TRPC5 channels undergo changes in gating properties during the activation-deactivation cycle

The goal of this study was to determine the morphological and sub-cellular mechanical effects of Rac activation on fibroblasts within 3-D collagen matrices. Corneal fibroblasts were plated at low density inside 100 microm thick fibrillar collagen matrices and cultured for 1-2 days in serum-free media. Time-lapse imaging was then performed using Nomarski DIC. After an acclimation period, perfusion was switched to media containing PDGF. In some experiments, Y-27632 or blebbistatin were used to inhibit Rho-kinase (ROCK) or myosin II, respectively. PDGF activated Rac and induced cell spreading, which resulted in an increase in cell length, cell area, and the number of pseudopodial processes. Tractional forces were generated by extending pseudopodia, as indicated by centripetal displacement and realignment of collagen fibrils. Interestingly, the pattern of pseudopodial extension and local collagen fibril realignment was highly dependent upon the initial orientation of fibrils at the leading edge. Following ROCK or myosin II inhibition, significant ECM relaxation was observed, but small displacements of collagen fibrils continued to be detected at the tips of pseudopodia. Taken together, the data suggests that during Rac-induced cell spreading within 3-D matrices, there is a shift in the distribution of forces from the center to the periphery of corneal fibroblasts. ROCK mediates the generation of large myosin II-based tractional forces during cell spreading within 3-D collagen matrices, however residual forces can be generated at the tips of extending pseudopodia that are both ROCK and myosin II-independent.     

Cells can use their transferrin receptors for locomotion.

Cells of the B lymphoblastoid cell line JY attach to substrata made of antibodies to the transferrin receptor. Many of these attached cells migrate considerable distances. JY cells also attach to an anti-integrin substratum (anti LFA-1), but on this surface they do not migrate. These results suggest that a circulating receptor--the transferrin receptor--can be used for locomotory purposes, whereas LFA-1, which is not endocytosed in these cells, cannot be used for locomotion. This indicates that the endocytotic cycle can drive cell locomotion.     

Stimulation of corneal differentiation by interaction between cell surface and extracellular matrix. I. Morphometric analysis of transfilter "induction".

The present study was undertaken to determine whether or not physical contact with the substratum is essential for the stimulatory effect of extracellular matrix (ECM) on corneal epithelial collagen synthesis. Previous studies showed that collagenous substrata stimulate isolated epithelia to produce three times as much collagen as they produce on noncollagenous substrate; killed collagenous substrata (e.g., lens capsule) are just as effective as living substrata (e.g., living lens) in promoting the production of new corneal stroma in vitro. In the experiments to be reported here, corneal epithelia were placed on one side of Nucleopore filters of different pore sizes and killed lens capsule on the other, with the expectation that contact of the reacting cells with the lens ECM should be limited by the number and size of the cell processes that can tranverse the pores. Transfilter cultures were grown for 24 h in [3H]proline-containing median and incorporation of isotope into hot trichloroacetic acid-soluble protein was used to measure corneal epithelial collagen production. Epithelial collagen synthesis increases directly as the size of the pores in the interposed filter increases and decreases as the thickness of the filter layer increases. Cell processes within Nucleopore filters were identified with the transmission electron microscope with difficulty; with the scanning electron microscope, however, the processes could easily be seen emerging from the undersurface of even 0.1-mum pore size filters. Morphometric techniques were used to show that cell surface area thus exposed to the underlying ECM is linearly correlated with enhancement of collagen synthesis. Epithelial cell processes did not pass through ultrathin (25-mum thick) 0.45-mum pore size Millipore filters nor did "induction" occur across them. The results are discussed in relation to current theories of embryonic tissue interaction.     

Morphology of fibroblasts in collagen gels: a study using 400 keV electron microscopy and computer graphics

We have used 400 kilovolt intermediate voltage electron microscopy (IVEM) to examine thick sections of fibroblasts cultured in collagen gels. In these 3D collagen lattices, the long, narrow pseudopodial extensions that extend out and make contact with the collagen matrix exhibit a complex topography not seen in the processes put out by cells moving on planar substrata. For this reason, sections 1 to 2 microns thick that enclose a whole cell process are more informative of the overall morphology of the interaction between cells and the collagen than are thin sections. To aid the discrimination of topography of cell processes in stereo views of micrographs, some cells were labeled with antibodies and protein A-colloidal gold conjugates. The gold particles provided clear 3D reference points for computer-aided reconstructions of membrane topography from tilt series of IVEM images. Our results confirm that cells that move through collagen lattices lack the well-spread morphology of their counterparts moving on glass. They are generally rather spindly with several long branching anterior pseudopodia. We found that the cell bodies and major pseudopodial processes were cylindrical, as one might expect of cells in a 3D environment, but at the leading edge of advancing pseudopodia there are small flat extensions similar to those seen in cells on glass. This similarity suggests that the lamellipodium is a basic type of protrusive structure used by fibroblasts during locomotion on all types of substratum. The flattened shape of lamellipodia may be part of the mechanism by which cells sense the orientation of fibrillar extracellular matrices during embryonic morphogenesis.     

Collagen modulates cell shape and cytoskeleton of embryonic corneal and fibroma fibroblasts: Distribution of actin, α-actinin,and myosin

In the embryo, fibroblasts migrating through extracellular matrices (ECM) are generally elongate in shape, exhibiting a leading pseudopodium with filopodial extensions, and a trailing cell process. Little is known about the mechanism of movement of embryonic cells in ECM, for studies of fibroblast locomotion in the past have been largely confined to observations of flattened cells grown on planar substrata. We confirm here that embryonic avian corneal fibroblasts migrating within hydrated collagen gels in vitro have the bipolar morphology of fibroblasts in vivo, and we show for the first time that highly flattened gerbil fibroma fibroblasts, grown as cell lines on planar substrata, can also respond to hydrated collagen gels by becoming elongate in shape. We demonstrate that the collagen-mediated change in cell shape is accompanied by dramatic rearrangement of the actin, α-actinin, and myosin components of the cytoskeleton. By immunofluorescence, the stress fibers of the flattened corneal fibroblasts grown on glass are seen to stain with antiactin, anti-α-actinin, and antimyosin, as has been reported for fibroma and other fibroblasts grown on glass. Stress fibers, adhesion plaques, and ruffles do not develop when the corneal or fibroma fibroblast is grown in ECM; these features seem to be a response to strong attachment of the cell underside to a planar substratum. When the fibroblasts are grown in ECM, antimyosin staining is distributed diffusely through the cytoplasm. Antiactin and anti-α-actinin stain the microfilamentous cell cortex strongly. We suggest that locomotion of the fibroblast in ECM is accompanied by adhesion of the cell to the collagen fibrils and may involve an interaction of the myosin-rich cytosol with the actin-rich filamentous cell cortex. Interestingly, the numerous filopodia that characterize the tips of motile pseudopodia of cells in ECM are very rich in actin and α-actinin, but seem to lack myosin; if filopodia use myosin to move, the interaction must be at a distance. Soluble collagen does not convert flattened fibroblasts on planar substrata to bipolar cells. Thus, the effect of collagen on the fibroblast cytoskeleton seems to depend on the presence of collagen fibrils in a gel surrounding the cell.     

Direct,dynamic assessment of cell-matrix interactions inside fibrillar collagen lattices

Cell mechanical behavior has traditionally been studied using 2-D planar elastic substrates. The goal of this study was to directly assess cell-matrix mechanical interactions inside more physiologic 3-D collagen matrices. Rabbit corneal fibroblasts transfected to express GFP-zyxin were plated at low density inside 100 micro m-thick type I collagen matrices. 3-D datasets of isolated cells were acquired at 1-3-min intervals for up to 5 h using fluorescent and Nomarski DIC imaging. Unlike cells on 2-D substrates, cells inside the collagen matrices had a bipolar morphology with thin pseudopodial processes, and without lamellipodia. The organization of the collagen fibrils surrounding each cell was clearly visualized using DIC. Using time-lapse color overlays of GFP and DIC images, displacement and/or realignment of collagen fibrils by focal adhesions could be directly visualized. During pseudopodial extension, new focal adhesions often formed in a line along collagen fibrils in front of the cell, while existing adhesions moved backward. This process generated tractional forces as indicated by the pulling in of collagen fibrils in front of the cell. Meanwhile, adhesions on both the dorsal and ventral surface of the cell body generally moved forward, resulting in contractile shortening along the pseudopodia and localized extracellular matrix (ECM) compression. Cytochalasin D induced rapid disassembly of focal adhesions, cell elongation, and ECM relaxation. This experimental model allows direct, dynamic assessment of cell-matrix interactions inside a 3-D fibrillar ECM. The data suggest that adhesions organize along actin-based contractile elements that are much less complex than the network of actin filaments that mechanically links lamellar adhesions on 2-D substrates.     

Changes in gravity inhibit lymphocyte locomotion through type I collagen

Summary Immunity relies on the circulation of lymphocytes through many different tissues including blood vessels, lymphatic channels, and lymphoid organs. The ability of lymphocytes to traverse the interstitium in both nonlymphoid and lymphoid tissues can be determined in vitro by assaying their capacity to locomote through Type I collagen. In an attempt to characterize potential causes of microgravity-induced immunosuppression, we investigated the effects of simulated microgravity on human lymphocyte function in vitro using a specialized rotating-wall vessel culture system developed at the Johnson Space Center. This very low shear culture system randomizes gravitational vectors and provides an in vitro approximation of microgravity. In the randomized gravity of the rotating-wall vessel culture system, peripheral blood lymphocytes did not locomote through Type I collagen, whereas static cultures supported normal movement. Although cells remained viable during the entire culture period, peripheral blood lymphocytes transferred to unit gravity (static culture) after 6 h in the rotating-wall vessel culture system were slow to recover and locomote into collagen matrix. After 72 h in the rotating-wall vessel culture system and an additional 72 h in static culture, peripheral blood lymphocytes did not recover their ability to locomote. Loss of locomotory activity in rotating-wall vessel cultures appears to be related to changes in the activation state of the lymphocytes and the expression of adhesion molecules. Culture in the rotating-wall vessel system blunted the ability of peripheral blood lymphocytes to respond to polyclonal activation with phytohemagglutinin. Locomotory response remained intact when peripheral blood lymphocytes were activated by anti-CD3 antibody and interleukin-2 prior to introduction into the rotating-wall vessel culture system. Thus, in addition to the systemic stress factors that may affect immunity, isolated lymphocytes respond to gravitational changes by ceasing locomotion through model interstitium. These in vitro investigations suggest that microgravity induces non-stress-related changes in cell function that may be critical to immunity. Preliminary analysis of locomotion in true microgravity revealed a substantial inhibition of cellular movement in Type I collagen. Thus, the rotating-wall vessel culture system provides a model for analyzing the microgravity-induced inhibition of lymphocyte locomotion and the investigation of the mechanisms related to lymphocyte movement.     

Identification of IL-8 as a locomotor attractant for activated human lymphocytes in mononuclear cell cultures with anti-CD3 or purified protein derivative of Mycobacterium tuberculosis.

On culture of human blood mononuclear cells for 24 to 48 h with anti-CD3 (aCD3) or purified protein derivative of Mycobacterium tuberculosis, chemoattractants are released into the medium which induce polarization and locomotion of activated (G1) lymphocytes but not resting lymphocytes. Here we show that, during a period of up to 72 h of culture, IL-8 is released in nanomolar quantities into the supernatant and that the lymphocyte chemoattractant activity of these supernatants is inhibited by incubation with anti-IL-8. Examination of the cultured mononuclear cells by immunofluorescence suggests that many monocytes, but almost no lymphocytes in aCD3 cultures contain IL-8 in cytoplasmic organelles, yet few monocytes direct from blood stained for IL-8. IL-8 is an attractant for only a small proportion (ca 10%) of lymphocytes direct from blood. The proportion of responding cells is increased after culture for 24 to 48 h in aCD3 or purified protein derivative of Mycobacterium tuberculosis, and these are a phenotypically distinct subpopulation consisting of large lymphocytes enriched for CD45RO. These cells respond to their own culture supernatants and to IL-8 in polarization assays and by invasion of collagen gels into which the attractants are incorporated. They also show orientation to a source of IL-8 in a chemotactic gradient. These responses are consistent with in vivo observations that the lymphocytes which migrate selectively into inflammatory sites are activated. The fact that many lymphocytes do not respond to IL-8 may reflect the diversity of migratory pathways shown by lymphocytes in vivo, the locomotion of small, recirculating, lymphocytes being regulated by other, unknown, locomotor stimuli.     

Endothelial cell protrusion and migration in three-dimensional collagen matrices

Many cells display dramatically different morphologies when migrating in 3D matrices vs. on planar substrata. How these differences arise and the implications they have on cell migration are not well understood. To address these issues, we examined the locomotive structure and behavior of bovine aortic endothelial cells (ECs) either inside 3D collagen gels or on 2D surfaces. Using time-lapse imaging, immunofluorescence, and confocal microscopy, we identified key morphological differences between ECs in 3D collagen gels vs. on 2D substrata, and also demonstrated important functional similarities. In 3D matrices, ECs formed cylindrical branching pseudopodia, while on 2D substrata they formed wide flat lamellae. Three distinct cytoplasmic zones were identified in both conditions: (i) a small, F-actin-rich, rapidly moving peripheral zone, (ii) a larger, more stable, intermediate zone characterized by abundant microtubules and small organelles, and (iii) a locomotively inert central zone rich in microtubules, and containing the larger organelles. There were few differences between 2D and 3D cells in the content and behavior of their peripheral and central zones, whereas major differences were seen in the shape and types of movements displayed by the intermediate zone, which appeared critical in distributing cell-matrix adhesions and directing cytoplasmic flow. This morphological and functional delineation of cytoplasmic zones provides a conceptual framework for understanding differences in the behavior of cells in 3D and 2D environments, and indicates that cytoskeletal structure and dynamics in the relatively uncharacterized intermediate zone may be particularly important in cell motility in general.     

Growth of Streptococcal Protoplasts and L-Colonies on Membrane Filters

Membrane filters (Millipore Corp.; pore sizes 1.2 to 0.22 mum) were placed on the surface of L-phase growth medium solidified with agar. The filter and the surrounding medium were inoculated with either protoplasts or stable broth-grown L-phase variants obtained from Streptococcus faecium strain F24. The L-phase inoculum gave rise to viable L-colonies on the filters and on the medium, whereas protoplasts gave colony formation only on the medium. However, when the Millipore filters were covered by a layer of solid L-phase medium, 75 mum or greater in depth, before inoculation with protoplasts, colony formation resulted but with atypical morphology. In contrast, inoculation of protoplasts on Nuclepore and Sartorius membrane filters did give rise to L-colonies on the surface and underneath the filters after 2 days of incubation at 37 C. Submicroscopic, viable L-phase elements produced during colony formation were capable of passing through membrane filters with pore channels as small as 0.22 mum; these elements required transfer from underneath the filters to fresh agar medium in order to develop into L-phase colonies. Membrane filters were also placed on the surface of L-phase growth medium solidified with gelatin. Inoculation of the filters and surrounding medium with a lysozyme-prepared protoplast suspension gave rise to streptococci on the surface of the filters and on the medium. However, inoculation with the stable broth-grown L-phase variants gave rise to atypical colonies on the medium and only small patches of abortive growth on the filters.     

Quantitative assessment of local collagen matrix remodeling in 3-D culture: the role of Rho kinase

The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling. Human corneal fibroblasts were plated inside 100-microm thick fibrillar collagen matrices and cultured for 24 h in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph. Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase (p<0.001). Fibroblasts generally had a bipolar morphology and intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. When Rho kinase was inhibited, cells had a more cortical f-actin distribution and dendritic morphology. Both local collagen fibril density and alignment were significantly reduced (p<0.01). Overall, the data suggests that Rho kinase-dependent contractile force generation leads to co-alignment of cells and collagen fibrils along the plane of greatest resistance, and that this process contributes to global matrix contraction.     

Cell Migration with Multiple Pseudopodia: Temporal and Spatial Sensing Models

Cell migration triggered by pseudopodia (or “false feet”) is the most used method of locomotion. A 3D finite element model of a cell migrating over a 2D substrate is proposed, with a particular focus on the mechanical aspects of the biological phenomenon. The decomposition of the deformation gradient is used to reproduce the cyclic phases of protrusion and contraction of the cell, which are tightly synchronized with the adhesion forces at the back and at the front of the cell, respectively. First, a steady active deformation is considered to show the ability of the cell to simultaneously initiate multiple pseudopodia. Here, randomness is considered as a key aspect, which controls both the direction and the amplitude of the false feet. Second, the migration process is described through two different strategies: the temporal and the spatial sensing models. In the temporal model, the cell “sniffs” the surroundings by extending several pseudopodia and only the one that receives a positive input will become the new leading edge, while the others retract. In the spatial model instead, the cell senses the external sources at different spots of the membrane and only protrudes one pseudopod in the direction of the most attractive one.     

Motility of IL-2-stimulated lymphocytes in neutral and acidified extracellular matrix.

The migration of lymphocytes through extracellular matrix (ECM) is an essential feature of the infiltration process. In the course of their extravasation into poorly perfused neoplastic lesions, lymphocytes often encounter regions of acidified ECM. This study was designed to determine whether lymphocyte adherence and motility in ECM are influenced by ambient pH. Murine splenic lymphocytes, activated by culture with high-titer IL-2, were allowed to migrate into three-dimensional gels of Type I collagen, a major component of interstitial stroma, or into Matrigel, a basement membrane model. After 18 hr at pH 7.1, the leading cell front traveled a mean distance of approx 475 microM into Type I collagen gel. Approx 50% of the cells remained nonadherent, 25% adhered to the gel surface, and 25% were motile (penetrated beneath the surface). At pH 6.7, the leading-front distance increased significantly, by a factor of 1.4X, but there was little change in the proportion of cells exhibiting nonadherence, surface adherence, or motility. The relative motilities of CD3+ and AsGM1+ subsets were also unaltered. It therefore appears that acidification of collagen matrix increases the locomotory activity of motile lymphocytes, but causes little recruitment of nonmotile lymphocytes into the motile pool. Similar results were obtained in experiments with Matrigel. The increased motility observed at pH 6.7 did not reflect breakdown or relaxation of matrix lattices, as measured by the passive diffusion of latex beads of defined diameter. Preincubation of lymphocytes at pH 6.7 did not alter their subsequent motility in pH 7.1 gels. The findings establish ambient pH as a microenvironmental variable which can influence lymphocyte migration through ECM. The weak acidity characteristic of certain tumor microenvironments may be a factor which encourages lymphocyte infiltration through tissue matrix. Treatments which alter intratumor pH could potentially be used to manipulate the infiltration process for immunotherapeutic benefit.     

Passage of Treponema pallidum Through Membrane Filters of Various Pore Sizes

The passage of Treponema pallidum through commercially available Millipore membrane filters of various pore sizes was examined. No microscopically detectable organisms passed through a filter with a pore diameter of 0.22 mum. As pore size was increased, progressively more organisms passed through. Motile organisms passed through filters to a greater extent than nonmotile ones; however, 22% of the motile and 50% of the nonmotile T. pallidum organisms did not pass through the largest pore diameter tested (14.0 mum). Filtration of T. pallidum suspensions through membrane filters may offer a way of separating the organisms from larger particles of debris which accompany their extraction from rabbit testicular syphilomas.     

Loss of signal transduction and inhibition of lymphocyte locomotion in a ground-based model of microgravity

Inflammatory adherence to, and locomotion through the interstitium is an important component of the immune response. Conditions such as microgravity and modeled microgravity (MMG) severely inhibit lymphocyte locomotion in vitro through gelled type I collagen. We used the NASA rotating wall vessel bioreactor or slow-turning lateral vessel as a prototype for MMG in ground-based experiments. Previous experiments from our laboratory revealed that when lymphocytes (human peripheral blood mononuclear cells [PBMCs]) were first activated with phytohemaglutinin followed by exposure to MMG, locomotory capacity was not affected. In the present study, MMG inhibits lymphocyte locomotion in a manner similar to that observed in microgravity. Phorbol myristate acetate (PMA) treatment of PBMCs restored lost locomotory capacity by a maximum of 87%. Augmentation of cellular calcium flux with ionomycin had no restorative effect. Treatment of lymphocytes with mitomycin C prior to exposure to MMG, followed by PMA, restored locomotion to the same extent as when nonmitomycin C-treated lymphocytes were exposed to MMG (80-87%), suggesting that deoxyribonucleic acid replication is not essential for the restoration of locomotion. Thus, direct activation of protein kinase C (PKC) with PMA was effective in restoring locomotion in MMG comparable to the normal levels seen in Ig cultures. Therefore, in MMG, lymphocyte calcium signaling pathways were functional, with defects occurring at either the level of PKC or upstream of PKC.     

Guanylyl cyclase protein and cGMP product independently control front and back of chemotaxing Dictyostelium cells

Chemotaxis of amoeboid cells is driven by actin filaments in leading pseudopodia and actin-myosin filaments in the back and at the side of the cell to suppress pseudopodia. In Dictyostelium, cGMP plays an important role during chemotaxis and is produced predominantly by a soluble guanylyl cyclase (sGC). The sGC protein is enriched in extending pseudopodia at the leading edge of the cell during chemotaxis. We show here that the sGC protein and the cGMP product have different functions during chemotaxis, using two mutants that lose either catalytic activity (sGCDelta cat) or localization to the leading edge (sGCDeltaN). Cells expressing sGCDeltaN exhibit excellent cGMP formation and myosin localization in the back of the cell, but they exhibit poor orientation at the leading edge. Cells expressing the catalytically dead sGCDelta cat mutant show poor myosin localization at the back, but excellent localization of the sGC protein at the leading edge, where it enhances the probability that a new pseudopod is made in proximity to previous pseudopodia, resulting in a decrease of the degree of turning. Thus cGMP suppresses pseudopod formation in the back of the cell, whereas the sGC protein refines pseudopod formation at the leading edge.     

A Model for a Correlated Random Walk Based on the Ordered Extension of Pseudopodia

Cell migration in the absence of external cues is well described by a correlated random walk. Most single cells move by extending protrusions called pseudopodia. To deduce how cells walk, we have analyzed the formation of pseudopodia by Dictyostelium cells. We have observed that the formation of pseudopodia is highly ordered with two types of pseudopodia: First, de novo formation of pseudopodia at random positions on the cell body, and therefore in random directions. Second, pseudopod splitting near the tip of the current pseudopod in alternating right/left directions, leading to a persistent zig-zag trajectory. Here we analyzed the probability frequency distributions of the angles between pseudopodia and used this information to design a stochastic model for cell movement. Monte Carlo simulations show that the critical elements are the ratio of persistent splitting pseudopodia relative to random de novo pseudopodia, the Left/Right alternation, the angle between pseudopodia and the variance of this angle. Experiments confirm predictions of the model, showing reduced persistence in mutants that are defective in pseudopod splitting and in mutants with an irregular cell surface.     

Neural crest migration in 3D extracellular matrix utilizes laminin, fibronectin, or collagen

The trunk neural crest originates by transformation of dorsal neuroepithelial cells into mesenchymal cells that migrate into embryonic interstices. Fibronectin (FN) is thought to be essential for the process, although other extracellular matrix (ECM) molecules are potentially important. We have examined the ability of three dimensional (3D) ECM to promote crest formation in vitro. Neural tubes from stage 12 chick embryos were suspended within gelling solutions of either basement membrane (BM) components or rat tail collagen, and the extent of crest outgrowth was measured after 22 hr. Fetal calf serum inhibits outgrowth in both gels and was not used unless specified. Neither BM gel nor collagen gel contains fibronectin. Extensive crest migration occurs into the BM gel, whereas outgrowth is less in rat tail collagen. Addition of fibronectin or embryo extract (EE), which is rich in fibronectin, does not increase the extent of neural crest outgrowth in BM, which is already maximal, but does stimulate migration into collagen gel. Removal of FN from EE with gelatin-Sepharose does not remove the ability of EE to stimulate migration. Endogenous FN is localized by immunofluorescence to the basal surface of cultured neural tubes, but is not seen in the proximity of migrating neural crest cells. Addition of the FN cell-binding hexapeptide GRGDSP does not affect migration into either the BM gel or the collagen gel with EE, although it does block spreading on FN-coated plastic. Thus, although crest cells appear to use exogenous fibronectin to migrate on planar substrata in vitro, they can interact with 3D collagenous matrices in the absence of exogenous or endogenous fibronectin. In BM gels, the laminin cell-binding peptide, YIGSR, completely inhibits migration of crest away from the neural tube, suggesting that laminin is the migratory substratum. Indeed, laminin as well as collagen and fibronectin is present in the embryonic ECM. Thus, it is possible that ECM molecules in addition to or instead of fibronectin may serve as migratory substrata for neural crest in vivo.     

Quantitative study of various factors influencing the migration of lymphocytes in vitro: glucocortico-steroid, PHA, cyclosporin A and heparin

Various agents known to influence the lymphocyte recirculation in vivo were investigated for their effect on the migration of lymphocytes in 3-D collagen gel. Both glucocortico-steroid and cyclosporin A were shown to inhibit the migration in a dose dependent manner. In contrast heparin and PHA treated lymphocytes migrated at a significantly faster rate.     

Phenotype modulation in non-adherent and adherent sublines of Walker carcinosarcoma cells: the role of cell-substratum contacts and microtubules in controlling cell shape, locomotion and cytoskeletal structure

We characterised two sublines of Walker carcinosarcoma cells generated by epigenetic changes. Subline 1 cells were mostly polarised and made no or only non-adhesive cell-substratum contacts. Subline 2 cells were spread, adhesive and mainly non-polar. Subline 1 cells migrate in a non-adhesive mode which is very efficient but operates only in a 3D environment, whereas subline 2 cells migrate in an adhesive mode, which is less efficient but works on 2D and 3D substrata. Nocodazole had little or no effect on shape, polarity and locomotion of subline 1 cells. In glass-adherent subline 2 cells, 10(-6)M nocodazole increased the proportion of polarised cells migrating in an adhesive mode and decreased adhesion to the substratum, whereas 10(-5)M nocodazole further reduced the contacts and the cells reverted to a non-adhesive mode of locomotion. When non-polar subline 2 cells were detached mechanically or by nocodazole, they became polarised and morphologically indistinguishable from non-adherent subline 1 cells. On more adhesive plastic substrata, subline 2 cells produced heterogeneous responses to nocodazole including loss of polarity. The phenotypes of Walker carcinosarcoma sublines have similarities with a broad range of cell types ranging from leucocytes to fibroblast-like cells, suggesting that these phenotypic differences can be controlled by the adhesive and contractile state rather than the cell type. Adhesion modulates contractility (isometric or isotonic contraction) and vice versa and this determines morphology (shape, F-actin, myosin and alpha-actinin), locomotion and responses to microtubule-disassembly. The model may be applied to analyse the mechanisms controlling the phenotype of cells in general.