Directed cell migration on fibronectin gradients: effect of gradient slope

The migration of human microvascular endothelial cells (hMEC) was measured on a range of fibronectin gradient slopes. hMEC drift speed increased with increasing gradient slope with no concurrent change in cellular persistence time or random cell speed. The frequency of discrete cellular motion in the gradient direction increased with gradient slope. Morphological polarization of cells on the gradients is also characterized and correlated with cellular drift speed. These experiments present the first demonstration of cellular response to changing haptotactic gradient slope using an in vitro system for the quantitative study of cell migration.     

A stochastic model for leukocyte random motility and chemotaxis based on receptor binding fluctuations

Two central features of polymorphonuclear leukocyte chemosensory movement behavior demand fundamental theoretical understanding. In uniform concentrations of chemoattractant, these cells exhibit a persistent random walk, with a characteristic "persistence time" between significant changes in direction. In chemoattractant concentration gradients, they demonstrate a biased random walk, with an "orientation bias" characterizing the fraction of cells moving up the gradient. A coherent picture of cell movement responses to chemoattractant requires that both the persistence time and the orientation bias be explained within a unifying framework. In this paper, we offer the possibility that "noise" in the cellular signal perception/response mechanism can simultaneously account for these two key phenomena. In particular, we develop a stochastic mathematical model for cell locomotion based on kinetic fluctuations in chemoattractant/receptor binding. This model can simulate cell paths similar to those observed experimentally, under conditions of uniform chemoattractant concentrations as well as chemoattractant concentration gradients. Furthermore, this model can quantitatively predict both cell persistence time and dependence of orientation bias on gradient size. Thus, the concept of signal "noise" can quantitatively unify the major characteristics of leukocyte random motility and chemotaxis. The same level of noise large enough to account for the observed frequency of turning in uniform environments is simultaneously small enough to allow for the observed degree of directional bias in gradients.     

Locomotion of CV-1 cytoplasts with or without a centrosome

The movement of cultured cells along the substratum is a convenient model for studying cell movement in the organism, occurring during embryogenesis, angiogenesis, metastasis, wound closure, etc. The moving cells must control their pseudopodial activity along the perimeter, regulate the attachment and reattachment to the substratum, and pull their body following pseudopodium during their movement along the substratum. As proven by numerous investigations, these processes directly depend on the actomyosin system of cells. The role of microtubules as components of cytoskeleton in cell locomotion still remains obscure. The role of microtubules in cell movement is commonly investigated using microtubule-destructive drugs. Therefore in the final results the accessory drug effect on, for example, signal cascades cannot be excluded. Another mode of action on the microtubule dynamics is centrosome removal from the cells, which is easily realized by their removal together with the nucleus. It has been shown that in cytoplasts of centrosome containing fibroblasts, dynamic instability of microtubules remains. Unlike, in non-centriolar cytoplasts tread milling is observed. Some literature evidence suggests that cytoplasts of cultured cells move along the substratum not worse that intact cells do. In this study cytoplasts with and without centrosome were obtained and identified, and parameters of movement along the substratum (speed, direction) were registered for both these two populations of cytoplasts, and for control intact cells and cells involved in the experiment. The model of experimental wound of monolayer was used, because it guaranteed cell synchronization in respect to movement direction and speed. Centrosome-containing CV-1 cytoplasts displayed radial microtubule array, and centrosome-lacking cytoplasts exhibited chaotic distribution of microtubules, which is characteristic of microtubule tread milling. In addition, both kinds of cytoplasts appeared to move along the substratum much slower than the whole cells. No difference was found is speed and keeping direction between centriolar and non-centriolar cytoplasts.     

Association between fibronectin receptor and the substratum: Spare receptors for cell adhesion

We have investigated the association of the recently described 140-kDa cell membrane receptor for fibronectin with the cytoskeleton or with substratum-bound fibronectin. Using a monospecific polyclonal antibody to the 140-kDa receptor, we have demonstrated that most of the receptor molecules are soluble in nonionic detergent either in suspension culture CHO cells or in CHO cells attached to and spread on a fibronectin-coated substratum. This may suggest that putative linkages of the receptor either to fibronectin or to detergent-insoluble cytoskeletal components are labile to nonionic detergent and thus are rather weak. Alternatively, it may mean that only a small fraction of the cell's receptors are needed to mediate adhesion. In order to explore this latter concept, we have coated substrata with various concentrations of PB1, a monoclonal antibody with a high affinity for fibronectin receptor. We demonstrate that coating the substratum with increasing concentrations of PB1 results in increasing amounts of 140-kDa receptor becoming bound to the substratum in detergent-insoluble form. However, the amount of receptor bound does not necessarily correlate with the degree of cell adhesion and spreading. Thus, coating the substratum with 5 μg/ml of PB1 results in essentially complete attachment and spreading of CHO cells, but only a small fraction of the 140-kDa receptor becomes substratum bound. Coating with 50 μg/ml of PB1 produces no further increase in cell adhesion and spreading, but results in the detergent-stable association of a large fraction of the total receptor pool with the substratum. These observations suggest the possibility of there being “spare” receptors for cell adhesion processes.     

Impaired neutrophil locomotion associated with hyperadhesiveness in a patient with Chédiak-Higashi syndrome

Neutrophils from a patient with Chédiak-Higashi (CH) syndrome exhibited defective directional locomotion in a gradient of activated plasma. Further analysis of the nature of this defect showed that CH neutrophils could respond normally to stimulation with f-Met-Leu-Phe, by increasing both their motility and polarization, provided the cells were kept in suspension. Contact with the substratum resulted in the loss of both motility and polarity in the majority of cells. CH neutrophils, in contrast to normal cells, did not respond chemokinetically to f-Met-Leu-Phe. Unstimulated random locomotion of CH neutrophils was also depressed, and this correlated with increased spreading on the substratum. Our results indicate that motility, locomotion and polarisation of CH neutrophils on the substratum are depressed because of excessive adhesion.     

Mechanically induced orientation of adult rat cardiac myocytes in vitro

Summary A population of freshly isolated adult rat cardiac myocytes is spatially oriented using a computerized mechanical cell stimulator device for tissue cultured cells. A continuous unidirectional stretch of the substratum at 60 to 400 μm/min for 120 to 30 min, respectively, during the cell attachment period in serum-free medium induces a significant three-fold increase in the number of rod-shaped myocytes oriented parallel to the direction of movement. The myocytes orient less well with unidirectional substratum stretching after their adhesion to the substratum. In contrast, adult myocytes plated onto a substratum undergoing continuous 10% stretch-relaxation cycling show no significant change in myocyte orientation or cytoskeletal organization. Orientation of rod-shaped myocytes is dependent on several factors other than the type of mechanical activity. These include: a) the speed of substratum movement; b) the final stretch amplitude; and c) the timing between initiation of substratum stretching and adhesion of myocytes to the substratum. Oriented adult rod shaped myocytes representing 65 to 70% of the total myocyte population in this model system can now be submitted to different patterns of repetitive mechanical stimulation for the study of stretch-induced alterations in cell growth and gene expression. This work was supported by grants AR36266, AR39998, and RR05818 from the National Institutes of Health, Bethesda, MD, and grant NAG2-414 from the National Aeronautics and Space Administration, Washington, DC. J.-L. Samuel was a recipient from the Foundation pour la Recherche Médicale.     

Modulation of cell migration by integrin-mediated cytoskeletal linkages and ligand-binding affinity

Integrin cell surface adhesion receptors play a central role in mediating cell migration. We have developed a model system consisting of CHO cells ectopically expressing the alpha IIb beta 3 integrin to study integrin affinity and cytoskeletal interactions during cell migration. The alpha IIb beta 3 integrins are suited for study of integrin receptors during cell migration because they are well characterized with respect to ligand binding, cytoskeletal interactions, and signal transduction, and mutants with altered receptor function are available. The alpha IIb beta 3 receptor specifically mediates migration of alpha IIb beta 3-transfected CHO cells. The migration of transfected CHO cells was studied on a fibrinogen substrate both by time lapse videomicroscopy and by random and haptotactic transwell assays. Haptotactic and random transwell assays measured distinct aspects of migration, with the random transwell assay correlating most closely with time lapse videomicroscopy. Mutations in the cytoplasmic domains that increase ligand affinity or activation of the alpha IIb beta 3 receptor into a high affinity state by the LIBS6 antibody decreased the migration rate. Likewise, mutations that increase cytoskeletal organization without affecting affinity also decreased the migration rate. In contrast, truncation of the beta chain, which alters cytoskeletal associations as assayed by absence of focal adhesions, decreased haptotactic migration while increasing random migration. These effects on the migration rate were partially compensated for by altering substrate concentration, demonstrating optimum substrate concentrations that supported maximal migration. For example, cells expressing integrins locked in the high affinity state showed maximal migration at lower substrate concentrations than cells expressing low affinity receptor. Together, these results implicate the strength of adhesion between cell and substrate, as modulated by receptor affinity, organization of adhesive complexes, and substrate concentration, as important regulators of cell migration rate. Further, we demonstrate a dominant effect of high affinity integrin in inhibiting migration regardless of the organization of adhesive complexes. These observations have potential implications for tumor metastasis and its therapy.     

Differential effects of laminin, intact type IV collagen, and specific domains of type IV collagen on endothelial cell adhesion and migration

Laminin and type IV collagen were compared for the ability to promote aortic endothelial cell adhesion and directed migration in vitro. Substratum-adsorbed IV promoted aortic endothelial cell adhesion in a concentration dependent fashion attaining a maximum level 141-fold greater than controls within 30 min. Aortic endothelial cell adhesion to type IV collagen was not inhibited by high levels (10(-3) M) of arginyl-glycyl-aspartyl-serine. In contrast, adhesion of aortic endothelial cells on laminin was slower, attaining only 53% of the adhesion observed on type IV collagen by 90 min. Type IV collagen when added to the lower well of a Boyden chamber stimulated the directional migration of aortic endothelial cells in a concentration dependent manner with a maximal response 6.9-fold over control levels, whereas aortic endothelial cells did not migrate in response to laminin at any concentration (.01-2.0 X 10(-7) M). Triple helix-rich fragments of type IV collagen were nearly as active as intact type IV collagen in stimulating both adhesion and migration whereas the carboxy terminal globular domain was less active at promoting adhesion (36% of the adhesion promoted by intact type IV collagen) or migration. Importantly, aortic endothelial cells also migrate to substratum adsorbed gradients of type IV collagen suggesting that the mechanism of migration is haptotactic in nature. These results demonstrate that the aortic endothelial cell adhesion and migration is preferentially promoted by type IV collagen compared with laminin, and has a complex molecular basis which may be important in angiogenesis and large vessel repair.     

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.     

Cells as Active Particles in Asymmetric Potentials: Motility under External Gradients

Cell migration is a crucial event during development and in disease. Mechanical constraints and chemical gradients can contribute to the establishment of cell direction, but their respective roles remain poorly understood. Using a microfabricated topographical ratchet, we show that the nucleus dictates the direction of cell movement through mechanical guidance by its environment. We demonstrate that this direction can be tuned by combining the topographical ratchet with a biochemical gradient of fibronectin adhesion. We report competition and cooperation between the two external cues. We also quantitatively compare the measurements associated with the trajectory of a model that treats cells as fluctuating particles trapped in a periodic asymmetric potential. We show that the cell nucleus contributes to the strength of the trap, whereas cell protrusions guided by the adhesive gradients add a constant tunable bias to the direction of cell motion.     

Cells as Active Particles in Asymmetric Potentials: Motility under External Gradients

Cell migration is a crucial event during development and in disease. Mechanical constraints and chemical gradients can contribute to the establishment of cell direction, but their respective roles remain poorly understood. Using a microfabricated topographical ratchet, we show that the nucleus dictates the direction of cell movement through mechanical guidance by its environment. We demonstrate that this direction can be tuned by combining the topographical ratchet with a biochemical gradient of fibronectin adhesion. We report competition and cooperation between the two external cues. We also quantitatively compare the measurements associated with the trajectory of a model that treats cells as fluctuating particles trapped in a periodic asymmetric potential. We show that the cell nucleus contributes to the strength of the trap, whereas cell protrusions guided by the adhesive gradients add a constant tunable bias to the direction of cell motion.     

A stochastic model for chemosensory cell movement: application to neutrophil and macrophage persistence and orientation

Two central features of leukocyte chemosensory movement behavior demand fundamental theoretical understanding. In uniform concentrations of chemoattractant, these cells exhibit a persistent random walk, with a characteristic “persistence time” between significant changes in direction. In chemoattractant concentration gradients, they demonstrate a biased random walk, with an “orientation bias” characterizing the fraction of cells moving up the gradient. A coherent picture of cell-movement responses to chemoattractant requires that both the persistence time and the orientation bias be explained within a unifying framework. In this paper we offer the possibility that “noise” in the cellular signal perception/response mechanism can simultaneously account for these two key phenomena. In particular, we report on a stochastic mathematical model for cell locomotion based on kinetic fluctuations in chemoattractant receptor binding. This model proves to be capable of stimulating cell paths similar to those observed experimentally for two cell types examined to date: neutrophils and alveolar macrophages, under conditions of uniform chemoattractant concentrations as well as chemoattractant concentration gradients. Further, this model can quantitatively predict both cell persistence time and dependence of orientation bias on gradient size. The model also successfully predicts that an increase in persistence time is associated with a decrease in orientation for typical system parameter values, as is observed for alveolar macrophages in comparison to neutrophils. Thus, the concept of signal “noise” can quantitatively unify the major characteristics of leukocyte random motility and chemotaxis. The same level of noise large enough to account for the observed frequency of turning in uniform environments is simultaneously small enough to allow for the observed degree of directional bias in gradients. This suggests that chemosensory cell movement behavior may be based on a “usefully” imperfect integrated signal response system, which allows both random and directed searches under appropriate conditions.     

Analyzing fish movement as a persistent turning walker

The trajectories of Kuhlia mugil fish swimming freely in a tank are analyzed in order to develop a model of spontaneous fish movement. The data show that K. mugil displacement is best described by turning speed and its auto-correlation. The continuous-time process governing this new kind of displacement is modelled by a stochastic differential equation of Ornstein–Uhlenbeck family: the persistent turning walker. The associated diffusive dynamics are compared to the standard persistent random walker model and we show that the resulting diffusion coefficient scales non-linearly with linear swimming speed. In order to illustrate how interactions with other fish or the environment can be added to this spontaneous movement model we quantify the effect of tank walls on the turning speed and adequately reproduce the characteristics of the observed fish trajectories.     

Modeling the kinetics of cell membrane spreading on substrates with ligand density gradient

An analytical model is developed for the effect of surface gradient in ligand density on the adhesion kinetics of a curved elastic membrane with mobile receptors. The displacement and speed of spreading at the edge of adhesion zone as well as the density profile of receptors along the membrane are predicted as a function of time. According to results, in the diffusion-controlled regime, the front edge displacement of adhesion zone and the rate of membrane spreading decreased with increasing the ligand density in a certain direction. Furthermore, the displacement of the edge of the adhesion zone did not scale with the square root of time, as observed on substrates with uniform ligand density.     

Extracellular matrix-mediated chemotaxis can impede cell migration

Cells use a combination of changes in adhesion, proteolysis and motility (directed and random) during the process of migration. Proteolysis of the extracellular matrix (ECM) results in thecreation of haptotactic gradients which cells use to move in a directed fashion. The proteolytic creation of these gradients also results in the production of digested fragments of ECM. In this study we show that in the human fibrosarcoma cell line HT1080, matrix metalloproteinase-2(MMP-2)-digested fragments of fibronectin exert a chemotactic pull stronger than that of undigested fibronectin. During invasion, this gradient of ECM fragments is established in the wake of an invading cell, running counter to the direction of invasion. The resultant chemotactic pull is anti-invasive, contrary to the traditional view of the role of chemotaxis in invasion. Uncontrolled ECM degradation by high concentrations of MMP can thus result in steep gradients of ECM fragments, which run against the direction of invasion. Consequently, the invasive potential of a cell depends on MMP production in a biphasic mannerimplying that MMP inhibitors will upregulate invasion in high-MMPexpressing cells. Hence the therapeutic use of protease inhibitors against tumours expressing high levels of MMP could produce an augmentation of invasion.     

Models of normal and transformed cell adhesion,and capping and locomotion in vitro

It is proposed that patching, capping and endocytosis, and cell locomotion are manifestations of a single process whereby the cell discards foreign materials. Capping results from the binding to the cell surface of particulate (or molecular) objects which cannot function as immovable substratum. This might be described as unsuccessful or abortive cell adhesion in that the particles adhere to the cell rather than the cell adhering to the substratum. Lateral particle movements on the cell surface membrane are effected by the submembranous microfilament-microtubule system, resulting in capping without displacement of the cell. Successful adhesion of the cell to a substratum renders capping and endocytosis impossible and the cell attempts to discard the substratum by mechanisms analogous to capping. The cell achieves this by lateral movement and detachment of the trailing edge.The concept of abortive adhesion leading to capping has been amplified by the development of molecular models of normal and neoplastic cell adhesion in vitro in the presence and absence of serum. In these models, the normal cells have molecule A (adhesion sites) on their surface; they can spread on the substratum in the absence of serum. In the presence of serum, the A molecules on the normal cell surface bind with B molecules in serum, which may be substratum-bound or free in suspension. Binding of free B molecules with cell surface A molecules results in blockage of adhesion sites; these are cleared via capping. New adhesion sites (A molecules) are produced at the active edges of the cell. Binding of cell surface A molecules with the substratum bound B molecules results in cell adhesion. Transformed cells do not have A molecules on their surface; they cannot spread in the absence of serum. The transformed cells may recruit A molecules from the serum to attain deformability and spreading.These models also relate to capping of gold or resin particles, cell locomotion and regulation of cell division, and lectin-induced agglutination of transformed cells.     

Coated pits and asialoglycoprotein receptors redistribute to the substratum during hepatocyte adhesion to galactoside surfaces

Rat hepatocytes bind in a sugar-specific and concentration-dependent manner to flat polyacrylamide matrices containing covalently attached galactosyl (Gal) groups. Previous studies (Weigel, P.H., J. Cell Biol. 87, 855, 1980) concluded that binding was likely mediated by the asialoglycoprotein receptor. Here we confirm that adhesion is mediated by this receptor, since cell binding is inhibited by antireceptor antibody and a threshold binding response is also observed when hepatocytes adhere to surfaces coated with asialoorosomucoid, a ligand for this receptor. Cells that had bound to a Gal surface and were then sheared from the surface left a membrane patch behind on the substratum. The cytoplasmic side of these plasma membrane patches was visualized on the substratum by indirect immunofluorescence using antireceptor antibody or anticlathrin antibody. The density of punctate coated pits, visualized with the latter antibody, was enriched in a circular membrane region of about 4 microns 2 area that mediated cell binding. This zone also contained concentrated receptors, although the staining pattern with antireceptor antibody was more uniform and less punctate. The results show that both asialoglycoprotein receptors and coated pits are redistributed at the substratum interface on hepatocytes bound to Gal surfaces.     

Measurement of the chemotaxis coefficient for human neutrophils in the under-agarose migration assay

Clinical and scientific investigations of leukocyte chemotaxis will be greatly aided by an ability to measure quantitative parameters characterizing the intrinsic random motility, chemokinetic, and chemotactic properties of cell populations responding to a given attractant. Quantities typically used at present, such as leading front distances, migrating cell numbers, etc., are unsatisfactory in this regard because their values are affected by many aspects of the assay system unrelated to cell behavioral properties. In this paper we demonstrate the measurement of cell migration parameters that do, in fact, characterize the intrinsic cell chemosensory movement responses using cell density profiles obtained in the linear under-agarose assay. These parameters are the random motility coefficient, mu, and the chemotaxis coefficient, chi, which appear in a theoretical expression for cell population migration. We propose a priori the dependence of chi on attractant concentration, based on an independent experimental correlation of individual cell orientation bias in an attractant gradient with a spatial difference in receptor occupancy. Our under-agarose population migration results are consistent with this proposition, allowing chemotaxis to be reliably characterized by a chemotactic sensitivity constant, chi 0, to which chi is directly proportional. Further, chi 0 has fundamental significance; it represents the reciprocal of the difference in number of bound receptors across cell dimensions required for directional orientation bias. In particular, for the system of human peripheral blood polymorphonuclear neutrophil leukocytes responding to FNLLP, we find that the chemotaxis coefficient is a function of attractant concentration, a following the expression: chi = chi 0NT0 f(a) S(a) Kd/(Kd + a)2 where Kd is the FNLLP-receptor equilibrium dissociation constant and NT0 is the total number of cell surface receptors for FNLLP. f(a) is the fraction of surface receptors remaining after down-regulation, and S(a) is the cell movement speed, both known functions of FNLLP concentration. We find that chi 0NT0 = 0.2 cm; according to a theoretical argument outlined in the Appendix this means that these cells exhibit 75% orientation toward higher attractant concentration when the absolute spatial difference in bound receptors is 0.0025NT0 over 10 micron. (For example, if NT0 = 50,000 this would correspond to a spatial difference of 125 bound receptors over 10 micron.) This result can be compared with estimates obtained from visual studies of individual neutrophils.