DAP-kinase is a mediator of endoplasmic reticulum stress-induced caspase activation and autophagic cell death.

Damage to endoplasmic reticulum (ER) homeostasis that cannot be corrected by the unfolded protein response activates cell death. Here, we identified death-associated protein kinase (DAPk) as an important component in the ER stress-induced cell death pathway. DAPk-/- mice are protected from kidney damage caused by injection of the ER stress-inducer tunicamycin. Likewise, the cell death response to ER stress-inducers is reduced in DAPk-/- primary fibroblasts. Both caspase activation and autophagy induction, events that are activated by ER stress and precede cell death, are significantly attenuated in the DAPk null cells. Notably, in this cellular setting, autophagy serves as a second cell killing mechanism that acts in concert with apoptosis, as the depletion of Atg5 or Beclin1 from fibroblasts significantly protected from ER stress-induced death when combined with caspase-3 depletion. We further show that ER stress promotes the catalytic activity of DAPk by causing dephosphorylation of an inhibitory autophosphorylation on Ser(308) by a PP2A-like phosphatase. Thus, DAPk constitutes a critical integration point in ER stress signaling, transmitting these signals into two distinct directions, caspase activation and autophagy, leading to cell death.

The role of sphingolipids in the maintenance of fibroblast morphology. The inhibition of protrusional activity, cell spreading, and cytokinesis induced by fumonisin B1 can be reversed by ganglioside GM3.

Previous studies demonstrated that inhibition of sphingolipid synthesis by the mycotoxin fumonisin B1 (FB1) disrupts axonal growth in cultured hippocampal neurons (Harel, R., and Futerman, A. H. (1993) J. Biol. Chem. 268, 14476-14481) by affecting the formation or stabilization of axonal branches (Schwarz, A., Rapaport, E., Hirschberg, K., and Futerman, A.H. (1995) J. Biol. Chem. 270, 10990-10998). We now demonstrate that long term incubation with FB1 affects fibroblast morphology and proliferation. Incubation of Swiss 3T3 cells with FB1 resulted in a decrease in synthesis of ganglioside GM3, the major glycosphingolipid in 3T3 fibroblasts and of sphingomyelin. The projected cell area of FB1-treated cells was approximately 45% less than control cells. FB1 had no affect on the organization of microtubules or intermediate filaments, but fewer actin-rich stress fibers were observed, and there was a loss of actin-rich lamellipodia at the leading edge. Three other processes involving the actin cytoskeleton, cytokinesis, microvilli formation, and the formation of long processes induced by protein kinase inhibitors, were all disrupted by FB1. All the effects of FB1 on cell morphology could be reversed by addition of ganglioside GM3 even in the presence of FB1, whereas the bioactive intermediates, sphinganine, sphingosine, and ceramide, were without effect. Finally, FB1 blocked cell proliferation and DNA synthesis in a reversible manner, although ganglioside GM3 could not reverse the effects of FB1 on cell proliferation. Together, these data suggest that ongoing sphingolipid synthesis is required for the assembly of both new membrane and of the underlying cytoskeleton.

Cell-adhesion to crystal surfaces. Adhesion-induced physiological cell death.

Cultured epithelial cells interact massively, rapidly and stereospecifically with the ¿011¿ faces of calcium (R,R)-tartrate tetrahydrate crystals. It was suggested that the massive rapid adhesion represents an exaggerated and isolated form of the first initial events in the attachment of cultured cells to conventional tissue culture surfaces (Hanein, et al., Cells and Materials, 5, 197-210; 1995). Attachment is however not followed by normal cell spreading and development of focal adhesions, but results in massive cell death. In this study, the fate of the crystal-bound cells was characterized by electron microscopy, flow cytometry and microscopic morphometry and was found to display the characteristics of physiological cell death. We show that the direct interaction with the highly homogenous and repetitive ¿011¿ faces per se does not trigger the transduction of lethal transmembrane signals. We suggest that the excessive direct interactions between the cell membrane and the crystal, by impairing cell motion, prevent the evolution of RGD-dependent cell adhesion. This implies that the deprivation of proper extracellular matrix (ECM)-receptor contacts of substrate-attached epithelial cells eventually triggers physiological cell death.

Focal adhesion formation by F9 embryonal carcinoma cells after vinculin gene disruption.

The assembly of focal adhesions was investigated in F9 embryonal carcinoma cells in which the expression of vinculin was eliminated by a targeted disruption of the vinculin gene. Vinculin-deficient F9 cells were capable of adhering to fibronectin-coated surfaces, though they displayed a reduced spreading compared to the parental cells. Transmission electron microscopy as well as interference reflection microscopy of live cells showed that vinculin-null F9 cells formed focal adhesions that were indistinguishable from those of the control cells. Fluorescent labeling for actin, talin, alpha-actinin, paxillin and phosphotyrosinated components indicated that the organization of all these focal contact-associated components was essentially identical in the vinculin-containing and vinculin-null cells. However, quantitative, digitized microscopy indicated that the intensity of fluorescence labeling in focal adhesions for alpha-actinin, talin and paxillin was significantly higher in cells lacking vinculin. The results suggest that there are multiple molecular mechanisms for the formation of focal adhesions in the absence of vinculin.

Selective interactions of cells with crystal surfaces. Implications for the mechanism of cell adhesion.

In this study we have characterized the mode of cell adhesion to calcite and calcium (R,R)-tartrate tetrahydrate crystals. The use of crystals as adhesion substrata was motivated by their well-established chemical nature and structurally defined surfaces. We show that calcite binds A6 Xenopus laevis epithelial cells rapidly and efficiently, most likely via surface-adsorbed proteins. Surface topology had only a limited effect on the adhesive interactions. Calcium (R,R)-tartrate tetrahydrate crystals exhibits two chemically equivalent, yet structurally distinct faces that differ mainly in the surface distribution of their lattice water molecules and charges. However, despite the gross similarity between the two faces striking differences were noted in their adhesive behavior. One of the faces was highly adhesive for cells, leading to protein-independent attachment and spreading followed by cell death. In contrast, cell adhesion to the other surface of tartrate was slow (> 24 h) and apparently mediated by RGD-containing protein(s). It was further shown that the latter face of tartrate crystals could be "conditioned" by long (24 h) incubation with serum-containing medium, after which it becomes highly adhesive. The results presented here indicate that crystal surfaces may serve as excellent, structurally defined, substrata for cell adhesion, that cell binding may occur directly or via RGD-containing proteins and that cell adhesion may be dramatically modulated by variations in surface structure. The implications of the results to the mechanism of cell-substratum adhesion are discussed.

Cingulin: characterization and localization.

Cingulin, a protein component associated with the tight junctions of chicken intestinal epithelium, has been purified to homogeneity by a new procedure and characterized. Purified cingulin is a heat-stable elongated dimer, composed of two polypeptides of Mr 108,000 (cingulin-108), with a Stokes' radius of approximately 15 nm, and a molecular length of 130 nm +/- 32 nm. Monoclonal antibodies were used to determine the tissue distribution and subcellular localization of cingulin in a variety of avian tissues and cultured cells. Indirect immunofluorescence analysis of semi-thin frozen sections demonstrated that cingulin is localized in the junctional complex of various polarized epithelia and in the endothelium, whereas it is essentially absent from mesenchymal and myogenic cells. In permeabilized and fixed cultured chick embryo kidney cells, the antibodies stained solely the regions of contacts between the epithelial cells. Double immunofluorescent labeling of these cells with anti-cingulin and anti-vinculin antibodies showed that cingulin is localized close to the vinculin-rich cytoskeletal belt associated with adherens junctions, but is absent from focal contacts and stress fibers. In cultured kidney cells, actin was detected mainly in stress fibers and in the peripheral junctional regions, where it showed a distribution similar to that of cingulin, suggesting that actin filaments may be part of the submembrane cytoskeleton at the level of the tight junction. Indirect immunoelectron microscopic labeling of ultrathin frozen sections of chicken intestine showed that cingulin is localized along the endofacial surfaces of the tight junction (zonula occludens), and is apparently excluded from the more basal zonula adhaerens, and from the desmosomes.

Cingulin, a new peripheral component of tight junctions.

The tight junction (Zonula occludens), a belt-like region of contact between cells of polarized epithelia, serves as a selective barrier to small molecules and as a total barrier to large molecules, and is involved in the separation between lumenal and basolateral compartments of the epithelium. In the electron microscope, tight junctions show focal regions of apparent fusion between the adjoining cell membranes, and freeze-fractured membranes display an elaborate network of branching and anastomosing strands. Very little is known about the molecular composition and architecture of tight junctions. The first specific zonula occludens-associated protein, designated ZO-1, has recently been identified in mammalian epithelial and endothelial cells. Here we describe the identification and purification of a new component of this junctional complex in avian brush-border cells, which we name cingulin. Cingulin is an acidic, heat-stable protein, with a highly elongated shape. Immunofluorescence and immunoelectron microscopy of brush-border cells with anti-cingulin antibodies show that cingulin is localized in the apical zone of the terminal web, at the endofacial surfaces of the zonula occludens.

Spatial and temporal relationships between vinculin and talin in the developing chicken gizzard smooth muscle.

The spatiotemporal relationships between vinculin and talin in developing chicken gizzard smooth muscle were investigated. Immunofluorescence and immunoelectron-microscopic labeling revealed that both proteins are associated with membrane-bound dense plaques in muscle cells; however, the most intense labeling for vinculin was located rather closer to the membrane than that for talin. The localization of vinculin and talin in embryonic chicken gizzards indicated that both are primarily cytoplasmic during the first 2 embryonic weeks. Only around days 16-18 does talin apparently become associated with the plasma membrane, this being concomitant with the appearance of distinct myofilament-bound dense plaques. Vinculin, on the other hand, remains primarily cytoplasmic and appears in the plaques only 1-3 days after hatching. It is thus proposed that the interactions of the dense plaque with myofilaments or with the membrane do not depend on the presence of vinculin in the plaque. Electrophoretic analyses indicated that, during development, there is no major change in the differential expression of specific vinculin isoforms. Quantitative immunoblotting analysis indicated that the vinculin content (relative to total extracted protein) is virtually constant during the last week of embryonic life. However, within 3 days of hatching, the vinculin concentration increases remarkably to over twice the embryonic level, and then slowly increases until it reaches the adult levels, which are three to four times higher than the embryonic level. The concentration of metavinculin (a 160-Kd vinculin-related protein) showed only a limited increase after hatching. We discuss the possible roles of vinculin and talin in the assembly of membrane-bound dense plaques during the different phases of smooth-muscle development.