Alleles of unc-33/CRMP exhibit defects during Caenorhabditis elegans epidermal morphogenesis.

BACKGROUND: Microtubule-associated proteins regulate the dynamics, organization, and function of microtubules, impacting a number of vital cellular processes. CRMPs have been shown to control microtubule assembly and axon outgrowth during neuronal differentiation. While many microtubule-associated proteins have been linked to roles in cell division and neuronal development, it is still unclear the complement that control the formation of parallel microtubule arrays in epithelial cells. RESULTS: Here we show through time-lapse DIC microscopy that Caenorhabditis elegans embryos homozygous for the weak loss-of-function allele unc-33(e204) progress more slowly through epidermal morphogenesis, while animals homozygous for strong loss-of-function alleles exhibit more embryonic lethality. Identification of two novel missense mutations in unc-33(e572), Val476Gly, and Ser731Thr, lead to computational approaches to determine the potential effects of these changes on UNC-33/CRMP structure. Molecular dynamics simulations show that for Asp389Asn and Arg502His, two other known missense mutations, local changes in protein-protein hydrogen bonding affect the stability of the protein. However, the Val476Gly/Ser731Thr combination does not alter the structure or energetics of UNC-33 drastically when compared to the wild-type protein. CONCLUSIONS: These results support a novel role for UNC-33/CRMP in C. elegans epidermal development and shed light on how individual amino acid changes cause a loss-of-function in UNC-33.

Synergistic effects of hmp-2/ß-catenin and sma-1/ßH-spectrin on epidermal morphogenesis in Caenorhabditis elegans.

The F-actin, spectrin, and microtubule cytoskeletons are important mediators of embryonic epidermal morphogenesis in Caenorhabditis elegans. While SMA-1/ßH-spectrin is known to organize actin bundles that connect to cadherin-based adhesions, the role of microtubules in the developing epidermis is not well understood. To determine if the spectrin cytoskeleton also plays a role in organizing epidermal microtubules, we conducted feeding RNA interference of four microtubule-associated protein genes in sma-1/ßH-spectrin null animals. Knockdown of apr-1, unc-33, unc-44, or cls-1 in sma-1(ru18) homozygotes did not reveal a genetic interaction; however, knockdown of hmp-2/ß-catenin in sma-1(ru18) synergistically increased embryonic lethality and epidermal defects.

Microtubules Inhibit E-Cadherin Adhesive Activity by Maintaining Phosphorylated p120-Catenin in a Colon Carcinoma Cell Model.

Tight regulation of cadherin-mediated intercellular adhesions is critical to both tissue morphogenesis during development and tissue homeostasis in adults. Cell surface expression of the cadherin-catenin complex is often directly correlated with the level of adhesion, however, examples exist where cadherin appears to be inactive and cells are completely non-adhesive. The state of p120-catenin phosphorylation has been implicated in regulating the adhesive activity of E-cadherin but the mechanism is currently unclear. We have found that destabilization of the microtubule cytoskeleton, independent of microtubule plus-end dynamics, dephosphorylates p120-catenin and activates E-cadherin adhesion in Colo 205 cells. Through chemical screening, we have also identified several kinases as potential regulators of E-cadherin adhesive activity. Analysis of several p120-catenin phosphomutants suggests that gross dephosphorylation of p120-catenin rather than that of specific amino acids may trigger E-cadherin adhesion. Uncoupling p120-catenin binding to E-cadherin at the membrane causes constitutive adhesion in Colo 205 cells, further supporting an inhibitory role of phosphorylated p120-catenin on E-cadherin activity.

Allosteric Regulation of E-Cadherin Adhesion.

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation.

Specific conserved C-terminal amino acids of Caenorhabditis elegans HMP-1/α-catenin modulate F-actin binding independently of vinculin.

Stable intercellular adhesions formed through the cadherin-catenin complex are important determinants of proper tissue architecture and help maintain tissue integrity during morphogenetic movements in developing embryos. A key regulator of this stability is α-catenin, which connects the cadherin-catenin complex to the actin cytoskeleton. Although the C-terminal F-actin-binding domain of α-catenin has been shown to be crucial for its function, a more detailed in vivo analysis of discrete regions and residues required for actin binding has not been performed. Using Caenorhabditis elegans as a model system, we have characterized mutations in hmp-1/α-catenin that identify HMP-1 residues 687-742 and 826-927, as well as amino acid 802, as critical to the localization of junctional proximal actin during epidermal morphogenesis. We also find that the S823F transition in a hypomorphic allele, hmp-1(fe4), decreases actin binding in vitro. Using hmp-1(fe4) animals in a mutagenesis screen, we were then able to identify 11 intragenic suppressors of hmp-1(fe4) that revert actin binding to wild-type levels. Using homology modeling, we show that these amino acids are positioned at key conserved sites within predicted α-helices in the C terminus. Through the use of transgenic animals, we also demonstrate that HMP-1 residues 315-494, which correspond to a putative mechanotransduction domain that binds vinculin in vertebrate αE-catenin, are not required during epidermal morphogenesis but may aid efficient recruitment of HMP-1 to the junction. Our studies are the first to identify key conserved amino acids in the C terminus of α-catenin that modulate F-actin binding in living embryos of a simple metazoan.

The secret life of α-catenin: moonlighting in morphogenesis.

Cadherin-based intercellular adhesions are important determinants of proper tissue architecture. These adhesions must be both stable and dynamic to maintain tissue integrity as cells undergo morphogenetic movements during development. The role of α-catenin in this process has been vigorously debated due to conflicting in vitro and in vivo evidence regarding its molecular mechanism of action. Recent data supports the classical view that α-catenin facilitates actin attachments at adherens junctions, but also suggests that α-catenin may act as a force transducer, and may have additional roles in the cytoplasm. These multiple functions for α-catenin converge on the regulation of adhesion and may help to explain its stable yet dynamic nature.

In vitro and in vivo reconstitution of the cadherin-catenin-actin complex from Caenorhabditis elegans.

The ternary complex of cadherin, beta-catenin, and alpha-catenin regulates actin-dependent cell-cell adhesion. alpha-Catenin can bind beta-catenin and F-actin, but in mammals alpha-catenin either binds beta-catenin as a monomer or F-actin as a homodimer. It is not known if this conformational regulation of alpha-catenin is evolutionarily conserved. The Caenorhabditis elegans alpha-catenin homolog HMP-1 is essential for actin-dependent epidermal enclosure and embryo elongation. Here we show that HMP-1 is a monomer with a functional C-terminal F-actin binding domain. However, neither full-length HMP-1 nor a ternary complex of HMP-1-HMP-2(beta-catenin)-HMR-1(cadherin) bind F-actin in vitro, suggesting that HMP-1 is auto-inhibited. Truncation of either the F-actin or HMP-2 binding domain of HMP-1 disrupts C. elegans development, indicating that HMP-1 must be able to bind F-actin and HMP-2 to function in vivo. Our study defines evolutionarily conserved properties of alpha-catenin and suggests that multiple mechanisms regulate alpha-catenin binding to F-actin.

The N- or C-terminal domains of DSH-2 can activate the C. elegans Wnt/beta-catenin asymmetry pathway.

Dishevelleds are modular proteins that lie at the crossroads of divergent Wnt signaling pathways. The DIX domain of dishevelleds modulates a beta-catenin destruction complex, and thereby mediates cell fate decisions through differential activation of Tcf transcription factors. The DEP domain of dishevelleds mediates planar polarity of cells within a sheet through regulation of actin modulators. In Caenorhabditis elegans asymmetric cell fate decisions are regulated by asymmetric localization of signaling components in a pathway termed the Wnt/beta-catenin asymmetry pathway. Which domain(s) of Disheveled regulate this pathway is unknown. We show that C. elegans embryos from dsh-2(or302) mutant mothers fail to successfully undergo morphogenesis, but transgenes containing either the DIX or the DEP domain of DSH-2 are sufficient to rescue the mutant phenotype. Embryos lacking zygotic function of SYS-1/beta-catenin, WRM-1/beta-catenin, or POP-1/Tcf show defects similar to dsh-2 mutants, including a loss of asymmetry in some cell fate decisions. Removal of two dishevelleds (dsh-2 and mig-5) leads to a global loss of POP-1 asymmetry, which can be rescued by addition of transgenes containing either the DIX or DEP domain of DSH-2. These results indicate that either the DIX or DEP domain of DSH-2 is capable of activating the Wnt/beta-catenin asymmetry pathway and regulating anterior-posterior fate decisions required for proper morphogenesis.

Quantitative analysis of clonal bone marrow CD19+ B cells: use of B cell lineage trees to delineate their role in the pathogenesis of light chain amyloidosis.

Light chain amyloidosis (AL) is a bone marrow (BM) plasma cell neoplasia with systemic deposition of Ig light chain amyloid fibrils. Here, we report the identification of clonal CD19 B cells in the BM and the use of a novel mathematical algorithm to generate B cell lineage trees of the clonal CD19 B cells and CD138 plasma cells from the BM of AL patients to delineate the relationship between these two clonal populations. The CD19+ clonal B cells in the BM of AL patients related to the clonal plasma cells represent a pre-plasma cell precursor population. The B cell lineage trees from AL patients also show significant differences in clonal diversification and antigenic selection compared to clones from normal, healthy controls. These data provide a robust example of the use of graphical quantification methods in delineating the role of neoplastic precursors in the pathogenesis of hematopoietic malignancies.