact, beginning at the two-cell stage, and with a dramatic increase in intensity after gastrulation when most cell proliferation has been completed. Strikingly, depletion of nematode beta-G spectrin by RNA-mediated interference to undetectable levels does not affect the establishment of structural and functional polarity in epidermis and intestine. Contrary to recent speculation, beta-G spectrin is not associated with internal membranes and depletion of beta-G spectrin was not associated with any detectable defects in secretion. Instead beta-G spectrin-deficient nematodes arrest as early larvae with progressive defects in the musculature and nervous system. Therefore, C. elegans beta-G spectrin is required for normal muscle and neuron function, but is dispensable for embryonic elongation and establishment of early epithelial polarity. We hypothesize that heteromeric spectrin evolved in metazoans in response to the needs of cells in the context of mechanically integrated tissues that can withstand the rigors imposed by an active organism.     

PAT-12, a potential anti-nematode target, is a new spectraplakin partner essential for Caenorhabditis elegans hemidesmosome integrity and embryonic morphogenesis

Caenorhabditis elegans embryonic elongation depends on both epidermal and muscle cells. The hemidesmosome-like junctions, commonly called fibrous organelles (FOs), that attach the epidermis to the extracellular matrix ensure muscle anchoring to the cuticular exoskeleton and play an essential role during elongation.To further define how hemidesmosomes might control elongation, we searched for factors interacting with the core hemidesmosome component, the spectraplakin homolog VAB-10. Using the VAB-10 plakin domain as bait in a yeast two-hybrid screen, we identified the novel protein T17H7.4. We also identified T17H7.4 in an independent bioinformatic search for essential nematode-specific proteins that could define novel anti-nematode drug or vaccine targets. Interestingly, T17H7.4 corresponds to the C. elegans equivalent of the parasitic OvB20 antigen, and has a characteristic hemidesmosome distribution. We identified two mutations in T17H7.4, one of which defines the uncharacterized gene pat-12, previously identified in screens for genes required for muscle assembly. Using isoform-specific GFP constructs, we showed that one pat-12 isoform with a hemidesmosome distribution can rescue a pat-12 null allele. We further found that lack of pat-12 affects hemidesmosome integrity, with marked defects at the apical membrane. PAT-12 defines a novel component of C. elegans hemidesmosomes, which is required for maintaining their integrity. We suggest that PAT-12 helps maintaining VAB-10 attachment with matrix receptors.     

Multiple ephrins control cell organization in C. elegans using kinase-dependent and -independent functions of the VAB-1 Eph receptor

Eph receptor (EphR) tyrosine kinases and their ephrin ligands mediate direct cell-to-cell signaling. The C. elegans genome encodes four potential GPI-modified ephrins (EFN-1 to -4) and one EphR (VAB-1). Single and multiple ephrin mutants reveal functions for EFN-1, EFN-2, and EFN-3 in epidermal cell organization that, in aggregate, mirror those of VAB-1. Ephrin mutants have defects in head morphology and enclosure of the embryo by the epidermis and identify ephrin-EphR signaling functions involved in aligning and fusing tail and head epidermal cells, respectively. Biochemical analyses indicate that EFN-1, EFN-2, and EFN-3 jointly activate the VAB-1 tyrosine kinase in vivo. Mutant phenotypes and expression pattern analysis suggest that multiple ephrins are involved in distinct aspects of kinase-dependent and kinase-independent VAB-1 signaling required for proper cell organization during development in C. elegans.     

The C. elegans F-spondin family protein SPON-1 maintains cell adhesion in neural and non-neural tissues

The F-spondin family of extracellular matrix proteins has been implicated in axon outgrowth, fasciculation and neuronal cell migration, as well as in the differentiation and proliferation of non-neuronal cells. In screens for mutants defective in C. elegans embryonic morphogenesis, we identified SPON-1, the only C. elegans member of the spondin family. SPON-1 is synthesized in body muscles and localizes to integrin-containing structures on body muscles and to other basement membranes. SPON-1 maintains strong attachments of muscles to epidermis; in the absence of SPON-1, muscles progressively detach from the epidermis, causing defective epidermal elongation. In animals with reduced integrin function, SPON-1 becomes dose dependent, suggesting that SPON-1 and integrins function in concert to promote the attachment of muscles to the basement membrane. Although spon-1 mutants display largely normal neurite outgrowth, spon-1 synergizes with outgrowth defective mutants, revealing a cryptic role for SPON-1 in axon extension. In motoneurons, SPON-1 acts in axon guidance and fasciculation, whereas in interneurons SPON-1 maintains process position. Our results show that a spondin maintains cell-matrix adhesion in multiple tissues.     

Characterization of loss-of-function and gain-of-function Eph receptor tyrosine kinase signaling in C. elegans axon targeting and cell migration

To understand how our brains function, it is necessary to know how neurons position themselves and target their axons and dendrites to their correct locations. Several evolutionarily conserved axon guidance molecules have been shown to help navigate axons to their correct target site. The Caenorhabditis elegans Eph receptor tyrosine kinase (RTK), VAB-1, has roles in early neuroblast and epidermal cell movements, but its roles in axon guidance are not well understood. Here, we report that mutations that disrupt the VAB-1 Eph receptor tyrosine kinase cause incompletely penetrant defects in axonal targeting and neuronal cell body positioning. The predominant axonal defect in vab-1 mutant animals was an overextension axon phenotype. Interestingly, constitutively active VAB-1 tyrosine kinase signaling caused a lack of axon outgrowth or an early termination phenotype, opposite to the loss-of-function phenotype. The combination of loss-of-function and gain-of-function analyses suggests that the VAB-1 Eph RTK is required for targeting or limiting axons and neuronal cells to specific regions, perhaps by transducing a repellent or stop cue.     

The Caenorhabditis elegans p120 catenin homologue,JAC-1, modulates cadherin-catenin function during epidermal morphogenesis

The cadherin-catenin complex is essential for tissue morphogenesis during animal development. In cultured mammalian cells, p120 catenin (p120ctn) is an important regulator of cadherin-catenin complex function. However, information on the role of p120ctn family members in cadherin-dependent events in vivo is limited. We have examined the role of the single Caenorhabditis elegans p120ctn homologue JAC-1 (juxtamembrane domain [JMD]-associated catenin) during epidermal morphogenesis. Similar to other p120ctn family members, JAC-1 binds the JMD of the classical cadherin HMR-1, and GFP-tagged JAC-1 localizes to adherens junctions in an HMR-1-dependent manner. Surprisingly, depleting JAC-1 expression using RNA interference (RNAi) does not result in any obvious defects in embryonic or postembryonic development. However, jac-1(RNAi) does increase the severity and penetrance of morphogenetic defects caused by a hypomorphic mutation in the hmp-1/alpha-catenin gene. In these hmp-1 mutants, jac-1 depletion causes failure of the embryo to elongate into a worm-like shape, a process that involves contraction of the epidermis. Associated with failed elongation is the detachment of actin bundles from epidermal adherens junctions and failure to maintain cadherin in adherens junctions. These results suggest that JAC-1 acts as a positive modulator of cadherin function in C. elegans.     

Distinct cell guidance pathways controlled by the Rac and Rho GEF domains of UNC-73/TRIO in Caenorhabditis elegans

The cytoskeleton regulator UNC-53/NAV2 is required for both the anterior and posterior outgrowth of several neurons as well as that of the excretory cell while the kinesin-like motor VAB-8 is essential for most posteriorly directed migrations in Caenorhabditis elegans. Null mutations in either unc-53 or vab-8 result in reduced posterior excretory canal outgrowth, while double null mutants display an enhanced canal extension defect, suggesting the genes act in separate pathways to control this posteriorly directed outgrowth. Genetic analysis of putative interactors of UNC-53 or VAB-8, and cell-specific rescue experiments suggest that VAB-8, SAX-3/ROBO, SLT-1/Slit, and EVA-1 are functioning together in the outgrowth of the excretory canals, while UNC-53 appears to function in a parallel pathway with UNC-71/ADAM. The known VAB-8 interactor, the Rac/Rho GEF UNC-73/TRIO operates in both pathways, as isoform specific alleles exhibit enhancement of the phenotype in double-mutant combination with either unc-53 or vab-8. On the basis of these results, we propose a bipartite model for UNC-73/TRIO activity in excretory canal extension: a cell autonomous function that is mediated by the Rho-specific GEF domain of the UNC-73E isoform in conjunction with UNC-53 and UNC-71 and a cell nonautonomous function that is mediated by the Rac-specific GEF domain of the UNC-73B isoform, through partnering with VAB-8 and the receptors SAX-3 and EVA-1.     

Regulated trafficking of the MSP/Eph receptor during oocyte meiotic maturation in C. elegans

BACKGROUND: In C. elegans, a sperm-sensing mechanism regulates oocyte meiotic maturation and ovulation, tightly coordinating sperm availability and embryo production; sperm release the major sperm protein (MSP) signal to trigger meiotic resumption. Meiotic arrest depends on the parallel function of the oocyte VAB-1 MSP/Eph receptor and somatic G protein signaling. MSP promotes meiotic maturation by antagonizing Eph receptor signaling and counteracting inhibitory inputs from the gonadal sheath cells. RESULTS: Here, we present evidence suggesting that in the absence of the MSP ligand, the VAB-1 Eph receptor inhibits meiotic maturation while either in or in transit to the endocytic-recycling compartment. VAB-1::GFP localization to the RAB-11-positive endocytic-recycling compartment is independent of ephrins but is antagonized by MSP signaling. Two negative regulators of oocyte meiotic maturation, DAB-1/Disabled and RAN-1, interact with the VAB-1 receptor and are required for its accumulation in the endocytic-recycling compartment in the absence of MSP or sperm (hereafter referred to as MSP/sperm). Inactivation of the endosomal recycling regulators rme-1 or rab-11.1 causes a vab-1-dependent reduction in the meiotic-maturation rate in the presence of MSP/sperm. Further, we show that Galpha(s) signaling in the gonadal sheath cells, which is required for meiotic maturation in the presence of MSP/sperm, affects VAB-1::GFP trafficking in oocytes. CONCLUSIONS: Regulated endocytic trafficking of the VAB-1 MSP/Eph receptor contributes to the control of oocyte meiotic maturation in C. elegans. Eph receptor trafficking in other systems may be influenced by the conserved proteins DAB-1/Disabled and RAN-1 and by crosstalk with G protein signaling in neighboring cells.