Eukaryotic initiation factor 6 selectively regulates Wnt signaling and beta-catenin protein synthesis.

Eukaryotic initiation factor 6 (eIF6), an essential protein important in ribosome biosynthesis and assembly, was identified as an interacting partner of the beta-catenin C terminus in the yeast two-hybrid assay. Independent studies identified Drosophila eIF6 (DeIF6) in a genetic screen designed to detect new genes involved in the regulation of the Wnt/Wg (wingless) pathway. Ectopic expression of DeIF6 in wing discs results in a Wg phenotype. Expression of eIF6 in adenomatous polyposis coli (APC)-mutant colon cancer cells, which express high levels of active beta-catenin, showed that eIF6 selectively inhibits the Wnt pathway at the level of beta-catenin protein independently of proteasomal degradation. Incorporation of radiolabeled amino acids into beta-catenin was selectively decreased in cells that overexpressed eIF6. A similar inverse relationship of the two proteins was observed in the APC(min/+) mouse intestine, in which beta-catenin levels are very high. Taken together these data reveal a link between eIF6 and Wnt signaling, perhaps at the level of ribosome recycling on beta-catenin mRNA.

Vinculin and cell-cell adhesion.

Vinculin, a 117-kDa protein, is a constituent of adhesion plaques and adherence junctions in non-muscle cells. We investigated the role of vinculin on the physical strength of cell-cell adhesion by conducting disaggregation assays on aggregates of parental wild-type F9 mouse embryonal carcinoma cells (clone BIM), two vinculin-depleted F9 cell lines, gamma 227 and gamma 229, and a reconstituted gamma 229 cell line (R3) that re-express vinculin. Immunoblotting demonstrated that the four cell lines used in the study had similar expressions of the cell-cell adhesion molecule E-cadherin and associated membrane proteins alpha- and beta-catenin. Double immunofluorescence analysis showed that, in contrast to the vinculin-null cell lines. BIM and R3 cells expressed abundant vinculin at the cell margins in adhesion plaques and in cell-cell margins that also contained actin. Laminar flow assays showed that both the vinculin-positive and vinculin-negative cell aggregates that were formed in culture in the course of 24 to 48 hours largely remained intact despite the imposition of shear flow at high shear rates. Since laminar flow imposed on cell aggregates act to separate cells from each other, our data indicate that F9 cells that were adherent to a substrate formed strong cell-cell adhesion bonds independent of vinculin expression. On the other hand, aggregates of vinculin-depleted gamma 229 and gamma 227 cells that were formed in suspension during a two-hour static incubation at 37 degrees C were desegregated more easily with the imposition of shear flow than the BIM and R3 cell aggregates formed under identical conditions. Loss of vinculin was associated with a reduction in cell-cell adhesion strength only among those cells lacking contact to a substrate. Overall, the results indicate that vinculin is not needed for forming strong cell-cell adhesion bonds between neighboring carcinoma cells which are adherent to the basal lamina.

Role of cytoskeleton and deformability in laminin-mediated cell rolling.

Recent mathematical models show that molecular events that mediate rolling interactions also have an impact on the stochastic features of rolling. In spherical cells, statistical fluctuations in cell displacement were shown to be an indication that only a few adhesion bonds are involved in rolling interactions. In this study, we investigated whether cell shape and cell deformability could also modulate the stochastic features of rolling. As an experimental model we considered the flow-initiated rolling of MCF-10 breast epithelial cells on laminin. The dynamic adhesion of MCF-10A cells to laminin, which involves integrin alpha 6 beta 4, occurs slow enough to allow for an accurate determination of the trajectories of rolling cells. The data from high-magnification videomicroscopy showed that cell shape, cell deformability, and the level of fluid shear stress were all strong determinants of the rolling velocity and the extent of fluctuations in the trajectory of rolling cells. MCF-10A cells with large surface projections rolled faster and wobbled more extensively than spherical cells under the same flow conditions. The extent of wobbling decreased and the variation of rolling velocity increased with increasing fluid shear stress. MCF-10A cells treated with cytochalasin B, which increased cell deformability and caused extensive blebbing without significantly altering surface expression of laminin integrins, reduced mean rolling velocity and increased its variance. Because leukocytes change shape as they roll in postcapillary blood venules at high shear rates, results indicate the need for further expanding the present biophysical models of rolling to the case of deformable cells.

Candida albicans adherence to a human oesophageal cell line.

The oesophageal epithelium appears to be one of the primary cell targets of Candida albicans in AIDS patients. To study this interaction, we have established an in vitro adherence assay using a human epithelial oesophageal cell line (HET1-A). When yeast cells were grown in 500 mM D-galactose, adherence increased significantly over cultures prepared in 500 mM D-glucose. In addition to HET1-A cells, adherence of the organism grown in D-galactose to human buccal epithelial cells and a murine alveolar macrophage cell line was also higher. Adherence of yeast cells to HET1-A cells was partially inhibited in the presence of D-glucosamine or N-acetyl-D-glucosamine, but not with D-mannose, D-glucose, L-fucose or D-galactose. Attachment to HET1-A cells was studied using scanning and transmission electron microscopy. Partial phagocytosis of adhering yeast cells was observed occasionally within the first 90 min following infection, as evidenced by the formation of HET1-A pseudopodia in instances of close contact with yeast cells. The influence of D-galactose on cell surface proteins was studied by analysing beta-mercaptoethanol-extracted proteins from yeast cells grown in either 500 mM D-galactose or D-glucose. From D-galactose-grown cells only, a glycoprotein of approximately 190 kDa was observed in Aurodye-stained SDS-PAGE gels and in Western blots using an immunoglobulin fraction (IgG) prepared from sera of rabbits infected with the organism. These studies demonstrate that C. albicans adheres to human oesophageal cells and may utilize cell surface proteins whose synthesis is nutritionally regulated.

Role of E-cadherin in the response of tumor cell aggregates to lymphatic, venous and arterial flow: measurement of cell-cell adhesion strength.

Defects in the expression or function of the calcium dependent cell-cell adhesion molecule E-cadherin are common in invasive, metastatic carcinomas. In the present study the response of aggregates of breast epithelial cells and breast and colon carcinoma cells to forces imposed by laminar flow in a parallel plate flow channel was examined. Although E-cadherin negative tumor cells formed cell aggregates in the presence of calcium, these were significantly more likely than E-cadherin positive cell aggregates to disaggregate in response to low shear forces, such as those found in a lymphatic vessel or venule (< 3.5 dyn/cm2). E-cadherin positive normal breast epithelial cells and E-cadherin positive breast tumor cell aggregates could not be disaggregated when exposed to shear forces in excess of those found in arteries (> 100 dyn/cm2). E-cadherin negative cancer cells which had been transfected with E-cadherin exhibited large increases in adhesion strength only if the expressed protein was appropriately linked to the cytoskeleton. These results show that E-cadherin negative tumor cells, or cells in which the adhesion molecule is present but is inefficiently linked to the cytoskeleton, are far more likely than E-cadherin positive cells to detach from a tumor mass in response to low shear forces, such as those found in a lymphatic vessel or venule. Since a primary route of dissemination of many carcinoma cells is to the local lymph nodes these results point to a novel mechanism whereby defects in cell-cell adhesion could lead to carcinoma cell dissemination.

Polarized functions and permeability properties of rat epididymal epithelial cells in vitro.

Cultured rat caput and cauda epididymidal epithelial cells are shown to exhibit polarized properties characteristic of functioning epithelia. When grown on plastic substrates coated with reconstituted basement membrane, confluent monolayers of cells from both regions formed domes characteristic of other transporting epithelia. Immunocytochemical localization of three proteins characteristically associated with epithelial junctional complexes revealed that uvomorulin, zonula occludens 1 and cingulin were present in cultured epididymal epithelial cells and that their distribution was similar to that in the epididymal epithelium in vivo. These three molecules were not found in epididymal stromal cells. Cells from both regions growing in two compartment chambers developed an electrical resistance across the monolayer with a magnitude characteristic of high resistance epithelia. The optimal plating density of cells was 0.75 x 10(6) cells cm-2. The presence of reconstituted basement membrane on the filters did not affect the resistance of the cells. Inulin passage from basal to apical chambers was less than 2% over 24 h. These results show that several polarized functions of epididymal epithelial cells can be maintained in culture and that this type of culture system is useful for studying the function of the epididymis in vitro.

Development of Sertoli cell junctional specializations and the distribution of the tight-junction-associated protein ZO-1 in the mouse testis.

Basally located tight junctions between Sertoli cells in the postpubertal testis are the largest and most complex junctional complexes known. They form at puberty and are thought to be the major structural component of the "blood-testis" barrier. We have now examined the development of these structures in the immature mouse testis in conjunction with immunolocalization of the tight-junction-associated protein ZO-1 (zonula occludens 1). In testes from 5-day-old mice, tight junctional complexes are absent and ZO-1 is distributed generally over the apicolateral, but not basal, Sertoli cell membrane. As cytoskeletal and reticular elements characteristic of the mature junction are recruited to the developing junctions, between 7 and 14 days, ZO-1 becomes progressively restricted to tight junctional regions. Immunogold labeling of ZO-1 on Sertoli cell plasma membrane preparations revealed specific localization to the cytoplasmic surface of tight junctional regions. In the mature animal, ZO-1 is similarly associated with tight junctional complexes in the basal aspects of the epithelium. In addition, it is also localized to Sertoli cell ectoplasmic specializations adjacent to early elongating, but not late, spermatids just prior to sperm release. Although these structures are not tight junctions, they do have a similar cytoskeletal arrangement, suggesting that ZO-1 interacts with the submembrane cytoskeleton. These results show that, in the immature mouse testis, ZO-1 is present on the Sertoli cell plasma membrane in the absence of recognizable tight junctions. In the presence of tight junctions, however, ZO-1 is found only at the sites of junctional specializations associated with tight junctions and with elongating spermatids.

Growth of a fish ear. II. Locations of newly proliferated sensory hair cells in the saccular epithelium of Astronotus ocellatus.

Tritiated thymidine was used to investigate the sites of postembryonic hair cell addition in adult fish Astonotus ocellatus (oscar), a species known to add large numbers of hair cells for several years after hatching. Several types of labeled cells, including newly proliferated sensory hair cells, were found throughout the saccular epithelium, with the majority of the cells some distance away from the edges of the epithelium. There was no evidence for an 'annular' addition of sensory hair cells in Astronotus.

Sensory and nonsensory ciliated cells in the ear of the sea lamprey, Petromyzon marinus.

The inner ear of the sea lamprey, Petromyzon marinus, was examined using scanning and transmission electron microscopy. Many of the nonsensory surfaces of the ear chamber are lined by numerous, noninnervated, multiciliated epithelial cells. Each multiciliated epithelial cell has 43-66 true cilia projecting from its apical surface into the lumen of the ear. Although the cilia leave the cell individually, all of the cilia from a single cell come together just above the apical cell surface and are held together by a cross-network of fibrillar material. The cell bodies of the multiciliated cells sit upon a basal lamina which overlies a collagen-filled matrix. Petromyzon has typical vertebrate sensory hair cells on the cristae of the two semicircular canals as well as on the main sensory epithelium, the macula communis. Cell bodies of the sensory hair cells are similar to hair cells of other vertebrates. However, unlike other fishes, the sensory hair cells in Petromyzon have striated organelles between the nucleus and the apical cell membrane. The hair cells are innervated by afferent and efferent nerve fibers.

Growth of a fish ear: 1. Quantitative analysis of hair cell and ganglion cell proliferation.

Proliferation (or addition) of inner ear sensory hair cells continues for a long time postembryonically in cartilaginous and bony fishes, and in amphibians. In contrast, proliferation only occurs during embryonic development in birds and mammals. However, detailed quantitative data on hair cell addition are not available for bony fishes. In order to quantify the extent of proliferation, we determined the number of sensory hair cells on the saccular sensory epithelium in specimens of the cichlid fish Astronotus ocellatus (the oscar) ranging from 2.0 to 19.0 cm in standard length (0.9-343 g). Ganglion cells were counted using serial sections of the saccular branch of the eighth nerve in animals of the same size range. The saccular macula of a 2.0 cm long (0.9 g) Astronotus contains approximately 5500 sensory hair cells; fish from 16 to 19 cm long have over 170 000 hair cells. The increase in number of sensory cells and the increase in both length and weight of the animals studied were statistically correlated (r2 = 0.8). The relative densities of saccular sensory cells in different epithelial regions remained constant in animals from 2.0 to 17 cm; in larger animals the cell density decreased somewhat. Based upon very conservative estimates of the rate of growth of Astronotus, we calculate that an average of 167 hair cells/day are added during the time when the cell population of the saccule increases. Ganglion cell number also increased approximately 4.8 times in the range of fish studied. The smallest animals in our study had about 150 ganglion cells per saccular epithelium, while the largest fish had over 600 ganglion cells. We estimate that the average ratio of hair cells to afferent fibers increases from about 30:1 in the smallest fish to over 300:1 in the largest animals.

The fine structure of the sacculus and lagena of a teleost fish.

The ultrastructure of the sensory epithelia in the auditory regions of the ear, the sacculus and lagena, were investigated in the blue gourami, Trichogaster trichopterus, using transmission and scanning electron microscopy. The sensory epithelium consists of sensory hair cells surrounded by supporting cells, both of which are quite similar to comparable cells found in other fishes. The apical surface of each sensory cell contains a ciliary bundle which varies in length in different epithelial regions. Tight junctions and one or more levels of desmosomes are located between supporting cells, and between sensory and supporting cells, just below the apical cell membrane. Peripheral to the actual sensory epithelium is a region of epithelial cells that resemble the supporting cells on the sensory epithelium itself. However, interspersed among these cells are other cells containing large numbers of mitochondria, extensive smooth endoplasmic reticulum, and large vacuoles. Investigations of the orientation patterns of the ciliary bundles on the sensory hair cells demonstrate that the lagena is typical of other Perciform fishes while the position of two of the four orientation groups normally found in the Perciform sacculus are quite different in Trichogaster from that found in other species. Comparisons of the ultrastructure of the sensory and supporting epithelia of Trichogaster and other fishes shows that, with the exception of the mitochondria-filled cells, there are no apparent significant interspecific differences with regard to ultrastructure of the sensory and supporting cells themselves, although there are differences in hair cell orientation patterns among the same fish groups.