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.

AlphaE-catenin regulates actin dynamics independently of cadherin-mediated cell-cell adhesion.

alphaE-catenin binds the cell-cell adhesion complex of E-cadherin and beta-catenin (beta-cat) and regulates filamentous actin (F-actin) dynamics. In vitro, binding of alphaE-catenin to the E-cadherin-beta-cat complex lowers alphaE-catenin affinity for F-actin, and alphaE-catenin alone can bind F-actin and inhibit Arp2/3 complex-mediated actin polymerization. In cells, to test whether alphaE-catenin regulates actin dynamics independently of the cadherin complex, the cytosolic alphaE-catenin pool was sequestered to mitochondria without affecting overall levels of alphaE-catenin or the cadherin-catenin complex. Sequestering cytosolic alphaE-catenin to mitochondria alters lamellipodia architecture and increases membrane dynamics and cell migration without affecting cell-cell adhesion. In contrast, sequestration of cytosolic alphaE-catenin to the plasma membrane reduces membrane dynamics. These results demonstrate that the cytosolic pool of alphaE-catenin regulates actin dynamics independently of cell-cell adhesion.

Bench to bedside and back again: molecular mechanisms of alpha-catenin function and roles in tumorigenesis.

The cadherin/catenin complex, comprised of E-cadherin, beta-catenin and alpha-catenin, is essential for initiating cell-cell adhesion, establishing cellular polarity and maintaining tissue organization. Disruption or loss of the cadherin/catenin complex is common in cancer. As the primary cell-cell adhesion protein in epithelial cells, E-cadherin has long been studied in cancer progression. Similarly, additional roles for beta-catenin in the Wnt signaling pathway has led to many studies of the role of beta-catenin in cancer. Alpha-catenin, in contrast, has received less attention. However, recent data demonstrate novel functions for alpha-catenin in regulating the actin cytoskeleton and cell-cell adhesion, which when perturbed could contribute to cancer progression. In this review, we use cancer data to evaluate molecular models of alpha-catenin function, from the canonical role of alpha-catenin in cell-cell adhesion to non-canonical roles identified following conditional alpha-catenin deletion. This analysis identifies alpha-catenin as a prognostic factor in cancer progression.

Geminin prevents rereplication during xenopus development.

To maintain a stable genome, it is essential that replication origins fire only once per cell cycle. The protein Geminin is thought to prevent a second round of DNA replication by inhibiting the essential replication factor Cdt1. Geminin also affects the development of several different organs by binding and inhibiting transcription factors and chromatin-remodeling proteins. It is not known if the defects in Geminin-deficient organisms are due to overreplication or to effects on cell differentiation. We previously reported that Geminin depletion in Xenopus causes early embryonic lethality due to a Chk1-dependent G(2) cell cycle arrest just after the midblastula transition. Here we report that expressing a non-Geminin-binding Cdt1 mutant in Xenopus embryos exactly reproduces the phenotype of geminin depletion. Expressing the same mutant in replication extracts induces a partial second round of DNA replication within a single S phase. We conclude that Geminin is required to suppress a second round of DNA replication in vivo and that the phenotype of Geminin-depleted Xenopus embryos is caused by abnormal Cdt1 regulation. Expressing a nondegradable Cdt1 mutant in embryos also reproduces the Geminin-deficient phenotype. In cell extracts, the nondegradable mutant has no effect by itself but augments the amount of rereplication observed when Geminin is depleted. We conclude that Cdt1 is regulated both by Geminin binding and by degradation.

Geminin has dimerization, Cdt1-binding, and destruction domains that are required for biological activity.

Geminin is an unstable regulatory protein that affects both cell division and cell differentiation. Geminin inhibits a second round of DNA synthesis during S and G(2) phase by binding the essential replication protein Cdt1. Geminin is also required for entry into mitosis, either by preventing replication abnormalities or by down-regulating the checkpoint kinase Chk1. Geminin overexpression during embryonic development induces ectopic neural tissue, inhibits eye formation, and perturbs the segmental patterning of the embryo. In order to define the structural and functional domains of the geminin protein, we generated over 40 missense and deletion mutations and tested their phenotypes in biological and biochemical assays. We find that geminin self-associates through the coiled-coil domain to form dimers and that dimerization is required for activity. Geminin contains a typical bipartite nuclear localization signal that is also required for its destruction during mitosis. Nondegradable mutants of geminin interfere with DNA replication in succeeding cell cycles. Geminin's Cdt1-binding domain lies immediately adjacent to the dimerization domain and overlaps it. We constructed two nonbinding mutants in this domain and found that they neither inhibited replication nor permitted entry into mitosis, indicating that this domain is necessary for both activities. We identified several missense mutations in geminin's Cdt1 binding domain that were deficient in their ability to inhibit replication yet were still able to allow mitotic entry, suggesting that these are separate functions of geminin.

Robust G1 checkpoint arrest in budding yeast: dependence on DNA damage signaling and repair.

Although most eukaryotes can arrest in G1 after ionizing radiation, the existence or significance of a G1 checkpoint in S. cerevisiae has been challenged. Previous studies of G1 response to chemical mutagens, X-ray or UV irradiation indicate that the delay before replication is transient and may reflect a strong intra-S-phase checkpoint. We examined the yeast response to double-stranded breaks in G1 using gamma irradiation. G1 irradiation induces repair foci on chromosome spreads and a Rad53 band shift characteristic of activation, which suggest an active DNA damage response. Consistent with a G1 arrest, bud emergence, spindle pole duplication and DNA replication are each delayed in a dose-dependent manner. Sensitivity to mating pheromone is prolonged to over 18 hours when G1 cells are lethally gamma or UV irradiated. Strikingly, G1 delay is the predominant response to continuous gamma irradiation at a dose that confers no loss of viability but delays cell division. Like the G2/M checkpoint, G1 delay is completely dependent on both RAD9 and RAD24 epistasis groups but independent of POL(epsilon). Lethally irradiated rad9 mutants rapidly exit G1 but perform a slow S phase, whereas rad17 and rad24 mutants are completely arrest deficient. Distinct from gamma irradiation, G1 arrest after UV is RAD14 dependent, suggesting that DNA damage processing is required for checkpoint activation. Therefore, as in the yeast G2/M checkpoint response, free DNA ends and/or single-stranded DNA are necessary and sufficient to induce a bona fide G1 checkpoint arrest.