A multiscale whole-cell theory for mechanosensitive migration on viscoelastic substrates

Increasing experimental evidence validates that both the elastic stiffness and viscosity of the extracellular matrix regulate mesenchymal cell behavior, such as the rational switch between durotaxis (cell migration to stiffer regions), anti-durotaxis (migration to softer regions), and adurotaxis (stiffness-insensitive migration). To reveal the mechanisms underlying the crossover between these motility regimes, we have developed a multiscale chemomechanical whole-cell theory for mesenchymal migration. Our framework couples the subcellular focal adhesion dynamics at the cell-substrate interface with the cellular cytoskeletal mechanics and the chemical signaling pathways involving Rho GTPase proteins. Upon polarization by the Rho GTPase gradients, our simulated cell migrates by concerted peripheral protrusions and contractions, a hallmark of the mesenchymal mode. The resulting cell dynamics quantitatively reproduces the experimental migration speed as a function of the uniform substrate stiffness and explains the influence of viscosity on the migration efficiency. In the presence of stiffness gradients and absence of chemical polarization, our simulated cell can exhibit durotaxis, anti-durotaxis, and adurotaxis respectively with increasing substrate stiffness or viscosity. The cell moves toward an optimally stiff region from softer regions during durotaxis and from stiffer regions during anti-durotaxis. We show that cell polarization through steep Rho GTPase gradients can reverse the migration direction dictated by the mechanical cues. Overall, our theory demonstrates that opposing durotactic behaviors emerge via the interplay between intracellular signaling and cell-medium mechanical interactions in agreement with experiments, thereby elucidating complex mechanosensing at the single-cell level.     

Multiscale mechanisms of cell migration during development: theory and experiment

Long-distance cell migration is an important feature of embryonic development, adult morphogenesis and cancer, yet the mechanisms that drive subpopulations of cells to distinct targets are poorly understood. Here, we use the embryonic neural crest (NC) in tandem with theoretical studies to evaluate model mechanisms of long-distance cell migration. We find that a simple chemotaxis model is insufficient to explain our experimental data. Instead, model simulations predict that NC cell migration requires leading cells to respond to long-range guidance signals and trailing cells to short-range cues in order to maintain a directed, multicellular stream. Experiments confirm differences in leading versus trailing NC cell subpopulations, manifested in unique cell orientation and gene expression patterns that respond to non-linear tissue growth of the migratory domain. Ablation experiments that delete the trailing NC cell subpopulation reveal that leading NC cells distribute all along the migratory pathway and develop a leading/trailing cellular orientation and gene expression profile that is predicted by model simulations. Transplantation experiments and model predictions that move trailing NC cells to the migratory front, or vice versa, reveal that cells adopt a gene expression profile and cell behaviors corresponding to the new position within the migratory stream. These results offer a mechanistic model in which leading cells create and respond to a cell-induced chemotactic gradient and transmit guidance information to trailing cells that use short-range signals to move in a directional manner.     

Differences in Caco-2 cell attachment, migration on collagen and fibronectin coated polyelectrolyte surfaces

Metastasis mechanisms depend on cell metabolism changes, migration and adhesion to different tissues. To understand their choice of interaction site, the tumoral cell adhesion to model surfaces was studied. The response of Caco-2 tumoral cells cultured on polyelectrolyte film-functionalized surfaces with or without adhesion proteins (fibronectin or collagen IV) was analyzed. Using the layer-by- layer method, multilayer films were prepared with cationic poly(allylamine hydrochloride) and anionic poly(sodium 4-styrenesulfonate) polyelectrolytes. Film surface wettability was evaluated. The electrochemical impedance spectroscopy analyses were carried out to control the elaborated surfaces on which Caco-2 tumoral cells were cultured. The cell velocity was studied by video-microscopy and a cell colorimetric assay (WST-1) was used to quantify cell viability. The film surface parameters as well as the protein nature and localization in the film were found to modulate cell response. Results demonstrated that the cancer cell motility and proliferation were higher when cultured onto pure collagen located above the polyelectrolyte film and that the reverse surprisingly was observed when proteins were inserted into the polyelectrolyte film. Data also showed that cell motility was correlated with a high charge transfer resistance (Rct) and a low surface free energy (SFE) polar component (electron donor character). This relationship was valid only for pure external proteins. Thus, fibronectin exhibited a low Rct and a high SFE polar component, which decreased cell motility and proliferation.     

Recap on cell migration

Most animal cells move cross-linked surface antigens to one pole of the cell, a phenomenon called 'capping'. It is closely related to the rearward movement of particles attached to their surface. Cap formation is one of the most accessible dynamic properties of cells and is closely related to how they move. Yet, how this occurs is unknown.     

Bacterial migration along solid surfaces.

An in vitro system was developed to study the migration of uropathogenic Escherichia coli strains. In this system an aqueous agar gel is placed against a solid surface, allowing the bacteria to migrate along the gel/solid surface interface. Bacterial strains as well as solid surfaces were characterized by means of water contact angle and zeta potential measurements. When glass was used as the solid surface, significantly different migration times for the strains investigated were observed. Relationships among the observed migration times of six strains, their contact angles, and their zeta potentials were found. Relatively hydrophobic strains exhibited migration times shorter than those of hydrophilic strains. For highly negatively charged strains shorter migration times were found than were found for less negatively charged strains. When the fastest-migrating strain with respect to glass was allowed to migrate along solid surfaces differing in hydrophobicity and charge, no differences in migration times were found. Our findings indicate that strategies to prevent catheter-associated bacteriuria should be based on inhibition of bacterial growth rather than on modifying the physicochemical character of the catheter surface.     

Bacterial migration along solid surfaces.

An in vitro system was developed to study the migration of uropathogenic Escherichia coli strains. In this system an aqueous agar gel is placed against a solid surface, allowing the bacteria to migrate along the gel/solid surface interface. Bacterial strains as well as solid surfaces were characterized by means of water contact angle and zeta potential measurements. When glass was used as the solid surface, significantly different migration times for the strains investigated were observed. Relationships among the observed migration times of six strains, their contact angles, and their zeta potentials were found. Relatively hydrophobic strains exhibited migration times shorter than those of hydrophilic strains. For highly negatively charged strains shorter migration times were found than were found for less negatively charged strains. When the fastest-migrating strain with respect to glass was allowed to migrate along solid surfaces differing in hydrophobicity and charge, no differences in migration times were found. Our findings indicate that strategies to prevent catheter-associated bacteriuria should be based on inhibition of bacterial growth rather than on modifying the physicochemical character of the catheter surface.     

A theory of ion transport across cell surfaces by a process analogous to electron transport across liquid-solid interfaces

A kinetic theory of ion transport across cell surfaces has been developed in a form analogous to the kinetic theory of electron transport across solid-liquid interfaces of biological particles. The ionic theory is based on the observation that, at least in one instance, the voltage-current behavior for ion conduction across a cell surface is describable by the Tafel equation, in analogy to the conduction of electrons across solid-liquid interfaces. The theory predicts that the kinetics of ion transport across cell surfaces should conform to the Elovich rate equation, which is shown to be true for various experimental data. The opinions and conclusions contained in this report are those of the author. They are not to be construed as necessarily reflecting the views or the endorsement of the Navy Department.     

Modification of Drosophila cell surfaces by concanavalin A

Summary The effect of Con A on the surface morphology of cultured cells of Drosophilia melanogaster growing on coverglasses was examined by scanning electron microscopy. With low lectin concentrations (5–10g/ml) surface filaments disappeared and the cells flattened and spread against the glass surface. Cytoplasmic fusion bridges were observed in areas where cells made contact. Concentrations of Con A ranging between 50–500 g/ml caused cell shrinkage and surface distortions without cell flattening and filament loss. These morphologic effects were not apparent if Con A binding sites were blocked by preincubation with -methyl-D-mannopyranoside before application to the cell cultures. However, once the Con A-mediated changes were in effect, the cells failed to show recovery when they were returned to growth medium and a majority of the cells on the coverglasses degenerated. Presumably the cells whose morphology appears unaffected by Con A treatment are the survivors that repopulate cultures returned to growth medium.Supported by Grants CA-12600 and CA 16619 awarded by the National Cancer Institute, DREW and in part by NIH Biomedical Sciences Grant No. RR-07050. CAA's participation in this project was supported by Training Grant No. 5T01-GM-71-17We wish to thank Dr. Imogene Schneider for providing the cell lines     

A quantitative high-throughput endothelial cell migration assay

By combining the use of BD Biosciences FluoroBlok membrane-based Boyden chambers with the Cellomics HCS ArrayScan, a more sensitive method for measuring cell migration has been developed. This assay is based on counting nuclei of migrated cells on the bottom of the filter rather than conventional approaches, which use measurement of total well fluorescence. This cell migration assay provides approximately 10-fold increased signal/background compared to conventional approaches and can be used to assess the effects of growth factors on endothelial cell migration and to screen chemical compounds for inhibitory effects on growth factor-mediated endothelial cell migration.     

Auto-reverse nuclear migration in bipolar mammalian cells on micropatterned surfaces

A novel assay based on micropatterning and time-lapse microscopy has been developed for the study of nuclear migration dynamics in cultured mammalian cells. When cultured on 10-20-microm wide adhesive stripes, the motility of C6 glioma and primary mouse fibroblast cells is diminished. Nevertheless, nuclei perform an unexpected auto-reverse motion: when a migrating nucleus approaches the leading edge, it decelerates, changes the direction of motion, and accelerates to move toward the other end of the elongated cell. During this process, cells show signs of polarization closely following the direction of nuclear movement. The observed nuclear movement requires a functioning microtubular system, as revealed by experiments disrupting the main cytoskeletal components with specific drugs. On the basis of our results, we argue that auto-reverse nuclear migration is due to forces determined by the interplay of microtubule dynamics and the changing position of the microtubule organizing center as the nucleus reaches the leading edge. Our assay recapitulates specific features of nuclear migration (cell polarization, oscillatory nuclear movement), while it allows the systematic study of a large number of individual cells. In particular, our experiments yielded the first direct evidence of reversive nuclear motion in mammalian cells, induced by attachment constraints.     

Immunological analysis of glycolipids on cell surfaces of cultured human tumor cell lines: expression of lactoneotetraosylceramide on tumor cell surfaces

Glycolipids of human cell lines of colonic adenocarcinoma (Colo 205 and BM 314), gastric tumor (AZ 521 and KATO-III), and lung tumor (A 549) were studied by the immunohistochemical fluorescence technique, flow cytometric analysis and immunostaining on thin layer chromatoplates with antibodies against gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), fucogangliotetraosylceramide (Fuc-Gg4Cer), blood group B active lipid, globopentaosylceramide (Gb5Cer) and lactoneotetraosylceramide (nLc4Cer). Anti-nLc4Cer antibody was the only antibody which reacted with all the tumor cell lines used. The glycolipid fractions of each cell line separated by Iatrobeads column chromatography were immunostained with the six antibodies mentioned above on thin layer plates. The presence of nLc4Cer was detected in all cell lines. On the other hand, Gg4Cer was detected in gastric tumor cell lines, and Gg3Cer was detected in AZ 521. Based on these results, the tumor cell lines were analyzed by flow cytometry using anti-nLc4Cer antibody. About 70% of total cells in each cell line were separated as nLc4Cer-expressing cells. The present findings, together with the occurrence of nLc4Cer in ascitic fluids of cancer patients (Taki, T., Kojima, S., Seto, H., Yamada, H., & Matsumoto, M. (1984) J. Biochem. 96, 1257-1265), suggest that nLc4Cer may be a tumor-associated lipid.     

Dynamics of cell deposition on surfaces.

The rate of deposition of particles onto a surface, in the presence of London, double-layer, and gravitational forces, is calculated in terms of the energy of interaction between cell and surface by assuming that Brownian motion over a potential energy barrier is the rate-determining step of the process.     

Specificity of human galectins on cell surfaces

Galectins are ß-galactoside-binding proteins sharing homology in amino acid sequence of their carbohydraterecognition domain. Their carbohydrate specificity outside cells has been studied previously. The main conclusion of these studies was that several levels of glycan ligand recognition exist for galectins: (i) disaccharide Galß1-4GlcNAc (LN, Nacetyllactosamine) binds stronger than ß-galactopyranose; (ii) substitution at 0 -2 and 0 -3 of galactose residue as well as core fragments (“right” from GlcNAc) provides significant increase in affinity; (iii) similarly glycosylated proteins can differ significantly in affinity to galectins. Information about the natural cellular receptors of galectins is limited. Until recently, it was impossible to study specificity of cell-bound galectins. A model based on controlled incorporation of a single protein into glycocalyx of cells and subsequent interaction of loaded cells with synthetic glycoprobes measured by flow cytometry made this possible recently. In this review, data about glycan specificity of proto-, chimera-, and tandem-repeat type galectins on the cell surface are systematized, and comparative analysis of the results with data on specificity of galectins in artificial systems was performed. The following conclusions from these studies were made: (i) cellular galectins have practically no ability to bind disaccharide LNⁿ, but display affinity to 3'-substituted oligolactosamines and oligomers LNⁿ; (ii) tandem-repeat type galectins recognize another disaccharide, namely Galß1-3GlcNAc (Le^c); (iii) on the cell surface, tandemrepeat type galectins conserve the ability to display high affinity to blood group antigens of ABH system; (iv) in general, when galectins are immersed into glycocalyx, they are more selective regarding glycan interactions. Thus, we conclude that competitive interaction of galectins with cell microenvironment (endogenous cell glycans) is the main factor providing selectivity of galectins in vivo.     

Regulation of integrin affinity on cell surfaces

Lymphocyte activation triggers adhesiveness of lymphocyte function-associated antigen-1 (LFA-1; integrin α(L)β(2)) for intercellular adhesion molecules (ICAMs) on endothelia or antigen-presenting cells. Whether the activation signal, after transmission through multiple domains to the ligand-binding αI domain, results in affinity changes for ligand has been hotly debated. Here, we present the first comprehensive measurements of LFA-1 affinities on T lymphocytes for ICAM-1 under a broad array of activating conditions. Only a modest increase in affinity for soluble ligand was detected after activation by chemokine or T-cell receptor ligation, conditions that primed LFA-1 and robustly induced lymphocyte adhesion to ICAM-1 substrates. By stabilizing well-defined LFA-1 conformations by Fab, we demonstrate the absolute requirement of the open LFA-1 headpiece for adhesiveness and high affinity. Interaction of primed LFA-1 with immobilized but not soluble ICAM-1 triggers energy-dependent affinity maturation of LFA-1 to an adhesive, high affinity state. Our results lend support to the traction or translational motion dependence of integrin activation.     

Epidermal cell migration on laminin-coated substrates

Summary Pieces of coverslip glass coated with various proteins were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for the migrating wound epithelium. Laminin, a protein that has been implicated as an epithelial-specific adhesin, was a moderately good migration substrate. Type-IV collagen, fibrinogen and fibronectin, however, were significantly better. Fetuin, myoglobin, and casein all proved to be very poor substrates, allowing practically no migration. The inability of fetuin, myoglobin, and casein to support migration is further evidence that the considerable migration that occurs on collagen (Donaldson et al. 1982) fibrinogen and fibronectin (Donaldson and Mahan 1983) and the moderate migration on laminin, is a relatively specific response to these proteins and is therefore of special significance. The fact that laminin is a poorer migration substrate than collagen, fibrinogen or fibronectin suggests that the absence of cell surface laminin that has been associated with epithelial movement in several studies (Stanley et al. 1981; Clark et al. 1982; Madri and Stenn 1982; Gulati et al. 1983) may promote motility by allowing epithelial cells to interact directly with other extracellular macromolecules.