SRGP-1/srGAP and AFD-1/Afadin stabilize HMP-1/α-Catenin at rosettes to seal internalization sites following gastrulation inC. elegans

SRGP-1/srGAP and AFD-1/Afadin stabilize HMP-1/α-Catenin at rosettes to seal internalization sites following gastrulation in C. elegans | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results SRGP-1/srGAP and AFD-1/Afadin stabilize HMP-1/α-Catenin at rosettes to seal internalization sites following gastrulation in C. elegans Joel M. Serre , Mark M. Slabodnick , Bob Goldstein , View ORCID Profile Jeff Hardin doi: https://doi.org/10.1101/2022.11.04.515144 Joel M. Serre a Program in Genetics, University of Wisconsin-Madison Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mark M. Slabodnick b Department of Biology, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina, 27599 USA c Department of Biology, Knox University , Galesburg, Illinois, 61401 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bob Goldstein b Department of Biology, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina, 27599 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jeff Hardin a Program in Genetics, University of Wisconsin-Madison d Department of Integrative Biology, University of Wisconsin-Madison Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jeff Hardin For correspondence: jdhardin{at}wisc.edu Abstract Full Text Info/History Metrics Preview PDF Abstract 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 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 C-terminus or F-BAR domain results in defects in rosette formation and defective clustering of HMP-1/α-catenin in surface cells during cleft closure. A mutant form of HMP-1 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 is not favored in this case, we sought another HMP-1 interactor that might be recruited when HMP-1 is constitutively open. A good candidate is AFD-1/Afadin, which genetically interacts with cadherin-based adhesion later during embryonic elongation. AFD-1 is prominently expressed at the vertex of neuroblast rosettes in wildtype, and depletion of AFD-1/Afadin increases cleft closure defects in srgp-1 and hmp-1 R551/554A backgrounds. We propose that SRGP-1 promotes nascent junction formation in rosettes; as junctions mature and sustain higher levels of tension, the M domain of HMP-1 opens, allowing maturing junctions to transition from recruitment of SRGP-1 to AFD-1. Our work identifies new roles for α-catenin interactors during a process crucial to metazoan development. Introduction Gastrulation is a hallmark of metazoan development that establishes the basic body plan [ 1 ]. In many organisms, internalization of founder cells that form the three primary germ layers, as well as primordial germ cells, occurs via detachment of the apical surfaces of individual cells from the embryo’s exterior [ 2 – 5 ]. Such internalization can involve an epithelial-mesenchymal transition (EMT), as cells dismantle their cell-cell junctional machinery and detach [ 6 , 7 ]; in other cases, a true EMT does not occur [ 5 , 8 ]. Neighboring cells that remain on the exterior must seal the breach left behind by internalizing cells, rearranging and making new cell-cell junctional connections as they do so. While cell internalization is essential for successful gastrulation in numerous organisms, most of the focus thus far has been on cellular events within internalizing cells; relatively less attention has been paid to neighboring cells that seal the embryonic exterior. The early C. elegans embryo is a useful model system for understanding changes in cell-cell adhesion associated with cell internalization. Gastrulation in C. elegans involves stereotypical events on the ventral surface of the embryo that internalize endodermal, mesodermal, and germ cell precursors [ 5 , 9 ]. The best studied of these events is the internalization of Ea and Ep, the endodermal precursors. Ea/p undergo myosin-mediated apical constriction [ 8 , 10 – 13 ]. Germ cell precursors rely on a different mechanism, involving cadherin-dependent “hitchhiking” [ 14 ]. Concomitant with internalization of Ea/p, neighboring cells have been observed to produced protrusions that may aid resealing of the embryo’s surface via active crawling [ 15 ]. Together with apical constriction of internalizing cells themselves, these movements are thought to aid cell internalization and simultaneous resealing of the ventral surface [ 12 , 15 ]. A ventral cleft forms on the surface of the embryo as the last sets of cells are internalized at the end of gastrulation. The ventral cleft is surrounded by neuroblasts derived from ABplp and ABprp in the posterior and ABalp and ABarp in the anterior. The ventral gastrulation cleft is subsequently closed via movements of ventral neuroblasts toward the ventral midline between 230 and 290 minutes postfertilization, causing the ventral cleft to disappear approximately one hour before the movements of ventral epidermal enclosure begin [ 16 , 17 ]. Failures in ventral cleft closure lead to highly penetrant failure of ventral enclosure (for reviews of this process, see [ 9 , 18 , 19 ]). Defects in the movement of neuroblasts to close the ventral cleft are observed in embryos defective in several cell signaling pathways, including those involving Eph/ephrin signaling [ 16 , 20 , 21 ], PTP-3/LAR (Leukocyte Common Antigen Related Receptor, a protein tyrosine phosphatase; [ 22 ]), semaphorin-2A/MAB-20/plexin signaling [ 23 , 24 ], and the C. elegans Kallmann syndrome ortholog kal-1 [ 25 , 26 ]. Such defects result in an enlarged or persistent ventral cleft; if the ventral cleft is not closed by the time of epidermal enclosure, enclosure movements are often disrupted. The motile events downstream of cell signaling at the ventral cleft are poorly understood; loss of function of the SCAR/WAVE gene wve-1 leads to significant defects in ventral neuroblast organization [ 27 ], suggesting that actin-based motility may be important for ventral neuroblast movement. Filopodial protrusions have been observed during cleft closure, but their significance is unclear [ 28 ]. As ventral neuroblasts move together, surrounding cells adjacent to the cleft must rearrange as the cleft closes [ 12 ]. After the events of ventral cleft closure, the neuroblasts that seal the cleft divide and rearrange to form part of the presumptive ventral nerve cord before the embryo begins to elongate into a vermiform shape [ 9 , 29 ]. Ventral neuroblasts later accumulate myosin foci and cadherin complex proteins [ 30 ]. Internalization of cells during gastrulation in C. elegans involves detachment of cells from their neighbors and establishment of new connections among cells remaining at the ventral surface, so changes in cell-cell adhesion must presumably occur during this process. The C. elegans cadherin/catenin complex (CCC) has been the focus of significant attention in this regard. The core components of the CCC, HMR-1/cadherin, HMP-2/β-catenin, and HMP-1/α-catenin, are present in the early embryo before gastrulation begins [ 11 , 14 , 31 ]. While there is not an essential requirement for cadherin-dependent adhesion during Ea/p internalization in otherwise wild-type embryos, there is a synergistic requirement for the cadherin complex when the L1CAM homologue SAX-7 or CED-5/DOCK180 is depleted [ 32 , 33 ]. Accumulation of CCC components at the interface between cells that internalize and those that remain on the surface has been proposed to aid recruitment of actomyosin contractile networks necessary for internalization [ 5 , 11 ], after engagement of an actomyosin-mediated “clutch” in Ea/p [ 13 ]. Much of the focus regarding the CCC during gastrulation has been on the internalizing cells, specifically Ea/p. Requirements for the CCC in subsequent internalizations have not been specifically analyzed, nor has the role of the CCC in resealing the ventral surface after internalization been assessed. We set out to investigate roles for the core CCC component, HMP-1, in these processes. In addition, we turned our attention to SRGP-1/srGAP, the lone slit/robo GTPase activating protein in C. elegans [ 28 , 34 ]. We showed previously that SRGP-1 is a modulator of cell-cell adhesion during the later events of ventral enclosure [ 28 ] and embryonic elongation [ 35 , 36 ]. In addition, however, srgp-1 knockdown in hmp-1 ( fe4 ) mutants leads to Gex ( G ut on the ex terior) phenotypes due to a failure to complete cleft closure [ 28 ], implicating it in the earlier events of internalization and ventral sealing at the end of gastrulation. SRGP-1 is a homolog of vertebrate Slit/Robo GTPase Activating Proteins (srGAPs), which have an N-terminal F-BAR domain that associates with curved membranes, a central RhoGAP domain, and an SH3 domain which has been shown to associate with various other factors such as WAVE, WASP, and Lamellipodin [ 37 – 39 ]. SRGP-1 in C. elegans does not contain an SH3 domain; nevertheless, we showed previously that the SRGP-1 C-terminus interacts with both the N-terminal half of SRGP-1 [ 28 ] and with HMP-1 [ 35 ]. Overexpression of the F-BAR domain of SRGP-1 leads to ectopic membrane tubulations. The C-terminus of SRGP-1 is required to recruit HMP-1 into these tubulations [ 28 ] and for normal HMP-1 dynamics [ 36 ], consistent with a role for the SRGP-1 C terminus is coordinating the interaction with HMP-1. Here we investigated the role of SRGP-1 prior to epidermal morphogenesis, as the ventral surface seals the final breaches due to cell internalization at the end of gastrulation. We found that SRGP-1 is required for normal cell behavior, cell morphology, and HMP-1 recruitment during this essential process. We also found that destabilizing salt bridge mutations within the M (middle) domain of HMP-1, which cause the M domain to remain in an extended state and abrogate binding by the SRGP-1 C terminus [ 35 ], are able to suppress SRGP-1 phenotypes. This suppression may be in part due to increased recruitment of components that interact with an open conformation of the M domain, including the C. elegans afadin homologue, AFD-1. Results Mutations in srgp-1 lead to cleft closure defects Our prior work established a role for SRGP-1 in the embryonic epidermis in C. elegans [ 28 , 35 ], but srGAPs in vertebrates were originally identified through their roles in the developing nervous system [ 39 – 43 ]. In C. elegans , a majority of neuroblasts are found on the ventral side of the embryo following gastrulation [ 9 , 17 ]. These neuroblasts must (1) adhere to one another to keep other tissues internalized during gastrulation (reviewed in [ 5 , 9 ]), (2) divide and rearrange to form part of the ventral nerve cord [ 29 ], and (3) act as a substrate for the epidermis, which undergoes epiboly during ventral enclosure [ 23 , 30 , 44 ]. Using 4D DIC microscopy, we observed that an appreciable percentage (11.1%) of homozygotes for srgp-1 ( jc72 ), a nonsense allele hereafter referred to as srgp-1 W122Stop ( Fig. 1A ), do not complete ventral cleft closure at the end of gastrulation, leading to endodermal precursors being extruded when the epidermis attempts to undergo epiboly and the contractions normally associated with embryonic elongation ( Figure 1B , second row). Download figure Open in new tab Figure 1 Genetic perturbation of srgp-1 leads to cleft closure defects. (A) A map depicting the domains of SRGP-1 including mutants used in this study ( srgp-1 W122Stop , srgp-1 R563A , srgp-1 ΔF-BAR , and srgp-1 ΔC ) (B) DIC images of embryos over 230 minutes. White dotted lines depict the ventral cleft. The first row depicts a typical wild-type embryo proceeding through cleft closure and into early elongation. srgp-1 W122Stop , srgp-1 ΔF-BAR , and srgp-1 ΔC mutants all display cleft closure failure, resulting in extruded endoderm (yellow dotted lines). Scale bar is 10 μm. (C) A graph depicting percentage cleft closure defects in wild-type and srgp-1 mutants. ****, p < 0.0001; *, p < 0.05. SRGP-1 has three major functional domains: (1) an N-terminal F-BAR domain, (2) a central GAP domain, and (3) an unstructured C-terminal region that is involved in protein-protein interactions (see Figure 1A , [ 28 , 34 , 35 , 45 ]) We explored whether one of these domains might be important for SRGP-1 function during cleft closure. Using CRISPR/Cas9 methodology, we generated the following alleles: srgp-1 Δ F-BAR , missense allele srgp-1 R563A , which prevents GAP activity [ 34 , 46 ], and srgp-1 ΔC , which deletes most of the region C-terminal to the GAP domain. Loss of the SRGP-1 F-BAR domain and C-terminal region both led to cleft closure defects following gastrulation ( Figure 1B ). The percentages of embryos that displayed cleft closure defects were similar between srgp-1 ΔC and srgp-1 W122Stop alleles; while we observed cleft closure defects in srgp-1 ΔF-BAR mutant embryos, the lower frequency did not rise to the level of statistical significance compared to wildtype, in which we did not observe cleft closure defects ( Figure 1C ). As in our previous studies examining the epidermal functions of SRGP-1 [ 28 ], we did not observe any obvious defects in embryos lacking SRGP-1 GAP functionality. These results suggest that important aspects of SRGP-1 function during cleft closure are mediated through its C terminus, with the F-BAR domain playing a supporting role. HMP-1 and SRGP-1 co-localize at the vertices of rosettes following the last internalization events of gastrulation Gastrulation in C. elegans involves the internalization of progenitor cells that generate endoderm, mesoderm, and germ line tissues. As these cells move into the interior, they undergo apical constriction. As they do so, neighboring cells form transient rosettes to cover the space vacated by the departing cells [ 12 ]. We examined endogenously tagged HMP-1::mScarlet-I and SRGP-1::mNeonGreen in living embryos, beginning with ventral cleft formation through the final internalization events of gastrulation, which occur after cleft closure ( Figure 2A ). Two rosettes form and resolve at this stage, involving cells born on the left and right sides of the ventral cleft ( Figure 2A , yellow dotted line; B, colored cells). At the vertex of the anterior rosette, where cells internalize, we observed a bright accumulation of HMP-1::mScarlet-I immediately after the internalization event ( Figure 2A , white arrowhead). Subsequent to the accumulation of HMP-1 in the anterior rosette, the posterior rosette resolved and elongated along the anterior-posterior axis, forming new cell contacts as it did so ( Figure 2A ; yellow arrowheads at 10 min indicate direction of cell movement by 20 min). Significantly, SRGP-1 also accumulated at vertices in both the anterior and posterior rosettes ( Fig. 2A , 0 min, arrowheads). Download figure Open in new tab Figure 2 Rosette formation leads to cell rearrangement during cleft closure. (A) An embryo expressing SRGP-1::mNeonGreen and HMP-1::mScarlet-I before, during, and after anterior rosette formation (ventral view), showing the cell rearrangements that take place to seal the ventral cleft. Times are relative to formation of the anterior rosette (t = 0 min). White dotted lines outline cells involved in rosette formation. Blue dotted lines indicate cells that internalize. White arrowhead indicates the vertex of the anterior rosette. Yellow arrows indicate the direction of cell movement at the posterior end of the embryo following anterior rosette formation. (B) Cell tracings of the embryos in (A). Internalizing cells are colored grey, cells that form rosettes are colored, all other cells are white. Scale bar is 10 μm. We previously demonstrated that homozygotes carrying a nonsense allele of srgp-1 display decreased HMP-1 junctional intensity [ 35 ]. We therefore sought to determine whether srgp-1 mutant backgrounds could influence the localization of HMP-1 during rosette formation, focusing on the anterior rosette. We examined localization of HMP-1::mScarlet-I within the anterior rosette before and after the final set of cell internalizations ( Figure 3 ; blue dotted line indicates internalizing cells). In a full-length, endogenously tagged srgp-1 background, HMP-1 accumulated at the vertex formed by the disappearance of internalizing cells ( Figure 3A , 0 min). In contrast, in srgp-1 ΔF-BAR mutants HMP-1 at the vertex failed to coalesce into a single cluster ( Figure 3B , 0 minutes). In addition, a stable rosette no longer formed, and the remaining neuroblasts instead coalesced into two rows with no central vertex ( Figure 3B , yellow lines). In srgp-1 ΔC mutants clusters of SRGP-1 accumulated within neuroblasts with no apparent pattern; while HMP-1 was still able to coalesce around the rosette, multiple clusters with accumulated HMP-1 were visible ( Figure 3C ). Taken together, these results indicate that both the SRGP-1 N- and C-terminal regions have important roles during cleft closure. The F-BAR domain appears to be important for organizing the tips of cells within rosettes into a single vertex, whereas the C-terminus may play an important role in spatially organizing and interacting with HMP-1 during this process. Download figure Open in new tab Figure 3 srgp-1 mutants display aberrant HMP-1::mScarlet-I aggregation and anterior rosette formation. Representative embryos expressing full-length or deleted SRGP-1::mNeonGreen, as well as HMP-1::mScarlet-I. ( A ) SRGP-1::mNeonGreen; ( B ) SRGP-1 ΔF-BAR ::mNeonGreen; (C) SRGP-1 ΔC ::mNeonGreen. Blue dotted lines indicate cell internalization that precedes formation of rosettes. White arrowheads indicate the vertex of the rosette following cell internalization. White dotted lines outline cells that contribute to the rosette; yellow dotted lines depict cells arranged around the vertex of the rosette. Scale bar is 5 μm. Destabilizing the M domain of HMP-1 suppresses cleft closure defects in srgp-1 mutants The M domain of HMP-1 forms a closed structure that is stabilized by multiple salt bridges ( Figure 4A ; [ 47 ]). Mutating Arginines 551 and 554 to alanines prevents two of these salt bridges from forming between MII and MIII; as a result, the HMP-1 M domain adopts a constitutively open conformation that prevents the recruitment of the SRGP-1 C-terminus [ 35 ]. Although the hmp-1 R551/554A mutation abrogates interaction with the C-terminus of SRGP-1, there is evidence in vertebrates that an extended conformation of α-catenin may activate actin binding and/or recruitment of other binding partners, including vinculin [ 48 – 53 ] and afadin [ 54 ]. We therefore assessed whether a constitutively open conformation of HMP-1 could bypass the requirement for SRGP-1 at the end of gastrulation. Download figure Open in new tab Figure 4 The hmp-1 R551/554A mutation suppresses defects due to srgp-1 loss of function and reduced actin binding ability of HMP-1. (A) A domain map of HMP-1 depicting the sites of the R551/554A and S823F variations. (B) Percentage cleft closure defects in srgp-1 and hmp-1 R551/554A mutants. (C) Fecundity of wildtype and various hmp-1 mutants at 20°C and 15°C. (D) Embryonic lethality of wildtype and various hmp-1 mutants at 20°C and 15°C. (E) Embryonic lethality of wildtype and various hmp-1 mutants subjected to control (empty vector L4440) or srgp-1 RNAi. (F) hmp-1 S823F and hmp-1 R551/554A; S823F hermaphrodites. Yellow arrowheads indicate abnormally shaped or swollen regions along the body. Red dotted lines indicate clubbed tails. Scale bar is 200 μm. ****, p < 0.0001; ***, p<0.001; **, p<0.01; *, p < 0.05. Mutants homozygous for hmp-1 R551/554A and either the srgp-1 W122Stop or the srpg-1 ΔC allele displayed fewer cleft closure defects compared to srgp-1 W122Stop or srpg-1 ΔC homozygotes with wild-type hmp-1; in contrast, there was no change in frequency of cleft closure defects in srgp-1 ΔF-BAR homozygotes when the salt bridge mutations were introduced ( Figure 4B ). These results suggest that an open conformation of the HMP-1 M domain can bypass some functions normally performed by the SRGP-1 C-terminus, but that the SRGP-1 F-BAR domain is still required, presumably independently of binding of the SRGP-1 C terminus to the HMP-1 M domain. To test if the salt bridge mutations in HMP-1 truly behave as gain-of-function mutations, we introduced these mutations into the hmp-1(fe4) background. The hmp-1 ( fe4 ) allele, hereafter referred to as hmp-1 S823F , replaces a serine with a phenylalanine (S823F) within the actin binding domain of HMP-1 and behaves as a hypomorph ( Figure 4A ; [ 55 , 56 ]). hmp-1 S823F homozygotes display morphogenetic failure and developmental arrest at a variety of stages, including during cleft closure. Using CRISPR/Cas9 we introduced the R551/554A mutations into the hmp-1 S823F background. Hermaphrodites homozygous for hmp-1 R551/554A; S823F on average laid more embryos and had minimal body morphology defects compared to hmp-1 S823F homozygotes ( Figure 4C,F ). hmp-1 R551/554A; S823F homozygous embryos also showed reduced lethality compared to embryos homozygous for hmp-1 S823F ( Figure 4D,E ). However, when we imaged hmp-1 R551/554A; S823F embryos, we found that they were more sensitive to mechanical pressure in 10% agar mounts and required mounting on 5% agar pads (Figure S1), which we used for subsequent experiments utilizing this allele. We also observed that hmp-1 S823F homozygotes exhibited increased embryonic lethality at colder temperatures ( Figure 4D ). We examined whether the hmp-1 R551/554A mutation suppressed hmp-1 S823F phenotypes at 15°C. The introduction of the salt bridge mutations partially suppressed embryonic lethality at 15°C, and, although it did not rise to the level of statistical significance, hmp-1 R551/554A;S823F homozygotes reared at 15°C had an increase in brood size compared to hmp-1 S823F homozygotes ( Figure 4C,D ). Taken together, these results indicate that the hmp-1 R551/554A mutation acts as a gain-of-function allele that can partially offset reduction of actin binding activity conferred by the C-terminal S823F mutation. We previously utilized hmp-1 S823F hypomorphic ( hmp-1(fe4)) homozygotes as a sensitized background to identify modulators of cadherin-dependent adhesion [ 57 ] and identified srgp-1 as a strong enhancer of embryonic lethality in the hmp-1 S823F background. RNAi knockdown of srgp-1 resulted in nearly total embryonic lethality, while srgp-1 knockdown by feeding RNAi in wild-type embryos had minimal effects. At least some of the synergistic lethality was caused by Gex phenotypes during ventral enclosure [ 28 ]. We therefore examined the synergistic effect of srgp-1 RNAi knockdown in hmp-1 R551/554A; S823F embryos. The salt bridge mutation was able to reduce the embryonic lethality of srgp-1(RNAi);hmp-1 S823F embryos significantly ( Figure 4E ). These results confirm that an open conformation of the HMP-1 M domain is able to bypass some requirements for SRGP-1, in addition to its ability to compensate for reduced actin binding activity mediated by the HMP-1 C terminus. Loss of afd-1/afadin function leads to increased frequency of cleft closure defects Conformational changes within the α-catenin M domain can affect its ability to recruit components that modulate cell adhesion. One such modulator in vertebrates is vinculin; when the αE-catenin M domain is extended, either via direct mechanical distension [ 58 – 60 ] or by introducing salt bridge mutations [ 51 ], its binding affinity for vinculin is increased. However, DEB-1, the vinculin homolog in C. elegans does not interact with HMP-1 [ 47 ], and its expression is confined to muscle cells during development [ 61 – 63 ], ruling it out as a candidate HMP-1 interactor that could be positively affected by the R551/554A salt bridge mutations in the context of cleft closure. Another candidate modulator is AFD-1/afadin. Vertebrate afadin can bind to αE-catenin [ 64 , 65 ]. While there is currently no published evidence for direct binding of the Drosophila afadin, Canoe, to α-catenin (M. Peifer and U. Tepass, pers. commun.), Canoe localizes to cell-cell junctions and modulates morphogenesis in a variety of contexts in Drosophila [ 66 – 71 ]. Moreover, we showed previously that AFD-1 can be co-immunoprecipitated with HMP-1 [ 72 ] and that loss of afd-1 function synergizes with the hmp-1 S823F mutation during later morphogenesis [ 57 ]. We therefore examined whether afd-1 loss of function showed genetic interactions with srgp-1 and with hmp-1 salt bridge mutations. We first determined whether afd-1 RNAi led to cleft closure defects and lethality in wild-type embryos or in embryos homozygous for the hmp-1 S823F allele ( Figure 5A-C ). Knockdown of srgp-1 or afd-1 led to comparable levels of lethality and cleft closure defects in otherwise wild-type embryos on agar mounts. Moreover, RNAi against either srpg-1 and afd-1 caused a significant increase in cleft closure defects in hmp-1 S823F embryos on plates or agar mounts ( Figures 5A-C ). Since both srgp-1 and afd-1 knockdown increased the frequency of cleft closure defects in hmp-1 S823F , we examined how afd-1 knockdown genetically interacted with srgp-1 loss of function and with the hmp-1 R551/554A mutation. In wild-type, hmp-1 R551/554A , and srgp-1 W122Stop backgrounds, afd-1 knockdown resulted in an increase in cleft closure defects ( Figure 5C ). In srgp-1 W122Stop ; hmp-1 R551/554A double mutants, loss of afd-1 resulted in a higher frequency of cleft closure defects than in hmp-1 R551/554A alone, but less than in srgp-1 W122Stop mutants alone, suggesting that there may be additional factors beyond AFD-1 that may stabilize HMP-1 when it adopts an open conformation. Download figure Open in new tab Figure 5 srgp-1 and hmp-1 genetically interact with afd-1 during cleft closure. (A) DIC images of hmp-1 S823F , hmp-1 S823F ; srgp-1(RNAi) , and hmp-1 S823F ; afd-1(RNAi) embryos over the course of 270 minutes. Dotted white lines outline the ventral cleft. Yellow dotted lines indicate extruded gut. Scale bar is 10 μm. (B) A stacked bar plot indicating the percentage of embryos that die during cleft closure, head enclosure, and elongation in various genetic backgrounds. afd-1 and srgp-1 knockdown in the hmp-1 S823F background both significantly increase the percentage of cleft closure defects. (C) Percent cleft closure defects in various genetic backgrounds with or without depletion of srgp-1 and afd-1 via RNAi. (D) Percentage of cleft closure defects in embryos treated with Control or afd-1 RNAi. ****, p < 0.0001; ***, p<0.001; *, p < 0.05. Computational work was previously used to engineer the actin binding domain of human αE-catenin to bind actin with higher affinity [ 50 ]. Using protein alignment, we identified the homologous amino acids in HMP-1 and generated hmp-1 QNLM676-679GSGS , which is predicted to bind actin with higher affinity (Figure S2A,B). Embryos homozygous for hmp-1 QNLM676-679GSGS have a low level of embryonic lethality, which causes developmental arrest at various embryonic stages, including during cleft closure. These phenotypes were suppressed by loss of function of srgp-1 and afd-1 (Figure S2C,D). These results suggest that HMP-1 stability and linkage to the actin network must be maintained within a dynamic range during processes that contribute to cleft closure. AFD-1 localizes to the vertex of the anterior rosette at the end of gastrulation Since genetic perturbation of afd-1 had consequences for cleft closure, we next assessed the localization of AFD-1 during cleft closure. We visualized mKate2::AFD-1 and HMP-1::GFP from cleft closure through rosette formation. While expression of mKate2::AFD-1 in the ventral neuroblasts was weak, we observed strong accumulation of AFD-1 at the vertex of the anterior rosette immediately following the final internalization events of gastrulation, which quickly dispersed as the rosette resolved ( Figure 6A ). We next examined HMP-1 accumulation and localization at the vertex of the anterior rosette in various mutant backgrounds ( Figure 6B ). Total accumulation of HMP-1::GFP increased in the hmp-1 R551/554A mutant background, but RNAi against afd-1 reduced total HMP-1 accumulation at the vertex, as well as the spatial extent of HMP-1 accumulation at the vertex. We did not find a significant change in AFD-1 accumulation in srgp-1 W122Stop mutants; while there was an increase in AFD-1 accumulation in hmp-1 R551/4A homozygotes, it did not quite rise to statistical significance (Figure S3). These results suggest that while SRGP-1 may play an important role in orienting cells during rosette formation and organizing HMP-1 around the vertex, AFD-1 is essential for normal HMP-1 accumulation at the vertex following the final internalization events of gastrulation. Unfortunately, we could not perform the converse experiment to address whether AFD-1 recruitment requires HMP-1 at this stage of development, because depletion of maternal and zygotic HMP-1 leads to catastrophic morphogenetic failure, including lack of cleft closure [ 31 , 73 ]. Download figure Open in new tab Figure 6 AFD-1 accumulates at the vertex of the anterior rosette. (A) A typical embryo expressing HMP-1::GFP and mKate2::AFD-1 before, during, and after anterior rosette formation. White dotted line outlines cells that form the rosette. Blue dotted lines mark the anterior cells that internalize prior to formation of the rosette. White arrowheads indicate the vertex of the rosette. Scale bar is 10 μm. (B) Images of HMP-1::GFP and mKate2::AFD-1 localization at the anterior rosette immediately following internalization in various genetic backgrounds. White dotted lines outline cells forming the rosette; yellow dotted lines indicate ventral cleft that remains open. Scale bar is 5 μm. (C) A graph depicting the total accumulation of HMP-1::GFP at the anterior rosette. (D) Graph depicting the area of HMP-1::GFP aggregation at the vertex of the anterior rosette. ****, p < 0.0001; ***, p<0.001; **, p<0.01; *, p < 0.05. Download figure Open in new tab Figure 7. Summary of the roles of SRGP-1 and AFD-1 in stabilizing HMP-1 at rosettes. (A) A protein map depicting the proposed functional roles of SRGP-1 domains. (B) Visual summary of HMP-1 and SRGP-1 distribution in cells in different srgp-1 mutant backgrounds. (C) Genetic pathways summarizing proposed routes by which HMP-1 is stabilized in wild-type and srgp-1 W122Stop ; hmp-1 R551/554A backgrounds Discussion Rosette formation during C. elegans gastrulation requires cadherin-based adhesion Ventral cleft closure is the culmination of gastrulation in the C. elegans embryo. It is essential for proper organization and cohesion of neuroblasts following gastrulation, which in turn is crucial for the embryo to survive the mechanical forces that operate during later morphogenesis [ 5 , 9 , 12 ]. Here we have characterized the cell rearrangements that accompany sealing of the ventral surface of the embryo, as cells on the surface change position to accommodate loss of cells that internalize near the end of gastrulation. Specifically, we have demonstrated that HMP-1/α-catenin and two of its functional modulators, SRGP-1/srGAP and AFD-1/afadin, facilitate the adhesion of cells during this critical stage in embryogenesis in the C. elegans embryo. Based on prior work, rosettes that form as a result of earlier cell internalization events in C. elegans appear similar [ 12 ], so insights gleaned from studying these later events will likely be useful in understanding other internalization events in the earlier embryo. Cell internalization is a common event during gastrulation in metazoan embryos, as cells destined for the embryo’s interior detach their apical surfaces from the embryo’s exterior [ 2 – 5 ]. Given the apical-to-basal axis of such movements during C. elegans gastrulation, internalization also bears similarities to other basal extrusion events, often triggered by apoptosis or cell crowding in a variety of epithelia (reviewed in [ 74 – 76 ]). In all these cases, however, relatively little attention has been paid to how the cells that remain on the surface seal breaches on the embryonic exterior left behind by internalizing cells. At least in some cases, such tissue sealing involves multicellular rosette formation. The geometry of these rosettes bears similarities to those associated with other morphogenetic processes, such as convergent extension [ 77 ]. An intriguing parallel to the rosettes we observed are those observed in the chick epiblast [ 3 , 78 , 79 ]. Although the functional significance of the rosette structures in the primitive streak is unclear, these may reflect similar events at sites where cells depart from the surface of the embryo during gastrulation. Rosettes in other contexts, such as during convergent extension in the Drosophila germband, involve modulation of adhesion complexes as cells change their connections to one another [ 80 , 81 ]. In contrast, little is known about adhesive changes among neighboring non-intercalating cells that seal gaps left behind by ingressing cells. In the case of sea urchin primary mesenchyme cells, which exhibit many aspects of standard epithelial-mesenchymal transition [ 82 ], cells lose cadherin-catenin complex components at the time of ingression [ 82 , 83 ]. The situation may be different in gastrulating cells in C. elegans and Drosophila neuroblasts; in the former, at least in the case of internalization of endodermal founder cells Ea and Ep, CCC components are transiently upregulated during apical constriction [ 11 ], while in the latter, post-translational loss of CCC components can be uncoupled from ingression events [ 84 ]. These differences indicate that while many processes may be conserved during internalization events, there may be a variety of mechanisms involved. Our results shed light on this relatively understudied process by demonstrating that rosette formation at the end of gastrulation in C. elegans requires a robust cell-cell adhesion machinery. Reduction in the ability of HMP-1/α-catenin to bind F-actin in hmp-1 S823F mutants leads to an increase in ventral cleft closure failure, as does loss of the HMP-1 binding partner, SRGP-1. The cells surrounding the position of the vacated cell at the end of ventral cleft closure form a rosette, which ultimately resolves as cells make new connections to one another at the site of sealing. Rosette formation is fostered by activating HMP-1/α-catenin We and others have shown that the α-catenin M domain engages in interactions that regulate the C-terminal F-actin binding region of α-catenins [ 50 , 85 ]. In this context it is striking that the hmp-1 R551/554A mutation suppresses phenotypes associated with the S823F mutation, which we have shown previously measurably decreases the F-actin binding activity of HMP-1 [ 86 ]. Previous intragenic suppressors all clustered in the C terminus of HMP-1, not in the M domain [ 86 ]. Our present results provide further evidence that the conformation of the M domain is relevant to the ability of HMP-1 to interact, either directly or indirectly, with the actin cytoskeleton. Rosette formation depends on proper HMP-1/a-catenin localization mediated by both the C-terminus and F-BAR domains of SRGP-1/srGAP Salt bridges in the M domain of mammalian αE-catenin stabilize the M domain in a “closed” conformation, reducing the likelihood of association of vinculin [ 51 , 59 ]. In C. elegans , however, we have shown previously that DEB-1/vinculin is confined to myoblasts in the early embryo, and that it does not bind HMP-1 [ 47 , 86 ], suggesting that HMP-1 interacts with other effectors in non-muscle cells. In addition to its utility in identifying intramolecular interactions that regulate HMP-1 activity, the hmp-1 S823F mutation has been useful as a sensitized background for identifying such functional interactors. Both SRGP-1/srGAP and AFD-1/afadin were identified in a genome-wide RNAi screen for such interactors [ 57 ]. Our previous analysis indicated that the C terminus of SRGP-1 can physically bind the HMP-1 M domain, but, unlike the case with vertebrate aE-catenin and vinculin, not when the HMP-1 M domain is fully extended. We also showed that both the N-terminal F-BAR and C-terminal domains of SRGP-1 are functionally important during elongation [ 36 ]. Our analysis here also revealed roles for the N- and C-terminal regions of SRGP-1 during ventral cleft closure. HMP-1 becomes highly concentrated at the tips of cells at rosette vertices in embryos expressing endogenously tagged, full-length SRGP-1. The greater severity of gross morphological defects in srgp-1 nonsense and C-terminal deletion mutants further suggests a more stringent requirement for the C terminus, which is lacking in both mutants, in stabilizing HMP-1. Since the SRGP-1 C terminus is intact in srgp-1 ΔF-BAR mutants, it is possible that, whereas SRGP-1 ΔF-BAR can no longer interact with the membrane directly to stabilize the CCC, when the N-terminus is absent SRGP-1 can still interact with HMP-1 in some functional capacity. In this case HMP-1 presumably exclusively relies on its association with the HMP-1/HMP-2/HMR-1 heterotrimeric complex to associate with the plasma membrane, which is less efficient in recruiting HMP-1 at sites of high membrane curvature, such as cell tips at rosette vertices. The likelihood of this possibility is strengthened by our observation that srgp-1 ΔF-BAR homozygous embryos exhibit less lethality than srgp-1 W122Stop and srgp-1 ΔC homozygotes. In our previous work we suggested that there may be a second region of SRGP-1, which lies N-terminal to the C-terminal region, that can interact with some junctional component— possibly including HMP-1 [ 28 , 36 ]; our present work is consistent with this possibility. A distinct role for the SRGP-1 N terminus is also suggested by our results. When the SRGP-1 F-BAR domain is deleted the tips of cells in the rosette are blunted and HMP-1 forms multiple aggregates in cells in the rosette, leading to less robust rosettes. In our previous work we observed reduced membrane curvature at the leading edge during ventral epidermal enclosure, suggesting that SRGP-1 promotes highly curved membranes [ 28 ]. Our present results are consistent with a similar role at nascent rosette vertices, which require that the plasma membranes of cell tips in the rosette adopt a high degree of curvature. SRGP-1 may either stabilize such highly curved regions of the plasma membrane or be recruited to such sites, leading to clustering of HMP-1 at such sites to stabilize nascent adhesions. The loss of normal HMP-1 accumulation at cell tips in hmp-1 ΔF-BAR mutants is consistent with this possibility and suggests that not only is membrane curvature adversely affected, but that HMP-1 recruitment to sites of high membrane curvature is reduced, with adverse effects on rosettes. The hmp-1 R551/554A mutation, which maintains the HMP-1 M domain in an open conformation [ 36 ], can suppress cleft closure defects caused by loss of the SRGP-1 C-terminus, but not those resulting from loss of the SRGP-1 F-BAR domain (see Figure 4B ). There are several potential explanations for this result. One possibility is that the C terminus of SRGP-1 regulates HMP-1 function beyond localization. For example, the C terminus of SRGP-1, once bound, could facilitate further opening and activation of the HMP-1 M domain, leading to recruitment of other binding partners. Constitutive opening of the HMP-1 M domain in hmp-1 R551/554A mutants could obviate this requirement. Alternatively, if the major role of the SRGP-1 C terminus is to fine-tune the localization or stability of HMP-1, the enhanced activity of a fully open HMP-1 could offset the quantitative loss of HMP-1 at nascent junctions in rosettes. Rosette formation is fostered by recruitment of AFD-1/afadin AFD-1 accumulation at the vertex of the anterior rosette is striking compared to the low levels of AFD-1 accumulation elsewhere at this stage of development, including the posterior rosette, within which we did not see a similar accumulation of AFD-1. This indicates that junctions at the anterior rosette vertex are unique among cell-cell adhesions between ventral cells in the embryo at this stage, and that AFD-1/afadin is crucial for stabilizing them. Vertebrate afadin and Drosophila Canoe are recruited to junctions under increased tension or to sites with increased cellular (and hence actomyosin) dynamics [ 87 – 89 ]. In C. elegans , during later development when the epidermis is under substantial mechanical tension, AFD-1 appears at epidermal cell-cell junctions [ 57 ], consistent with tension-induced recruitment at that stage. That AFD-1 is recruited to the tips of cells in the anterior rosette vertex at the end of gastrulation suggests that the tips of these cells likewise experience increased tension. As the internalization event that forms the anterior rosette concludes, multiple cells must converge to create new contact points, which are susceptible to mechanical failure. A similar accumulation of AFD-1 is not observed in the posterior rosette, however. Notably, the cells of the posterior rosette undergo rapid extension shortly after rosette formation, whereas the anterior rosette persists. Work in Drosophila has demonstrated a necessity for Canoe localization to maintain tricellular junctions experiencing high tension; however, prolonged and continued accumulation of Canoe at junctions prevents vertex resolution during cell rearrangement [ 90 ]. If AFD-1 works in a similar fashion in C. elegans , this could imply that AFD-1 is required to stabilize the anterior rosette under higher mechanical loads. We also found that, as is the case for srgp-1 , loss of afd-1 function leads to ventral cleft closure defects that can be suppressed via the hmp-1 R551/554A mutation. Moreover, simultaneous depletion of SRGP-1 and AFD-1 leads to synergistic ventral cleft closure defects. One reasonable model that accounts for this data is that, while SRGP-1 fosters the initial recruitment of HMP-1 to nascent contact sites within rosettes, AFD-1 subsequently stabilizes more mature adhesions, allowing them to withstand tension prior to rosette resolution. In this case, forcing HMP-1 into an open conformation may be able to bypass functional requirements for SRGP-1 by increasing the stability of adhesions through additional AFD-1 recruitment. It remains unclear whether AFD-1 can directly interact with HMP-1 in the way that their vertebrate counterparts do [ 54 , 65 ], or if AFD-1 is recruited to cell-cell adhesion sites through other effectors that in turn depend on an open conformation of HMP-1. Since defects in afd-1 (RNAi); srpg-1 W122Stop double loss-of-function embryos are still suppressed by hmp-1 R551/554A , there may be additional mechanisms that are stimulated by an open conformation of the HMP-1 M domain. In conclusion, this work has clarified how cadherin-dependent adhesion between non-internalizing neighbors of internalizing cells, supported by SRGP-1/srGAP and AFD-1/afadin, stabilizes nascent cell-cell adhesions following the internalization events of gastrulation. Future work focused on identifying other factors that play a role in anterior rosette formation and that dissects the mechanisms through which SRGP-1, AFD-1, and HMP-1 work together in this process should continue to clarify the cellular events of tissue sealing following internalization. Contributions JMS designed, implemented, and analyzed experiments, designed and generated the novel srgp-1 and hmp-1 CRISPR alleles, and wrote and edited drafts of the manuscript. MMS generated the afd-1 knock-in allele. BG commented on the manuscript. JH oversaw experimental design, implementation, and analysis and edited the manuscript. Materials and Methods Strains and genetics C. elegans were maintained using standard methods. Bristol N2 was used as wildtype. A complete list of strains and genotypes used in this manuscript can be found in Supplementary Table 1. DIC imaging Four dimensional DIC movies were collected on either a Nikon Optiphot-2 microscope connected to a QiCAM camera (QImaging) or an Olympus BX50 microscope connected to a Scion CFW-1512M camera (Scion Corp.) using Micro-Manager software (v. 1.42) [ 91 , 92 ]. ImageJ plugins ( https://worms.zoology.wisc.edu/research/4d/4d.html ) were used to compress and view DIC movies. All embryos were mounted on 10% agar pads in M9 solution unless otherwise specified. Confocal imaging Embryos were dissected from adult hermaphrodites and mounted onto 10% agar pads in M9 solution and imaged. For fluorescence imaging, a Dragonfly 500 spinning disc confocal microscope (Andor Corp.), mounted on a Leica DMi8 microscope, equipped with an iXon-EMCCD camera and controlled by Fusion software (Andor Corp.) was used to collect images using 0.21 μm slices with a 63×/1.3 NA glycerol Leica objective at 20°C. CRISPR/Cas9 genome editing All novel knock-in and deletion alleles with jc## designation were generated via plasmid-based CRISPR/Cas9 [ 93 ] using repair templates cloned by SapTrap cloning [ 94 ]. Small substitution mutations were made via marker-free genome editing [ 95 ]. Guides, homology arm primers, and single-stranded repair templates for all CRISPR/Cas9 editing can be found in Supplementary Table 2. Injection RNAi Injection RNAi was performed by synthesizing double-stranded RNA (dsRNA) using a T7 Megascript kit (Invitrogen). The templates for srgp-1 and control RNAi were obtained from a feeding library [ 96 ]. pIC386 was used as a template for production of afd-1 dsRNA. dsRNA was injected at a concentration of 2μg/μL in nuclease free water. L4 worms were injected and aged overnight before embryos were dissected from mature adults for imaging. Quantification and analysis Percentage cleft closure defects were measured from embryos mounted for DIC imaging. Embryonic lethality was quantified by dividing the number of unhatched embryos laid on a plate by the total number of embryos on the plate from a single hermaphrodite. Total accumulation (integrated signal) and aggregation size for HMP-1::GFP were measured by drawing a circle around GFP signal at the vertex immediately following the internalization event. Statistical analysis Data from control and experimental groups were compared using one-way ANOVA with Tukey post hoc testing to assess significance between individual groups. All statistical analyses were carried out in Prism (GraphPad Corp.). Acknowledgements Some strains were provided by the C. elegans Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). JS and JH were supported by NIH grants R01GM058038, R01GM127687, and R35GM145312. BG was supported by NIH grant R35GM134838. MMS was supported by NIH grant F32GM119348 References 1. ↵ Solnica-Krezel L , Sepich DS. Gastrulation: making and shaping germ layers . Annu Rev Cell Dev Biol . 2012 ; 28 : 687 – 717 . Epub 20120709. doi: 10.1146/annurev-cellbio-092910-154043 . PubMed PMID: 22804578 . OpenUrl CrossRef PubMed 2. ↵ Byrum CA. An analysis of hydrozoan gastrulation by unipolar ingression . Dev Biol . 2001 ; 240 ( 2 ): 627 – 40 . doi: 10.1006/dbio.2001.0484 . PubMed PMID: 11784088 . OpenUrl CrossRef PubMed 3. ↵ Wagstaff LJ , Bellett G , Mogensen MM , Munsterberg A. Multicellular rosette formation during cell ingression in the avian primitive streak . Dev Dyn . 2008 ; 237 ( 1 ): 91 – 6 . doi: 10.1002/dvdy.21390 . PubMed PMID: 18069691 . OpenUrl CrossRef PubMed 4. Shook DR , Keller R. Epithelial type, ingression, blastopore architecture and the evolution of chordate mesoderm morphogenesis . J Exp Zool B Mol Dev Evol . 2008 ; 310 ( 1 ): 85 – 110 . doi: 10.1002/jez.b.21198 . PubMed PMID: 18041055 . OpenUrl CrossRef PubMed 5. ↵ Goldstein B , Nance J. Caenorhabditis elegans Gastrulation: A Model for Understanding How Cells Polarize, Change Shape, and Journey Toward the Center of an Embryo . Genetics . 2020 ; 214 ( 2 ): 265 – 77 . doi: 10.1534/genetics.119.300240 . PubMed PMID: 32029580 ; PubMed Central PMCID: PMCPMC7017025 . OpenUrl Abstract / FREE Full Text 6. ↵ Mareel M , Bracke M , Van Roy F , Vakaet L. Expression of E-cadherin in embryogenetic ingression and cancer invasion . Int J Dev Biol . 1993 ; 37 ( 1 ): 227 – 35 . PubMed PMID: 8507565 . OpenUrl PubMed 7. ↵ Wu SY , Ferkowicz M , McClay DR. Ingression of primary mesenchyme cells of the sea urchin embryo: a precisely timed epithelial mesenchymal transition . Birth Defects Res C Embryo Today . 2007 ; 81 ( 4 ): 241 – 52 . doi: 10.1002/bdrc.20113 . PubMed PMID: 18228256 . OpenUrl CrossRef PubMed 8. ↵ Harrell JR , Goldstein B. Internalization of multiple cells during C. elegans gastrulation depends on common cytoskeletal mechanisms but different cell polarity and cell fate regulators . Dev Biol . 2011 ; 350 ( 1 ): 1 – 12 . Epub 20100926. doi: 10.1016/j.ydbio.2010.09.012 . PubMed PMID: 20875815 ; PubMed Central PMCID: PMCPMC3022094 . OpenUrl CrossRef PubMed 9. ↵ Chisholm AD , Hardin J. Epidermal morphogenesis . WormBook . 2005 : 1 – 22 . Epub 20051201. doi: 10.1895/wormbook.1.35.1 . PubMed PMID: 18050408 ; PubMed Central PMCID: PMCPMC4781537 . OpenUrl CrossRef PubMed 10. ↵ Lee JY , Marston DJ , Walston T , Hardin J , Halberstadt A , Goldstein B. Wnt/Frizzled signaling controls C. elegans gastrulation by activating actomyosin contractility . Curr Biol . 2006 ; 16 ( 20 ): 1986 – 97 . doi: 10.1016/j.cub.2006.08.090 . PubMed PMID: 17055977 ; PubMed Central PMCID: PMCPMC2989422 . OpenUrl CrossRef PubMed Web of Science 11. ↵ Marston DJ , Higgins CD , Peters KA , Cupp TD , Dickinson DJ , Pani AM , et al. MRCK-1 Drives Apical Constriction in C. elegans by Linking Developmental Patterning to Force Generation . Curr Biol . 2016 ; 26 ( 16 ): 2079 – 89 . Epub 20160721. doi: 10.1016/j.cub.2016.06.010 . PubMed PMID: 27451898 ; PubMed Central PMCID: PMCPMC4996705 . OpenUrl CrossRef PubMed 12. ↵ Pohl C , Tiongson M , Moore JL , Santella A , Bao Z. Actomyosin-based self-organization of cell internalization during C. elegans gastrulation . BMC Biol . 2012 ; 10 : 94 . Epub 20121130. doi: 10.1186/1741-7007-10-94 . PubMed PMID: 23198792 ; PubMed Central PMCID: PMCPMC3583717 . OpenUrl CrossRef PubMed 13. ↵ Roh-Johnson M , Shemer G , Higgins CD , McClellan JH , Werts AD , Tulu US , et al. Triggering a cell shape change by exploiting preexisting actomyosin contractions . Science . 2012 ; 335 ( 6073 ): 1232 – 5 . Epub 20120209. doi: 10.1126/science.1217869 . PubMed PMID: 22323741 ; PubMed Central PMCID: PMCPMC3298882 . OpenUrl Abstract / FREE Full Text 14. ↵ Chihara D , Nance J. An E-cadherin-mediated hitchhiking mechanism for C. elegans germ cell internalization during gastrulation . Development . 2012 ; 139 ( 14 ): 2547 – 56 . Epub 20120606. doi: 10.1242/dev.079863 . PubMed PMID: 22675206 ; PubMed Central PMCID: PMCPMC3383229 . OpenUrl Abstract / FREE Full Text 15. ↵ Nance J , Priess JR. Cell polarity and gastrulation in C. elegans . Development . 2002 ; 129 ( 2 ): 387 – 97 . doi: 10.1242/dev.129.2.387 . PubMed PMID: 11807031 . OpenUrl CrossRef PubMed Web of Science 16. ↵ George SE , Simokat K , Hardin J , Chisholm AD. The VAB-1 Eph receptor tyrosine kinase functions in neural and epithelial morphogenesis in C. elegans . Cell . 1998 ; 92 ( 5 ): 633 – 43 . doi: 10.1016/s0092-8674(00)81131-9 . PubMed PMID: 9506518 . OpenUrl CrossRef PubMed Web of Science 17. ↵ Sulston JE , Schierenberg E , White JG , Thomson JN. The embryonic cell lineage of the nematode Caenorhabditis elegans . Developmental Biology . 1983 ; 100 ( 1 ): 64 – 119 . doi: 10.1016/0012-1606(83)90201-4 . OpenUrl CrossRef PubMed Web of Science 18. ↵ Carvalho CA , Broday L. Game of Tissues: How the Epidermis Thrones C. elegans Shape . J Dev Biol . 2020 ; 8 ( 1 ). Epub 20200309. doi: 10.3390/jdb8010007 . PubMed PMID: 32182901 ; PubMed Central PMCID: PMCPMC7151205 . OpenUrl CrossRef PubMed 19. ↵ Vuong-Brender TT , Yang X , Labouesse M. C. elegans Embryonic Morphogenesis . Curr Top Dev Biol . 2016 ; 116 : 597 – 616 . Epub 20160201. doi: 10.1016/bs.ctdb.2015.11.012 . PubMed PMID: 26970644 . OpenUrl CrossRef PubMed 20. ↵ Chin-Sang ID , George SE , Ding M , Moseley SL , Lynch AS , Chisholm AD. The ephrin VAB-2/EFN-1 functions in neuronal signaling to regulate epidermal morphogenesis in C. elegans . Cell . 1999 ; 99 ( 7 ): 781 – 90 . doi: 10.1016/s0092-8674(00)81675-x . PubMed PMID: 10619431 . OpenUrl CrossRef PubMed Web of Science 21. ↵ Chin-Sang ID , Moseley SL , Ding M , Harrington RJ , George SE , Chisholm AD. The divergent C. elegans ephrin EFN-4 functions inembryonic morphogenesis in a pathway independent of the VAB-1 Eph receptor . Development . 2002 ; 129 ( 23 ): 5499 – 510 . doi: 10.1242/dev.00122 . PubMed PMID: 12403719 . OpenUrl Abstract / FREE Full Text 22. ↵ Harrington RJ , Gutch MJ , Hengartner MO , Tonks NK , Chisholm AD. The C. elegans LAR-like receptor tyrosine phosphatase PTP-3 and the VAB-1 Eph receptor tyrosine kinase have partly redundant functions in morphogenesis . Development . 2002 ; 129 ( 9 ): 2141 – 53 . doi: 10.1242/dev.129.9.2141 . PubMed PMID: 11959824 . OpenUrl Abstract / FREE Full Text 23. ↵ Ikegami R , Simokat K , Zheng H , Brown L , Garriga G , Hardin J , et al. Semaphorin and Eph receptor signaling guide a series of cell movements for ventral enclosure in C. elegans . Curr Biol . 2012 ; 22 ( 1 ): 1 – 11 . Epub 20111222. doi: 10.1016/j.cub.2011.12.009 . PubMed PMID: 22197242 ; PubMed Central PMCID: PMCPMC4306670 . OpenUrl CrossRef PubMed 24. ↵ Nakao F , Hudson ML , Suzuki M , Peckler Z , Kurokawa R , Liu Z , et al. The PLEXIN PLX-2 and the ephrin EFN-4 have distinct roles in MAB-20/Semaphorin 2A signaling in Caenorhabditis elegans morphogenesis . Genetics . 2007 ; 176 ( 3 ): 1591 – 607 . Epub 20070516. doi: 10.1534/genetics.106.067116 . PubMed PMID: 17507686 ; PubMed Central PMCID: PMCPMC1931547 . OpenUrl Abstract / FREE Full Text 25. ↵ Hudson ML , Kinnunen T , Cinar HN , Chisholm AD. C. elegans Kallmann syndrome protein KAL-1 interacts with syndecan and glypican to regulate neuronal cell migrations . Dev Biol . 2006 ; 294 ( 2 ): 352 – 65 . Epub 20060503. doi: 10.1016/j.ydbio.2006.02.036 . PubMed PMID: 16677626 . OpenUrl CrossRef PubMed 26. ↵ Rugarli EI , Di Schiavi E , Hilliard MA , Arbucci S , Ghezzi C , Facciolli A , et al. The Kallmann syndrome gene homolog in C. elegans is involved in epidermal morphogenesis and neurite branching . Development . 2002 ; 129 ( 5 ): 1283 – 94 . doi: 10.1242/dev.129.5.1283 . PubMed PMID: 11874923 . OpenUrl Abstract / FREE Full Text 27. ↵ Withee J , Galligan B , Hawkins N , Garriga G. Caenorhabditis elegans WASP and Ena/VASP proteins play compensatory roles in morphogenesis and neuronal cell migration . Genetics . 2004 ; 167 ( 3 ): 1165 – 76 . doi: 10.1534/genetics.103.025676 . PubMed PMID: 15280232 ; PubMed Central PMCID: PMCPMC1470955 . OpenUrl Abstract / FREE Full Text 28. ↵ Zaidel-Bar R , Joyce MJ , Lynch AM , Witte K , Audhya A , Hardin J. The F-BAR domain of SRGP-1 facilitates cell-cell adhesion during C. elegans morphogenesis . J Cell Biol . 2010 ; 191 ( 4 ): 761 – 9 . Epub 20101108. doi: 10.1083/jcb.201005082 . PubMed PMID: 21059849 ; PubMed Central PMCID: PMCPMC2983056 . OpenUrl Abstract / FREE Full Text 29. ↵ Shah PK , Tanner MR , Kovacevic I , Rankin A , Marshall TE , Noblett N , et al. PCP and SAX-3/Robo Pathways Cooperate to Regulate Convergent Extension-Based Nerve Cord Assembly in C. elegans . Dev Cell . 2017 ; 41 ( 2 ): 195 – 203 e3 . doi: 10.1016/j.devcel.2017.03.024 . PubMed PMID: 28441532 ; PubMed Central PMCID: PMCPMC5469364 . OpenUrl CrossRef PubMed 30. ↵ Wernike D , Chen Y , Mastronardi K , Makil N , Piekny A. Mechanical forces drive neuroblast morphogenesis and are required for epidermal closure . Dev Biol . 2016 ; 412 ( 2 ): 261 – 77 . Epub 20160227. doi: 10.1016/j.ydbio.2016.02.023 . PubMed PMID: 26923492 . OpenUrl CrossRef PubMed 31. ↵ Costa M , Raich W , Agbunag C , Leung B , Hardin J , Priess JR. A putative catenin-cadherin system mediates morphogenesis of the Caenorhabditis elegans embryo . J Cell Biol . 1998 ; 141 ( 1 ): 297 – 308 . doi: 10.1083/jcb.141.1.297 . PubMed PMID: 9531567 ; PubMed Central PMCID: PMCPMC2132712 . OpenUrl Abstract / FREE Full Text 32. ↵ Grana TM , Cox EA , Lynch AM , Hardin J. SAX-7/L1CAM and HMR-1/cadherin function redundantly in blastomere compaction and non-muscle myosin accumulation during Caenorhabditis elegans gastrulation . Dev Biol . 2010 ; 344 ( 2 ): 731 – 44 . Epub 20100531. doi: 10.1016/j.ydbio.2010.05.507 . PubMed PMID: 20515680 ; PubMed Central PMCID: PMCPMC2914123 . OpenUrl CrossRef PubMed 33. ↵ Sawyer JM , Glass S , Li T , Shemer G , White ND , Starostina NG , et al. Overcoming redundancy: an RNAi enhancer screen for morphogenesis genes in Caenorhabditis elegans . Genetics . 2011 ; 188 ( 3 ): 549 – 64 . Epub 20110428. doi: 10.1534/genetics.111.129486 . PubMed PMID: 21527776 ; PubMed Central PMCID: PMCPMC3176534 . OpenUrl Abstract / FREE Full Text 34. ↵ Neukomm LJ , Frei AP , Cabello J , Kinchen JM , Zaidel-Bar R , Ma Z , et al. Loss of the RhoGAP SRGP-1 promotes the clearance of dead and injured cells in Caenorhabditis elegans . Nat Cell Biol . 2011 ; 13 ( 1 ): 79 – 86 . Epub 20101219. doi: 10.1038/ncb2138 . PubMed PMID: 21170032 ; PubMed Central PMCID: PMCPMC3808961 . OpenUrl CrossRef PubMed Web of Science 35. ↵ Serre JM , Lucas B , Martin SCT , Heier JA , Shao X , Hardin J. A C. elegans srGAP is a novel α-catenin M domain-binding protein that strengthens cadherin-dependent adhesion during morphogenesis . Development. in revision . 36. ↵ Serre JM , Lucas B , Martin SCT , Heier JA , Shao X , Hardin J. C. elegans srGAP is an alpha-catenin M domain-binding protein that strengthens cadherin-dependent adhesion during morphogenesis . Development . 2022 ; 149 ( 18 ). Epub 20220920. doi: 10.1242/dev.200775 . PubMed PMID: 36125129 . OpenUrl CrossRef PubMed 37. ↵ Guerrier S , Coutinho-Budd J , Sassa T , Gresset A , Jordan NV , Chen K , et al. The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis . Cell . 2009 ; 138 ( 5 ): 990 – 1004 . doi: 10.1016/j.cell.2009.06.047 . PubMed PMID: 19737524 ; PubMed Central PMCID: PMCPMC2797480 . OpenUrl CrossRef PubMed Web of Science 38. Endris V , Haussmann L , Buss E , Bacon C , Bartsch D , Rappold G. SrGAP3 interacts with lamellipodin at the cell membrane and regulates Rac-dependent cellular protrusions . J Cell Sci . 2011 ; 124 ( Pt 23 ): 3941 – 55 . Epub 20111208. doi: 10.1242/jcs.077081 . PubMed PMID: 22159416 . OpenUrl Abstract / FREE Full Text 39. ↵ Lucas B , Hardin J. Mind the (sr)GAP - roles of Slit-Robo GAPs in neurons, brains and beyond . J Cell Sci . 2017 ; 130 ( 23 ): 3965 – 74 . Epub 20171102. doi: 10.1242/jcs.207456 . PubMed PMID: 29097383 ; PubMed Central PMCID: PMCPMC5769592 . OpenUrl Abstract / FREE Full Text 40. Endris V , Wogatzky B , Leimer U , Bartsch D , Zatyka M , Latif F , et al. The novel Rho-GTPase activating gene MEGAP/ srGAP3 has a putative role in severe mental retardation . Proc Natl Acad Sci U S A . 2002 ; 99 ( 18 ): 11754 – 9 . Epub 20020823. doi: 10.1073/pnas.162241099 . PubMed PMID: 12195014 ; PubMed Central PMCID: PMCPMC129341 . OpenUrl Abstract / FREE Full Text 41. Wong K , Ren X-R , Huang Y-Z , Xie Y , Liu G , Saito H , et al. Signal Transduction in Neuronal Migration . Cell . 2001 ; 107 ( 2 ): 209 – 21 . doi: 10.1016/s0092-8674(01)00530-x . OpenUrl CrossRef PubMed Web of Science 42. Foletta VC , Brown FD , Scott Young Iii W. Cloning of rat ARHGAP4/C1, a RhoGAP family member expressed in the nervous system that colocalizes with the Golgi complex and microtubules . Molecular Brain Research . 2002 ; 107 ( 1 ): 65 – 79 . doi: 10.1016/s0169-328x(02)00448-5 . OpenUrl CrossRef PubMed 43. ↵ Katoh M , Katoh M. Identification and characterization of human FCHSD1 and FCHSD2 genes in silico . International Journal of Molecular Medicine . 2004 . doi: 10.3892/ijmm.13.5.749 . OpenUrl CrossRef 44. ↵ Fotopoulos N , Wernike D , Chen Y , Makil N , Marte A , Piekny A. Caenorhabditis elegans anillin (ani-1) regulates neuroblast cytokinesis and epidermal morphogenesis during embryonic development . Dev Biol . 2013 ; 383 ( 1 ): 61 – 74 . Epub 20130907. doi: 10.1016/j.ydbio.2013.08.024 . PubMed PMID: 24016757 . OpenUrl CrossRef PubMed 45. ↵ Neukomm LJ , Zeng S , Frei AP , Huegli PA , Hengartner MO. Small GTPase CDC-42 promotes apoptotic cell corpse clearance in response to PAT-2 and CED-1 in C. elegans . Cell Death Differ . 2014 ; 21 ( 6 ): 845 – 53 . Epub 20140314. doi: 10.1038/cdd.2014.23 . PubMed PMID: 24632947 ; PubMed Central PMCID: PMCPMC4013519 . OpenUrl CrossRef PubMed 46. ↵ Barrett T , Xiao B , Dodson EJ , Dodson G , Ludbrook SB , Nurmahomed K , et al. The structure of the GTPase-activating domain from p50rhoGAP . Nature . 1997 ; 385 ( 6615 ): 458 – 61 . doi: 10.1038/385458a0 . PubMed PMID: 9009196 . OpenUrl CrossRef PubMed Web of Science 47. ↵ Kang H , Bang I , Jin KS , Lee B , Lee J , Shao X , et al. Structural and functional characterization of Caenorhabditis elegans alpha-catenin reveals constitutive binding to beta-catenin and F-actin . J Biol Chem . 2017 ; 292 ( 17 ): 7077 – 86 . Epub 20170315. doi: 10.1074/jbc.M116.769778 . PubMed PMID: 28298447 ; PubMed Central PMCID: PMCPMC5409474 . OpenUrl Abstract / FREE Full Text 48. ↵ Pang SM , Le S , Kwiatkowski AV , Yan J. Mechanical stability of alphaT-catenin and its activation by force for vinculin binding . Mol Biol Cell . 2019 ; 30 ( 16 ): 1930 – 7 . Epub 20190718. doi: 10.1091/mbc.E19-02-0102 . PubMed PMID: 31318313 ; PubMed Central PMCID: PMCPMC6727763 . OpenUrl CrossRef PubMed 49. Seddiki R , Narayana G , Strale PO , Balcioglu HE , Peyret G , Yao M , et al. Force-dependent binding of vinculin to alpha-catenin regulates cell-cell contact stability and collective cell behavior . Mol Biol Cell . 2018 ; 29 ( 4 ): 380 – 8 . Epub 20171227. doi: 10.1091/mbc.E17-04-0231 . PubMed PMID: 29282282 ; PubMed Central PMCID: PMCPMC6014167 . OpenUrl Abstract / FREE Full Text 50. ↵ Ishiyama N , Sarpal R , Wood MN , Barrick SK , Nishikawa T , Hayashi H , et al. Force-dependent allostery of the alpha-catenin actin-binding domain controls adherens junction dynamics and functions . Nat Commun . 2018 ; 9 ( 1 ): 5121 . Epub 20181130. doi: 10.1038/s41467-018-07481-7 . PubMed PMID: 30504777 ; PubMed Central PMCID: PMCPMC6269467 . OpenUrl CrossRef PubMed 51. ↵ Barrick S , Li J , Kong X , Ray A , Tajkhorshid E , Leckband D. Salt bridges gate alpha-catenin activation at intercellular junctions . Mol Biol Cell . 2018 ; 29 ( 2 ): 111 – 22 . Epub 20171115. doi: 10.1091/mbc.E17-03-0168 . PubMed PMID: 29142072 ; PubMed Central PMCID: PMCPMC5909925 . OpenUrl Abstract / FREE Full Text 52. Yao M , Qiu W , Liu R , Efremov AK , Cong P , Seddiki R , et al. Force-dependent conformational switch of alpha-catenin controls vinculin binding . Nat Commun . 2014 ; 5 : 4525 . Epub 20140731. doi: 10.1038/ncomms5525 . PubMed PMID: 25077739 . OpenUrl CrossRef PubMed 53. ↵ Yonemura S , Wada Y , Watanabe T , Nagafuchi A , Shibata M. alpha-Catenin as a tension transducer that induces adherens junction development . Nat Cell Biol . 2010 ; 12 ( 6 ): 533 – 42 . Epub 20100509. doi: 10.1038/ncb2055 . PubMed PMID: 20453849 . OpenUrl CrossRef PubMed Web of Science 54. ↵ Sakakibara S , Mizutani K , Sugiura A , Sakane A , Sasaki T , Yonemura S , et al. Afadin regulates actomyosin organization through alphaE-catenin at adherens junctions . J Cell Biol . 2020 ; 219 ( 5 ). doi: 10.1083/jcb.201907079 . PubMed PMID: 32227204 ; PubMed Central PMCID: PMCPMC7199863 . OpenUrl CrossRef PubMed 55. ↵ Maiden SL , Hardin J. The secret life of alpha-catenin: moonlighting in morphogenesis . J Cell Biol . 2011 ; 195 ( 4 ): 543 – 52 . doi: 10.1083/jcb.201103106 . PubMed PMID: 22084304 ; PubMed Central PMCID: PMCPMC3257527 . OpenUrl Abstract / FREE Full Text 56. ↵ Pettitt J , Cox EA , Broadbent ID , Flett A , Hardin J. The Caenorhabditis elegans p120 catenin homologue, JAC-1, modulates cadherin-catenin function during epidermal morphogenesis . J Cell Biol . 2003 ; 162 ( 1 ): 15 – 22 . doi: 10.1083/jcb.200212136 . PubMed PMID: 12847081 ; PubMed Central PMCID: PMCPMC2172718 . OpenUrl Abstract / FREE Full Text 57. ↵ Lynch AM , Grana T , Cox-Paulson E , Couthier A , Cameron M , Chin-Sang I , et al. A genome-wide functional screen shows MAGI-1 is an L1CAM-dependent stabilizer of apical junctions in C . elegans. Curr Biol . 2012 ; 22 ( 20 ): 1891 – 9 . Epub 20120913. doi: 10.1016/j.cub.2012.08.024 . PubMed PMID: 22981773 ; PubMed Central PMCID: PMCPMC3482306 . OpenUrl CrossRef PubMed 58. ↵ Xu XP , Pokutta S , Torres M , Swift MF , Hanein D , Volkmann N , et al. Structural basis of alphaE-catenin-F-actin catch bond behavior . Elife . 2020 ; 9 . Epub 20200911. doi: 10.7554/eLife.60878 . PubMed PMID: 32915141 ; PubMed Central PMCID: PMCPMC7588230 . OpenUrl CrossRef PubMed 59. ↵ Li J , Newhall J , Ishiyama N , Gottardi C , Ikura M , Leckband DE , et al. Structural Determinants of the Mechanical Stability of alpha-Catenin . J Biol Chem . 2015 ; 290 ( 31 ): 18890 – 903 . Epub 20150612. doi: 10.1074/jbc.M115.647941 . PubMed PMID: 26070562 ; PubMed Central PMCID: PMCPMC4521009 . OpenUrl Abstract / FREE Full Text 60. ↵ Terekhova K , Pokutta S , Kee YS , Li J , Tajkhorshid E , Fuller G , et al. Binding partner-and force-promoted changes in alphaE-catenin conformation probed by native cysteine labeling . Sci Rep . 2019 ; 9 ( 1 ): 15375 . Epub 20191025. doi: 10.1038/s41598-019-51816-3 . PubMed PMID: 31653927 ; PubMed Central PMCID: PMCPMC6814714 . OpenUrl CrossRef PubMed 61. ↵ Barstead RJ , Waterston RH. The basal component of the nematode dense-body is vinculin . Journal of Biological Chemistry . 1989 ; 264 ( 17 ): 10177 – 85 . doi: 10.1016/s0021-9258(18)81782-3 . OpenUrl Abstract / FREE Full Text 62. Lecroisey C , Brouilly N , Qadota H , Mariol MC , Rochette NC , Martin E , et al. ZYX-1, the unique zyxin protein of Caenorhabditis elegans, is involved in dystrophin-dependent muscle degeneration . Mol Biol Cell . 2013 ; 24 ( 8 ): 1232 – 49 . Epub 20130220. doi: 10.1091/mbc.E12-09-0679 . PubMed PMID: 23427270 ; PubMed Central PMCID: PMCPMC3623643 . OpenUrl Abstract / FREE Full Text 63. ↵ Liu Q , Jones TI , Bachmann RA , Meghpara M , Rogowski L , Williams BD , et al. C. elegans PAT-9 is a nuclear zinc finger protein critical for the assembly of muscle attachments . Cell Biosci . 2012 ; 2 ( 1 ): 18 . Epub 20120522. doi: 10.1186/2045-3701-2-18 . PubMed PMID: 22616817 ; PubMed Central PMCID: PMCPMC3419604 . OpenUrl CrossRef PubMed 64. ↵ Tachibana K , Nakanishi H , Mandai K , Ozaki K , Ikeda W , Yamamoto Y , et al. Two cell adhesion molecules, nectin and cadherin, interact through their cytoplasmic domain-associated proteins . J Cell Biol . 2000 ; 150 ( 5 ): 1161 – 76 . doi: 10.1083/jcb.150.5.1161 . PubMed PMID: 10974003 ; PubMed Central PMCID: PMCPMC2175253 . OpenUrl Abstract / FREE Full Text 65. ↵ Pokutta S , Drees F , Takai Y , Nelson WJ , Weis WI. Biochemical and structural definition of the l-afadin-and actin-binding sites of alpha-catenin . J Biol Chem . 2002 ; 277 ( 21 ): 18868 – 74 . Epub 20020320. doi: 10.1074/jbc.M201463200 . PubMed PMID: 11907041 ; PubMed Central PMCID: PMCPMC3368618 . OpenUrl Abstract / FREE Full Text 66. ↵ Schmidt A , Lv Z , Grosshans J. ELMO and Sponge specify subapical restriction of Canoe and formation of the subapical domain in early Drosophila embryos . Development . 2018 ; 145 ( 2 ). Epub 20180126. doi: 10.1242/dev.157909 . PubMed PMID: 29361564 . OpenUrl Abstract / FREE Full Text 67. Manning LA , Perez-Vale KZ , Schaefer KN , Sewell MT , Peifer M. The Drosophila Afadin and ZO-1 homologues Canoe and Polychaetoid act in parallel to maintain epithelial integrity when challenged by adherens junction remodeling . Mol Biol Cell . 2019 ; 30 ( 16 ): 1938 – 60 . Epub 20190612. doi: 10.1091/mbc.E19-04-0209 . PubMed PMID: 31188739 ; PubMed Central PMCID: PMCPMC6727765 . OpenUrl CrossRef PubMed 68. Walther RF , Burki M , Pinal N , Rogerson C , Pichaud F. Rap1, Canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor . J Cell Sci . 2018 ; 131 ( 6 ). Epub 20180326. doi: 10.1242/jcs.207779 . PubMed PMID: 29507112 ; PubMed Central PMCID: PMCPMC5897711 . OpenUrl Abstract / FREE Full Text 69. Slovakova J , Speicher S , Sanchez-Soriano N , Prokop A , Carmena A. The actin-binding protein Canoe/AF-6 forms a complex with Robo and is required for Slit-Robo signaling during axon pathfinding at the CNS midline . J Neurosci . 2012 ; 32 ( 29 ): 10035 – 44 . doi: 10.1523/JNEUROSCI.6342-11.2012 . PubMed PMID: 22815517 ; PubMed Central PMCID: PMCPMC6621277 . OpenUrl Abstract / FREE Full Text 70. Ma Z , Li P , Hu X , Song H. Polarity protein Canoe mediates overproliferation via modulation of JNK, Ras-MAPK and Hippo signalling . Cell Prolif . 2019 ; 52 ( 1 ): e12529 . Epub 20181017. doi: 10.1111/cpr.12529 . PubMed PMID: 30328653 ; PubMed Central PMCID: PMCPMC6430484 . OpenUrl CrossRef PubMed 71. ↵ Matsuo T , Takahashi K , Kondo S , Kaibuchi K , Yamamoto D. Regulation of cone cell formation by Canoe and Ras in the developing Drosophila eye . Development . 1997 ; 124 ( 14 ): 2671 – 80 . doi: 10.1242/dev.124.14.2671 . PubMed PMID: 9226438 . OpenUrl Abstract 72. ↵ Callaci S , Morrison K , Shao X , Schuh AL , Wang Y , Yates JR , 3rd . , et al. Phosphoregulation of the C . elegans cadherin-catenin complex. Biochem J . 2015 ; 472 ( 3 ): 339 – 52 . Epub 20151006. doi: 10.1042/BJ20150410 . PubMed PMID: 26443865 ; PubMed Central PMCID: PMCPMC4663164 . OpenUrl Abstract / FREE Full Text 73. ↵ Raich WB , Agbunag C , Hardin J. Rapid epithelial-sheet sealing in the Caenorhabditis elegans embryo requires cadherin-dependent filopodial priming . Curr Biol . 1999 ; 9 ( 20 ): 1139 – 46 . doi: 10.1016/S0960-9822(00)80015-9 . PubMed PMID: 10531027 . OpenUrl CrossRef PubMed Web of Science 74. ↵ Mitchell SJ , Rosenblatt J. Early mechanical selection of cell extrusion and extrusion signaling in cancer . Curr Opin Cell Biol . 2021 ; 72 : 36 – 40 . Epub 20210524. doi: 10.1016/j.ceb.2021.04.005 . PubMed PMID: 34034216 ; PubMed Central PMCID: PMCPMC8869604 . OpenUrl CrossRef PubMed 75. Tada M. The morphogenetic changes that lead to cell extrusion in development and cell competition . Dev Biol . 2021 ; 477 : 1 – 10 . Epub 20210511. doi: 10.1016/j.ydbio.2021.05.003 . PubMed PMID: 33984304 . OpenUrl CrossRef PubMed 76. ↵ Villars A , Levayer R. Collective effects in epithelial cell death and cell extrusion . Curr Opin Genet Dev . 2022 ; 72 : 8 – 14 . Epub 20211006. doi: 10.1016/j.gde.2021.09.004 . PubMed PMID: 34626896 . OpenUrl CrossRef PubMed 77. ↵ Harding MJ , McGraw HF , Nechiporuk A. The roles and regulation of multicellular rosette structures during morphogenesis . Development . 2014 ; 141 ( 13 ): 2549 – 58 . doi: 10.1242/dev.101444 . PubMed PMID: 24961796 ; PubMed Central PMCID: PMCPMC4067956 . OpenUrl Abstract / FREE Full Text 78. ↵ Chuai M , Weijer CJ. The mechanisms underlying primitive streak formation in the chick embryo . Curr Top Dev Biol . 2008 ; 81 : 135 – 56 . doi: 10.1016/S0070-2153(07)81004-0 . PubMed PMID: 18023726 . OpenUrl CrossRef PubMed 79. ↵ Rozbicki E , Chuai M , Karjalainen AI , Song F , Sang HM , Martin R , et al. Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation . Nat Cell Biol . 2015 ; 17 ( 4 ): 397 – 408 . doi: 10.1038/ncb3138 . PubMed PMID: 25812521 ; PubMed Central PMCID: PMCPMC4886837 . OpenUrl CrossRef PubMed 80. ↵ Bertet C , Sulak L , Lecuit T. Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation . Nature . 2004 ; 429 ( 6992 ): 667 – 71 . doi: 10.1038/nature02590 . PubMed PMID: 15190355 . OpenUrl CrossRef PubMed Web of Science 81. ↵ Blankenship JT , Backovic ST , Sanny JS , Weitz O , Zallen JA. Multicellular rosette formation links planar cell polarity to tissue morphogenesis . Dev Cell . 2006 ; 11 ( 4 ): 459 – 70 . doi: 10.1016/j.devcel.2006.09.007 . PubMed PMID: 17011486 . OpenUrl CrossRef PubMed Web of Science 82. ↵ Saunders LR , McClay DR. Sub-circuits of a gene regulatory network control a developmental epithelial-mesenchymal transition . Development . 2014 ; 141 ( 7 ): 1503 – 13 . Epub 20140305. doi: 10.1242/dev.101436 . PubMed PMID: 24598159 ; PubMed Central PMCID: PMCPMC3957374 . OpenUrl Abstract / FREE Full Text 83. ↵ Miller JR , McClay DR. Changes in the pattern of adherens junction-associated beta-catenin accompany morphogenesis in the sea urchin embryo . Dev Biol . 1997 ; 192 ( 2 ): 310 – 22 . doi: 10.1006/dbio.1997.8739 . PubMed PMID: 9441670 . OpenUrl CrossRef PubMed Web of Science 84. ↵ Simoes S , Oh Y , Wang MFZ , Fernandez-Gonzalez R , Tepass U. Myosin II promotes the anisotropic loss of the apical domain during Drosophila neuroblast ingression . J Cell Biol . 2017 ; 216 ( 5 ): 1387 – 404 . Epub 20170331. doi: 10.1083/jcb.201608038 . PubMed PMID: 28363972 ; PubMed Central PMCID: PMCPMC5412560 . OpenUrl Abstract / FREE Full Text 85. ↵ Shao X , Lucas B , Strauch J , Hardin J. The adhesion modulation domain of Caenorhabditis elegans alpha-catenin regulates actin binding during morphogenesis . Mol Biol Cell . 2019 ; 30 ( 17 ): 2115 – 23 . Epub 20190612. doi: 10.1091/mbc.E19-01-0018 . PubMed PMID: 31188702 ; PubMed Central PMCID: PMCPMC6743470 . OpenUrl CrossRef PubMed 86. ↵ Maiden SL , Harrison N , Keegan J , Cain B , Lynch AM , Pettitt J , et al. Specific conserved C-terminal amino acids of Caenorhabditis elegans HMP-1/alpha-catenin modulate F-actin binding independently of vinculin . J Biol Chem . 2013 ; 288 ( 8 ): 5694 – 706 . Epub 20121227. doi: 10.1074/jbc.M112.438093 . PubMed PMID: 23271732 ; PubMed Central PMCID: PMCPMC3581367 . OpenUrl Abstract / FREE Full Text 87. ↵ Choi W , Acharya BR , Peyret G , Fardin MA , Mege RM , Ladoux B , et al. Remodeling the zonula adherens in response to tension and the role of afadin in this response . J Cell Biol . 2016 ; 213 ( 2 ): 243 – 60 . doi: 10.1083/jcb.201506115 . PubMed PMID: 27114502 ; PubMed Central PMCID: PMCPMC5084271 . OpenUrl Abstract / FREE Full Text 88. Sawyer JK , Choi W , Jung KC , He L , Harris NJ , Peifer M. A contractile actomyosin network linked to adherens junctions by Canoe/afadin helps drive convergent extension . Mol Biol Cell . 2011 ; 22 ( 14 ): 2491 – 508 . Epub 20110525. doi: 10.1091/mbc.E11-05-0411 . PubMed PMID: 21613546 ; PubMed Central PMCID: PMCPMC3135475 . OpenUrl Abstract / FREE Full Text 89. ↵ Sawyer JK , Harris NJ , Slep KC , Gaul U , Peifer M. The Drosophila afadin homologue Canoe regulates linkage of the actin cytoskeleton to adherens junctions during apical constriction . J Cell Biol . 2009 ; 186 ( 1 ): 57 – 73 . doi: 10.1083/jcb.200904001 . PubMed PMID: 19596848 ; PubMed Central PMCID: PMCPMC2712996 . OpenUrl Abstract / FREE Full Text 90. ↵ Yu HH , Zallen JA. Abl and Canoe/Afadin mediate mechanotransduction at tricellular junctions . Science . 2020 ; 370 ( 6520 ). doi: 10.1126/science.aba5528 . PubMed PMID: 33243859 ; PubMed Central PMCID: PMCPMC8559527 . OpenUrl Abstract / FREE Full Text 91. ↵ Edelstein A , Amodaj N , Hoover K , Vale R , Stuurman N. Computer control of microscopes using microManager . Curr Protoc Mol Biol . 2010 ;Chapter 14 :Unit 14 20 . Epub 2010/10/05. doi: 10.1002/0471142727.mb1420s92 . PubMed PMID: 20890901 ; PubMed Central PMCID: PMCPMC3065365 . OpenUrl CrossRef PubMed 92. ↵ Edelstein AD , Tsuchida MA , Amodaj N , Pinkard H , Vale RD , Stuurman N. Advanced methods of microscope control using μManager software . Journal of Biological Methods . 2014 ; 1 ( 2 ): 10 . doi: 10.14440/jbm.2014.36 . OpenUrl CrossRef 93. ↵ Dickinson DJ , Goldstein B. CRISPR-based methods for Caenorhabditis elegans genome engineering . Genetics . 2016 ; 202 ( 3 ): 885 – 901 . doi: 10.1534/genetics.115.182162 . PubMed PMID: 26953268 ; PubMed Central PMCID: PMCPMC4788126 . OpenUrl Abstract / FREE Full Text 94. ↵ Schwartz ML , Jorgensen EM. SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans . Genetics . 2016 ; 202 ( 4 ): 1277 – 88 . Epub 2016/02/04. doi: 10.1534/genetics.115.184275 . PubMed PMID: 26837755 ; PubMed Central PMCID: PMCPMC4905529 . OpenUrl Abstract / FREE Full Text 95. ↵ Arribere JA , Bell RT , Fu BX , Artiles KL , Hartman PS , Fire AZ. Efficient marker-free recovery of custom genetic modifications with CRISPR/Cas9 in Caenorhabditis elegans . Genetics . 2014 ; 198 ( 3 ): 837 – 46 . Epub 2014/08/28. doi: 10.1534/genetics.114.169730 . PubMed PMID: 25161212 ; PubMed Central PMCID: PMCPMC4224173 . OpenUrl Abstract / FREE Full Text 96. ↵ Kamath RS , Ahringer J. Genome-wide RNAi screening in Caenorhabditis elegans . Methods . 2003 ; 30 ( 4 ): 313 – 21 . doi: 10.1016/s1046-2023(03)00050-1 . PubMed PMID: 12828945 . OpenUrl CrossRef PubMed Web of Science Back to top Previous Next Posted November 07, 2022. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following SRGP-1/srGAP and AFD-1/Afadin stabilize HMP-1/α-Catenin at rosettes to seal internalization sites following gastrulation in C. elegans Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share SRGP-1/srGAP and AFD-1/Afadin stabilize HMP-1/α-Catenin at rosettes to seal internalization sites following gastrulation in C. elegans Joel M. Serre , Mark M. Slabodnick , Bob Goldstein , Jeff Hardin bioRxiv 2022.11.04.515144; doi: https://doi.org/10.1101/2022.11.04.515144 Share This Article: Copy Citation Tools SRGP-1/srGAP and AFD-1/Afadin stabilize HMP-1/α-Catenin at rosettes to seal internalization sites following gastrulation in C. elegans Joel M. Serre , Mark M. Slabodnick , Bob Goldstein , Jeff Hardin bioRxiv 2022.11.04.515144; doi: https://doi.org/10.1101/2022.11.04.515144 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Developmental Biology Subject Areas All Articles Animal Behavior and Cognition (7969) Biochemistry (18623) Bioengineering (14765) Bioinformatics (44155) Biophysics (22458) Cancer Biology (19585) Cell Biology (26737) Clinical Trials (138) Developmental Biology (13900) Ecology (20880) Epidemiology (2067) Evolutionary Biology (25315) Genetics (16100) Genomics (23400) Immunology (18608) Microbiology (42209) Molecular Biology (17939) Neuroscience (92895) Paleontology (693) Pathology (2969) Pharmacology and Toxicology (5063) Physiology (8064) Plant Biology (15901) Scientific Communication and Education (2091) Synthetic Biology (4538) Systems Biology (10184) Zoology (2376) window.__CF$cv$params={r:'a37801d0edfb2858',t:'MTc4ODgwODE1OA==',u:'01a07d466a387c91b8c1d5cd561caa41',ut:'VwLUQkUnRZD5eMRQSAhUzkTK6y2HsbIEwsFVjWeKjTM-1788808161-1.2.1.1-LnNoD7MTduhcRma.J1oq2PiXtZ_Zf_BscDvN2.886voqRtc4nOuwyMjvhA5Lx3njGKfJwNgn5_FJAjGuhmGEZZd4iopcNMF7vNIYk9tffB4',i:60};(function(){if(!document.body)return;var s=document.createElement('script');s.src='/cdn-cgi/challenge-platform/scripts/precursor/main.js';document.head.appendChild(s);})();