Actin cytoskeleton of extraembryonic endoderm and teratocarcinoma-derived endoderm cells

Distinct F-actin- and myosin-containing stress fibers were observed in situ in many endoderm cells of parietal yolk sacs from 11-day mouse embryos. In visceral endoderm (VE) such fibers were not seen, and F-actin was concentrated in the cell periphery. Correspondingly, in electron microscopy ventral cell membrane-associated bundles of microfilaments were revealed in the periphery of parietal endoderm (PE) cells but not in VE cells. Both PE and VE cells formed stress fibers in primary cultures. Undifferentiated F9 embryonal carcinoma cells formed only short actin spikes and fibrils irrespective of growth substratum. In PE-like derivatives of F9 cells, on the other hand, distribution of F-actin was markedly affected by the growth substratum: They formed distinct stress fibers when plated on fibronectin but did not when plated on gelatin. Similarly, in teratocarcinoma-derived PE cells (PYS-2) adhesion to fibronectin induced the formation of distinct bundles of F-actin and plaques of vinculin. The results suggest that the susceptibility of teratocarcinoma cell actin cytoskeleton to the influence of molecular composition of surrounding matrix is developmentally regulated. On the other hand, the reason for the presence of stress fibers in PE cells and for their absence in VE cells is unclear.     

Modulation of survival and proliferation of BSC-1 cells through changes in spreading behavior caused by the tumor-promoting phorbol ester TPA

The effect of a tumor-promoting phorbol ester on spreading behavior was investigated to clarify the involvement of the interactions between cells and substratum in the maintenance of cell viability and the control of cell proliferation. BSC-1 cells did not spread and lost cell viability after a 24-h incubation in the absence of calf serum. Addition of calf serum initially induced radial spreading and then polarized spreading, with the formation on stress fibers and focal contact-like structure, and enhanced survival. Vitronectin also induced both radial spreading and polarized spreading, and enhanced cell survival. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced radial spreading with actin ribbons in the absence of serum. It improved the survival of cells attached to the substratum, but not in suspension. TPA suppressed polarized spreading, formation of stress fibers and of focal contact-like structure, and cell proliferation, in the presence of serum. Phorbol did not have any effect. These results suggest that enhancement of radial spreading and inhibition of polarized spreading of BSC-1 cells by TPA are closely related to the enhancement of cell survival and inhibition of cell growth.     

Vimentin in human erythroleukemia (HEL) cells is modulated with differentiation inducers

The expression of vimentin intermediate filaments (IFs) was studied in a human erythroleukemia cell line (HEL), exposed to a variety of differentiation-inducing agents. These cells grow normally in suspension and show a heterogenous expression of vimentin immunoreactivity. In the presence of retinoic acid the fibrillar vimentin immunoreactivity diminished rapidly, while it was increased when the cells were exposed to hemin or butyric acid. In the presence of a tumor promoter (TPA), the HEL cells maintained their heterogenous vimentin immunoreactivity, but some cells showed large bundles of cytoplasmic vimentin fibrils. Upon exposure to TPA the cells spread on a growth substratum covered with human plasma fibronectin (Fn). Many of the spread cells totally lacked vimentin IFs. The present results show that vimentin expression in HEL cells is rapidly and differentially modulated upon exposure to the different inducing agents.     

Close and focal contact adhesions of fibroblasts to a fibronectin-containing matrix

The fibroblast cell line Balb/c 3T3 makes both close and tight-focal adhesive contacts with the plasma fibronectin (pFn)-coated tissue culture substratum. Detachment of these cells mediated by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid leaves tight-focal contacts and a subset of close contacts as substratum-attached material (SAM). The enrichment of heparan sulfate (HS) proteoglycan (HS-PG) in SAM under specific attachment conditions, as well as the recent demonstration of HS binding to pFn or cellular Fn, has evolved a series of experiments with the selective HS-binding protein platelet factor-4 to analyze the requirement of the HS-binding activity of pFn in the formation of these two types of adhesive contacts. In addition, the cell-binding domain (CBD) of pFn, which recognizes an unidentified cell surface receptor, has been isolated free of HS-binding domains after proteolytic cleavage of pFn. These functional studies indicate that the binding of pFn on the substratum to cell surface HS-PG is necessary and sufficient to generate close contacts with transmembrane signaling of this proteoglycan to reorganize lengthy microfilament bundles in the cytoplasm. Cells spread incompletely on CBD alone, form only close contacts, and reorganize highly concentrated actin filaments only in their spiky projections. Furthermore, the formation of tight-focal contacts with associated stress fibers appears to require both reactions of pFn, binding to cell surface HS-PG and to its unidentified receptor. The importance of HS-PG in these functional studies has led to its better biochemical characterization in SAM. Initially formed close contacts of these cells contain principally a large HS-PG that aggregates into high-molecular-weight complexes by some unknown mechanism. With time, there are two different mechanisms of catabolism of HS-PG, one of which includes the liberation of single-chain HS. The importance of these changes in the HS-PG in SAM are now being analyzed with regard to the formation and disappearance of close and tight-focal adhesions.     

Early molecular events in the assembly of the focal adhesion-stress fiber complex during fibroblast spreading

Cell adhesion to the extracellular matrix triggers the formation of integrin-mediated contact and reorganization of the actin cytoskeleton. Examination of nascent adhesions, formed during early stages of fibroblast spreading, reveals a variety of forms of actin-associated matrix adhesions. These include: (1). small ( approximately 1 microm), dot-like, integrin-, vinculin-, paxillin-, and phosphotyrosine-rich structures, with an F-actin core, broadly distributed over the ventral surfaces of the cells; (2). integrin-, vinculin-, and paxillin-containing "doublets" interconnected by short actin bundles; (3). arrays of actin-vinculin complexes. Such structures were formed by freshly plated cells, as well as by cells recovering from latrunculin treatment. Time-lapse video microscopy of such cells, expressing GFP-actin, indicated that long actin cables are formed by an end-to-end lining-up and apparent fusion of short actin bundles. All these structures were prominent during cell spreading, and persisted for up to 30-60 min after plating. Upon longer incubation, they were gradually replaced by stress fibers, associated with focal adhesions at the cell periphery. Direct examination of paxillin and actin reorganization in live cells revealed alignment of paxillin doublets, forming long and highly dynamic actin bundles, undergoing translocation, shortening, splitting, and convergence. The mechanisms underlying the assembly and reorganization of actin-associated focal adhesions and the involvement of mechanical forces in regulating their dynamic properties are discussed.     

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.     

The removal of extracellular fibronectin from areas of cell-substrate contact

Immunofluorescent labeling for fibronectin was largely excluded from sites of closest contact between spreading chicken gizzard fibroblasts and the substratum. This was observed by double immunofluorescent labeling of fixed cells for fibronectin and vinculin, a smooth muscle intracellular protein that is specifically associated with focal adhesion plaques, in conjunction with interference-reflection microscopy. When the cells were plated on a fibronectin-coated substratum they adhered to its surface and rapidly spread on it. The immunofluorescent labeling for fibronectin in those cultures (after fixation and triton permeabilization) was usually absent from the newly formed, vinculin-containing focal adhesion plaques. We have found, however, that the accessibility to the cell-substrate gap at the focal adhesion plaques is limited and therefore a more direct approach was adopted. We have found that cells spreading on a substrate coated with rhodamine-labeled fibronectin progressively removed the underlying protein from the substrate. The removal of fibronectin involved at least two distinct mechanisms. Part of the substrate-associated fibronectin was removed from small areas and displaced toward the cell center. The arrowhead-shaped areas from which fibronectin was removed often coincided with vinculin-rich focal contacts. We observed, however, many areas where focal contacts were found over unperturbed fibronectin carpet, as well as fibronectin-free areas with no overlapping focal contacts. The possibilities that fibronectin is actively displaced from areas of cell-substrate contact, that the focal adhesion plaques are transiently associated with these areas and their implications on the dynamics of cell spreading and locomotion are discussed. The second route of fibronectin removal from the substrate was endocytosis. The rhodamine-labeled fibronectin was found in the cells in a partial or transient association with clathrin-containing structures.     

Arachidonate initiated protein kinase C activation regulates HeLa cell spreading on a gelatin substrate by inducing F-actin formation and exocytotic upregulation of β1 Integrin

HeLa cell spreading on a gelatin substrate requires the activation of protein kinase C (PKC), which occurs as a result of cell-attachment-induced activation of phospholipase A2 (PLA2) to produce arachidonic acid (AA) and metabolism of AA by lipoxyginase (LOX). The present study examines how PKC activation affects the actin- and microtubule-based cytoskeletal machinery to facilitate HeLa cell spreading on gelatin. Cell spreading on gelatin is contingent on PKC induction of both actin polymerization and microtubule-facilitated exocytosis, which is based on the following observations. There is an increase in the relative content of filamentous (F)-actin during HeLa cell spreading, and treating HeLa cells with PKC-activating phorbol esters such as 12-O-tetradecanoyl phorbol 13-acetate (TPA) further increases the relative content of F-actin and the rate and extent to which the cells spread. Conversely, inhibition of PKC by calphostin C blocked both cell spreading and the increase of F-actin content. The increased F-actin content induced by PKC activators also was observed in suspension cells treated with TPA, and the kinetics of F-actin were similar to that for PKC activation. In addition, PKCϵ, which is the PKC isoform most involved in regulating HeLa cell spreading in response to AA production, is more rapidly translocated to the membrane in response to TPA treatment than is the increase in F-actin. Blocking the activities of either PLA2 or LOX inhibited F-actin formation and cell spreading, both of which were reversed by TPA treatment. This result is consistent with AA and a LOX metabolite of AA as being upstream second messengers of activation of PKC and its regulation of F-actin formation and cell spreading. PKC appears to activate actin polymerization in the entire body of the cell and not just in the region of cell-substrate adhesion because activated PKC was associated not only with the basolateral plasma membrane domain contacting the culture dish but also with the apical plama membrane domain exposed to the culture medium and with an intracellular membrane fraction. In addition to the facilitation of F-actin formation, activation of PKC induces the exocytotic upregulation of β1 integrins from an intracellular domain to the cell surface, possibly in a microtubule-dependent manner because the upregulation is inhibited by Nocodazole. The results support the concept that cell-attachment-induced AA production and its metabolism by LOX results in the activation of PKC, which has a dual role in regulating the cytoskeletal machinery during HeLa cell spreading. One is through the formation of F-actin that induces the structural reorganization of the cells from round to spread, and the other is the exocytotic upregulation of collagen receptors to the cell surface to enhance cell spreading. J. Cell. Physiol. 173:361–370, 1997. © 1997 Wiley-Liss, Inc.     

Latent TGF-beta binding protein LTBP-2 decreases fibroblast adhesion to fibronectin

We have analyzed the effects of latent TGF-beta binding protein 2 (LTBP-2) and its fragments on lung fibroblast adhesion. Quantitative cell adhesion assays indicated that fibroblasts do not adhere to full-length LTBP-2. Interestingly, LTBP-2 had dominant disrupting effects on the morphology of fibroblasts adhering to fibronectin (FN). Fibroblasts plated on LTBP-2 and FN substratum exhibited less adherent morphology and displayed clearly decreased actin stress fibers than cells plated on FN. These cells formed, instead, extensive membrane ruffles. LTBP-2 had no effects on cells adhering to collagen type I. Fibroblasts adhered weakly to the NH2-terminal fragment of LTBP-2. Unlike FN, this fragment did not augment actin stress fiber formation. Interestingly, the adhesion-mediating and cytoskeleton-disrupting effects were localized to the same NH2-terminal proline-rich region of LTBP-2. LTBP-2 and its antiadhesive fragment bound to FN in vitro, and the antiadhesive fragment associated with the extracellular matrix FN fibrils. These observations reveal a potentially important role for LTBP-2 as an antiadhesive matrix component.     

Cytoskeleton and adhesion patterns of cultured chick embryo chondrocytes during cell spreading and Rous sarcoma virus transformation

The cytoskeleton and the adhesion complex of chick embryo chondrocytes maintained in vitro have been studied by fluorescence and interference reflection microscopy during the process of cell spreading. The pattern of actin-containing microfilaments and the distribution of vinculin speckles on adhesion plaques have been found to change as a function of the culture time. Newly plated chondrocytes adhere to the substratum mostly around a peripheral ring-like region and show a complex tridimensional array of microfilaments. When chondrocytes flatten, they develop stress fibres and show a diffuse system of vinculin-containing adhesion plaques scattered over the entire ventral side of the cells. Upon infection with Rous sarcoma virus (RSV) chondrocytes display one or more actin-containing ruffles located on the dorsal side similar to the 'actin flowers' earlier described in other cell types. These structures have been found to accumulate vinculin too. In chondrocytes infected with two td-ts mutants of RSV, 'actin flowers' have been found to persist at the restrictive temperature. At this temperature, however, in the majority of cells, stress fibres and adhesion plaques reappear.     

Differential binding to dorsal and ventral cell surfaces of fibroblasts: effect on collagen phagocytosis

Matrix remodeling by phagocytic fibroblasts is essential for growth and development but the regulatory processes are undefined. We evaluated the impact of spreading on the binding step of collagen phagocytosis with a novel culture system that more closely replicates phagocytosis in vivo than previous models. 3T3 cells were plated on collagen-coated beads, thereby loading only ventral surfaces (adhesion with spreading), or were allowed to spread on collagen films and then loaded with beads on their dorsal surfaces (adhesion without spreading). Ventral surfaces bound three-fold more beads than dorsal surfaces which was accompanied by accelerated phagosomal maturation. Arp3 and cortactin, markers of the actin-associated spreading machinery, strongly accumulated around ventrally but not dorsally loaded beads, suggesting that spreading contributes to enhanced binding of ventral surfaces. Further, ventral surfaces exhibited two-fold more free alpha2beta1 integrins, the major collagen receptors. Notably, compared to cells spread on collagen substrates, spreading cells exhibited a three-fold higher alpha2beta1 mobile fraction which was correlated with limited engagement of ventral receptors by actin filaments. Thus integrin ligation by actin filaments regulates the mobility of collagen receptors which in turn mediates the enhanced binding of collagen beads on spreading surfaces.     

ADHESION-DEPENDENT F-ACTIN PATTERN INAMOEBA PROTEUSAS A COMMON FEATURE OF AMOEBAE AND THE METAZOAN MOTILE CELLS

Adhesion and movement ofAmoeba proteusare both dependent on the appropriate arrangement of the F-actin cytoskeleton and on the presence of the cell nucleus. In this study the F-actin organization was examined by routine FITC-phalloidin staining and confocal laser microscopy in intact amoebae and in their nucleated and anucleated fragments, at different levels of cell adherence to the substratum. In the adhering and migrating intact cells and nucleated cell fragments dot-like aggregates of F-actin are scattered over the ventral side at sites close to the substratum. In the case of de-adhesion of nucleated specimens this pattern disappears and F-actin is accumulated in the cell centre and/or dispersed in the cytoplasm. The same actin distribution, without ventral dots, is found in the anucleated fragments which usually fail to attach to the substratum. Re-adhesion of anucleated fragments, induced by a modified substratum or spontaneous, is accompanied by restoration of actin dots at the lower cell side. It is concluded that: (1) adhering specimens ofA. proteusdisplay the same dot-like actin pattern on the ventral cell side, as many metazoan motile cells; (2) organization or disorganization of this pattern may occur independently of the presence of the cell nucleus, under the control of cell adhesion to the substratum.     

Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading

To learn more about the possible role of the coated pits endocytic pathway in cell adhesion, we studied attachment and spreading of fibroblasts whose coated pits were disrupted by depletion of intercellular potassium. Fibroblasts incubated in suspension in potassium-free medium lost 80% of their intracellular potassium within 10 min and showed disrupted coated pits based on fluorescence staining of clathrin. Potassium-depleted cells attached and spread on fibronectin-coated substrata over the same time course (15 min-2 h) as control cells. Unlike controls, however, potassium-depleted fibroblasts attained a radial morphology with circumferentially organized actin filament bundles and were unable to make the transition to a polarized morphology with stress fibers. In the radially spread fibroblasts, fibronectin receptors and vinculin colocalized in focal adhesion sites and appeared to be membrane insertion points for circumferentially arranged actin filament bundles, but these sites were much smaller than the focal adhesion plaques in polarized cells. The effects of potassium depletion on cell adhesion were reversible. Within 1 h after switching K(+)-depleted fibroblasts to medium containing KCl, cells developed a polarized morphology with actin stress fibers inserting into focal adhesion plaques. Coated pits also reformed on the cell surface during this time. Because formation of focal adhesion plaques preceded reappearance of clathrin-coated pits at the cell margins, it seems unlikely that coated pits play a direct role in adhesion plaque assembly. Polarization of fibroblasts upon addition of KCl was inhibited by ouabain showing that intracellular potassium was required for activity. Polarization also was inhibited when potassium-depleted cells were switched to potassium-containing medium under hypertonic or acidified conditions, both of which have been shown to inhibit receptor- mediated endocytosis. Our results suggest that the coated pit endocytic pathway is not required for initial attachment, spreading, and formation of focal adhesions by fibroblasts, but may play a role in cell polarization.     

The Role of Carboxy-Terminal Glycosaminoglycan-binding Domain of Vitronectin in Cytoskeletal Organization and Migration of Endothelial Cells

Vitronectin is a major cell adhesion molecule present in the subendothelial matrix that mediates the attachment and spreading of a variety of cells. The carboxy-terminal end of vitronectin has a consensus sequence for glycosaminoglycan-binding. To define the functional role of this domain, we generated fragments of vitronectin that lack the glycosaminoglycan-binding domain by formic acid cleavage of plasma-derived vitronectin. In addition, we also generated similar recombinant fragments of vitronectin as glutathione S-transferase fusion proteins in E. coll. These fragments were tested for their ability to support the adhesion of human umbilical vein endothelial cells. These fragments promoted endothelial cell adhesion, reaching half maximal activity at 2-5 μg/well compared to plasma-derived vitronectin which reached at 0.2 μg/well. However, the cells that adhered to these fragments did not develop well-formed focal adhesion plaques and actin stress fibers. In addition, these fragments were poorly chemotactic for endothelial cell migration when compared to intact plasma-derived vitronectin in a modified Boyden chamber assay. The present studies show that carboxy-terminal glycosaminoglycan-binding domain of vitronectin is essential for proper cytoskeletal organization and migration of endothelial cells on vitronectin substratum.     

The Role of Carboxy-Terminal Glycosaminoglycan-binding Domain of Vitronectin in Cytoskeletal Organization and Migration of Endothelial Cells

Vitronectin is a major cell adhesion molecule present in the subendothelial matrix that mediates the attachment and spreading of a variety of cells. The carboxy-terminal end of vitronectin has a consensus sequence for glycosaminoglycan-binding. To define the functional role of this domain, we generated fragments of vitronectin that lack the glycosaminoglycan-binding domain by formic acid cleavage of plasma-derived vitronectin. In addition, we also generated similar recombinant fragments of vitronectin as glutathione S-transferase fusion proteins in E. coll. These fragments were tested for their ability to support the adhesion of human umbilical vein endothelial cells. These fragments promoted endothelial cell adhesion, reaching half maximal activity at 2-5 μg/well compared to plasma-derived vitronectin which reached at 0.2 μg/well. However, the cells that adhered to these fragments did not develop well-formed focal adhesion plaques and actin stress fibers. In addition, these fragments were poorly chemotactic for endothelial cell migration when compared to intact plasma-derived vitronectin in a modified Boyden chamber assay. The present studies show that carboxy-terminal glycosaminoglycan-binding domain of vitronectin is essential for proper cytoskeletal organization and migration of endothelial cells on vitronectin substratum.     

Adhesion-induced intracellular signalling in endothelial cells depends on the nature of the matrix

The adhesion of a human microvascular endothelial cell line to its own matrix was studied in comparison with adhesion of the same cells to fibronectin or thrombospondin-1. These endothelial cells adhered preferentially to their matrix whereas an equal cell number was attached to fibronectin or thrombospondin-1. The adhesion of cells to thrombospondin-1 was mediated by the N-terminal heparin binding domain of thrombospondin-1 as shown by the use of a recombinant fragment, N18. Cells adhering to their matrix displayed a morphology and a cytoskeleton organization very similar to that observed in vivo with an apical immunostaining for actin stress fibers and a fine basal labeling for vinculin. Cells on fibronectin were extensively spread and rapidly assembled stress fibers and focal contacts. Cells adherent to thrombospondin-1 presented large lamellae rich in actin but devoid of vinculin and only few actin fibers were observed. Depending on the substratum used, adhering endothelial cells displayed also different tyrosine phosphorylation patterns on electrophoresis. Our observations indicate that endothelial cells adhering to their matrix present an activation state intermediate between that induced by a "hyperadhesive" protein like fibronectin and that generated by a moderate, indeed anti-adhesive, protein like thrombospondin-1.     

Dorsoventral differences in cell-cell interactions modulate the motile behaviour of cells from the Xenopus gastrula.

When groups of cells from the inner marginal zone (mesendoderm) of the early Xenopus gastrula are placed on a fibronectin-coated substratum, the explants of the dorsal region spread into monolayers whereas those from the ventral region, though they adhere to the substratum, do not show this spreading reaction. This different behaviour is not reflected in the in vitro behaviour of the respective cells kept in isolation. No difference between dorsal and ventral cells was observed, when they were tested for lamellipodia-driven spreading, movement over the substratum or properties of integrin- and cadherin-mediated adhesion. However, cell contacts between individual dorsal cells are significantly less stable than those between ventral cells. The higher flexibility of the cell-cell contacts seems to determine the spreading behaviour of the dorsal explants, which includes lamellipodia-driven outward movement of the peripheral cells, rearrangements of the cells, building up a horizontal tension within the aggregate and intercalation of cells from above into the bottom layer. Ventral explants lack these properties. Staining for F-actin revealed a decisive difference of the supracellular organisation of the cytoskeleton that underlies the morphology of the different types of explants. Evidence for a higher flexibility of cell-cell contacts in the dorsal mesendoderm was also obtained in SEM studies on gastrulating embryos. Dorsal mesendodermal cells show stronger protrusive activity as compared to ventral mesendodermal cells. The meaning of these observations for the mechanisms of morphogenetic movements during gastrulation is central to the discussion.     

Activation of macrophage-like cells by multiple grooved substrata. Topographical control of cell behaviour.

We studied the influence of substrata topography on the behaviour of murine P388D1 macrophage cell line. Cells were plated on plain fused silica substrata or substrata with microfabricated grooves of varying depth and width. Cell spread area, elongation, orientation and F-actin content were measured on plain substratum and 6 sets of gratings. The speed and persistence of cell movement were also studied. We found that patterned substrata substantially activated cell spreading and elongation and significantly increased the persistence and speed of cell movement, shallow grooves being more effective than deep ones. The contact of cells with micropatterned substrata significantly increased the F-actin content in cells. The sensitivity of LPS (lipopolisaccharide) stimulated and unstimulated macrophages to topographical cues was also compared.