TIAM-1 regulates polarized protrusions during dorsal intercalation in the Caenorhabditis elegans embryo through both its GEF and N-terminal domains.

Mediolateral cell intercalation is a morphogenetic strategy used throughout animal development to reshape tissues. Dorsal intercalation in the Caenorhabditis elegans embryo involves the mediolateral intercalation of two rows of dorsal epidermal cells to create a single row that straddles the dorsal midline, and thus is a simple model to study cell intercalation. Polarized protrusive activity during dorsal intercalation requires the C. elegans Rac and RhoG orthologs CED-10 and MIG-2, but how these GTPases are regulated during intercalation has not been thoroughly investigated. In this study, we characterized the role of the Rac-specific guanine nucleotide exchange factor (GEF) TIAM-1 in regulating actin-based protrusive dynamics during dorsal intercalation. We found that TIAM-1 can promote formation of the main medial lamellipodial protrusion extended by intercalating cells through its canonical GEF function, whereas its N-terminal domains function to negatively regulate the generation of ectopic filiform protrusions around the periphery of intercalating cells. We also show that the guidance receptor UNC-5 inhibits these ectopic filiform protrusions in dorsal epidermal cells and that this effect is in part mediated via TIAM-1. These results expand the network of proteins that regulate basolateral protrusive activity during directed rearrangement of epithelial cells in animal embryos.

TIAM-1 regulates polarized protrusions during dorsal intercalation in the C. elegans embryo through both its GEF and N-terminal domains.

Mediolateral cell intercalation is a morphogenetic strategy used throughout animal development to reshape tissues. Dorsal intercalation in the C. elegans embryo involves the mediolateral intercalation of two rows of dorsal epidermal cells to create a single row that straddles the dorsal midline, and so is a simple model to study cell intercalation. Polarized protrusive activity during dorsal intercalation requires the C. elegans Rac and RhoG orthologs CED-10 and MIG-2, but how these GTPases are regulated during intercalation has not been thoroughly investigated. In this study, we characterize the role of the Rac-specific guanine nucleotide exchange factor (GEF), TIAM-1, in regulating actin-based protrusive dynamics during dorsal intercalation. We find that TIAM-1 can promote protrusion formation through its canonical GEF function, while its N-terminal domains function to negatively regulate this activity, preventing the generation of ectopic protrusions in intercalating cells. We also show that the guidance receptor UNC-5 inhibits ectopic protrusive activity in dorsal epidermal cells, and that this effect is in part mediated via TIAM-1. These results expand the network of proteins that regulate basolateral protrusive activity during directed cell rearrangement. Summary statement: TIAM-1 activates the Rac pathway to promote protrusion formation via its GEF domain, while its N-terminal domains suppress ectopic protrusions during dorsal intercalation in the C. elegans embryo.

FARL-11 (STRIP1/2) is required for sarcomere and sarcoplasmic reticulum organization in C. elegans.

Protein phosphatase 2A (PP2A) functions in a variety of cellular contexts. PP2A can assemble into four different complexes based on the inclusion of different regulatory or targeting subunits. The B''' regulatory subunit "striatin" forms the STRIPAK complex consisting of striatin, a catalytic subunit (PP2AC), striatin-interacting protein 1 (STRIP1), and MOB family member 4 (MOB4). In yeast and Caenorhabditis elegans, STRIP1 is required for formation of the endoplasmic reticulum (ER). Because the sarcoplasmic reticulum (SR) is the highly organized muscle-specific version of ER, we sought to determine the function of the STRIPAK complex in muscle using C. elegans. CASH-1 (striatin) and FARL-11 (STRIP1/2) form a complex in vivo, and each protein is localized to SR. Missense mutations and single amino acid losses in farl-11 and cash-1 each result in similar sarcomere disorganization. A missense mutation in farl-11 shows no detectable FARL-11 protein by immunoblot, disruption of SR organization around M-lines, and altered levels of the SR Ca+2 release channel UNC-68.

SRGP-1/srGAP and AFD-1/afadin stabilize HMP-1/⍺-catenin at rosettes to seal internalization sites following gastrulation in C. elegans.

A hallmark of gastrulation is the establishment of germ layers by internalization of cells initially on the exterior. In C. elegans the end of gastrulation is marked by the closure of the ventral cleft, a structure formed as cells internalize during gastrulation, and the subsequent rearrangement of adjacent neuroblasts that remain on the surface. We found that a nonsense allele of srgp-1/srGAP leads to 10-15% cleft closure failure. Deletion of the SRGP-1/srGAP C-terminal domain led to a comparable rate of cleft closure failure, whereas deletion of the N-terminal F-BAR region resulted in milder defects. Loss of the SRGP-1/srGAP C-terminus or F-BAR domain results in defects in rosette formation and defective clustering of HMP-1/⍺-catenin in surface cells during cleft closure. A mutant form of HMP-1/⍺-catenin with an open M domain can suppress cleft closure defects in srgp-1 mutant backgrounds, suggesting that this mutation acts as a gain-of-function allele. Since SRGP-1 binding to HMP-1/⍺-catenin is not favored in this case, we sought another HMP-1 interactor that might be recruited when HMP-1/⍺-catenin is constitutively open. A good candidate is AFD-1/afadin, which genetically interacts with cadherin-based adhesion later during embryonic elongation. AFD-1/afadin is prominently expressed at the vertex of neuroblast rosettes in wildtype, and depletion of AFD-1/afadin increases cleft closure defects in srgp-1/srGAP and hmp-1R551/554A/⍺-catenin backgrounds. We propose that SRGP-1/srGAP promotes nascent junction formation in rosettes; as junctions mature and sustain higher levels of tension, the M domain of HMP-1/⍺-catenin opens, allowing maturing junctions to transition from recruitment of SRGP-1/srGAP to AFD-1/afadin. Our work identifies new roles for ⍺-catenin interactors during a process crucial to metazoan development.

FARL-11 (STRIP1/2) is Required for Sarcomere and Sarcoplasmic Reticulum Organization in C. elegans.

Protein phosphatase 2A (PP2A) functions in a variety of cellular contexts. PP2A can assemble into four different complexes based on the inclusion of different regulatory or targeting subunits. The B''' regulatory subunit "striatin" forms the STRIPAK complex consisting of striatin, a catalytic subunit (PP2AC), striatin interacting protein 1 (STRIP1), and MOB family member 4 (MOB4). In yeast and C. elegans, STRIP1 is required for formation of the endoplasmic reticulum (ER). Since the sarcoplasmic reticulum (SR) is the highly organized muscle-specific version of ER, we sought to determine the function of the STRIPAK complex in muscle using C. elegans . CASH-1 (striatin) and FARL-11 (STRIP1/2) form a complex in vivo , and each protein is localized to SR. Missense mutations and single amino acid losses in farl-11 and cash-1 each result in similar sarcomere disorganization. A missense mutation in farl-11 shows no detectable FARL-11 protein by immunoblot, disruption of SR organization around M-lines, and altered levels of the SR Ca +2 release channel UNC-68. Summary: Protein phosphatase 2A forms a STRIPAK complex when it includes the targeting B''' subunit "striatin" and STRIP1. STRIP1 is required for formation of ER. We show that in muscle STRIP1 is required for organization of SR and sarcomeres.

TES-1/Tes and ZYX-1/Zyxin protect junctional actin networks under tension during epidermal morphogenesis in the C. elegans embryo.

LIM-domain-containing repeat (LCR) proteins are recruited to strained actin filaments within stress fibers in cultured cells,1,2,3 but their roles at cell-cell junctions in living organisms have not been extensively studied. Here, we show that the Caenorhabditis elegans LCR proteins TES-1/Tes and ZYX-1/Zyxin are recruited to apical junctions during embryonic elongation when junctions are under tension. In genetic backgrounds in which embryonic elongation fails, junctional recruitment is severely compromised. The two proteins display complementary patterns of expression: TES-1 is expressed in lateral (seam) epidermal cells, whereas ZYX-1 is expressed in dorsal and ventral epidermal cells. tes-1 and zyx-1 mutant embryos display junctional F-actin defects. The loss of either protein strongly enhances morphogenetic defects in hypomorphic mutant backgrounds for cadherin/catenin complex (CCC) components. The LCR regions of TES-1 and ZYX-1 are recruited to stress fiber strain sites (SFSSs) in cultured vertebrate cells. Together, these data establish TES-1 and ZYX-1 as components of a multicellular, tension-sensitive system that stabilizes the junctional actin cytoskeleton during embryonic morphogenesis.

C. elegans srGAP is an α-catenin M domain-binding protein that strengthens cadherin-dependent adhesion during morphogenesis.

The cadherin-catenin complex (CCC) is central to embryonic development and tissue repair, yet how CCC binding partners function alongside core CCC components remains poorly understood. Here, we establish a previously unappreciated role for an evolutionarily conserved protein, the slit-robo GTPase-activating protein SRGP-1/srGAP, in cadherin-dependent morphogenetic processes in the Caenorhabditis elegans embryo. SRGP-1 binds to the M domain of the core CCC component, HMP-1/α-catenin, via its C terminus. The SRGP-1 C terminus is sufficient to target it to adherens junctions, but only during later embryonic morphogenesis, when junctional tension is known to increase. Surprisingly, mutations that disrupt stabilizing salt bridges in the M domain block this recruitment. Loss of SRGP-1 leads to an increase in mobility and decrease of junctional HMP-1. In sensitized genetic backgrounds with weakened adherens junctions, loss of SRGP-1 leads to late embryonic failure. Rescue of these phenotypes requires the C terminus of SRGP-1 but also other domains of the protein. Taken together, these data establish a role for an srGAP in stabilizing and organizing the CCC during epithelial morphogenesis by binding to a partially closed conformation of α-catenin at junctions.

The Lamellipodin homologue MIG-10 is not essential for dorsal intercalation in the embryonic epidermis of the C. elegans embryo.

Dorsal intercalation of the embryonic epidermis in the Caenorhabditis elegans embryo is a promising system for genetic analysis of convergent extension, a conserved process in animal embryos. We sought to identify functionally important actin regulators in dorsal epidermal cells. A promising candidate is MIG-10, the single MIG-10/RIAM/Lamellipodin (MRL) family member in C. elegans. We endogenously tagged all mig-10 isoforms with mNeonGreen and analyzed mig-10 mutants using 4-dimensional microscopy. MIG-10::mNG is expressed prominently in muscle progenitors but is not detectable in the dorsal epidermis. mig-10(ct41) homozygotes complete dorsal intercalation in a manner indistinguishable from wildtype, indicating MIG-10 is not essential during dorsal intercalation.

Imaging Epidermal Cell Rearrangement in the C. elegans Embryo.

The Caenorhabditis elegans embryo is well suited for analysis of directed cell rearrangement via modern microscopy, due to its simple organization, short generation time, transparency, invariant lineage, and the ability to generate engineered embryos expressing various fluorescent proteins. This chapter provides an overview of routine microscopy techniques for imaging dorsal intercalation, a convergent extension-like morphogenetic movement in the embryonic epidermis of C. elegans, including making agar mounts, low-cost four-dimensional (4D) Nomarski microscopy, laser microsurgery, and 4D fluorescence microscopy using actin and junctional fusion proteins, as well as tissue-specific promoters useful for studying dorsal intercalation.

The adhesion modulation domain of Caenorhabditis elegans α-catenin regulates actin binding during morphogenesis.

Maintaining tissue integrity during epidermal morphogenesis depends on α-catenin, which connects the cadherin complex to F-actin. We show that the adhesion modulation domain (AMD) of Caenorhabditis elegans HMP-1/α-catenin regulates its F-actin-binding activity and organization of junctional-proximal actin in vivo. Deleting the AMD increases F-actin binding in vitro and leads to excess actin recruitment to adherens junctions in vivo. Reducing actin binding through a compensatory mutation in the C-terminus leads to improved function. Based on the effects of phosphomimetic and nonphosphorylatable mutations, phosphorylation of S509, within the AMD, may regulate F-actin binding. Taken together, these data establish a novel role for the AMD in regulating the actin-binding ability of an α-catenin and its proper function during epithelial morphogenesis.

Cell rearrangement induced by filopodial tension accounts for the late phase of convergent extension in the sea urchin archenteron.

George Oster was a pioneer in using mechanical models to interrogate morphogenesis in animal embryos. Convergent extension is a particularly important morphogenetic process to which George Oster gave significant attention. Late elongation of the sea urchin archenteron is a classic example of convergent extension in a monolayered tube, which has been proposed to be driven by extrinsic axial tension due to the activity of secondary mesenchyme cells. Using a vertex-based mechanical model, we show that key features of archenteron elongation can be accounted for by passive cell rearrangement due to applied tension. The model mimics the cell elongation and the Poisson effect (necking) that occur in actual archenterons. We also show that, as predicted by the model, ablation of secondary mesenchyme cells late in archenteron elongation does not result in extensive elastic recoil. Moreover, blocking the addition of cells to the base of the archenteron late in archenteron elongation leads to excessive cell rearrangement consistent with tension-induced rearrangement of a smaller cohort of cells. Our mechanical simulation suggests that responsive rearrangement can account for key features of archenteron elongation and provides a useful starting point for designing future experiments to examine the mechanical properties of the archenteron.

Mind the (sr)GAP - roles of Slit-Robo GAPs in neurons, brains and beyond.

The Slit-Robo GTPase-activating proteins (srGAPs) were first identified as potential Slit-Robo effectors that influence growth cone guidance. Given their N-terminal F-BAR, central GAP and C-terminal SH3 domains, srGAPs have the potential to affect membrane dynamics, Rho family GTPase activity and other binding partners. Recent research has clarified how srGAP family members act in distinct ways at the cell membrane, and has expanded our understanding of the roles of srGAPs in neuronal and non-neuronal cells. Gene duplication of the human-specific paralog of srGAP2 has resulted in srGAP2 family proteins that may have increased the density of dendritic spines and promoted neoteny of the human brain during crucial periods of human evolution, underscoring the importance of srGAPs in the unique sculpting of the human brain. Importantly, srGAPs also play roles outside of the nervous system, including during contact inhibition of cell movement and in establishing and maintaining cell adhesions in epithelia. Changes in srGAP expression may contribute to neurodevelopmental disorders, cancer metastasis and inflammation. As discussed in this Review, much remains to be discovered about how this interesting family of proteins functions in a diverse set of processes in metazoans and the functional roles srGAPs play in human disease.

Caenorhabditis elegans SORB-1 localizes to integrin adhesion sites and is required for organization of sarcomeres and mitochondria in myocytes.

We have identified and characterized sorb-1, the only sorbin and SH3 domain-containing protein family member in Caenorhabditis elegans SORB-1 is strongly localized to integrin adhesion complexes in larvae and adults, including adhesion plaques and dense bodies (Z-disks) of striated muscles and attachment plaques of smooth muscles. SORB-1 is recruited to the actin-binding, membrane-distal regions of dense bodies via its C-terminal SH3 domains in an ATN-1(α-actinin)- and ALP-1(ALP/Enigma)-dependent manner, where it contributes to the organization of sarcomeres. SORB-1 is also found in other tissues known to be under mechanical stress, including stress fibers in migratory distal tip cells and the proximal gonad sheath, where it becomes enriched in response to tissue distention. We provide evidence for a novel role for sorbin family proteins: SORB-1 is required for normal positioning of the mitochondrial network in muscle cells. Finally, we demonstrate that SORB-1 interacts directly with two other dense body components, DEB-1(vinculin) and ZYX-1(zyxin). This work establishes SORB-1 as a bona fide sorbin family protein-one of the late additions to the dense body complex and a conserved regulator of body wall muscle sarcomere organization and organelle positioning.

Cell-cell adhesion in metazoans relies on evolutionarily conserved features of the α-catenin·ß-catenin-binding interface.

Stable tissue integrity during embryonic development relies on the function of the cadherin·catenin complex (CCC). The Caenorhabditis elegans CCC is a useful paradigm for analyzing in vivo requirements for specific interactions among the core components of the CCC, and it provides a unique opportunity to examine evolutionarily conserved mechanisms that govern the interaction between α- and ß-catenin. HMP-1, unlike its mammalian homolog α-catenin, is constitutively monomeric, and its binding affinity for HMP-2/ß-catenin is higher than that of α-catenin for ß-catenin. A crystal structure shows that the HMP-1·HMP-2 complex forms a five-helical bundle structure distinct from the structure of the mammalian α-catenin·ß-catenin complex. Deletion analysis based on the crystal structure shows that the first helix of HMP-1 is necessary for binding HMP-2 avidly in vitro and for efficient recruitment of HMP-1 to adherens junctions in embryos. HMP-2 Ser-47 and Tyr-69 flank its binding interface with HMP-1, and we show that phosphomimetic mutations at these two sites decrease binding affinity of HMP-1 to HMP-2 by 40-100-fold in vitro. In vivo experiments using HMP-2 S47E and Y69E mutants showed that they are unable to rescue hmp-2(zu364) mutants, suggesting that phosphorylation of HMP-2 on Ser-47 and Tyr-69 could be important for regulating CCC formation in C. elegans Our data provide novel insights into how cadherin-dependent cell-cell adhesion is modulated in metazoans by conserved elements as well as features unique to specific organisms.

Structural and functional characterization of Caenorhabditis elegans α-catenin reveals constitutive binding to ß-catenin and F-actin.

Intercellular epithelial junctions formed by classical cadherins, ß-catenin, and the actin-binding protein α-catenin link the actin cytoskeletons of adjacent cells into a structural continuum. These assemblies transmit forces through the tissue and respond to intracellular and extracellular signals. However, the mechanisms of junctional assembly and regulation are poorly understood. Studies of cadherin-catenin assembly in a number of metazoans have revealed both similarities and unexpected differences in the biochemical properties of the cadherin·catenin complex that likely reflect the developmental and environmental requirements of different tissues and organisms. Here, we report the structural and biochemical characterization of HMP-1, the Caenorhabditis elegans α-catenin homolog, and compare it with mammalian α-catenin. HMP-1 shares overall similarity in structure and actin-binding properties, but displayed differences in conformational flexibility and allosteric regulation from mammalian α-catenin. HMP-1 bound filamentous actin with an affinity in the single micromolar range, even when complexed with the ß-catenin homolog HMP-2 or when present in a complex of HMP-2 and the cadherin homolog HMR-1, indicating that HMP-1 binding to F-actin is not allosterically regulated by the HMP-2·HMR-1 complex. The middle (i.e. M) domain of HMP-1 appeared to be less conformationally flexible than mammalian α-catenin, which may underlie the dampened effect of HMP-2 binding on HMP-1 actin-binding activity compared with that of the mammalian homolog. In conclusion, our data indicate that HMP-1 constitutively binds ß-catenin and F-actin, and although the overall structure and function of HMP-1 and related α-catenins are similar, the vertebrate proteins appear to be under more complex conformational regulation.

CDC-42 Orients Cell Migration during Epithelial Intercalation in the Caenorhabditis elegans Epidermis.

Cell intercalation is a highly directed cell rearrangement that is essential for animal morphogenesis. As such, intercalation requires orchestration of cell polarity across the plane of the tissue. CDC-42 is a Rho family GTPase with key functions in cell polarity, yet its role during epithelial intercalation has not been established because its roles early in embryogenesis have historically made it difficult to study. To circumvent these early requirements, in this paper we use tissue-specific and conditional loss-of-function approaches to identify a role for CDC-42 during intercalation of the Caenorhabditis elegans dorsal embryonic epidermis. CDC-42 activity is enriched in the medial tips of intercalating cells, which extend as cells migrate past one another. Moreover, CDC-42 is involved in both the efficient formation and orientation of cell tips during cell rearrangement. Using conditional loss-of-function we also show that the PAR complex functions in tip formation and orientation. Additionally, we find that the sole C. elegans Eph receptor, VAB-1, functions during this process in an Ephrin-independent manner. Using epistasis analysis, we find that vab-1 lies in the same genetic pathway as cdc-42 and is responsible for polarizing CDC-42 activity to the medial tip. Together, these data establish a previously uncharacterized role for polarized CDC-42, in conjunction with PAR-6, PAR-3 and an Eph receptor, during epithelial intercalation.

Another morphogenetic movement on the map: Charting dorsal intercalation in C. elegans.

Dorsal intercalation is a coordinated cell migration event that rearranges hypodermal cells during C. elegans embryogenesis, and that resembles cell intercalation in many systems from flies to mice. Despite its conservation, the molecular mechanisms that govern dorsal intercalation in worms have remained elusive. Here, we comment on our recent publication, Walck-Shannon et al.,(1) which begins to spatially map the molecular requirements for intercalation. First, we provide a historical perspective on the factors that have previously hampered the study of dorsal intercalation. Next, we provide a summary of the molecular pathways identified in Walck-Shannon et al.,(1) pointing out surprises along the way. Finally, we consider the potential conservation of the molecular pathway we described and discuss future questions surrounding dorsal intercalation. Despite the challenges, dorsal intercalation is a process poised to advance our understanding of cell intercalation during morphogenesis throughout the animal kingdom.

Regulating cell-cell junctions from A to Z.

Epithelial sheets often present a "cobblestone" appearance, but the mechanisms underlying the dynamics of this arrangement are unclear. In this issue, Choi et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201506115) show that afadin and ZO-1 regulate tension and maintain zonula adherens architecture in response to changes in contractility.

Phosphoregulation of the C. elegans cadherin-catenin complex.

Adherens junctions play key roles in mediating cell-cell contacts during tissue development. In Caenorhabditis elegans embryos, the cadherin-catenin complex (CCC), composed of the classical cadherin HMR-1 and members of three catenin families, HMP-1, HMP-2 and JAC-1, is necessary for normal blastomere adhesion, gastrulation, ventral enclosure of the epidermis and embryo elongation. Disruption of CCC assembly or function results in embryonic lethality. Previous work suggests that components of the CCC are subject to phosphorylation. However, the identity of phosphorylated residues in CCC components and their contributions to CCC stability and function in a living organism remain speculative. Using mass spectrometry, we systematically identify phosphorylated residues in the essential CCC subunits HMR-1, HMP-1 and HMP-2 in vivo. We demonstrate that HMR-1/cadherin phosphorylation occurs on three sites within its ß-catenin binding domain that each contributes to CCC assembly on lipid bilayers. In contrast, phosphorylation of HMP-2/ß-catenin inhibits its association with HMR-1/cadherin in vitro, suggesting a role in CCC disassembly. Although HMP-1/α-catenin is also phosphorylated in vivo, phosphomimetic mutations do not affect its ability to associate with other CCC components or interact with actin in vitro. Collectively, our findings support a model in which distinct phosphorylation events contribute to rapid CCC assembly and disassembly, both of which are essential for morphogenetic rearrangements during development.