Immobilization of Saccharomyces cerevisiae by adhesion: treatment of the cells by Al ions

The adsorption of aluminum ions by Saccharomyces cerevisiae has been investigated by determining adsorption isotherms and electrophoretic mobility. The adsorption of aluminum ensures a neutralization of the cell surface charge and allows adhesion of the cells to glass and polycarbonate. Glass slides have been taken as a negatively charged model support, allowing the authors to study in detail the process of adhesion. The cells are simply pretreated by an aluminum solution near pH 4. Bringing the Al-pretreated cells in contact with the support by sedimentation and washing the support and sediment makes it possible to obtain a single, dense, regular layer of cells adhering strongly to the support. Adhesion can also be obtained from a suspension flowing parallel to a vertical support, provided the flow velocity is sufficiently small; the amount of cells immobilized per unit support area is about one-half that obtained by sedimentation. The immobilized cells show a specific activity for ethanol production from glucose which is similar to cells in suspension.     

Adhesion of Azospirillum brasilense to polystyrene: Influence of ionic strength through protein adsorption

The influence of ionic strength on the adhesion of Azospirillum brasilense to polystyrene has been examined by comparing water and phosphate buffer saline (PBS) as suspending media. Polystyrene supports analysed by X‐ray photoelectron spectroscopy (XPS) after adhesion in PBS for 2 h or 24 h and detachment of adhering cells showed a higher protein surface concentration, reflected by the N/C atomic concentration ratio, compared to supports analysed after adhesion in water. It was shown that PBS both favours protein release by the cells into the solution and enhances the tendency of proteins to adsorb at the support surface.

After 2 h contact time, the increase in the concentration of adsorbed proteins in PBS was related to an increase in adhesion density. However, the observation that the adhesion density after 24 h was lower in PBS than in water indicated that the amount of proteins adsorbed at the support surface controls cell adhesion in a complex way. In PBS, a thick layer of proteinaceous material retaining the bacterial cells is formed; this leads to underestimation of the density of adhering cells as well as to a heterogeneous adhesion pattern and to a relatively low adhesion density due to detachment of pellicles upon rinsing.

The ionic strength thus influences bacterial adhesion in a more subtle way than simply through double layer interactions between the cells and the support.     

Immobilized IL-8 triggers progressive activation of neutrophils rolling in vitro on P-selectin and intercellular adhesion molecule-1

The chemokine IL-8 is found on the luminal side of vascular endothelial cells, where it is postulated to be immobilized during inflammation. In this study, we observed that immobilized IL-8 can stimulate neutrophils to firmly adhere to a substrate containing ICAM-1 in a static adhesion assay. Soluble IL-8 was then perfused over neutrophils rolling on P-selectin (P-sel) and ICAM-1, confirming that IL-8 in solution can quickly cause rolling neutrophils to arrest. To mimic a blood vessel wall with IL-8 expressed on the luminal surface of endothelial cells, IL-8 was immobilized along with P-sel and ICAM-1 at defined site densities to a surface. Neutrophils rolled an average of 200 microm on surfaces of P-sel, ICAM-1, and IL-8 before firmly adhering through ICAM-1-beta(2) integrin interactions at 2 dynes/cm(2) wall shear stress. Increasing the density of IL-8 from 60 to 350 sites/microm(2) on the surface decreased by 50% the average distance and time the neutrophils rolled before becoming firmly adherent. Temporal dynamics of ICAM-1-beta(2) integrin interactions of rolling neutrophils following IL-8 exposure suggest the existence of two classes of beta(2) integrin-ICAM-1 interactions, a low avidity interaction with a 65% increase in pause times as compared with P-sel-P-sel glycoprotein ligand-1 interactions, and a high avidity interaction with pause times 400% greater than the selectin interactions. Based on the proportionality between IL-8 site density and time to arrest, it appears that neutrophils may need to sample a critical number of IL-8 molecules presented by the vessel wall before forming a sufficient number of high avidity beta(2) integrin bonds for firm adhesion.     

Dot assay for determining adhesive interactions between yeasts and bacteria under controlled hydrodynamic conditions

Candida belongs to the normal human microflora and are found adhering to a number of human body tissues as well as to a variety of biomaterials implants. Often, yeasts adhere in association with bacteria, but to date there is no definitive assay to investigate adhesive interactions between yeasts and bacteria adhering on surfaces. Although we recently described the use of a parallel plate flow chamber to this purpose [Millsap, K.W., Bos, R., Van der Mei, H.C., Busscher, H.J., 1998. Adhesive interactions between medically important yeasts and bacteria. FEMS Microbiol. Rev. 21, 321–336], the method was slow and evaluation of a large number of strains showed major biological variation between experiments. Here, we describe a new assay for the simultaneous determination of the adhesive interactions between yeasts and different bacterial strains on a surface under controlled hydrodynamic conditions. On an acrylic surface, the presence of adhering bacteria suppressed adhesion of Candida albicans ATCC 10261 to various degrees, depending on the bacterial strain involved. Suppression of C. albicans ATCC 10261 adhesion was strongest by Actinomyces naeslundii T14V-J1, while adhering Streptococcus gordonii NCTC 7869 caused the weakest suppression of yeast adhesion. When adhering yeasts and bacteria were challenged with the high detachment force of a passing liquid–air interface, the majority of the yeasts detached, while C. albicans adhering on the control, bare polymethylmethacrylate surface formed aggregates. Summarizing, this study presents a new method to determine suggested adhesive interactions between yeasts and adhering bacteria under controlled hydrodynamic conditions. However, the results seem to indicate that these adhesive interactions may well not exist, but that instead different bacterial strains have varying abilities to discourage yeast adhesion.     

Flow-induced detachment of red blood cells adhering to surfaces by specific antigen-antibody bonds.

Fixed spherical swollen human red blood cells of blood type B adhering on a glass surface through antigen-antibody bonds to monoclonal mouse antihuman IgM, adsorbed or covalently linked on the surface, were detached by known hydrodynamic forces created in an impinging jet. The dynamic process of detachment of the specifically bound cells was recorded and analyzed. The fraction of adherent cells remaining on the surface decreased with increasing hydrodynamic force. For an IgM coverage of 0.26%, a tangential force on the order of 100 pN was able to detach almost all of the cells from the surface within 20 min. After a given time of exposure to hydrodynamic force, the fraction of adherent cells remaining increased with time, reflecting an increase in adhesion strength. The characteristic time for effective aging was approximately 4 h. Results from experiments in which the adsorbed antibody molecules were immobilized through covalent coupling and from evanescent wave light scattering of adherent cells, imply that deformation of red cells at the contact area was the principal cause for aging, rather than local clustering of the antibody through surface diffusion. Experiments with latex beads specifically bound to red blood cells suggest that, instead of breaking the antigen-antibody bonds, antigen molecules were extracted from the cell membrane during detachment.     

Tailing of Survival Curves of Bacillus licheniformis Spores Treated with Hydrogen Peroxide

An attempt has been made to rule out possible causes of artefacts in establishing survival curves of Bacillus licheniformis spores heated (30–80 °C) in 4.4 mol/l hydrogen peroxide (pH 2.0). A tailing phenomenon apparent as that of a suspension of spores produced by routine subculture was obtained with those grown-up from a single spore selected by micromanipulation. No spore fraction differing in size or density could be separated from the whole population. The tail was not due to decomposition of hydrogen peroxide, protective effect by other spores, release of protective factors, or temperature heterogeneity during treatment. Changing from an open vessel to a closed tube did not influence the tailing. The only apparent artefact was therefore the formation of clumps under the conditions of the treatment. Since the spore catalase was demonstrated to be highly resistant, it was concluded that a spore could be protected against hydrogen peroxide by the catalase of the other spores in the clump. Conditions resembling those arising in spore suspensions could occur under industrial conditions, for example in sterilizing surfaces contaminated with aggregates of Bacillus spores.     

Modulation of chondrocyte adhesion to collagen by echistatin

Primary chondrocytes from quail embryo epiphysis (quail epiphyseal chondrocytes, QEC) can grow either in suspension or in monolayer. In this study, the adhesion of QEC to collagen II was used as a model to study the regulation of the ligand-binding activity of integrin receptors that allows these cells to undergo a rapid transition from suspension to an adherent state. Preincubation of suspension QEC (QECSP) with the disintegrin echistatin increased by 40% their adhesion to collagen II. An inverse relationship between immobilized collagen density and echistatin-induced increase of chondrocyte adhesion was observed, thus suggesting that the disintegrin acts by increasing the ligand-binding affinity of collagen receptor(s). Further, echistatin activity does not appear to depend upon a direct binding of the disintegrin to collagen receptor(s). In fact, immobilized anti-beta1 antibodies, but not immobilized echistatin, served as effective binding sites for QECSP. Echistatin failed to stimulate chondrocyte adhesion to collagen in the presence of metabolic inhibitors, while an activating anti-beta1 antibody was still effective. Thus, echistatin may promote cell adhesion by interfering with energy-dependent signals that keep the collagen receptor(s) in a low-affinity state. Adhesion experiments performed in the presence of pharmacological inhibitors indicate that phosphatidyl inositol 3-kinase (PI3-K)/protein kinase C (PKC) and protein kinase A (PKA) pathways may transmit opposing signals on chondrocyte adhesion, and that collagen receptors are kept in a low-affinity state by PI3-kinase/PKC signalling. Since echistatin is a high-affinity ligand for alphavbeta3 integrin, the effect of the function-blocking anti-alphavbeta3 antibody LM609 was investigated. Like echistatin, LM609 stimulated chondrocyte adhesion to collagen and failed to support their attachment. Therefore, our data suggest that alphavbeta3-antagonists might regulate the binding activity of the beta1 collagen receptor, which in turn leads to the rapid transition of chondrocytes from suspension to an adherent state.     

Expression of CD44 confers a new adhesive phenotype on transfected cells

The function of the CD44 glycoprotein as an adhesion molecule was directly tested by transformation of a CD44 cDNA into mouse fibroblasts. This cDNA was expressed as a heavily modified cell surface protein reactive with monoclonal antibodies that recognize glycoproteins now identified in primates as CD44. Independent transfectants exhibited a new self-adhesive phenotype, forming large aggregates when placed in suspension. In variants derived from a clone of cells, aggregation competence segregated with expression of the transfected gene. This CD44-mediated adhesion was blocked specifically by monoclonal antibodies binding one immunologically defined region of CD44. Nontransfected L cells did not self-aggregate but were capable of adhering to the transfectants, indicating that at least one ligand for this adhesion molecule is expressed by mouse fibroblasts.     

Correlation between cell substrate attachment in vitro and cell surface heparan sulfate affinity for fibronectin and collagen

Heparan sulfate glycosaminoglycan, isolated from the cell surface of nonadhering murine myeloma cells (P3X63-Ag8653), does not bind to plasma fibronectin, but binds partially to collagen type I, as assayed by affinity chromatography with proteins immobilized on cyanogen bromide-activated Sepharose 4B. Identical results were obtained when myeloma heparan sulfate was cochromatographed, on the same fibronectin and collagen columns, with cell surface heparan sulfates collagen columns, with cell surface heparan sulfates from adhering Swiss mouse 3T3 and SV3T3 cells. These latter heparan sulfates do, however, bind to both fibronectin and collagen, as reported earlier (Stamatoglou, S.C., and J.M. Keller, 1981, Biochim. Biophys. Acta., 719:90-97). Cell adhesion assays established that hydrated collagen substrata can support myeloma cell attachment, but fibronectin cannot. Saturation of the heparan sulfate binding sites on the collagen substrata with heparan sulfate or heparin, prior to cell inoculation, abolished the ability to support cell adhesion, whereas chondroitin 4 sulfate, chondroitin 6 sulfate, and hyaluronic acid had no effect.     

A kinetic study of embryonic cell adhesion

A new assay is described for measuring the kinetics of adhesion of cells to collecting aggregates. The parameter measured is the percentage of cells in suspension adhering to a large number of collecting aggregates per unit time. Using this assay on trypsinized cells it is shown that several hours of recovery are required before the maximum adhesion rate is reached. The recovery period is sensitive to cycloheximide and to low temperature (4°C). As the cells approach the maximum adhesion rate, their collection to aggregates becomes increasingly insensitive to the addition of cycloheximide; this adhesion remains sensitive to low temperature. Specificity studies show that the rate of adhesion of embryonic cells to collecting aggregates is highest between cells and aggregates of the same histotype. This rate is affected by the embryonic age of the cells. Finally, when cells of a tissue are fractionated on Ficoll gradients, it is shown that subpopulations of cells adhere to collecting aggregates at different rates.     

Direct observations of cooperative effects in oral streptococcal adhesion to glass by analysis of the spatial arrangement of adhering bacteria

The spatial arrangement of two strains of oral bacteria adhering on glass was studied in order to investigate cooperative effects in their adhesion mechanisms. Streptococcus salivarius HB was a strain which possessed several classes of fibrillar surface appendages, whereas on the cell surface of S. mutants NS no surface appendages could be identified. The bacteria were deposited from a flowing suspension with various buffer concentrations on the bottom glass plate of a parallel plate flow cell and were observed directly with a video camera mounted on a phase contrast microscope. The positions of all adhering bacteria were determined by means of automated real time image analysis and subsequently employed for calculating radial and angular pair distribution functions. Pair distribution functions indicate the average relative number density of bacteria around one deposited bacterium as a function of the radial distance or the angular orientation relative to the flow direction. From the calculated pair distribution functions of both bacterial strains it was concluded that cooperative effects contributed to the adhesion of S. salivarius HB, but not to adhesion of S. mutants NS. It was suggested that these cooperative effects originate from the surface appendages of S. salivarius HB.     

Tissue Factor-Expressing Tumor Cells Can Bind to Immobilized Recombinant Tissue Factor Pathway Inhibitor under Static and Shear Conditions In Vitro

Mammary tumors and malignant breast cancer cell lines over-express the coagulation factor, tissue factor (TF). High expression of TF is associated with a poor prognosis in breast cancer. Tissue factor pathway inhibitor (TFPI), the endogenous inhibitor of TF, is constitutively expressed on the endothelium. We hypothesized that TF-expressing tumor cells can bind to immobilized recombinant TFPI, leading to arrest of the tumor cells under shear in vitro. We evaluated the adhesion of breast cancer cells to immobilized TFPI under static and shear conditions (0.35 – 1.3 dyn/cm²). We found that high-TF-expressing breast cancer cells, MDA-MB-231 (with a TF density of 460,000/cell), but not low TF-expressing MCF-7 (with a TF density of 1,400/cell), adhered to recombinant TFPI, under static and shear conditions. Adhesion of MDA-MB-231 cells to TFPI required activated factor VII (FVIIa), but not FX, and was inhibited by a factor VIIa-blocking anti-TF antibody. Under shear, adhesion to TFPI was dependent on the TFPI-coating concentration, FVIIa concentration and shear stress, with no observed adhesion at shear stresses greater than 1.0 dyn/cm². This is the first study showing that TF-expressing tumor cells can be captured by immobilized TFPI, a ligand constitutively expressed on the endothelium, under low shear in vitro. Based on our results, we hypothesize that TFPI could be a novel ligand mediating the arrest of TF-expressing tumor cells in high TFPI-expressing vessels under conditions of low shear during metastasis.     

Minimal adhesion surface area in tangentially loaded digital contacts

The stick-to-slip transition of a fingertip in contact with a planar surface does not occur instantaneously. As the tangential load increases, portions of the skin adhere while others slip, giving rise to an evolution of the contact state, termed partial slip. We develop a quasi-static model that predicts that if the coefficient of kinetic friction is larger than the coefficient of static friction, then the stuck surface area diminishes as the tangential load increases until reaching a 'minimal adhesion surface area' where it vanishes abruptly. This phenomenon was observed in recently measured finger-slip image data (André et al., 2011) that were processed by an optic flow detection algorithm. We examined the results of 10 trials. Four of them exhibited the minimal adhesion surface area phenomenon, four of them did not, and two were inconclusive.     

Differential Involvement of ERK2 and p38 in platelet adhesion to collagen

We investigated the role of two MAP kinases, ERK2 and p38, in platelet adhesion and spreading over collagen matrix in static and blood flow conditions. P38 was involved in collagen-induced platelet adhesion and spreading in static adhesion conditions, whereas ERK2 was not. In blood flow conditions, with shear rates of 300 or 1500 s(-1), ERK2 and p38 displayed differential involvement in platelet adhesion, depending on the presence or absence of the von Willebrand factor (vWF). Low collagen coverage densities (0.04 microg/cm2) did not support vWF binding. During perfusions over this surface, platelet adhesion was not affected by the inhibition of ERK2 phosphorylation by PD 98059. However, abolishing p38 activation by SB 203580 treatment reduced platelet adhesion by 67 +/- 9% at 300 s(-1) and 56 +/- 2% at 1500 s(-1). In these conditions, the p38 activity required for platelet adhesion depends on the alpha2beta1 collagen receptor. At higher collagen coverage densities (0.8 microg/cm2) supporting vWF binding, the inhibition of ERK2 activity by PD 98059 decreased adhesion by 47 +/- 6% at 300 s(-1) and 72 +/- 3% at 1500 s(-1), whereas p38 inhibition had only a small effect. The ERK2 activity required for platelet adhesion was dependent on the interaction of vWF with GPIb. In conclusion, ERK2 and p38 have complementary effects in the control of platelet adhesion to collagen in a shear stress-dependent manner.     

Adhesion and surface-aggregation of Candida albicans from saliva on acrylic surfaces with adhering bacteria as studied in a parallel plate flow chamber

Adhesive interactions between Candida albicans and oral bacteria are generally thought to play a crucial role in the microbial colonization of denture acrylic, which may lead to denture stomatitis. This study investigated the influence of saliva on the adhesive interactions between C. albicans and Streptococcus sanguis or Actinomyces naeslundii on denture acrylic. First, bacteria were allowed to adhere to the acrylic surface from a flowing suspension, and subsequently yeasts were flowed over the acrylic surface. The organisms were assayed in the presence or absence of human whole saliva. All experiments were carried out in a parallel plate flow chamber and enumeration was done in situ with an image analysis system. In the absence of adhering bacteria, adhesion of C. albicans from buffer was more extensive than from saliva. However, in the presence of adhering bacteria, yeast adhesion from saliva was increased with respect to adhesion of yeasts from buffer, indicating that specific salivary components constitute a bridge between bacteria and yeasts. In all cases, yeast aggregates consisting of 3 to 5 yeast cells were observed adhering to the surface. A surface physico-chemical analysis of the microbial cell surfaces prior to and after bathing the microorganisms in saliva, suggests that this bridging is mediated by acid-base interactions since all strains show a major increase in electron-donating surface free energy parameters upon bathing in saliva, with no change in their zeta potentials. The surface physico-chemical analysis furthermore suggests that S. sanguis and A. naeslundii may use a different mechanism for adhesive interactions with C. albicans in saliva.     

Tumor cell haptotaxis on immobilized N-acetylglucosamine gradients

Polyacrylamide surfaces covalently derivatized with quantifiable gradients of glycosides superimposed on a uniform adhesive background of coimmobilized Arg-Gly-Asp-containing adhesion peptide were synthesized. Substrate-directed cell redistribution (haptotaxis) was measured by seeding derivatized surfaces uniformly with B16F10 murine melanoma cells. After 4-32 hr, cells on gradients of N-acetylglucosamine (GlcNAc) redistributed markedly; higher cell densities were found at gel positions having a higher immobilized GlcNAc density. In contrast, cells seeded on otherwise identical gels having a uniform concentration of immobilized GlcNAc, or on gels having gradients of glucose or galactose, did not redistribute. Soluble inhibitors containing nonreducing terminal GlcNAc (but not those with terminal GalNAc or Gal) blocked redistribution on immobilized GlcNAc gradients. Redistribution was not affected by the presence or absence of serum in the medium. An affinity-purified antibody against beta-1,4-galactosyltransferase, a GlcNAc-binding protein reported to be expressed on B16F10 cell surfaces, attenuated GlcNAc-directed redistribution. When cells were seeded on surfaces derivatized with various uniform densities of immobilized GlcNAc coimmobilized with an invariant density of immobilized Arg-Gly-Asp-peptide, neither cell attachment nor proliferation rate were enhanced on the gels having a higher GlcNAc density. These data indicate that the redistribution on immobilized GlcNAc gradients was due to cell motility. Although gels derivatized with Arg-Gly-Asp-peptide alone supported strong B16F10 cell adhesion, surfaces derivatized with uniform high concentrations of GlcNAc did not. We conclude that cell recognition of substratum gradients that support, at best, weak adhesion (GlcNAc) on an otherwise uniform strongly adhesive background (Arg-Gly-Asp-peptide) may be sufficient to direct cell migration.     

Microenvironment Modulates Osteogenic Cell Lineage Commitment in Differentiated Embryonic Stem Cells

Background

Due to their self-renewal, embryonic stem cells (ESCs) are attractive cells for applications in regenerative medicine and tissue engineering. Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best. It is possible that the traditional culture methods, which have been used, are not optimal and that other approaches must be explored.

Methodology/Principal Findings

ESCs were differentiated into osteoblast lineage using a micro-mass approach. In response to osteogenic differentiation medium, many cells underwent apoptosis, while others left the micro-mass, forming small aggregates in suspension. These aggregates were cultured in three different culture conditions (adhesion, static suspension, and stirred suspension), then examined for osteogenic potential in vitro and in vivo. In adhesion culture, ESCs primed to become osteoblasts recommitted to the adipocyte lineage in vitro. In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification. In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells.

Conclusions/Significance

We demonstrate that microenvironment modulates cell fate and subsequent tissue formation during ESC differentiation. For effective tissue engineering using ESCs, it is important to develop optimized cell culture/differentiation conditions based upon the influence of microenvironment.     

Adhesion of lymphoid cells to CD44-specific substrata: the consequences of attachment depend on the ligand

Interactions between cell-surface adhesion receptors and immobilized specific substrata can exert profound effects on cell morphology. Using phase-contrast microscopy, we show that CD44-expressing mouse lymphoid cells display a spread morphology when adhering to CD44-specific monoclonal antibody (mAb) immobilized on plastic. This spread morphology is different from that of these same cells when adhering to immobilized hyaluronan, the natural ligand of CD44. Morphometric measurements, in combination with intracellular actin staining and fluorescence microscopy, revealed that the adhesion of lymphoid cells to hyaluronan required essentially no cytoskeletal reorganization and resulted in no fundamental change in morphology. On the other hand, cells adhering to immobilized CD44-specific mAb rearranged their actin structure and established multiple membrane contact sites (spread). Cell spreading on antibody, but not attachment to hyaluronan, was inhibited by cytoskeleton-disrupting agents. Transfection of CD44-negative lymphoid cells with full-length and tailless CD44 enabled these cells to bind to both immobilized hyaluronan and mAb. However, the transfectant lacking the cytoplasmic tail of CD44 spread only transiently on the antibody-coated surface. Our results suggest that CD44 may mediate lymphocyte attachment to its carbohydrate ligand hyaluronan by mechanisms broadly similar to those used by selectins. When immobilized CD44-specific antibody is the ligand, however, CD44 may regulate the activity of the cytoskeleton by mechanisms broadly similar to those used by integrins. In the latter case, the cytoplasmic domain of CD44 contributes to cell spreading.     

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, γ227 and γ229, and a reconstituted γ229 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 α- and β-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 vinculinnegative 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 γ229 and γ227 cells that were formed in suspension during a two-hour static incubation at 37°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.     

Embryo development from discrete cell aggregates inIpomoea Batatas (L.) Lam. in response to structural polarity

Summary High numbers of embryos are difficult to obtain in liquid cultures of sweet potato (Ipomoea batatas (L.) Lam.) because discrete cell aggregates, produced through calli fragmentation, do not support embryo growth. In an effort to demonstrate that embryo development is possible from discrete cell aggregates, we compared embryo formation from cell aggregates 250–355 μm in diameter cultured either in suspension in liquid medium, on agar solidified medium, or immobilized on alginate beads floated in liquid medium. Embryos were initiated but remained arrested in their globular stage on cell aggregates cultured in suspension. Embryos developed to the torpedo stage from cell aggregates cultured on solidified medium and from cell aggregates anchored on alginate beads. Thus, embryos continued to develop beyond the globular stage when a structural polarity, which led probably to the establishment of a physiological polarity, was created. The production of sweet potato embryos in liquid culture can be improved by using alginate beads or culture conditions and protocols leading to the release during calli fragmentation of polarized individual cell aggregates. This work was supported in part by a IFAS/Gas Research Institute cooperative grant. Florida Agriculture Experiment Station Journal Series 9297