Biophysical integration of effects of epidermal growth factor and fibronectin on fibroblast migration

Cell migration is regulated simultaneously by growth factors and extracellular matrix molecules. Although information is continually increasing regarding the relevant signaling pathways, there exists little understanding concerning how these pathways integrate to produce the biophysical processes that govern locomotion. Herein, we report the effects of epidermal growth factor (EGF) and fibronectin (Fn) on multiple facets of fibroblast motility: locomotion speed, membrane extension and retraction activity, and adhesion. A surprising finding is that EGF can either decrease or increase locomotion speed depending on the surface Fn concentration, despite EGF diminishing global cell adhesion at all Fn concentrations. At the same time, the effect of EGF on membrane activity varies from negative to positive to no-effect as Fn concentration and adhesion range from low to high. Taking these effects together, we find that EGF and Fn regulate fibroblast migration speed through integration of the processes of membrane extension, attachment, and detachment, with each of these processes being rate-limiting for locomotion in sequential regimes of increasing adhesivity. Thus, distinct biophysical processes are shown to integrate for overall cell migration responses to growth factor and extracellular matrix stimuli.     

Low concentrations of fibrinogen increase cell migration speed on fibronectin/fibrinogen composite cables

Optimal cell migration rate in a given direction (velocity) is a function of speed and directional persistence. Migration speed has been reported to be a function of adhesion strength such that optimal cell migration occurs where the cell is able to form enough stable attachments for good traction while allowing attachments at the trailing end to be broken during locomotion. This is particularly important in peripheral nerve regeneration where rapid Schwann cell recruitment across the injury site will lead to better functional recovery and reduced end organ atrophy. The aim here was to investigate the effects of changing adhesion properties of Fn materials by adding fibrinogen in order to design an optimal material for repair processes. Cell migration on Fn/Fg-cables increased with increasing content of %Fg to a peak cell migration velocity (Schwann cells) of 49 microm/h, at 50% Fg. Further increases in Fg content hindered cell migration. Vinculin-rich attachment plaques were reduced in a dose-dependent manner as the content of %Fg was increased whilst cells at the optimum Fg proportion for cell migration were moderately well spread. These results support the idea that optimum cell migration rates occur at intermediate attachment conditions, in this case at 50% Fg. These results show that incorporation of Fg into Fn-based materials will enhance the speed of Schwann cell migration and this would be likely to improve peripheral nerve regeneration. Indeed, directionally aligned Fn-based materials can now be engineered to give optimal cell velocity during repair cell recruitment in a range of tissue repair or tissue engineering applications.     

Polarity, protrusion-retraction dynamics and their interplay during keratinocyte cell migration.

Keratinocyte migration on a two-dimensional substrate can be split into four distinct phases: cell extension, attachment, contraction, and detachment. It is preceded by polarization of the cell which leads to a functional asymmetry observable by the formation of a leading lamella. In this work variation of fibronectin coating concentrations and competitive inhibition with RGD peptides are used to investigate the dependency of polarization, migration, lamella dynamics, and ruffling on substrate adhesiveness. Looking at migrating human epidermal keratinocytes with a well-defined polarity we find that a fibronectin-coating concentration of 10 microg/cm(2) stimulates migration and ruffling speed twofold, whereas protrusion speed increases only by 20% (compared to 2.5 microg/cm(2) fibronectin). Nonpolar cells show a constant migration and ruffling speed independent of the amount of fibronectin. In contrast protrusion speeds of polar and nonpolar cells are equal. Treatment of cells on 10 microg/cm(2) fibronectin with 1 mg/ml GRGDS reduces the characteristic migration, protrusion, and ruffling speed of polar cells which corresponds to lowering the effective coating concentration to under 5 microg/cm(2). The probability of being polarized (quantified by a polarity index) increases with increasing fibronectin concentration. However, addition of soluble RGD on 10 microg/cm(2) fibronectin does not simply reduce the polarity index like one would expect from the corresponding changes in the other motility parameters, but it remains unchanged.     

Micron-scale positioning of features influences the rate of polymorphonuclear leukocyte migration

Microfabrication technology was used to create regular arrays of micron-size holes (2 microm x 2 microm x 210 nm) on fused quartz and photosensitive polyimide surfaces. The patterned surfaces, which possessed a basic structural element of a three-dimensional (3-D) network (i.e., spatially separated mechanical edges), were used as a model system for studying the effect of substrate microgeometry on neutrophil migration. The edge-to-edge spacing between features was systematically varied from 6 microm to 14 microm with an increment of 2 microm. In addition, collagen was used to coat the patterned quartz surfaces in an attempt to change the adhesive properties of the surfaces. A radial flow detachment assay revealed that cell adhesion was the strongest on the quartz surface (approximately 50% cell attached), whereas it was relatively weaker on polyimide and collagen-coated quartz (approximately 25% cell attached). Cell adhesion to each substrate was not affected either by the presence of holes or by the spacing between holes. A direct visualization assay showed that neutrophil migration on each patterned surface could be characterized as a persistent random walk; the dependence of the random motility coefficient (mu) as a function of spacing was biphasic with the optimal spacing at approximately 10 microm on each substrate. The presence of evenly distributed holes at the optimal spacing of 10 microm enhanced mu by a factor of 2 on polyimide, a factor of 2.5 on collagen-coated quartz, and a factor of 10 on uncoated quartz. The biphasic dependence on the mechanical edges of neutrophil migration on 2-D patterned substrate was strikingly similar to that previously observed during neutrophil migration within 3-D networks, suggesting that microfabricated materials provide relevant models of 3-D structures with precisely defined physical characteristics. In addition, our results demonstrate that the microgeometry of a substrate, when considered separately from adhesion, can play a significant role in cell migration.     

Cell-substratum adhesion strength as a determinant of hepatocyte aggregate morphology

Cultured hepatocytes typically form multicellular aggregates which are either monolayered or spheroidal in morphology. We propose that the aggregate morphology resulting from a particular cell-substratum interaction has a biophysical basis: when cell contractile forces are greater than cell-substratum adhesion forces, spheroidal aggregates form; when cell contractile forces are weaker than cell-substratum adhesion forces, cells remain essentially spread and form monolayered aggregates. We tested this hypothesis by systematically varying the morphology of hepatocellular aggregates formed on substrata coated with a series of different concentrations of Matrigel, and correlating aggregate morphology with the cell-substratum adhesion strength measured in a shear flow detachment assay. Aggregate morphology was binary-spheroidal aggregates formed at low Matrigel concentrations and monolayered aggregates formed at high Matrigel concentrations. Cell-substratum adhesion strength was similarly binary, with low adhesion strengths correlated with spheroidal aggregates and high adhesion strengths correlated with formation of monolayered aggregates. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 415-426, 1997.     

Strain-induced orientation response of endothelial cells: effect of substratum adhesiveness and actin-myosin contractile level

Endothelial cells subjected to cyclic stretching change orientation so as to be aligned perpendicular to the direction of applied strain in a magnitude and time-dependent manner. Although this type of response is not the same as motility, it could be governed by motility-related factors such as substratum adhesiveness and actin-myosin contractile level. To examine this possibility, human aortic endothelial cells (HAEC) were uniaxially, cyclically stretched on silicone rubber membranes coated with various concentrations of fibronectin, collagen type IV and laminin to produce differing amounts of adhesiveness (measured using a radial flow detachment assay). Cells were subjected to 10% pure cyclic uniaxial stretching for three hours at a rate of 10%/sec. Time-lapse images revealed that cells underwent large morphological changes without moving. For each type of protein there was a parabolic dependence on initial adhesiveness with optimal cell orientation occurring at very similar adhesive strengths. The effect of actin-myosin contractile level was examined by stretching cells treated with different doses of 2,3-butanedione monoxime (BDM) and Blebbistatin. Each drug induced a dose-dependent decrease in orientation angles after three hours of cyclic stretching. Furthermore, cell and stress fiber orientations were tightly coupled for untreated and Blebbistatin-treated cells but were uncoupled for BDM-treated cells. Even though orientation response to cyclic stretching is not a spontaneous motile response, it is determined, in large part, by the same factors that affect spontaneous motility--the cell-substratum adhesiveness and actin-myosin contractile level.     

Computational model for cell migration in three-dimensional matrices

Although computational models for cell migration on two-dimensional (2D) substrata have described how various molecular and cellular properties and physiochemical processes are integrated to accomplish cell locomotion, the same issues, along with certain new ones, might contribute differently to a model for migration within three-dimensional (3D) matrices. To address this more complicated situation, we have developed a computational model for cell migration in 3D matrices using a force-based dynamics approach. This model determines an overall locomotion velocity vector, comprising speed and direction, for individual cells based on internally generated forces transmitted into external traction forces and considering a timescale during which multiple attachment and detachment events are integrated. Key parameters characterize cell and matrix properties, including cell/matrix adhesion and mechanical and steric properties of the matrix; critical underlying molecular properties are incorporated explicitly or implicitly. Model predictions agree well with experimental results for the limiting case of migration on 2D substrata as well as with recent experiments in 3D natural tissues and synthetic gels. Certain predicted features such as biphasic behavior of speed with density of matrix ligands for 3D migration are qualitatively similar to their 2D counterparts, but new effects generally absent in 2D systems, such as effects due to matrix sterics and mechanics, are now predicted to arise in many 3D situations. As one particular sample manifestation of these effects, the optimal levels of cell receptor expression and matrix ligand density yielding maximal migration are dependent on matrix mechanical compliance.     

Polarity, Protrusion–Retraction Dynamics and Their Interplay during Keratinocyte Cell Migration

Keratinocyte migration on a two-dimensional substrate can be split into four distinct phases: cell extension, attachment, contraction, and detachment. It is preceded by polarization of the cell which leads to a functional asymmetry observable by the formation of a leading lamella. In this work variation of fibronectin coating concentrations and competitive inhibition with RGD peptides are used to investigate the dependency of polarization, migration, lamella dynamics, and ruffling on substrate adhesiveness. Looking at migrating human epidermal keratinocytes with a well-defined polarity we find that a fibronectin-coating concentration of 10 μg/cm² stimulates migration and ruffling speed twofold, whereas protrusion speed increases only by 20% (compared to 2.5 μg/cm² fibronectin). Nonpolar cells show a constant migration and ruffling speed independent of the amount of fibronectin. In contrast protrusion speeds of polar and nonpolar cells are equal. Treatment of cells on 10 μg/cm² fibronectin with 1 mg/ml GRGDS reduces the characteristic migration, protrusion, and ruffling speed of polar cells which corresponds to lowering the effective coating concentration to under 5 μg/cm². The probability of being polarized (quantified by a polarity index) increases with increasing fibronectin concentration. However, addition of soluble RGD on 10 μg/cm² fibronectin does not simply reduce the polarity index like one would expect from the corresponding changes in the other motility parameters, but it remains unchanged.     

Mathematical model for the effects of adhesion and mechanics on cell migration speed.

Migration of mammalian blood and tissue cells over adhesive surfaces is apparently mediated by specific reversible reactions between cell membrane adhesion receptors and complementary ligands attached to the substratum. Although in a number of systems these receptors and ligand molecules have been isolated and identified, a theory capable of predicting the effects of their properties on cell migration behavior currently does not exist. We present a simple mathematical model for elucidating the dependence of cell speed on adhesion-receptor/ligand binding and cell mechanical properties. Our model can be applied to propose answers to questions such as: does an optimal adhesiveness exist for cell movement? How might changes in receptor and ligand density and/or affinity affect the rate of migration? Can cell rheological properties influence movement speed? This model incorporates cytoskeletal force generation, cell polarization, and dynamic adhesion as requirements for persistent cell movement. A critical feature is the proposed existence of an asymmetry in some cell adhesion-receptor property, correlated with cell polarity. We consider two major alternative mechanisms underlying this asymmetry: (a) a spatial distribution of adhesion-receptor number due to polarized endocytic trafficking and (b) a spatial variation in adhesion-receptor/ligand bond strength. Applying a viscoelastic-solid model for cell mechanics allows us to represent one-dimensional locomotion with a system of differential equations describing cell deformation and displacement along with adhesion-receptor dynamics. In this paper, we solve these equations under the simplifying assumption that receptor dynamics are at a quasi-steady state relative to cell locomotion. Thus, our results are strictly valid for sufficiently slow cell movement, as typically observed for tissue cells such as fibroblasts. Numerical examples relevant to experimental systems are provided. Our results predict how cell speed might vary with intracellular contractile force, cell rheology, receptor/ligand kinetics, and receptor/ligand number densities. A biphasic dependence is shown to be possible with respect to some of the system parameters, with position of the maxima essentially governed by a balance between transmitted contractile force and adhesiveness. We demonstrate that predictions for the two alternative asymmetry mechanisms can be distinguished and could be experimentally tested using cell populations possessing different adhesion-receptor numbers.     

Calreticulin modulates cell adhesiveness via regulation of vinculin expression

Calreticulin is an ubiquitous and highly conserved high capacity Ca(2+)- binding protein that plays a major role in Ca2+ storage within the lumen of the ER. Here, using L fibroblast cell lines expressing different levels of calreticulin, we show that calreticulin plays a role in the control of cell adhesiveness via regulation of expression of vinculin, a cytoskeletal protein essential for cell-substratum and cell-cell attachments. Both vinculin protein and mRNA levels are increased in cells overexpressing calreticulin and are downregulated in cells expressing reduced level of calreticulin. Abundance of actin, talin, alpha 5 and beta 1 integrins, pp125 focal adhesion kinase, and alpha-catenin is not affected by the differential calreticulin expression. Overexpression of calreticulin increases both cell- substratum and cell-cell adhesiveness of L fibroblasts that, most surprisingly, establish vinculin-rich cell-cell junctions. Upregulation of calreticulin also affects adhesion-dependent phenomena such as cell motility (which decreases) and cell spreading (which increases). Downregulation of calreticulin brings about inverse effects. Cell adhesiveness is Ca2+ regulated. The level of calreticulin expression, however, has no effect on either the resting cytoplasmic Ca2+ concentration or the magnitude of FGF-induced Ca2+ transients. Calreticulin, however, participates in Ca2+ homeostasis as its level of expression affects cell viability at low concentrations of extracellular Ca2+. Consequently, we infer that it is not the Ca2+ storage function of calreticulin that affects cell adhesiveness. Neither endogenous calreticulin nor overexpressed green fluorescent protein-calreticulin construct can be detected outside of the ER. Since all of the adhesion-related effects of differential calreticulin expression can be explained by its regulation of vinculin expression, we conclude that it is the ER-resident calreticulin that affects cellular adhesiveness.     

Quantitative relationships between single-cell and cell-population model parameters for chemosensory migration responses of alveolar macrophages to C5a

Phenomenological parameters from a mathematical model of cell motility are used to quantitatively characterize chemosensory migration responses of rat alveolar macrophages migrating to C5a in the linear under-agarose assay, simultaneously at the levels of both single cells and cell populations. This model provides theoretical relationships between single-cell and cell-population motility parameters. Our experiments offer a critical test of these theoretical linking relationships, by comparison of results obtained at the cell population level to results obtained at the single-cell level. Random motility of a cell population is characterized by the random motility coefficient, mu (analogous to a particle diffusion coefficient), whereas single-cell random motility is described by cell speed, s, and persistence time, P (related to the period of time that a cell moves in one direction before changing direction). Population chemotaxis is quantified by the chemotactic sensitivity, chi 0, which provides a measure of the minimum attractant gradient necessary to elicit a specified chemotactic response. Single-cell chemotaxis is characterized by the chemotactic index, CI, which ranges from 0 for purely random motility to 1 for perfectly directed motility. Measurements of cell number versus migration distance were analyzed in conjunction with the phenomenological model to determine the population parameters while paths of individual cells in the same experiment were analyzed in order to determine the single-cell parameters. The parameter mu shows a biphasic dependence on C5a concentration with a maximum of 1.9 x 10(-8) cm2/sec at 10(-11) M C5a and relative minima of 0.86 x 10(-8) cm2/sec at 10(-7) M C5a and 1.1 x 10(-8) cm2/sec in the absence of Ca; s and P remain fairly constant with C5a concentration, with s ranging from 2.1 to 2.5 microns/min and P varying from 22 to 32 min. chi 0 is equal to 1.0 x 10(-6) cm/receptor for all C5a concentrations tested, corresponding to 60% correct orientation for a difference of 500 bound C5a receptors across a 20 microns cell length. The maximum CI measured was 0.2. Values for the population parameters mu and chi 0 were calculated from single-cell parameter values using the aforementioned theoretical linking relationships. The values of mu and chi 0 calculated from single-cell parameters agreed with values of mu and chi 0 determined independently from population migrations, over the full range of C5a concentrations, confirming the validity of the linking equations. Experimental confirmation of such relationships between single-cell and cell-population parameters has not previously been reported.     

Inhibition of cell-substratum attachment of cultured rat heart cells by protein synthesis inhibitors.

Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were not dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachment completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.     

Inhibition of cell-substratum attachment of cultured rat heart cells by protein synthesis inhibitors

Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were most dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachement completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.     

Recombinant human uteroglobin/CC10 inhibits the adhesion and migration of primary human endothelial cells via specific and saturable binding to fibronectin

Uteroglobin (UG) or Clara Cell 10 kDa protein (CC10) is a small, stable, epithelial secretory anti-inflammatory protein. Uteroglobin has been shown to inhibit neointimal formation in vivo after balloon angioplasty through an unknown mechanism. An interaction between UG and plasma fibronectin (Fn) has been demonstrated in mice. Since Fn plays a key role in endothelial cell (EC) migration and angiogenesis, we investigated whether recombinant human UG (rhUG) affects EC migration via Fn binding. In this report, we show a saturable binding of rhUG to Fn depending on Fn conformation and that rhUG is covalently cross-linked to Fn by transglutaminase (TGase). Additionally, our study highlights that rhUG can also bind to exogenously added or self-secreted Fn on the membrane of human primary microvascular endothelial cells (HMVEC), although these complexes are weakly associated with the plasmalemma. Upon the interaction with Fn in solid phase, rhUG strongly inhibits HMVEC attachment on Fn, but not on other ECM proteins. Consequently, rhUG also inhibits cell migration in a dose dependent fashion (I.C.50 = 65 nM) and hinders the "wound healing" in vitro. The small size, stability and human tolerability of rhUG suggest that rhUG in slow-release form or genetically delivered could be used in humans to modulate cell/Fn interactions in the context of tumor microenvironment or in the context of inflammation and fibrosis.     

Fibronectin distribution during the transdifferentiation and proliferation of eye cells after retinal detachment and removal of the crystalline lens in tritons

Expression of fibronectin (Fn) during eye tissue regeneration in the newt after retinal detachment and lens removal was studied by immunohistochemistry. Proliferation of cells involved in eye tissue regeneration was studied using autoradiography. Fn was detected around the cell membranes of undifferentiated proliferating and migrating cells in ciliary body of the iris and growth zone of the retina. Redistribution of Fn was observed in proliferating cells of the dorsal iris participating in lens regeneration. Fn appeared on the apical surface of proliferating redifferentiating pigment epithelium (PE) cells at the periphery of the eye and over the whole surface of proliferating PE cells in the central part of the eye. The Fn level in the Bruch's membrane decreased in the area of transdifferentiating cells detachment from PE layer (in the lower part of the eye) but continued to be stable in the area of PE cell redifferentiation (at the periphery of the eye). The role of Fn is discussed in relation to transdifferentiation, proliferation and migration of cells in the regenerating eye.     

Calreticulin affects cell adhesiveness through differential phosphorylation of insulin receptor substrate-1

Cellular adhesion to the underlying substratum is regulated through numerous signaling pathways. It has been suggested that insulin receptor substrate 1 (IRS-1) is involved in some of these pathways, via association with and activation of transmembrane integrins. Calreticulin, as an important endoplasmic reticulum-resident, calcium-binding protein with a chaperone function, plays an obvious role in proteomic expression. Our previous work showed that calreticulin mediates cell adhesion not only by affecting protein expression but also by affecting the state of regulatory protein phosphorylation, such as that of c-src. Here, we demonstrate that calreticulin affects the abundance of IRS-1 such that the absence of calreticulin is paralleled by a decrease in IRS-1 levels and the unregulated overexpression of calreticulin is accompanied by an increase in IRS-1 levels. These changes in the abundance of calreticulin and IRS-1 are accompanied by changes in cell-substratum adhesiveness and phosphorylation, such that increases in the expression of calreticulin and IRS-1 are paralleled by an increase in focal contact-based cellsubstratum adhesiveness, and a decrease in the expression of these proteins brings about a decrease in cell-substratum adhesiveness. Wild type and calreticulin-null mouse embryonic fibroblasts (MEFs) were cultured and the IRS-1 isoform profile was assessed. Differences in morphology and motility were also quantified. While no substantial differences in the speed of locomotion were found, the directionality of cell movement was greatly promoted by the presence of calreticulin. Calreticulin expression was also found to have a dramatic effect on the phosphorylation state of serine 636 of IRS-1, such that phosphorylation of IRS-1 on serine 636 increased radically in the absence of calreticulin. Most importantly, treatment of cells with the RhoA/ROCK inhibitor, Y-27632, which among its many effects also inhibited serine 636 phosphorylation of IRS-1, had profound effects on cell-substratum adhesion, in that it suppressed focal contacts, induced extensive close contacts, and increased the strength of adhesion. The latter effect, while counterintuitive, can be explained by the close contacts comprising labile bonds but in large numbers. In addition, the lability of bonds in close contacts would permit fast locomotion. An interesting and novel finding is that Y-27632 treatment of MEFs releases them from contact inhibition of locomotion, as evidenced by the invasion of a cell’s underside by the thin lamellae and filopodia of a cell in close apposition.     

Characteristics of a BHK cell variant defective in the cell-substratum contact process

In this paper we document the phenotypic characteristics of a novel BHK cell adhesion variant designated FN-2. Unlike parental cells, FN-2 cells did not attach to fibronectin (pFN)-coated dishes, even after 4-hr incubations on dishes treated with 100 micrograms/ml of pFN. Mixing experiments with the variant and parental cells revealed that the parental cells attached normally in the presence of a ninefold excess of variant cells and the variant cells failed to attach in the presence of a ninefold excess of parental cells. Therefore, the defect in FN-2 cells could not be explained by secretion of a factor inhibiting attachment or lack of secretion of a factor required for attachment. Also, the inability of FN-2 cells to attach to pFN-coated dishes could not be explained by an absence of cell pFN receptors since the variant cells bound normal numbers of small (ca. 0.8 micron) pFN-coated latex beads, although they phagocytosed the beads poorly compared to parental cells. Also, the variant cells were not able to bind large (5.7 or 16.8 microns) pFN-coated beads. When tested on dishes coated with ligands that, unlike fibronectin, have a high affinity for cell surface receptors, e.g., lectins and anti-BHK antibodies, FN-2 cells were observed to attach at a rate similar to that of parental cells but spread much more slowly. The phenotypic characteristics of FN-2 cells suggest that they are deficient in what previously has been called the "cell contact" process in cell adhesion. It is proposed that the cell contact process is the initial formation by an individual cell of a sufficient number of cell-substratum bonds to resist the shear forces operationally used to define "attachment," and that more cell-substratum bonds are necessary for cell attachment to large substrata (dishes or large beads) than for attachment to small substrata (small beads). The molecular defect in FN-2 cells was studied by electroblotting analysis. A high molecular weight (ca. 370 kd) glycoprotein detected by blotting with anti-BHK antibodies and ConA that was present in parental cell membranes was reduced or absent in the variant cells.     

A von Willebrand factor-derived heparin-binding peptide regulates cell--substrate adhesive strength and chemokinesis behavior

The ability of a soluble heparin-binding oligopeptide sequence derived from the von Willebrand factor (vWF) to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells was assessed using a novel glass microsphere centrifugation assay and automated time-lapse fluorescence videomicroscopy, respectively. Treatment of cells grown on fibronectin-coated substrates with the heparin-binding peptide resulted in the disassembly of focal adhesions, as assessed by immunohistochemical staining. These observations were consistent with six-fold decrease in cell--substrate adhesive strength (P<0.001), a biphasic effect on migration speed (P<0.05), as well as a dose-dependent reduction in the percentage of motile cells and the cell dispersion coefficient (mu=S(2)T/2). The specificity of this response to the vWF-derived heparin-binding peptide was supported by the absence of an observed effect in the presence of either a scrambled peptide or a consensus heparin-binding peptide sequence of similar heparin affinity. These data support the notion that competitive interactions between cell surface heparan sulfates with heparin-binding peptide domains located in soluble peptide fragments may modulate chemokinetic cell migration behavior and other adhesion-related processes.     

Attachment, spreading and growth of VERO cells on microcarriers for the optimization of large scale cultures

The VERO cell attachment, spreading and growth were measured as a function of the substrate and temperature used for cell cultivation, the presence of fetal calf serum (FCS) in the medium and the initial cell inoculum used for cultivation on MCs. The data show that the cell attachment kinetics were comparable at RT or 37v°C, a higher rate of cell attachment occurred to MCs and the presence of FCS inhibited the cell attachment to glass or plastic but not to MCs. The cell spreading, in general higher at 37v°C, was dependent on the presence of FCS, comparable on glass or plastic substrate and lower on MCs. The spread of VERO cells over MCs was fully dependent on the presence of FCS and decreases progressively with a delayed addition of FCS into the medium. The cell detachment by trypsin was slower from MCs and the cells recovered showed lower viability and reattachment. Better results of detachment, viability and reattachment were obtained by treatment with the trypsin at pH of 8 instead of 7. The lower was the number of cells/MC for the initial inoculum, the higher was the percent of unoccupied MCs (with 1 cell/MC we had 35.6% of unoccupied MCs), which were shown to remain uncovered during the whole period of culture. With an initial inoculum of 4, 6 and 8 VERO cells/MC, respectively 46%, 76% and 83% of the MCs were totally covered by cells after 7 days, the cultures showing at this time, respectively, 5.1 2 10⁵, 8.8 2 10⁵ and 1.8 2 10⁶ cells/ml, which represented a biomass production of respectively 8.5x, 9.7x and 15.5x. When compared to 175 cm² T-flasks, using the same amount of medium, a VERO cell culture on 2 mg/ml of MCs offers about 10 times more available surface for cell growth and allowed the obtention of 7 times more cells. The optimization procedures concerning initial steps of VERO cell cultures, such as the attachment, spreading and growth as a function of parameters like initial cell inoculum and medium supplementation are of special interest mainly due to the perspective of a large use of VERO cell cultures for human viral vaccine production.     

Regulation of protein L-isoaspartyl methyltransferase by cell-matrix interactions: involvement of integrin alphavbeta3, PI 3-kinase, and the proteasome.

The enzyme L-isoaspartyl methyltransferase (PIMT) is known to repair damaged proteins that have accumulated abnormal aspartyl residues during cell aging. However, little is known about the mechanisms involved in the regulation of PIMT expression. Here we report that PIMT expression in bovine aortic endothelial cells is regulated by cell detachment and readhesion to a substratum. During cell detachment, the PIMT level was rapidly and strongly increased and correlated with a stimulation of protein synthesis. Aside from endothelial cells, PIMT levels were also regulated by cell adhesion in various cancer cell lines. The upregulation of PIMT expression could be prevented by an anti-alphavbeta3 antibody (LM609) or by a cyclic RGD peptide (XJ735) specific to integrin alphavbeta3, indicating that this integrin was likely involved in PIMT regulation. Moreover, we found that PIMT expression returned to the basal level when cells were replated on a substratum after detachment, though downregulation of PIMT expression could be partly prevented by the PI3K inhibitors LY294002 and wortmannin, as well as by the proteasome inhibitors MG-132, lactacystin, and beta-lactone. These findings support the assumption that the PIMT level was downregulated by proteasomal degradation, involving the PI3K pathway, during cell attachment. This study reports new insights on the molecular mechanisms responsible for the regulation of PIMT expression in cells. The regulation of PIMT level upon cell-substratum contact suggests a potential role for PIMT in biological processes such as wound healing, cell migration, and tumor metastasis dissemination.