Global cell sorting is mediated by local cell-cell interactions in the C. elegans embryo

The Caenorhabditis elegans embryo achieves pattern formation by sorting cells into coherent regions before morphogenesis is initiated. The sorting of cells is coupled to their fate. Cells move extensively relative to each other to reach their correct position in the body plan. Analyzing the mechanism of cell sorting in in vitro culture experiments using 4D microscopy, we show that all AB-derived cells sort only according to their local neighbors, and that all cells are able to communicate with each other. The directions of cell movement do not depend on a cellular polarity but only on local cell-cell interactions; in experimental situations, this allows even the reversal of the polarity of whole regions of the embryo. The work defines a new mechanism of pattern formation we call "cell focusing".     

glp-1 and lin-12, genes implicated in distinct cell-cell interactions in C. elegans, encode similar transmembrane proteins

Genomic DNA closely related in sequence to lin-12, a gene that specifies certain cell fates during C. elegans development, was isolated from a C. elegans library by low stringency hybridization. DNA sequencing of genomic and cDNA clones predicts the new sequence to encode an integral membrane protein that shares three repeated amino acid sequence motifs with the lin-12 product and the Drosophila Notch product: an epidermal growth factor-like motif, the "lin-12/Notch Repeat," and a motif present in two yeast gene products that have cell cycle dependent functions. Austin and Kimble (see accompanying paper) present evidence that this sequence corresponds to glp-1, a gene implicated in cell-cell interactions distinct from those involving lin-12. Possible implications of the predicted structure of the glp-1 product with respect to these cell-cell interactions are discussed.     

Cell-matrix interactions and cell-cell junctions during epithelial histo-morphogenesis in the developing mouse incisor.

The continuously growing rodent incisor develops mainly along its antero-posterior axis. The labio-lingual asymmetry which characterizes this tooth is initiated at the cap stage and increases further during the cap to bell transition (ED14 to ED16) when histogenesis of the enamel organ proceeds. Histology, transmission electron microscopy (TEM), and immunostaining were used to document the changes in the basement membrane (BM) as well as the modifications of epithelial cell-matrix and cell-cell interactions during this period. The expression of plakoglobin, desmoglein and E-cadherin at ED14 suggested that the main cell-cell junctional complexes were adherens junctions. The expression of desmoglein and TEM observations suggested a progressive antero-posterior stabilization of the enamel organ by means of desmosomes from ED14 to ED18. alpha6 integrin, BP 230 and laminin gamma2 chain were all expressed in the developing incisor but were not always co-distributed. Immunostaining and TEM suggested that only primitive type II hemidesmosomes were present. At ED14, cells of the enamel knot (EK) did not show any specific expression for antigens involved in cell-cell interaction. However, strong staining for the laminin gamma2 chain characterized the BM in contact with EK cells. The BM in the labial part of the cervical loop demonstrated ultrastructural changes: the presence of loops of the lamina densa in this region preceeded the differential expression of the integrin alpha6 subunit and that of the laminin gamma2 chain in the labial/lingual parts of the cervical loop. Apoptosis was transiently observed in the contiguous mesenchyme. This affected osteoblasts and also nerve cells close to the labial part of the cervical loop.     

ADIP,a novel Afadin- and alpha-actinin-binding protein localized at cell-cell adherens junctions

Afadin is an actin filament (F-actin)-binding protein that is associated with the cytoplasmic tail of nectin, a Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecule. Nectin and afadin strictly localize at cell-cell adherens junctions (AJs) undercoated with F-actin bundles and are involved in the formation of AJs in cooperation with E-cadherin in epithelial cells. In epithelial cells of afadin (-/-) mice and (-/-) embryoid bodies, the proper organization of AJs is markedly impaired. However, the molecular mechanism of how the nectin-afadin system is associated with the E-cadherin-catenin system or functions in the formation of AJs has not yet been fully understood. Here we identified a novel afadin-binding protein, named ADIP (afadin DIL domain-interacting protein). ADIP consists of 615 amino acids with a calculated M(r) of 70,954 and has three coiled-coil domains. Northern and Western blot analyses in mouse tissues indicated that ADIP was widely distributed. Immunofluorescence and immunoelectron microscopy revealed that ADIP strictly localized at cell-cell AJs undercoated with F-actin bundles in small intestine absorptive epithelial cells. This localization pattern was the same as those of afadin and nectin. ADIP was undetectable at cell-matrix AJs. ADIP furthermore bound alpha-actinin, an F-actin-bundling protein known to be indirectly associated with E-cadherin through its direct binding to alpha-catenin. These results indicate that ADIP is an afadin- and alpha-actinin-binding protein that localizes at cell-cell AJs and may have two functions. ADIP may connect the nectin-afadin and E-cadherin-catenin systems through alpha-actinin, and ADIP may be involved in organization of the actin cytoskeleton at AJs through afadin and alpha-actinin.     

Competition between cell-substratum interactions and cell-cell interactions.

Clusterin, a glycoprotein which elicits the aggregation of a wide variety of cells (Fritz, I. B., and Burdy, K.:J. Cell Physiol., 140:18-28, 1989), has been utilized to investigate some of the factors modulating the competition between cell-substratum interactions and cell-cell interactions. We compared the responses to clusterin by anchorage-independent cells (erythrocytes) with those by anchorage-dependent TM4 cells (a cell line derived from neonatal mouse testis cells). Cells were maintained in culture in the presence of various substrata chosen to enhance cell-substratum interactions (laminin-coated wells), or to diminish cell-substratum interactions (agarose-coated wells). Results obtained showed that the aggregation of erythrocytes elicited by clusterin was independent of the nature of the substratum. In contrast, clusterin addition resulted in aggregation of anchorage-dependent TM4 cells only when TM4 cell-substratum interactions were weak. Thus, clusterin did not aggregate TM4 cells plated upon a laminin substratum, but readily aggregated TM4 cells plated upon an agarose-coated substratum, independent of the sequence of addition of cells and clusterin to the culture dish. We utilized YIGSR, a peptide which competes with laminin for laminin receptors, to determine the possible role of laminin receptors on TM4 cells in the competition between cell-substratum interactions and cell-cell interactions. The presence of YIGSR did not alter responses of erythrocytes to clusterin under all conditions examined. In contrast, the responses of TM4 cells to clusterin were greatly changed. YIGSR addition resulted in the inhibition of aggregation of TM4 cells otherwise elicited by clusterin. YIGSR also prevented attachment of TM4 cells to a laminin-coated surface, but this was reversed by the presence of clusterin. We discuss the possible roles of clusterin and laminin in altering the balance in the competition between cell to cell interactions and cell to substratum interactions.     

Cellular interactions in early C. elegans embryos

In normal development both the anterior and posterior blastomeres in a 2-cell C. elegans embryo produce some descendants that become muscles. We show that cellular interactions appear to be necessary in order for the anterior blastomere to produce these muscles. The anterior blastomere does not produce any muscle descendants after either the posterior blastomere or one of the daughters of the posterior blastomere is removed from the egg. Moreover, we demonstrate that a daughter of the anterior blastomere that normally does not produce muscles appears capable of generating muscles when interchanged with its sister, a cell that normally does produce muscles. Embryos develop normally after these blastomeres are interchanged, suggesting that cellular interactions play a major role in determining the fates of some cells in early embryogenesis.     

The role of cell-cell interactions in postembryonic development of the Caenorhabditis elegans germ line.

This review addresses the role of cell-cell interactions in the development of the Caenorhabditis elegans germ line: specifically, the relative contributions of germ-line-soma interactions versus autonomous processes are considered. Current knowledge of the interacting cell types and the genes essential for various aspects of germ-line development is discussed.     

The C. elegans TspanC8 tetraspanin TSP-14 exhibits isoform-specific localization and function

Tetraspanin proteins are a unique family of highly conserved four-pass transmembrane proteins in metazoans. While much is known about their biochemical properties, the in vivo functions and distribution patterns of different tetraspanin proteins are less understood. Previous studies have shown that two paralogous tetraspanins that belong to the TspanC8 subfamily, TSP-12 and TSP-14, function redundantly to promote both Notch signaling and bone morphogenetic protein (BMP) signaling in C. elegans. TSP-14 has two isoforms, TSP-14A and TSP-14B, where TSP-14B has an additional 24 amino acids at its N-terminus compared to TSP-14A. By generating isoform specific knock-ins and knock-outs using CRISPR, we found that TSP-14A and TSP-14B share distinct as well as overlapping expression patterns and functions. While TSP-14A functions redundantly with TSP-12 to regulate body size and embryonic and vulva development, TSP-14B primarily functions redundantly with TSP-12 to regulate postembryonic mesoderm development. Importantly, TSP-14A and TSP-14B exhibit distinct subcellular localization patterns. TSP-14A is localized apically and on early and late endosomes. TSP-14B is localized to the basolateral cell membrane. We further identified a di-leucine motif within the N-terminal 24 amino acids of TSP-14B that serves as a basolateral membrane targeting sequence, and showed that the basolateral membrane localization of TSP-14B is important for its function. Our work highlights the diverse and intricate functions of TspanC8 tetraspanins in C. elegans, and demonstrates the importance of dissecting the functions of these important proteins in an intact living organism.     

Feedback interactions between cell-cell adherens junctions and cytoskeletal dynamics in newt lung epithelial cells

To test how cell-cell contacts regulate microtubule (MT) and actin cytoskeletal dynamics, we examined dynamics in cells that were contacted on all sides with neighboring cells in an epithelial cell sheet that was undergoing migration as a wound-healing response. Dynamics were recorded using time-lapse digital fluorescence microscopy of microinjected, labeled tubulin and actin. In fully contacted cells, most MT plus ends were quiescent; exhibiting only brief excursions of growth and shortening and spending 87.4% of their time in pause. This contrasts MTs in the lamella of migrating cells at the noncontacted leading edge of the sheet in which MTs exhibit dynamic instability. In the contacted rear and side edges of these migrating cells, a majority of MTs were also quiescent, indicating that cell-cell contacts may locally regulate MT dynamics. Using photoactivation of fluorescence techniques to mark MTs, we found that MTs in fully contacted cells did not undergo retrograde flow toward the cell center, such as occurs at the leading edge of motile cells. Time-lapse fluorescent speckle microscopy of fluorescently labeled actin in fully contacted cells revealed that actin did not flow rearward as occurs in the leading edge lamella of migrating cells. To determine if MTs were required for the maintenance of cell-cell contacts, cells were treated with nocodazole to inhibit MTs. After 1-2 h in either 10 microM or 100 nM nocodazole, breakage of cell-cell contacts occurred, indicating that MT growth is required for maintenance of cell-cell contacts. Analysis of fixed cells indicated that during nocodazole treatment, actin became reduced in adherens junctions, and junction proteins alpha- and beta-catenin were lost from adherens junctions as cell-cell contacts were broken. These results indicate that a MT plus end capping protein is regulated by cell-cell contact, and in turn, that MT growth regulates the maintenance of adherens junctions contacts in epithelia.     

Repression of cell-cell fusion by components of the C. elegans vacuolar ATPase complex

Cell-cell fusion initiates fertilization, sculpts tissues during animal development, reprograms stem cells to new differentiated states, and may be a key step in cancer progression. While cell fusion is tightly regulated, the mechanisms that limit fusion to appropriate partners are unknown. Here, we report that the fus-1 gene is essential to repress fusion of epidermal cells in C. elegans: in severe fus-1 mutants, all epidermal cells, except the lateral seam cells, inappropriately fuse into a single large syncytium. This hyperfusion requires EFF-1, an integral membrane protein essential for fusion of epidermal cells into discrete syncytia. FUS-1 is localized to the apical plasma membrane in all epidermal cells potentiated to undergo fusion, whereas it is virtually undetectable in nonfusing seam cells. fus-1 encodes the e subunit of the vacuolar H(+)-ATPase (V-ATPase), and loss of other V-ATPase subunits also causes widespread hyperfusion. These findings raise the possibility of manipulating cell fusion by altering V-ATPase activity.     

The F-BAR domain of SRGP-1 facilitates cell-cell adhesion during C. elegans morphogenesis

Robust cell-cell adhesion is critical for tissue integrity and morphogenesis, yet little is known about the molecular mechanisms controlling cell-cell junction architecture and strength. We discovered that SRGP-1 is a novel component of cell-cell junctions in Caenorhabditis elegans, localizing via its F-BAR (Bin1, Amphiphysin, and RVS167) domain and a flanking 200-amino acid sequence. SRGP-1 activity promotes an increase in membrane dynamics at nascent cell-cell contacts and the rapid formation of new junctions; in addition, srgp-1 loss of function is lethal in embryos with compromised cadherin-catenin complexes. Conversely, excess SRGP-1 activity leads to outward bending and projections of junctions. The C-terminal half of SRGP-1 interacts with the N-terminal F-BAR domain and negatively regulates its activity. Significantly, in vivo structure-function analysis establishes a role for the F-BAR domain in promoting rapid and robust cell adhesion during embryonic closure events, independent of the Rho guanosine triphosphatase-activating protein domain. These studies establish a new role for this conserved protein family in modulating cell-cell adhesion.     

N-cadherin association with lipid rafts regulates its dynamic assembly at cell-cell junctions in C2C12 myoblasts

Cadherins are homophilic cell-cell adhesion molecules implicated in cell growth, differentiation, and organization into tissues during embryonic development. They accumulate at cell-cell contact sites and act as adhesion-activated signaling receptors. Here, we show that the dynamic assembly of N-cadherin at cell-cell contacts involves lipid rafts. In C2C12 myoblasts, immunofluorescence and biochemical experiments demonstrate that N-cadherin present at cell-cell contacts is colocalized with lipid rafts. Disruption of lipid rafts leads to the inhibition of cell-cell adhesion and disorganization of N-cadherin-dependent cell-cell contacts without modifying the association of N-cadherin with catenins and its availability at the plasma membrane. Fluorescent recovery after photobleaching experiments demonstrate that at the dorsal plasma membrane, lipid rafts are not directly involved in the diffusional mobility of N-cadherin. In contrast, at cell-cell junctions N-cadherin association with lipid rafts allows its stabilization enabling the formation of a functional adhesive complex. We show that lipid rafts, as homophilic interaction and F-actin association, stabilize cadherin-dependent adhesive complexes. Homophilic interactions and F-actin association of N-cadherin are both required for its association to lipid rafts. We thus identify lipid rafts as new regulators of cadherin-mediated cell adhesion.     

The C. elegans MAGI-1 protein is a novel component of cell junctions that is required for junctional compartmentalization

Cell junctions are essential to maintain polarity and tissue integrity. Epithelial cell junctions are composed of distinct sub-compartments that together ensure the strong adhesion between neighboring cells. In Caenorhabditis elegans epithelia, the apical junction (CeAJ) forms a single electron-dense structure, but at the molecular level it is composed of two sub-compartments that function redundantly and localize independently as two distinct but adjacent circumferential rings on the lateral plasma membrane. While investigating the role of the multi PDZ-domain containing protein MAGI-1 during C. elegans epidermal morphogenesis, we found that MAGI-1 localizes apical to both the Cadherin/Catenin (CCC) and AJM-1/DLG-1 (DAC) containing sub-domains. Removal of MAGI-1 function causes a loss of junctional compartmentalization along the lateral membrane and reduces the overall robustness of cell-cell adhesion mediated by either type of cell junctions. Our results suggest that MAGI-1 functions as an “organizer” that ensures the correct segregation of different cell adhesion complexes into distinct domains along the lateral plasma membrane. Thus, the formation of stable junctions requires the proper distribution of the CCC and DAC adhesion protein complexes along the lateral plasma membrane.     

System-wide mapping of peptide-GPCR interactions in C. elegans

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A novel family of C. elegans snRNPs contains proteins associated with trans-splicing

In many Caenorhabditis elegans pre-mRNAs, the RNA sequence between the 5' cap and the first 3' splice site is replaced by trans-splicing a short spliced leader (SL) from the Sm snRNP, SL1. C. elegans also utilizes a similar Sm snRNP, SL2, to trans-splice at sites between genes in polycistronic pre-mRNAs from operons. How do SL1 and SL2 snRNPs function in different contexts? Here we show that the SL1 snRNP contains a complex of SL75p and SL21p, which are homologs of novel proteins previously reported in the Ascaris SL snRNP. Interestingly, we show that the SL2 snRNP does not contain these proteins. However, SL75p and SL26p, a paralog of SL21p, are components of another Sm snRNP that contains a novel snRNA species, Sm Y. Knockdown of SL75p is lethal. However, knockdown of either SL21p or SL26p alone leads to cold-sensitive sterility, whereas knockdown of both SL21p and SL26p is lethal. This suggests that these two proteins have overlapping functions even though they are associated with different classes of snRNP. These phenotypic relationships, along with the association of SL26p with SL75p, imply that, like the SL1 RNA/Sm/SL75p/SL21p complex, the Sm Y/Sm/SL75p/SL26p complex is associated with trans-splicing.     

Frizzled signaling and cell-cell interactions in planar polarity.

The function of the Frizzled pathway is essential for the formation of the array of distally pointing hairs found on the Drosophila wing. Previous research found that regulating the subcellular location for hair initiation controlled hair polarity. Recent work argues a graded Frizzled-dependent signal results in the accumulation of the Frizzled, Dishevelled and Flamingo proteins along the distal edge of the wing cells. This cortical mark leads to the local activation of downstream gene products and the subsequent activation of the cytoskeleton to form a hair.