Laminin and fibronectin promote the haptotactic migration of B16 mouse melanoma cells in vitro

The migration of tumor cells through basement membranes and extracellular matrices is an integral component of tumor invasion and metastasis. Laminin and fibronectin are two basement membrane- and extracellular matrix-associated noncollagenous glycoproteins that have been shown to promote both cell adhesion and motility. Purified preparations of laminin and fibronectin stimulated the directed migration of B16 murine metastatic melanoma cells in vitro as assessed in modified Boyden chambers. The stimulation of migration occurred over a concentration range of 1-100 micrograms/ml of laminin or fibronectin, with a peak response occurring between 12.5 and 25 micrograms/ml. The maximal response of these cells was 80-120-fold higher than control migration. Affinity-purified antibody preparations specifically abrogated the migration of these cells in response to the respective proteins. Tumor cells in suspension were preincubated in physiologic levels of plasma fibronectin prior to assay to partially mimic what occurs when a metastasizing cell is in the blood stream. This preincubation with plasma fibronectin had no effect on the subsequent migration of cells in response to either laminin or fibronectin. Furthermore, experiments using filters precoated with fibronectin or laminin indicated that these cells could migrate by haptotaxis to these two proteins. We conclude that tumor cell migration in response to such noncollagenous adhesive glycoproteins could be an important aspect in the invasion and metastasis of certain malignant cell types.     

Annulate lamellae: comparison of antigenic epitopes of annulate lamellae membranes with the nuclear envelope

Laminin derived from the Engelbreth-Holm-Swarm (EHS) tumor and a lamininlike molecule synthesized by RN22 Schwannoma cells both stimulate rapid neurite outgrowth, consistent with a common neurite-promoting site. However, antilaminin antisera can only inhibit the activity of the EHS laminin. The blocking antibodies in such sera are directed against the terminal heparin-binding domain of the laminin long arm (Edgar, D., R. Timpl, and H. Thoenen. 1984. EMBO [Eur. Mol. Biol. Organ.] J. 3: 1463-1468). These epitopes are demonstrated by immunoblotting to be part of the A chain and to be absent in RN22 laminin, showing (through metabolic labeling) that the cells synthesized little if any 440-kD A chain. This indicates that the antibody inhibition was probably due to steric hindrance, a common neurite-promoting site, apparently not being antigenic in native molecules. Antibodies raised against a 25-kD proteolytic fragment derived from the long arm of laminin were then used as probes to identify other potential neurite-promoting structures. Although these antibodies do not cross-react with native laminin, they recognized the B chains of denatured EHS and RN22 molecules on immunoblots. The antibodies also bound to the large proteolytic fragment, derived from the long arm of laminin that contains the neurite-promoting site, thus inhibiting its activity. Taken together, these results point to the localization of normally nonantigenic, defined, B chain sequences within or close to the neurite-promoting site of laminin.     

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.     

Production of laminin and fibronectin by Schwannoma cells: cell-protein interactions in vitro and protein localization in peripheral nerve in vivo

We studied a rat Schwannoma cell line (RN22F) to determine if it produced the basement membrane glycoproteins laminin and fibronectin, and how it interacted with these proteins in vitro. We used antisera to laminin and fibronectin for immunoprecipitation experiments and immunocytochemical localization at the electron microscope level. Polyacrylamide gels of antilaminin immunoprecipitates of conditioned medium and solubilized Schwannoma cells contained bands of reduced Mr 200,000 and 150,000. Antilaminin immunoprecipitates of conditioned medium contained nonreduced bands of 850,000 daltons and 150,000, and immunoprecipitates of solubilized cells contained nonreduced bands of 850,000, 400,000, 200,000, and 150,000 daltons. Antifibronectin immunoprecipitates of conditioned medium contained a reduced band of 220,000 daltons, and nonreduced bands of 440,000 and 220,000 daltons. Radio-labeled protein was not detected in antifibronectin immunoprecipitates of solubilized cells. By immunocytochemistry, laminin was found along the cell surface in a continuous band, whereas fibronectin was only sparsely distributed along the cell surface. In cell adhesion assays, Schwannoma cells bound preferentially to laminin- coated substrates as compared to fibronectin or noncoated substrates. A number of Schwannoma cells displayed a curved and elongated morphology on laminin substrates, as compared to a uniformly spread morphology on fibronectin, and a round, nonspread morphology on noncoated substrates. Immunofluorescent staining showed laminin in the endoneurium and perineurium and fibronectin predominantly in the perineurium of mouse sciatic nerve in vivo. The production of laminin and fibronectin by Schwann cells may be important in the development and myelination of peripheral nerves, and the proper regeneration of axons following nerve injury.     

Laminin inhibits human keratinocyte migration

A quantitative migration assay for human keratinocytes was developed to assess the influence of extracellular matrix molecules on cell motility independently from their effect on cell proliferation. Fibronectin and collagen types I and IV markedly promoted keratinocyte migration, but albumin, type V collagen, and heparan sulfate proteoglycan had little effect. In contrast, laminin inhibited keratinocyte motility and dramatically reduced type IV collagen-induced migration in a concentration-dependent manner. Laminin was not toxic, since it had no apparent effect on morphology, growth, or ability of cells to be passaged. Laminin, a major component of the lamina lucida, may inhibit motility of keratinocytes in vivo. Absence of contact with laminin, which accompanies wounding, may facilitate motility and healing in the epidermis.     

Signal transduction for chemotaxis and haptotaxis by matrix molecules in tumor cells

Transduction of signals initiating motility by extracellular matrix (ECM) molecules differed depending on the type of matrix molecule and whether the ligand was in solution or bound to a substratum. Laminin, fibronectin, and type IV collagen stimulated both chemotaxis and haptotaxis of the A2058 human melanoma cell line. Peak chemotactic responses were reached at 50-200 nM for laminin, 50-100 nM for fibronectin, and 200-370 nM for type IV collagen. Checkerboard analysis of each attractant in solution demonstrated a predominantly directional (chemotactic) response, with a minor chemokinetic component. The cells also migrated in a concentration-dependent manner to insoluble step gradients of substratum-bound attractant (haptotaxis). The haptotactic responses reached maximal levels at coating concentrations of 20 nM for laminin and type IV collagen, and from 30 to 45 nM for fibronectin. Pretreatment of cells with the protein synthesis inhibitor, cycloheximide (5 micrograms/ml), resulted in a 5-30% inhibition of both chemotactic and haptotactic responses to each matrix protein, indicating that de novo protein synthesis was not required for a significant motility response. Pretreatment of cells with 50-500 micrograms/ml of synthetic peptides containing the fibronectin cell-recognition sequence GRGDS resulted in a concentration-dependent inhibition of fibronectin-mediated chemotaxis and haptotaxis (70-80% inhibition compared to control motility); negative control peptide GRGES had only a minimal effect. Neither GRGDS nor GRGES significantly inhibited motility to laminin or type IV collagen. Therefore, these results support a role for the RGD-directed integrin receptor in both types of motility response to fibronectin. After pretreatment with pertussis toxin (PT), chemotactic responses to laminin, fibronectin, and type IV collagen were distinctly different. Chemotaxis to laminin was intermediate in sensitivity; chemotaxis to fibronectin was completely insensitive; and chemotaxis to type IV collagen was profoundly inhibited by PT. In marked contrast to the inhibition of chemotaxis, the hepatotactic responses to all three ligands were unaffected by any of the tested concentrations of PT. High concentrations of cholera toxin (CT; 10 micrograms/ml) or the cAMP analogue, 8-Br-cAMP (0.5 mM), did not significantly affect chemotactic or haptotactic motility to any of the attractant proteins, ruling out the involvement of cAMP in the biochemical pathway initiating motility in these cells. The sensitivity of chemotaxis induced by laminin and type IV collagen, but not fibronectin, to PT indicates the involvement of a PT-sensitive G protein in transduction of the signals initiating motility to soluble laminin and type IV collagen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lectin KM+-induced neutrophil haptotaxis involves binding to laminin

The lectin KM+ from Artocarpus integrifolia, also known as artocarpin, induces neutrophil migration by haptotaxis. The interactions of KM+ with both the extracellular matrix (ECM) and neutrophils depend on the lectin ability to recognize mannose-containing glycans. Here, we report the binding of KM+ to laminin and demonstrate that this interaction potentiates the KM+-induced neutrophil migration. Labeling of lung tissue by KM+ located its ligands on the endothelial cells, in the basement membrane, in the alveolus, and in the interstitial connective tissue. Such labeling was inhibited by 400 mM D-mannose, 10 mM Manalpha1-3[Manalpha1-6]Man or 10 microM peroxidase (a glycoprotein-containing mannosyl heptasaccharide). Laminin is a tissue ligand for KM+, since both KM+ and anti-laminin antibodies not only reacted with the same high molecular mass components of a lung extract, but also determined colocalized labeling in basement membranes of the lung tissue. The relevance of the KM+-laminin interaction to the KM+ property of inducing neutrophil migration was evaluated. The inability of low concentrations of soluble KM+ to induce human neutrophil migration was reversed by coating the microchamber filter with laminin. So, the interaction of KM+ with laminin promotes the formation of a substrate-bound KM+ gradient that is able to induce neutrophil haptotaxis.     

Embryonic heart mesenchymal cell migration in laminin

Laminin, a large glycoprotein and major component of basement membranes, influences cell adhesion, migration, morphology, and differentiation. A peptide sequence, YIGSR, from the B1 chain of laminin has been found to correspond to an active site for cell adhesion. We report here that cardiac mesenchymal cells migrate vigorously within three-dimensional gels of laminin and that the YIGSR peptide will completely abolish this migratory activity. In contrast, migration of the mesenchymal cells into three-dimensional gels composed of collagen or collagen + laminin is not effected by YIGSR or other peptides (GRGDS, GRGDTP) reported to mediate cellular adhesion.     

Vitronectin-induced haptotaxis of vascular smooth muscle cells in vitro.

Vitronectin, a multifunctional glycoprotein present in the plasma and interstitial tissues, has recently been found to be localized in atherosclerotic lesions. In this study we examined the effects of vitronectin on the migration of cultured bovine aortic smooth muscle cells using a modified Boyden chamber assay. The cells migrated to fluid-phase vitronectin in a concentration-dependent fashion. The cells also migrated to membrane filter surfaces precoated with vitronectin for a few minutes in the absence of additional vitronectin in the fluid phase, suggesting that this substance binds easily to the filters and stimulates cell migration by haptotaxis under the conditions described. These observations suggest that vitronectin deposited in the intima may be involved in the pathogenesis of atherosclerosis by recruiting smooth muscle cells from the media into the intima.     

Synthetic pentapeptide from the B1 chain of laminin promotes B16F10 melanoma cell migration

Laminin is a basement membrane-specific glycoprotein that promotes cell adhesion, proliferation, differentiation, and tumor cell migration. Synthetic peptides from the amino acid sequence deduced from a cDNA clone of the B1 chain of laminin were tested for their ability to promote the migration of B16F10 melanoma cells. A peptide, CDPGYIGSR, that is able to mediate epithelial cell attachment to laminin was found to promote migration, and the constituent pentapeptide YIGSR was also active but to a lesser degree. This nine-amino acid peptide blocked migration of melanoma cells to laminin but had no effect on migration to fibronectin. These data suggest that the cell-binding site and migration site on laminin share a common sequence that is unique to laminin.     

Direct evidence that cancer cell locomotion contributes importantly to invasion

This study was undertaken to clarify whether active locomotion of cancer cells is important for their ability to invade. The most rapidly moving cells were isolated from a cultured murine parent fibrosarcoma by successive cycles of migration through a micropore membrane. Cells were isolated by unstimulated locomotion and by haptotaxis to laminin, and the selected cells did indeed constitute rapidly locomoting subpopulations. These cells invaded biological tissues more efficiently than did the unselected parent cells. The cells selected by haptotaxis to laminin invaded most rapidly through amnion with basement membranes (containing laminin). Cancer cell haptotaxis to laminin in basement membranes thus promotes penetration of these tissue barriers. These results show in a direct manner that cancer cell locomotion is in fact important in invasion of biological tissues.     

Amphibian neural crest cell migration on purified extracellular matrix components: a chondroitin sulfate proteoglycan inhibits locomotion on fibronectin substrates

The ability of purified extracellular matrix components to promote the initial migration of amphibian neural crest (NC) cells was quantitatively investigated in vitro. NC cells migrated avidly on fibronectin (FN), displaying progressively more extensive dispersion at increasing amounts of material incorporated in the substrate. In contrast, dispersion on laminin substrates was optimal at low protein concentrations but strongly reduced at high concentrations. NC cells were unable to migrate on substrates containing a high molecular mass chondroitin sulfate proteoglycan (ChSP). When proteolytic peptides, representing isolated functional domains of the FN molecule, were tested as potential migration substrates, the cell binding region of the molecule (105 kD) was found to be as active as the intact FN. A 31- kD heparin-binding fragment also stimulated NC cell migration, whereas NC cells dispersed to a markedly lower extent on the isolated collagen- binding domain (40 kD), or the latter domain linked to the NH2-terminal part of the FN molecule. Migration on the intact FN was partially inhibited by antibodies directed against the 105- and 31-kD fragments, respectively; dispersion was further decreased when the antibodies were used in combination. Addition of the ChSP to the culture medium dramatically perturbed NC cell migration on substrates of FN, as well as of 105- or 31-kD fragments. However, preincubation of isolated cells or substrates with ChSP followed by washing did not affect NC cell movement. The use of substrates consisting of different relative amounts of ChSP and the 105-kD peptide revealed that ChSP counteracted the motility-promoting activity of the 105-kD FN fragment in a concentration-dependent manner also when bound to the substrate. Our results indicate that NC cell migration on FN involves two separate domains of the molecule, and that ChSP can modulate the migratory behavior of NC cells moving along FN-rich pathways and may therefore influence directionally and subsequent localization of NC cells in the embryo.     

Rac Regulates Vascular Endothelial Growth Factor Stimulated Motility

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood.

Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF.

These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.     

Rac Regulates Vascular Endothelial Growth Factor Stimulated Motility

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood.

Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF.

These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.     

The short arm of the laminin gamma2 chain plays a pivotal role in the incorporation of laminin 5 into the extracellular matrix and in cell adhesion

Laminin 5 is a basement membrane component that actively promotes adhesion and migration of epithelial cells. Laminin 5 undergoes extracellular proteolysis of the gamma2 chain that removes the NH(2)-terminal short arm of the polypeptide and reduces the size of laminin 5 from 440 to 400 kD. The functional consequence of this event remains obscure, although lines of evidence indicate that cleavage of the gamma2 chain potently stimulated scattering and migration of keratinocytes and cancer cells. To define the biological role of the gamma2 chain short arm, we expressed mutated gamma2 cDNAs into immortalized gamma2-null keratinocytes. By immunofluorescence and immunohistochemical studies, cell detachment, and adhesion assays, we found that the gamma2 short arm drives deposition of laminin 5 into the extracellular matrix (ECM) and sustains cell adhesion. Our results demonstrate that the unprocessed 440-kD form of laminin 5 is a biologically active adhesion ligand, and that the gamma2 globular domain IV is involved in intermolecular interactions that mediate integration of laminin 5 in the ECM and cell attachment.     

Thrombospondin-induced tumor cell migration: haptotaxis and chemotaxis are mediated by different molecular domains

Thrombospondin induces the migration of human melanoma and carcinoma cells. Using a modified Boyden chamber assay, tumor cells migrated to a gradient of soluble thrombospondin (chemotaxis). Checkerboard analysis indicated that directional migration was induced 27-fold greater than stimulation of random motility. Tumor cells also migrated in a dose-dependent manner to a gradient of substratum-bound thrombospondin (haptotaxis). A series of human melanoma and carcinoma cells were compared for their relative motility stimulation by thrombospondin haptotaxis vs. chemotaxis. Some cell lines exhibited a stronger haptotactic response compared to their chemotactic response while other lines exhibited little or no migration response to thrombospondin. Human A2058 melanoma cells which exhibit a strong haptotactic and chemotactic response to thrombospondin were used to study the structural domains of thrombospondin required for the response. Monoclonal antibody C6.7, which binds to the COOH-terminal region of thrombospondin, inhibited haptotaxis in a dose-dependent optimal manner. C6.7 had no significant effect on thrombospondin chemotaxis. In contrast, monoclonal antibody A2.5, heparin, and fucoidan, which bind to the NH2-terminal heparin-binding domain of thrombospondin, inhibited thrombospondin chemotaxis but not haptotaxis. Monoclonal antibody A6.1 directed against the internal core region of thrombospondin had no significant effect on haptotaxis or chemotaxis. Synthetic peptides GRGDS (50 micrograms/ml), but not GRGES, blocked tumor cell haptotaxis on fibronectin, but had minimal effect on thrombospondin or laminin haptotaxis. The 140-kD fragment of thrombospondin lacking the heparin-binding amino-terminal region retained the property to fully mediate haptotaxis but not chemotaxis. When the COOH region of the 140-kD fragment, containing the C6.7-binding site, was cleaved off, the resulting 120-kD fragment (which retains the RGDA sequence) failed to induce haptotaxis. Separate structural domains of thrombospondin are therefore required for tumor cell haptotaxis vs. chemotaxis. This may have implications during hematogenous cancer metastases formation.     

The effects of interleukin-8 on airway smooth muscle contraction in cystic fibrosis

Background

Clara cells are the epithelial progenitor cell of the small airways, a location known to be important in many lung disorders. Although migration of alveolar type II and bronchiolar ciliated epithelial cells has been examined, the migratory response of Clara cells has received little attention.

Methods

Using a modification of existing procedures for Clara cell isolation, we examined mouse Clara cells and a mouse Clara-like cell line (C22) for adhesion to and migration toward matrix substrate gradients, to establish the nature and integrin dependence of migration in Clara cells.

Results

We observed that Clara cells adhere preferentially to fibronectin (Fn) and type I collagen (Col I) similar to previous reports. Migration of Clara cells can be directed by a fixed gradient of matrix substrates (haptotaxis). Migration of the C22 cell line was similar to the Clara cells so integrin dependence of migration was evaluated with this cell line. As determined by competition with an RGD containing-peptide, migration of C22 cells toward Fn and laminin (Lm) 511 (formerly laminin 10) was significantly RGD integrin dependent, but migration toward Col I was RGD integrin independent, suggesting that Clara cells utilize different receptors for these different matrices.

Conclusion

Thus, Clara cells resemble alveolar type II and bronchiolar ciliated epithelial cells by showing integrin mediated pro-migratory changes to extracellular matrix components that are present in tissues after injury.     

Thrombospondin stimulates motility of human neutrophils

Polymorphonuclear leukocytes (PMNs) migrate to sites of inflammation or injury in response to chemoattractants released at those sites. The presence of extracellular matrix (ECM) proteins at these sites may influence PMN accumulation at blood vessel walls and enhance their ability to move through tissue. Thrombospondin (TSP), a 450-kD ECM protein whose major proteolytic fragments are a COOH-terminal 140-kD fragment and an NH2-terminal heparin-binding domain (HBD), is secreted by platelets, endothelial cells, and smooth muscle cells. TSP binds specifically to PMN surface receptors and has been shown, in other cell types, to promote directed movement. TSP in solution at low concentrations (30-50 nM) "primed" PMNs for f-Met-Leu-Phe (fMLP)- mediated chemotaxis, increasing the response two- to fourfold. A monoclonal antibody against the HBD of TSP totally abolished this priming effect suggesting that the priming activity resides in the HBD of TSP. Purified HBD retains the priming activity of TSP thereby corroborating the antibody data. TSP alone, in solution at high concentrations (0.5-3.0 microM), stimulated chemotaxis of PMNs and required both the HBD and the 140-kD fragment of TSP. In contrast to TSP in solution, TSP bound to nitrocellulose filters in the range of 20- 70 pmol stimulated random locomotion of PMNs. The number of PMNs migrating in response to bound TSP was approximately two orders of magnitude greater than the number of cells that exhibited chemotaxis in response to soluble TSP or fMLP. Monoclonal antibody C6.7, which recognizes an epitope near the carboxyl terminus of TSP, blocked migration stimulated by bound TSP, suggesting that the activity resides in this domain. Using proteolytic fragments, we demonstrated that bound 140-kD fragment, but not HBD, promoted migration of PMNs. Therefore, TSP released at injury sites, alone or in synergy with chemotactic peptides like fMLP, could play a role in directing PMN movement.     

A steering model of endothelial sheet migration recapitulates monolayer integrity and directed collective migration

Cells in endothelial cell monolayers maintain a tight barrier between blood and tissue, but it is not well understood how endothelial cells move within monolayers, pass each other, migrate when stimulated with growth factor, and also retain monolayer integrity. Here, we develop a quantitative steering model based on functional classes of genes identified previously in a small interfering RNA (siRNA) screen to explain how cells locally coordinate their movement to maintain monolayer integrity and collectively migrate in response to growth factor. In the model, cells autonomously migrate within the monolayer and turn in response to mechanical cues resulting from adhesive, drag, repulsive, and directed steering interactions with neighboring cells. We show that lateral-drag steering explains the local coordination of cell movement and the maintenance of monolayer integrity by allowing closure of small lesions. We further demonstrate that directional steering of cells at monolayer boundaries, combined with adhesive steering of cells behind, can explain growth factor-triggered collective migration into open space. Together, this model provides a mechanistic explanation for the observed genetic modularity and a conceptual framework for how cells can dynamically maintain sheet integrity and undergo collective directed migration.