Characterization of eukaryotic cell surfaces prior to and after serum protein adsorption by X-ray photoelectron spectroscopy

Elemental surface concentration ratios N/C, O/C, and P/C of fibroblasts, HELA epithelial cells, and smooth muscle cells, prior to and after washing in the absence or presence of serum proteins, were determined by X-ray photoelectron spectroscopy. Cell surfaces appeared to adsorb hardly any serum proteins, and the relatively high P/C, as compared to N/C and O/C, elemental surface concentration ratio indicated that the cell surfaces consisted mainly of the phospholipid bilayer, with little or no proteins present. The lack of adsorption of serum proteins to the cell surfaces seems at odds with the common notion that cells require adhesive proteins in order to adhere and spread. However, the adsorption behavior of cellularly produced proteins may be completely different, particularly since they seem to be able to displace adsorbed serum proteins from biomaterials surfaces. Interestingly, only HELA epithelial cells (a tumor cell line) appeared to adsorb a very small amount of proteins.     

Characterization of eukaryotic cell surfaces prior to and after serum protein adsorption by X-ray photoelectron spectroscopy. Fibroblasts, HELA epithelial, and smooth muscle cells.

Elemental surface concentration ratios N/C,O/C, and P/C of fibroblasts, HELA epithelial cells, and smooth muscle cells, prior to and after washing in the absence or presence of serum proteins, were determined by X-ray photoelectron spectroscopy. Cell surfaces appeared to adsorb hardly any serum proteins, and the relatively high P/C, as compared to N/C and O/C, elemental surface concentration ratio indicated that the cell surfaces consisted mainly of the phospholipid bilayer, with little or no proteins present. The lack of adsorption of serum proteins to the cell surfaces seems at odds with the common notion that cells require adhesive proteins in order to adhere and spread. However, the adsorption behavior of cellularly produced proteins may be completely different, particularly since they seem to be able to displace adsorbed serum proteins from biomaterials surfaces. Interestingly, only HELA epithelial cells (a tumor cell line) appeared to adsorb a very small amount of proteins.     

"Gap guidance" of fibroblasts and epithelial cells by discontinuous edged surfaces

Cell adhesion, shape, and directed migration are some of the fundamental processes underlying tissue development and organization. The setting of geometric limits on cellular behavior has led to the hypothesis that a continuous edge is required to elongate a cell and guide its direction of movement. The aim of this study was to examine the validity of this hypothesis by examining the response of human gingival fibroblasts and periodontal ligament epithelial cells, to microfabricated surfaces that incorporate discontinuous edges. Cell response was assessed through spreading, morphology, cytoskeletal organization, and time-lapse microscopy, on substrata with a pattern of repeated open boxes with gaps at the corners. Fibroblasts attached and spread within 6 h, adopting either a square, triangular, or diagonally elongated morphology. Epithelial cells took longer to adhere, but were observed to adopt morphologies similar to those of the fibroblasts. Addition of colcemid or cytochalasin-D attenuated the orientation and alignment of both fibroblasts and epithelial cells. Fibroblasts and epithelial cell migration was guided diagonally in their movement through gaps in the square pattern, demonstrating that a continuous edge is not a prerequisite for guided cell migration.     

Time Lapse Filming of Cellular Interactions in Organ Culture

A new method of time lapse filming has been developed for the investigation of organ cultures. This method reveals the 3-dimensional structure of the living tissue.
In Paper I the interaction of non-malignant fibroblasts and macrophages with natural membranes is studied. These cells spread on the mesothelial and epithelial surfaces of the membranes. The cell membranes exhibit ruffled movements and glide smoothly over the surface of the normal mesothelial tissues without penetration of the surface layers.     

Locomotory activity of epithelial cells in culture.

The movement of epithelial cells in vitro has been studied with time lapse cinemicrography, micromanipulation, marking of the cell surface, and electron microscopy. The cells, in contrast to fibroblasts, spread as contiguous sheets. Locomotion results primarily from the activity of the marginal cells, as determined by the extent and location of cell adhesions to the plane substratum. The locomotory activity of epithelial cells as members of a sheet is similar to that of chick heart fibroblasts, consisting of a fluctuation of the flattened free edge, a backward movement of particles adhering to the upper surface of the lamellipodium, ruffling, blebbing, and microspike activity. Of these, only the first two are invariably associated with movement. These phenomena are discussed in relation to the mechanism of epithelial cell movement. The basic differences between epithelial cells and fibroblasts, as far as locomotory and adhesive properties are concerned, are the tendency of isolated epithelial cells to bleb more vigorously than fibroblasts and the more extensive and apparently stronger lateral adhesion of epithelial cells.     

灭活的青春双歧杆菌对人大肠癌细胞的粘附

针对灭活的青春双歧杆菌DM850 4与人大肠癌CCL 2 2 9细胞之间的粘附现象及粘附机制进行研究。结果发现灭活的双歧杆菌具有与活菌相同的粘附定植能力 ,两者粘附于体外培养的肠上皮细胞均依赖于耗尽培养上清 (SCS)的存在。青春双歧杆菌粘附素有可能是存在于细胞壁中及分泌至SCS中的脂磷壁酸 (LTA)。LTA与细菌细胞壁耐热蛋白相互粘连 ,并且伸出胞壁之外。此外 ,肠上皮细胞表面的粘附素受体可能为糖类或糖蛋白。     

Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion

The morphology and cytoskeletal structure of fibroblasts, endothelial cells, and neutrophils are documented for cells cultured on surfaces with stiffness ranging from 2 to 55,000 Pa that have been laminated with fibronectin or collagen as adhesive ligand. When grown in sparse culture with no cell-cell contacts, fibroblasts and endothelial cells show an abrupt change in spread area that occurs at a stiffness range around 3,000 Pa. No actin stress fibers are seen in fibroblasts on soft surfaces, and the appearance of stress fibers is abrupt and complete at a stiffness range coincident with that at which they spread. Upregulation of alpha5 integrin also occurs in the same stiffness range, but exogenous expression of alpha5 integrin is not sufficient to cause cell spreading on soft surfaces. Neutrophils, in contrast, show no dependence of either resting shape or ability to spread after activation when cultured on surfaces as soft as 2 Pa compared to glass. The shape and cytoskeletal differences evident in single cells on soft compared to hard substrates are eliminated when fibroblasts or endothelial cells make cell-cell contact. These results support the hypothesis that mechanical factors impact different cell types in fundamentally different ways, and can trigger specific changes similar to those stimulated by soluble ligands.     

Atomic force microscopy investigation of fibroblasts infected with wild-type and mutant murine leukemia virus (MuLV)

NIH 3T3 cells were infected in culture with the oncogenic retrovirus, mouse leukemia virus (MuLV), and studied using atomic force microscopy (AFM). Cells fixed with glutaraldehyde alone, and those postfixed with osmium tetroxide, were imaged under ethanol according to procedures that largely preserved their structures. With glutaraldehyde fixation alone, the lipid bilayer was removed and maturing virions were seen emerging from the cytoskeletal matrix. With osmium tetroxide postfixation, the lipid bilayer was maintained and virions were observable still attached to the cell surfaces. The virions on the cell surfaces were imaged at high resolution and considerable detail of the arrangement of protein assemblies on their surfaces was evident. Infected cells were also labeled with primary antibodies against the virus env surface protein, followed by secondary antibodies conjugated with colloidal gold particles. Other 3T3 cells in culture were infected with MuLV containing a mutation in the gPr80(gag) gene. Those cells were observed by AFM not to produce normal MuLV on their surfaces, or at best, only at very low levels. The cell surfaces, however, became covered with tubelike structures that appear to result from a failure of the virions to properly undergo morphogenesis, and to fail in budding completely from the cell's surfaces.     

Immunocytochemistry of cell surface heparan sulfate proteoglycan in mouse tissues. A light and electron microscopic study

The core protein of the proteoglycan at the cell surface of NMuMG mouse mammary epithelial cells bears both heparan and chondroitin sulfate chains and is recognized by the monoclonal antibody 281-2. Using this antibody and the peroxidase-antiperoxidase staining technique in adult mouse tissues, we found that the antibody recognizes the antigen in a highly restricted distribution, staining a variety of epithelial cells but no cells derived from embryonic mesoderm or neural crest. The antibody fails to stain any stromal (mesenchymal) or neuronal cells, with the exception of plasma cells and Leydig cells. Squamous and transitional epithelia stain intensely over their entire surfaces, whereas cuboidal and columnar epithelia stain moderately and only at the lateral surface of the basal cells. Within squamous and transitional epithelial tissues that undergo physiological regeneration (e.g., epidermis), the most superficial and differentiated cell types fail to stain. Within glandular and branched epithelia (e.g., pancreas), the secretory alveolar cells fail to stain. When evaluated by electron microscopy, granular deposits of stain are seen on the plasma membrane, especially on lateral surfaces, but none are noted within the cells or the basement membrane. These results indicate that in adult tissues the core protein of this heparan sulfate-rich proteoglycan is expressed almost exclusively at epithelial cell surfaces. Expression appears to be lost as the cells become either mature or highly differentiated.     

Determinants of the developing oral flora in normal newborns.

The ability of Streptococcus species to selectively adhere to the oral epithelial cells of newborns was studied in vitro. On day 1 of life, mucosal cells from normal infants demonstrated selective attraction for the natural distribution of streptococci that would soon colonize these surfaces. Streptococcus salivarius and Streptococcus mitis adhered well in vitro to scraped cells from cheek and tongue surfaces. Streptococcus mutans, on the other hand, exhibited feeble or no adherence to cheek or tongue cells. Adherence of Escherichia coli to oral epithelial cells was also studied. The ability of strains of E. coli to adhere to cheek and tongue cells correlated solely with the presence of cell surface substances, probably pili. These observations, made on infants at the critical moment of their developing flora, strengthen the hypothesis that the ability of bacteria to adhere to surfaces is an important determinant of their ecological place in the oral microflora.     

Spreading of normal and transformed fibroblasts in dense cultures

Cell spreading in dense cultures of normal mouse embryo fibroblasts and of the two lines of mouse transformed fibroblasts was examined by electron microscopy. The mean number of cell layers in culture and cell population density per unit area of the substrate were detetmined; the mean area of the cell projection on the substratum was found from these data.Normal fibroblasts formed multilayefed sheet in dense culture. The cells in this sheet were well-spread. These cells formed thin lamellae (lamellar cytoplasm) over the surface of other cells and over the intercellular substance. The mean cell area in dense culture was not smaller than that of the cell spread on the substratum in sparse culture.Dense cultures of two transformed lines (M 22 and L) had differing morphologies: cultures of one line (M 22) were multilayered, those of the other line (L) were monolayered. Decreased spreading and almost complete (M 22) or complete (L) absence of lamellar cytoplasm were characteristic of both transformed lines. The mean area of the cell in dense cultures of both lines was several times smaller than that of their normal progenitors.It is concluded that similar reactions leading to the spreading accompanied by the formation of lamellar cytoplasm can be induced by the contact of fibroblast with various surfaces: that of the substratum in sparse culture, that of other cells and of intercellular structures in dense culture. Deficiency of these reactions characteristic for transformed fibroblasts may be responsible for abnormal morphology of their cultures.     

Variants of polyoma-transformed BHK21 cells unresponsive to fibronectin

To investigate the relation between cell-substratum adhesion and cell-spreading we have isolated variants of anchorage-independent cells which fail to adhere to fibronectin. The variants are poorly adhesive both to fibronectin and serum, show dramatically altered morphology in culture and are unable to spread on any protein-coated surface yet tested.     

Role of fibronectin in the adhesion of Friend erythroleukemia cells

Friend Erythroleukemia Cells (FLC) (745 A and FW clones), normally growing in suspension, tend to adhere to fibroblast monolayers, but not to epithelial cells. Co-cultivation of FLC with Human Embryo Fibroblasts (HEF) resulted in the selection of adhesive Friend Cells. After 16 subcultures, we were able to isolate clones of adhesive FLC that grow in monolayer on plastic tissue culture plates. Both the binding of FLC to fibroblasts and of the adhesive clones to the plastic surfaces is completely suppressed by fibronectin antiserum, thus suggesting that fibronectin is responsible for FLC adhesion. Adhesive FLC clones maintain the ability to differentiate upon induction by DMSO.     

Copolymerization of 2-carboxyisopropylacrylamide with N-isopropylacrylamide accelerates cell detachment from grafted surfaces by reducing temperature

Acrylic acid (AAc) has been utilized to introduce reactive carboxyl groups to a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). However, AAc introduction shifts the copolymer phase transition temperatures higher and dampens the steep homopolymer phase transition with increasing AAc content. We previously synthesized 2-carboxyisopropylacrylamide (CIPAAm) having both a similar side chain structure to IPAAm and a functional carboxylate group in order to overcome these shortcomings. In the present study, these copolymers, grafted onto cell culture plastic, were assessed for cell adhesion control using their phase transition. AAc introduction to PIPAAm-grafted surfaces resulted in excessive surface hydration and hindered cell spreading in culture at 37 degrees C. In contrast, CIPAAm-containing copolymer-grafted surfaces exhibited relatively weak hydrophobicity similar to both homopolymer PIPAAm-grafted surfaces as well as commercial ungrafted tissue culture polystyrene dish surfaces. Cells adhered and spread well on these surfaces at 37 degrees C in culture. As observed previously on PIPAAm-grafted surfaces, cells were spontaneously detached from the copolymer-grafted surfaces by reducing culture temperature. Cell detachment was accelerated on the CIPAAm copolymer-grafted surfaces compared to pure IPAAm surfaces, suggesting that hydrophilic carboxyl group microenvironment in the monomer and polymer is important to accelerate grafted surface hydration below the lower critical solution temperature, detaching cells.     

Distribution of solid liposomes on the surface of an epithelial cell layer

A study was made of the adhesion of liposomes, composed of dipalmitoyl- or di-stearoylphosphatidycholine, on the surface of epithelial cells in culture. Sodium fluorescein was entrapped in liposomes for their visualization by fluorescence microscopy. It is found that sonicated unilamellar liposomes adhere predominantly along the sheet margins. Multilamellar liposomes and lipid-coated carmine particles adhere over the whole cellular surface. However, their adhesion along sheet margins was stronger, as evidenced by a brief trypsin treatment. A prolonged trypsin treatment removed all types of liposomes from the cell surface. After the cells were partly detached from each other, small liposomes readily adhered to the newly accessible cell margins. The existence of special lipid membrane-binding proteins on the cell surface is suggested.     

Substrate rigidity regulates the formation and maintenance of tissues

The ability of cells to form tissues represents one of the most fundamental issues in biology. However, it is unclear what triggers cells to adhere to one another in tissues and to migrate once a piece of tissue is planted on culture surfaces. Using substrates of identical chemical composition but different flexibility, we show that this process is controlled by substrate rigidity: on stiff substrates, cells migrate away from one another and spread on surfaces, whereas on soft substrates they merge to form tissue-like structures. Similar behavior was observed not only with fibroblastic and epithelial cell lines but also explants from neonatal rat hearts. Cell compaction on soft substrates involves a combination of weakened adhesions to the substrate and myosin II-dependent contractile forces that drive cells toward one another. Our results suggest that tissue formation and maintenance is regulated by differential mechanical signals between cell-cell and cell-substrate interactions, which in turn elicit differential contractile forces and adhesions to determine the preferred direction of cell migration and association.     

Spreading of human fibroblasts in serum-free medium: Inhibition by dithiothreitol and the effect of cold insoluble globulin (plasma fibronectin)

We have tested the effect of dithiothreitol (DTT) treatment on the initial spreading of human fibroblasts in serum-free medium in tissue culture dishes. Cell spreading was inhibited following treatment of these cells with 10 mM DTT. Inhibition occurred when the cells were treated at 37°C but not at 4° and was reversible metabolically but not by the addition of sulfhydryl oxidizing reagents. The inhibition was overcome when DTT-treated human fibroblasts were plated on cold insoluble globulin (plasma fibronectin)—coated dishes. Under these conditions spreading appeared to be completely normal, including the formation of focal adhesions. Analysis of the fibronectin concentrations in the human fibroblasts following DTT treatment indicated that there was little decrease in the absolute level of activity as determined in a biological assay for BHK cell spreading on culture dishes. Analysis of the fibronectin distribution on the DTT-treated human fibroblasts by indirect immunofluorescence using a specific anti-CIG antiserum revealed that fibronectin was no longer deposited onto the culture dish surfaces. Even when the DTT-treated human fibroblasts spread in the presence of fetal calf serum, the cell fibronectin remained for the most part in a perinuclear location. These results indicate that DTT treatment of human fibroblasts prevents the normal translocation of fibronectin from a perinuclear location to the surface of the culture dish. This study further supports our hypothesis that the initial spreading in serum-free medium of fibroblasts from cell strains depends upon secretion of fibronectin onto the culture dish surface.     

A simplified technique for the short-term tissue culture of rabbit corneal cells

Summary A technique for the short-term culture of pure populations of rabbit corneal endothelial and epithelial cells has been developed. Rabbit corneas were placed on concave agarose surfaces, treated briefly with a solution of trypsin and ethylenediamine tetracetic acid, and transferred, either epithelial cell surface or endothelial cell surface down, to microscope slide culture chambers. Within 6 to 12 h the epithelial cells or endothelial cells attached to the slide chamber surface and the cornea was removed, leaving behind a pure population of cells which spread out and grew to fill the surface of the slide chamber. This technique provides a simple and economic means for the reproducible initiation of primary cultures of rabbit corneal epithelial and endothelial cells for us in a variety of experiments. This study was supported in part by Public Health Service grants EY03150, EY02580, and EY02377 from the National Eye Institute, National Institutes of Health, Bethesda, MD, and a Foreign Fellowship (Dr. Xie) from Research to Prevent Blindness, Inc., New York, NY.     

The distribution of solid liposomes of different size on the surface of epithelial cells

Interaction of large multilamellar and small sonicated liposomes with epithelial sheets in culture has been studied. Liposomes were comprised of dipalmitoyl- or distearoylphosphatidylcholine and were solid at 37 degrees. Sodium fluorescein was entrapped in liposomes and their localization on the cell surface was studied by fluorescence microscopy. The distribution of liposomes depends on their size. Large liposomes adhere uniformly all over the surface of the sheet, which proved to be nonadhesive either for other cells or for inert particles. Small liposomes adhere mostly along the margins of the sheet. When the cells are partly detached from each other by mild EDTA (ethylene diamine tetraacetic acid) treatment, the newly accessible cell margins become capable of binding small liposomes. The binding of both types of liposomes is trypsin-sensitive; however large liposomes, bound along the edges of the cellular sheet, are removed slower than those adhering to the inner parts of the sheet. Competition experiments show that large and small liposomes bind to the same sites on the surface of the sheet. Liposome-acceptor proteins are suggested to exist on the surface of epithelial cells. We suppose that these proteins are concentrated along the cell margins and are involved in cell-cell contacts.