Inability of newt epidermal cells to migrate over concanavalin A-coated substrates

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 migrating epidermal cells as they attempted to form a wound epithelium. Despite the fact that concanavalin A (Con A) receptors could be demonstrated on newt epidermal cells with fluorescein isothiocyanate (FITC)-conjugated lectin, Con A-coated implants supported practically no migration, an even poorer response than the modest amount of migration that occurred on uncoated glass. Coomassie blue staining verified that the lectin formed a complete film over the glass, and peroxidase binding assays showed that even after several hours in the wound, the Con A binding sites for mannose were still available. Migration on fibrinogen-coated glass (a good migration substrate) was not affected by placing the implants next to Con A-coated implants. Thus, the failure to migrate on Con A cannot be explained by soluble Con A effects from lectin leaching off the implants. These data suggest that linkages between cell surface mannose and the substrate are not part of the strategy by which newt epidermal cells migrate.     

Emigration of bilayered epidermal cell sheets from tadpole tails (Xenopus laevis)

Summary Migration of bilayered epidermal cell sheets out of explants of tadpole tails (Xenopus laevis) were investigated with time-lapse cinemicrography using reflection-contrast optics. Cell-sheet formation begins beneath the explant in a region where it is closely attached to the coverslip. A single basal cell extends a lamellipodium through the outer (surface) epidermal layer and starts moving in a direction free of attached cells. This cell remains connected to the following basal cell, which the also extends a lamellipodium onto the glass. The cell sheet develops as increasingly more adjacent basal cells start to migrate. Surface cells do not actively locomote but they remain attached to the basal cells and to adjacent surface cells. Thus, they are transported as an intact cell layer, and consequently the in situ arrangement of the tadpole epidermis is largely preserved in the cell sheet, i.e., basal cells adhere to the substratum and are covered by outer cells (surface cells) which face the culture medium. Basal cells extend lamellae beneath the rear end of the preceding cell, which is slightly fifted off the substratum. The direction of locomotion is determined by the frontal cells. Cell-sheet enlargement and locomotion cease when all the epidermal cells facing the coverslip have left the explant, and the cell sheet and epidermis covering the explant form a continuous layer.     

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.     

Epidermal cell migration during wound healing in Dugesia lugubris

The epidermal cells that migrate over the surface during the wound closure stage of head regeneration in Dugesia lugubris s.l. were examined by scanning electron microscopy. The effect of cytochalasin B on epidermal cell migration was also examined. During the first few hours after decapitation epidermal cells at the edges of the wound showed significant changes of shape related to the process of migration that was accomplished approximately 10 h after wounding. Flattening of the marginal cells was associated with active epidermal spreading throughout the healing period. Suitable support for migrating cells appeared to be a rhabditic network attached to the wound tissue. Epidermal cell migration was inhibited by cytochalasin B. These results demonstrate that the basis for cell movement in planarians is similar to that of many other systems.     

Lectin binding to cell surfaces: comparisons between normal and migrating corneal epithelium

Comparisons were made between cell surfaces of normal and migrating corneal epithelium of the rat by localizing and/or quantifying concanavalin A (Con A) and wheat germ agglutinin (WGA) binding. Our results indicate that apical cell surfaces of the leading edge of a migrating sheet of epithelium differ from those of normal epithelium and that the various cell layers within the stratified normal epithelium have different lectin-binding characteristics. Three methods of monitoring lectin binding to cell surfaces were employed. Based on ferritin-conjugated Con A, ferritin-conjugated WGA, and [3H]Con A binding, apical cell membranes of migrating epithelia bind more Con A and WGA than do apical membranes of superficial cells of normal stratified epithelia. With both fluorescein isothiocyanate (FITC)-Con A and -WGA, membranes of all the cells of the leading edge of the migrating sheet fluoresce intensely. FITC-Con A binding of normal stratified epithelium is relatively uniform through all cell layers with no discernible staining of the apical membrane of superficial cells. With FITC-WGA, however, fluorescence is present only on basal cell layers but not on superficial cells. These data demonstrate that apical cell surface sugars on a sheet of epithelium migrating to cover a wound differ from the apical cell surface sugars of normal epithelium. As indicated by FITC-WGA binding, cells of the migrating sheet have cell surface characteristics similar to basal cells of normal epithelia. Perhaps, upon wounding, the leading edge of the migrating sheet is derived from the basal cell population of the normal stratified epithelium, or perhaps there is an alteration in cell surface glycoproteins as the cells become migratory.     

Cell polarity during wound healing in an insect epidermis

The insect integument displays uniform posterior orientation of cuticular denticles or bristles formed by the epidermal cells. We want to understand how cell polarities become uniformly oriented in the plane of the epidermal sheet. Here we test whether directed cell migration disturbs the orientation of denticles. Burning a circular area of epidermal cells beneath the cuticle causes cells to migrate into the resulting wound and the cuticle pattern observed after the subsequent moult depends on the time interval between burning and ecdysis. After a short wound-healing period cuticular protrusions tend to point away from the wound. With increasing would healing periods they tend to point more and more towards the wound centre. These results suggest that the migrating cells tend to orient cuticular protrusions in the direction of cell movement while continued cell movement will bend nascent cuticular protrusions outwards. Cell shape may also determine denticle orientation. I propose that the asymmetric localization of cell components known to determine the orientation of cell migration may also determine denticle orientation in insect epidermal cells.     

Collective cell migration of primary zebrafish keratocytes

Fish keratocytes are an established model in single cell motility but little is known about their collective migration. Initially, sheets migrate from the scale at ~145 μm/h but over the course of 24 h the rate of leading edge advance decreases to ~23 μm/h. During this period, leader cells retain their ability to migrate rapidly when released from the sheet and follower cell area increases. After the addition of RGD peptide, leader cell lamellae are lost, altering migratory forces within the sheet, resulting in rapid retraction. Leader and follower cell states interconvert within minutes with changes in cell–cell adhesions. Leader cells migrate as single cells when they detach from the leading edge and single cells appear to become leader cells if they rejoin the sheet. Follower cells rapidly establish leader cell morphology during closing of holes formed during sheet expansion and revert to follower cell morphology after hole-closure. Inhibition of Rho associated kinase releases leader cells and halts advancement of the leading edge suggesting an important role for the intercellular actomyosin cable at the leading edge. In addition, the presence of the stationary scale orients direction of sheet migration which is characterized by a more uniform advance of the leading edge than in some cell line systems. These data establish fish keratocyte explant cultures as a collective cell migration system and suggest that cell–cell interactions determine the role of keratocytes within the migrating sheet.     

Induced expression of syndecan in healing wounds

We have studied the expression of an integral cell surface proteoglycan, syndecan, during the healing of cutaneous wounds, using immunohistochemical and in situ hybridization methods. In normal mouse skin, both syndecan antigen and mRNA were found to be expressed exclusively by epidermal and hair follicle cells. After incision and subsequent suturing, remarkably increased amounts of syndecan on the cell surfaces of migrating and proliferating epidermal cells and on hair follicle cells adjacent to wound margins were noted. This increased syndecan expression was shown to be a consequence of greater amounts of syndecan mRNA. Induction was observed already 1 d after wounding, was most significant at the time of intense cell proliferation, and was still observable 14 d after incision. The migrating cells of the leading edge of the epithelium also showed enhanced syndecan expression, although clearly less than that seen in the proliferating epithelium. The merging epithelial cells at the site of incision showed little or no syndecan expression; increased syndecan expression, however, was detected during later epithelial stratification. When wounds were left unsutured, in situ hybridization experiments also revealed scattered syndecan-positive signals in the granulation tissue near the migrating epidermal sheet. By immunohistochemical analysis, positive staining in granulation tissue was observed around vascular endothelial cells in a subpopulation of growing capillaries. Induction of syndecan in granulation tissue both at the protein and mRNA levels was temporally and spatially highly restricted. Granulation tissue, which formed in viscose cellulose sponge cylinders placed under the skin of rats, was also found to produce 3.4 and 2.6 kb mRNA species of syndecan similar to that observed in the normal murine mammary epithelial cell line, NMuMG. These results suggest that syndecan may have a unique and important role as a cell adhesion and a growth factor-binding molecule not only during embryogenesis but also during tissue regeneration in mature tissues.     

Silk Film Topography Directs Collective Epithelial Cell Migration

The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization.     

Polarization of ADAM17‐driven EGFR signalling in electric field‐guided collective migration of epidermal sheets

Endogenous electric field is considered to play an important role in promoting collective migration of epidermis to the wound centre. However, most studies are focused on the effect of bioelectric field on the movement and migration of single epithelial cell; the molecular mechanisms about collective migration of epidermal monolayers remain unclear. Here, we found that EFs dramatically promoted the collective migration of HaCaT cells towards the anode, activated the sheddase activity of ADAM17 and increased the phosphorylation level of EGFR. Moreover, EGFR phosphorylation and HB‐EGF shedding level were significantly decreased by the ADAM17 inhibitor TAPI‐2 or siADAM17 under EFs, which subsequently attenuated the directed migration of HaCaT sheets. Notably, the inhibition of EF‐regulated collective migration by siADAM17 was rescued by addition of recombinant HB‐EGF. Furthermore, we observed that F‐actin was dynamically polarized along the leading edge of the migrated sheets under EFs and that this polarization was regulated by ADAM17/HB‐EGF/EGFR signalling. In conclusion, our study indicated that ADAM17 contributed to the collective directional movement of the epidermal monolayer by driving HB‐EGF release and activating EGFR under EFs, and this pathway also mediated the polarization of F‐actin in migrating sheets, which is essential in directional migration.     

Myosin II activity is required for functional leading-edge cells and closure of epidermal sheets in fish skin ex vivo

Re-epithelialization in skin wound healing is a process in which epidermal sheets grow and close the wound. Although the actin–myosin system is thought to have a pivotal role in re-epithelialization, its role is not clear. In fish skin, re-epithelialization occurs around 500 μm/h and is 50 times faster than in mammalian skin. We had previously reported that leading-edge cells of the epidermal outgrowth have both polarized large lamellipodia and “purse string”-like actin filament cables in the scale-skin culture system of medaka fish, Oryzias latipes (Cell Tissue Res, 2007). The actin purse-string (APS) is a supracellular contractile machinery in which adherens junctions (AJs) link intracellular myosin II-including actin cables between neighboring cells. In this study, we developed a modified “face-to-face” scale-skin culture system as an ex vivo model to study epidermal wound healing, and examined the role of the actin–myosin system in the rapid re-epithelialization using a myosin II ATPase inhibitor, blebbistatin. A low level of blebbistatin suppressed the formation of APS and induced the dissociation of keratocytes from the leading edge without attenuating the growth of the epidermal sheet or the migration rate of solitary keratocytes. AJs in the superficial layer showed no obvious changes elicited by blebbistatin. However, two epidermal sheets without APSs did not make a closure with each other, which was confirmed by inhibiting the connecting AJs between the superficial layers. These results suggest that myosin II activity is required for functional leading-edge cells and for epidermal closure.     

ERK activation propagates in epithelial cell sheets and regulates their migration during wound healing

In epithelial cell movements, which occur during wound healing or embryonic morphogenesis, sheets of cells move together as a unit. Molecular mechanisms that regulate this sheet movement have been largely unknown, although cell locomotion or movement mechanisms for individual cells, such as for fibroblastic cells, have been extensively studied. Here, we show that, during wound healing, sheets of MDCK epithelial cells migrate coordinately as a unit, and wound-induced activation of ERK MAP kinase (ERK1/2) propagates in cell sheets in accordance with the cell sheet movement. Inhibition of ERK1/2 activation by specific MEK inhibitors or by expressing dominant-negative ERK2 results in marked inhibition of the sheet movement during wound healing, and inhibition of the cell sheet movement by disrupting actin cytoskeleton suppresses propagation of ERK1/2 activation. These results indicate that cell movement and ERK1/2 activation form a positive feedback loop, which facilitates cell sheet migration. Moreover, we find that Src family kinase inhibitors suppress both cell migration and propagation of ERK1/2 activation, suggesting that Src family kinase may participate in this feedback loop. Interestingly, neither cell sheet migration as a unit nor migration-dependent propagation of ERK1/2 activation occurs during wound healing in fibroblastic 3Y1 cells. Thus, our results identify specific requirements of ERK1/2 MAP kinase for epithelial cell sheet movement.     

Cell movement is guided by the rigidity of the substrate

Directional cell locomotion is critical in many physiological processes, including morphogenesis, the immune response, and wound healing. It is well known that in these processes cell movements can be guided by gradients of various chemical signals. In this study, we demonstrate that cell movement can also be guided by purely physical interactions at the cell-substrate interface. We cultured National Institutes of Health 3T3 fibroblasts on flexible polyacrylamide sheets coated with type I collagen. A transition in rigidity was introduced in the central region of the sheet by a discontinuity in the concentration of the bis-acrylamide cross-linker. Cells approaching the transition region from the soft side could easily migrate across the boundary, with a concurrent increase in spreading area and traction forces. In contrast, cells migrating from the stiff side turned around or retracted as they reached the boundary. We call this apparent preference for a stiff substrate "durotaxis." In addition to substrate rigidity, we discovered that cell movement could also be guided by manipulating the flexible substrate to produce mechanical strains in the front or rear of a polarized cell. We conclude that changes in tissue rigidity and strain could play an important controlling role in a number of normal and pathological processes involving cell locomotion.     

The subsets of keratinocytes responsible for covering open wounds in neonatal rat skin

Full-thickness excisional wounds were made on the dorsal skin of 1-day-old rats to elucidate from where the cells move into the defect and what kinds of cells they are. Immunohistochemical analyses of the wound sites revealed that the following two subsets of keratinocytes were the major contributors to reepithelialization: first, the cells at the forefront of the migrating epithelium, termed leading edge cells, which expressed K14 keratin, known as basal cell-specific keratin, but not K6 or K10 keratins, so that they had probably moved from the basal cell layer; and, second, the cells tentatively termed immature spinous cells, which expressed K14 and K6 but not K10, and formed an ingrowth region following the leading edge cells. These two kinds of cells moved to the open wound area, as a multilayered cell sheet. Fluorescent phalloidin staining experiments indicated that actin filaments became concentrated in the leading edge cells within 6 h postwounding (PW), whereas weak signals of actin filaments were detected in the immature spinous cells. Taken together, the present findings support the view that wound covering in neonatal rat skin is accomplished by a movement en masse of keratinocytes from the bottom half of the surrounding epidermis.     

Nonmuscle myosin II localization is regulated by JNK during Drosophila larval wound healing

We investigated cell shape changes during wound closure in the Drosophila larval epidermis. During reepithelialization, epidermal cells permanently change shape from pentagonal or hexagonal to irregular forms. This process requires zipper, a gene encoding the Drosophila nonmuscle myosin II heavy chain. Following wounding, myosin II is localized at the wound margin and at the rear end of individual cells located within several rows from the wound hole. The c-Jun N-terminal kinase (JNK) pathway is essential for this myosin II localization. These results suggest that not only the wound leading edge but also the cells lying distal to the leading edge cells actively participate in epithelial cell sheet migration during wound hole closure.     

Signaling pathways directing the movement and fusion of epithelial sheets: lessons from dorsal closure in Drosophila

Wound healing in embryos and various developmental events in metazoans require the spreading and fusion of epithelial sheets. The complex signaling pathways regulating these processes are being pieced together through genetic, cell biological, and biochemical approaches. At present, dorsal closure of the Drosophila embryo is the best-characterized example of epithelial sheet movement. Dorsal closure involves migration of the lateral epidermal flanks to close a hole in the dorsal epidermis occupied by an epithelium called the amnioserosa. Detailed genetic studies have revealed a network of interacting signaling molecules regulating this process. At the center of this network is a Jun N-terminal kinase cascade acting at the leading edge of the migrating epidermis that triggers signaling by the TGF-beta superfamily member Decapentaplegic and which interacts with the Wingless pathway. These signaling modules regulate the cytoskeletal reorganization and cell shape change necessary to drive dorsal closure. Activation of this network requires signals from the amnioserosa and input from a variety of proteins at cell-cell junctions. The Rho family of small GTPases is also instrumental, both in activation of signaling and regulation of the cytoskeleton. Many of the proteins regulating dorsal closure have been implicated in epithelial movement in other organisms, and dorsal closure has emerged as an ideal model system for the study of the migration and fusion of epithelial sheets.     

Enhancement of vinculin synthesis by migrating stratified squamous epithelium

A 110-115-kD protein is present at levels 27-fold higher in migratory epithelium in the rat cornea than in stationary epithelium. This protein represents 2.7% of the total protein in migratory epithelium 6-h postabrasion wound and 0.1% of the total protein in stationary epithelium. Our findings demonstrate that this 110-115-kD protein is vinculin. In Western blots comparing proteins from migratory and control epithelium, antibody against vinculin cross-reacted with the 110-115-kD protein. Using immunoslot blots, vinculin was determined to be present at maximal levels 6 h postabrasion wound, at levels 22- and 8-fold higher than control at 18 and 48 h, respectively, returning to control levels 72 h postwounding. Vinculin was also localized by indirect immunohistochemistry in migrating corneal epithelium. 3-mm scrape wounds were allowed to heal in vivo for 20 h. In flat mounts of these whole wounded corneas, vinculin was localized as punctate spots in the leading edge of migrating epithelium. In cryostat sections, vinculin was localized as punctate spots along the basal cell membranes of the migrating sheet adjacent to the basement membrane and in patches between cells as well as diffusely throughout the cell. Only very diffuse localization with occasional punctate spots between adjacent superficial cells was present in stationary epithelium. The increased synthesis of vinculin during migration and the localization of vinculin at the leading edge of migratory epithelium suggest that vinculin may be involved in cell-cell and cell-substrate adhesion as the sheet of epithelium migrates to cover a wound.     

Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

Background

The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen.

Results

Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion.

Conclusions

The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0039-2) contains supplementary material, which is available to authorized users.     

Regulation of fibronectin expression and splicing in migrating epithelial cells: migrating MDCK cells produce a lesser amount of, but more active, fibronectin

Previously we have demonstrated that in MDCK epithelial cells not only transforming growth factor-beta (TGF-beta) but also hepatocyte growth factor/scatter factor (HGF/SF) regulates fibronectin (FN) splicing by increasing the ratio of EDA-containing FN (EDA+ FN) mRNA to EDA-minus FN (EDA- FN) mRNA (EDA+/EDA- ratio). EDA+ FN is known to be upregulated in tissues where cells actively migrate, such as those during morphogenesis, wound healing, and tumorigenesis. However, a direct association between cell migration and FN splicing at the EDA region has never been investigated. In this work, we have shown by using an in vitro wound migration assay that migrating epithelial cells regulate FN production and splicing differently compared to nonmigrating cells. Wounds were introduced as migration stimuli into the 10-day-old confluent cell sheet, where the EDA+/EDA- ratio and FN mRNA expression levels were stable. In migrating cells at the wound edge, the FN mRNA level decreased by 0.73-fold and the EDA+/EDA- ratio increased by 1.32-fold when compared with nonmigrating cells apart from the wound edge. HGF/SF significantly stimulated cell migration at the wound edge and concomitantly decreased the FN mRNA level by 0.60-fold and increased the EDA+/EDA- ratio by 1.84-fold in migrating cells. In nonmigrating cells apart from the wound edge, FN mRNA expression and splicing were not influenced by either wound stimulation or HGF/SF. EDA+ FN stimulates cell migration more effectively than EDA- FN and thus is considered to be a more active variant of FN. Taken together, migrating MDCK cells appear to regulate FN mRNA expression and splicing to produce a lesser amount of, but more active, FN.     

A new dimension in suspension fusion techniques with polyethylene glycol.

Polyethylene glycol (PEG) induces the hybridization of mammalian cells at a much higher frequency when the cells are attached to a substrate during treatment than when the cells are treated in suspension. Since many cell types, e.g., lymphocytes, cannot attach to a substrate, a new technique for the PEG-induced fusion of cells in suspension was developed. This technique, referred to as "pancake fusion," is based on the centrifugation of suspended cells onto a coverslip and the PEG treatment of the cells on the coverslip as if they were attached to a substrate. With this technique, the frequency of hybridization of human white blood cells, which are incapable of attaching to a substrate, can be greatly increased.