Signaling pathways and cell mechanics involved in wound closure by epithelial cell sheets

BACKGROUND: Sheets of cells move together as a unit during wound healing and embryonic tissue movements, such as those occurring during gastrulation and neurulation. We have used epithelial wound closure as a model system for such movements and examined the mechanisms of closure and the importance of the Rho family of Ras-related small GTPases in this process. RESULTS: Wounds induced in Madin-Darby canine kidney (MDCK) epithelial cell monolayers close by Rac- and phosphoinositide-dependent cell crawling, with formation of lamellipodia at the wound margin, and not by contraction of a perimarginal actomyosin purse-string. Although Rho-dependent actin bundles usually form at the margin, neither Rho activity nor formation of these structures is required for wound closure to occur at a normal rate. Cdc42 activity is also not required for closure. Inhibition of Rho or Cdc42 results, however, in statistically significant decreases in the regularity of wound closure, as determined by the ratio of wound margin perimeter over the remaining denuded area at different times. The Rac-dependent force generation for closure is distributed over several rows of cells from the wound margin, as inhibition of motility in the first row of cells alone does not inhibit closure and can be compensated for by generation of motile force in cells behind the margin. Furthermore, we observed high levels of Rac-dependent actin assembly in the first few rows of cells from the wound margin. CONCLUSIONS: Wounds in MDCK cell sheets do not close by purse-string contraction but by a crawling behavior involving Rac, phosphoinositides and active movement of multiple rows of cells. This finding suggests a new distributed mode of signaling and movement that, nevertheless, resembles individual cell motility. Although Rho and Cdc42 activities are not required for closure, they have a role in determining the regularity of closure.     

Emergence of HGF/SF-Induced Coordinated Cellular Motility

Collective cell migration plays a major role in embryonic morphogenesis, tissue remodeling, wound repair and cancer invasion. Despite many decades of extensive investigations, only few analytical tools have been developed to enhance the biological understanding of this important phenomenon. Here we present a novel quantitative approach to analyze long term kinetics of bright field time-lapse wound healing. Fully-automated spatiotemporal measures and visualization of cells' motility and implicit morphology were proven to be sound, repetitive and highly informative compared to single-cell tracking analysis. We study cellular collective migration induced by tyrosine kinase-growth factor signaling (Met-Hepatocyte Growth Factor/Scatter Factor (HGF/SF)). Our quantitative approach is applied to demonstrate that collective migration of the adenocarcinoma cell lines is characterized by simple morpho-kinetics. HGF/SF induces complex morpho-kinetic coordinated collective migration: cells at the front move faster and are more spread than those further away from the wound edge. As the wound heals, distant cells gradually accelerate and enhance spread and elongation -resembling the epithelial to mesenchymal transition (EMT), and then the cells become more spread and maintain higher velocity than cells located closer to the wound. Finally, upon wound closure, front cells halt, shrink and round up (resembling mesenchymal to epithelial transition (MET) phenotype) while distant cells undergo the same process gradually. Met inhibition experiments further validate that Met signaling dramatically alters the morpho-kinetic dynamics of the healing wound. Machine-learning classification was applied to demonstrate the generalization of our findings, revealing even subtle changes in motility patterns induced by Met-inhibition. It is concluded that activation of Met-signaling induces an elaborated model in which cells lead a coordinated increased motility along with gradual differentiation-based collective cell motility dynamics. Our quantitative phenotypes may guide future investigation on the molecular and cellular mechanisms of tyrosine kinase-induced coordinate cell motility and morphogenesis in metastasis.     

Nocodazole, vinblastine and taxol at low concentrations affect fibroblast locomotion and saltatory movements of organelles

Microtubules (MTs) are essential for the maintenance of asymmetric cell shape and motility of fibroblasts. MTs are considered to function as rails for organelle transport to the leading edge. We investigated the relationship between the motility of Vero fibroblasts and saltatory movements of particles in their lamella Fibroblasts extended their leading edges into the experimental wound at a rate of 20+/-11 microm/h. Intracellular particles in the front parts of the polarized fibroblasts moved saltatorily mainly along the long axis of the cells. MT depolymerization induced by the nocodazole at a high concentration (1.7 microM) resulted in the inhibition of both fibroblast motility and saltatory movements of the particles. Taxol (1 microM) inhibited the fibroblast locomotion but not the saltatory movements. The saltatory movement pattern was disorganized by taxol by decreasing the portion of longitudinal saltations and consequently by increasing the part of saltations perpendicular to the cell long axis. This effect may be explained by disorganization of the MT network resulting from the inhibition of dynamic instability. To further investigate the relationships between the MT dynamics instability, saltatory movements, and fibroblast locomotion, we treated fibroblasts with microtubule drugs at low concentration (nocodazole, 170 nM; vinblastine, 50 nM; and taxol, 50 nM). All these drugs induced rapid disorganization of the saltatory movements and decreased the rate of cell locomotion. Simultaneously, the amount of acetylated (stable) MTs increased. The treatment also induced reversible changes in the actin meshwork. We suggest that decrease in the fibroblast locomotion rate in the case of MT stabilization occurred because of the appearance of numerous free MTs. Saltations along free MTs are poorly organized and, as a result, the number of organelles reaching the fibroblast leading edge decreases.     

Simultaneous quantification of cell motility and protein-membrane-association using active contours

We present a new method for the quantification of dynamic changes in fluorescence intensities at the cell membrane of moving cells. It is based on an active contour method for cell-edge detection, which allows tracking of changes in cell shape and position. Fluorescence intensities at specific cortical subregions can be followed in space and time and correlated with cell motility. The translocation of two GFP tagged proteins (CRAC and GRP1) from the cytosol to the membrane in response to stimulation with the chemoattractant cAMP during chemotaxis of Dictyostelium cells and studies of the spatio-temporal dynamics of this process exemplify the method: We show that the translocation can be correlated with motility parameters and that quantitative differences in the rate of association and dissociation from the membrane can be observed for the two PH domain containing proteins. The analysis of periodic CRAC translocation to the leading edge of a cell responding to natural cAMP waves in a mound demonstrates the power of this approach. It is not only capable of tracking the outline of cells within aggregates in front of a noisy background, but furthermore allows the construction of spatio-temporal polar plots, capturing the dynamics of the protein distribution at the cell membrane within the cells' moving co-ordinate system. Compilation of data by means of normalised polar plots is suggested as a future tool, which promises the so-far impossible practicability of extensive statistical studies and automated comparison of complex spatio-temporal protein distribution patterns.     

Patterns of living beta-actin movement in wounded human coronary artery endothelial cells exposed to shear stress.

We previously demonstrated that physiologic levels of shear stress enhance endothelial repair. Cell spreading and migration, but not proliferation, were the major mechanisms accounting for the increases in wound closure rate (Albuquerque et al., 2000, Am. J. Physiol. Heart Circ. Physiol. 279, H293-H302). However, the patterns and movements of beta-actin filaments responsible for cell motility and translocation in human coronary artery endothelial cells (HCAECs) have not been previously investigated under physiologic flow. HCAECs transfected with beta-actin-GFP were cultured on type I collagen-coated coverslips. Confluent cell monolayers were subjected to laminar shear stress of 12 dynes/cm(2) for 18 h in a parallel-plate flow chamber to attain cellular alignment and then wounded by scraping with a metal spatula and subsequently exposed to a laminar shear stress of 20 dynes/cm(2) (S-W-sH) or static (S-W-sT) conditions. Time-lapse imaging and deconvolution microscopy was performed during the first 3 h after imposition of S-W-sH or S-W-sT conditions. The spatial and temporal dynamics of beta-actin-GFP motility and translocation during wound closure in HCAEC monolayers were analyzed under both conditions. Compared with HCAEC under S-W-sT conditions, our data show that HCAEC under S-W-sH conditions demonstrated greater beta-actin-GFP motility, filament and clumping patterns, and filament arcs used during cellular attachment and detachment. These findings demonstrate intriguing patterns of beta-actin organization and movement during wound closure in HCAEC exposed to physiological flow.     

Calcium signaling during convergent extension in Xenopus

BACKGROUND: During Xenopus gastrulation, cell intercalation drives convergent extension of dorsal tissues. This process requires the coordination of motility throughout a large population of cells. The signaling mechanisms that regulate these movements in space and time remain poorly understood. RESULTS: To investigate the potential contribution of calcium signaling to the control of morphogenetic movements, we visualized calcium dynamics during convergent extension using a calcium-sensitive fluorescent dye and a novel confocal microscopy system. We found that dramatic intercellular waves of calcium mobilization occurred in cells undergoing convergent extension in explants of gastrulating Xenopus embryos. These waves arose stochastically with respect to timing and position within the dorsal tissues. Waves propagated quickly and were often accompanied by a wave of contraction within the tissue. Calcium waves were not observed in explants of the ventral marginal zone or prospective epidermis. Pharmacological depletion of intracellular calcium stores abolished the calcium dynamics and also inhibited convergent extension without affecting cell fate. These data indicate that calcium signaling plays a direct role in the coordination of convergent extension cell movements. CONCLUSIONS: The data presented here indicate that intercellular calcium signaling plays an important role in vertebrate convergent extension. We suggest that calcium waves may represent a widely used mechanism by which large groups of cells can coordinate complex cell movements.     

Keratinocyte growth factor promotes cell motility during alveolar epithelial repair in vitro

Epithelia play a key role as protective barriers, and mechanisms of repair are crucial for restoring epithelial barrier integrity, especially in the lung. Cell spreading and migration are the first steps of reepithelialization. Keratinocyte growth factor (KGF) plays a key role in lung epithelial repair and protects against various injuries. We hypothesized that KGF may protect the lung not only by inducing proliferation but also by promoting epithelial repair via enhanced epithelial cell migration. In an in vitro wound-healing model, we found that KGF enhanced wound closure by 33%. KGF acted primarily by inducing lamellipodia emission (73.2 +/- 3.9% of KGF-treated cells had lamellipodia vs 61.3 +/- 3.4% of control cells) and increasing their relative surface area (59 +/- 2.7% with KGF vs 48 +/- 2.0% in controls). KGF reduced cytoskeleton stiffness as measured by magnetic twisting cytometry and increased cell motility (5.8 +/- 0.42 microm/h with KGF vs 3.7 +/- 0.41 microm/h in controls). KGF-increased cell motility was associated with increased fibronectin deposition during wound closure and with fibronectin reorganization into fibrils at the rear of the cells. Taken together, our findings strongly suggest that KGF may promote epithelial repair through several mechanisms involved in cell migration.     

Actin-dependent,retrograde motility of surface-attached beads and aggregating pigment granules in dissociated teleost retinal pigment epithelial cells

Teleost retinal pigment epithelial (RPE) cells contain pigment granules within apical projections which undergo actin-dependent, bi-directional motility. Dissociated RPE cells in culture attach to the substrate and extend apical projections in a radial array from the central cell body. Pigment granules within projections can be triggered to aggregate or disperse by the presence or absence of 1 mM cAMP. Aminated, fluorescent latex beads attached to the dorsal surface of apical projections and moved in the retrograde direction, towards the cell body. Bead rates on RPE cells with aggregating or fully aggregated pigment granules were 2.2 +/- 0.5 and 2.6 +/- 0.2 microm/min (mean +/- SEM), respectively, similar to rates of aggregating (retrograde) pigment granule movement (2.0 +/- 0.4 microm/min). Bead rates were slightly slower on cells with fully dispersed or dispersing pigment granules (1.5 +/- 0.1 and 1.5 +/- 0.4 microm/min). Movements of surface-attached beads and aggregating pigment granules were closely correlated in the distal portions of apical projections, but were more independent of each other in proximal regions of the projections. The actin disrupting drug, cytochalasin D (CD), reversibly halted retrograde bead movements, suggesting that motility of surface-attached particles is actin-dependent. In contrast, the microtubule depolymerizing drug, nocodazole, had no effect on retrograde bead motility. The similar characteristics and actin-dependence of retrograde bead movements and aggregating pigment granules suggest a correlation between these two processes.     

In vitro dynamics of chromatin organization and migration

The organization of eukaryotic chromatin is not static but changes as a function of cell status during processes such as proliferation, differentiation, and migration. DNA quantification has not been used extensively to investigate chromatin dynamics in combination with cellular migration. In this context, an optimized DNA-specific, nonperturbant method has been developed for studying chromatin organization, using the fluorescent vital bisbenzimidazole probe Hoechst 33342: this property has been described by Hamori et al. (1980). Computer-assisted image analysis was used to follow migratory activity and chromatin organization of L929 fibroblasts during in vitro wound healing. Cell movements were analyzed using an optical flow technique, which consists in the calculation of the velocity field of cells and nuclear movements in the frame. This system allows the correlation of cell migration and position in the cell cycle. It makes it possible to study chromatin dynamics using a quantitative analysis of nuclear differentiation reorganization (nuclear texture) and to correlate this with migration characteristics. The present system would be of interest for studying cell-extracellular matrix interactions using differing substrates, and also the migratory response to chemotactic factors. Such a model is a prerequisite for gaining better understanding of drug action.     

Physical Model of the Dynamic Instability in an Expanding Cell Culture

Collective cell migration is of great significance in many biological processes. The goal of this work is to give a physical model for the dynamics of cell migration during the wound healing response. Experiments demonstrate that an initially uniform cell-culture monolayer expands in a nonuniform manner, developing fingerlike shapes. These fingerlike shapes of the cell culture front are composed of columns of cells that move collectively. We propose a physical model to explain this phenomenon, based on the notion of dynamic instability. In this model, we treat the first layers of cells at the front of the moving cell culture as a continuous one-dimensional membrane (contour), with the usual elasticity of a membrane: curvature and surface-tension. This membrane is active, due to the forces of cellular motility of the cells, and we propose that this motility is related to the local curvature of the culture interface; larger convex curvature correlates with a stronger cellular motility force. This shape-force relation gives rise to a dynamic instability, which we then compare to the patterns observed in the wound healing experiments.     

Vascular sprout formation entails tissue deformations and VE-cadherin-dependent cell-autonomous motility

Embryonic and fetal vascular sprouts form within constantly expanding tissues. Nevertheless, most biological assays of vascular spouting are conducted in a static mechanical milieu. Here we study embryonic mouse allantoides, which normally give raise to an umbilical artery and vein. However, when placed in culture, allantoides assemble a primary vascular network. Unlike other in vitro assays, allantoic primordial vascular cells are situated on the upper surface of a cellular layer that is engaged in robust spreading motion. Time-lapse imaging allows quantification of primordial vascular cell motility as well as the underlying mesothelial tissue motion. Specifically, we calculate endothelial cell-autonomous motion by subtracting the tissue-level mesothelial motion from the total endothelial cell displacements. Formation of new vascular polygons is hindered by administration of function-blocking VE-cadherin antibodies. Time-lapse recordings reveal that (1) cells at the base of sprouts normally move distally “over” existing sprout cells to form new tip-cells; and (2) loss of VE-cadherin activity prevents this motile behavior. Thus, endothelial cell-cell-adhesion-based motility is required for the advancement of vascular sprouts within a moving tissue environment. To the best of our knowledge, this is the first study that couples endogenous tissue dynamics to assembly of vascular networks in a mammalian system.     

Controlled damage in thick specimens by multiphoton excitation

Controlled damage by light energy has been a valuable tool in studies of cell function. Here, we show that the Ti:Sapphire laser in a multiphoton microscope can be used to cause localized damage within unlabeled cells or tissues at greater depths than previously possible. We show that the damage is due to a multiphoton process and made wounds as small as 1 microm in diameter 20 microm from the surface. A characteristic fluorescent scar allows monitoring of the damage and identifies the wound site in later observations. We were able to lesion a single axon within a bundle of nerves, locally interrupt organelle transport within one axon, cut dendrites in a zebrafish embryo, ablate a mitotic pole in a sea urchin egg, and wound the plasma membrane and nuclear envelope in starfish oocytes. The starfish nucleus collapsed approximately 1 h after wounding, indicating that loss of compartmentation barrier makes the structure unstable; surprisingly, the oocyte still completed meiotic divisions when exposed to maturation hormone, indicating that the compartmentalization and translocation of cdk1 and its regulators is not required for this process. Multiphoton excitation provides a new means for producing controlled damage deep within tissues or living organisms.     

Localized elasticity measured in epithelial cells migrating at a wound edge using atomic force microscopy

Restoration of lung homeostasis following injury requires efficient wound healing by the epithelium. The mechanisms of lung epithelial wound healing include cell spreading and migration into the wounded area and later cell proliferation. We hypothesized that mechanical properties of cells vary near the wound edge, and this may provide cues to direct cell migration. To investigate this hypothesis, we measured variations in the stiffness of migrating human bronchial epithelial cells (16HBE cells) approximately 2 h after applying a scratch wound. We used atomic force microscopy (AFM) in contact mode to measure the cell stiffness in 1.5-microm square regions at different locations relative to the wound edge. In regions far from the wound edge (>2.75 mm), there was substantial variation in the elastic modulus in specific cellular regions, but the median values measured from multiple fields were consistently lower than 5 kPa. At the wound edge, cell stiffness was significantly lower within the first 5 microm but increased significantly between 10 and 15 microm before decreasing again below the median values away from the wound edge. When cells were infected with an adenovirus expressing a dominant negative form of RhoA, cell stiffness was significantly decreased compared with cells infected with a control adenovirus. In addition, expression of dominant negative RhoA abrogated the peak increase in stiffness near the wound edge. These results suggest that cells near the wound edge undergo localized changes in cellular stiffness that may provide signals for cell spreading and migration.     

Patterns of Living β-Actin Movement in Wounded Human Coronary Artery Endothelial Cells Exposed to Shear Stress

We previously demonstrated that physiologic levels of shear stress enhance endothelial repair. Cell spreading and migration, but not proliferation, were the major mechanisms accounting for the increases in wound closure rate (Albuquerque et al., 2000, Am. J. Physiol. Heart Circ. Physiol. 279, H293–H302). However, the patterns and movements of β-actin filaments responsible for cell motility and translocation in human coronary artery endothelial cells (HCAECs) have not been previously investigated under physiologic flow. HCAECs transfected with β-actin-GFP were cultured on type I collagen-coated coverslips. Confluent cell monolayers were subjected to laminar shear stress of 12 dynes/cm² for 18 h in a parallel-plate flow chamber to attain cellular alignment and then wounded by scraping with a metal spatula and subsequently exposed to a laminar shear stress of 20 dynes/cm² (S-W-sH) or static (S-W-sT) conditions. Time-lapse imaging and deconvolution microscopy was performed during the first 3 h after imposition of S-W-sH or S-W-sT conditions. The spatial and temporal dynamics of β-actin-GFP motility and translocation during wound closure in HCAEC monolayers were analyzed under both conditions. Compared with HCAEC under S-W-sT conditions, our data show that HCAEC under S-W-sH conditions demonstrated greater β-actin-GFP motility, filament and clumping patterns, and filament arcs used during cellular attachment and detachment. These findings demonstrate intriguing patterns of β-actin organization and movement during wound closure in HCAEC exposed to physiological flow.     

Development of micropost force sensor array with culture experiments for determination of cell traction forces

Cell traction forces (CTFs) are critical for cell motility and cell shape maintenance. As such, they play a fundamental role in many biological processes such as angiogenesis, embryogenesis, inflammation, and wound healing. To determine CTFs at the sub-cellular level with high sensitivity, we have developed high density micropost force sensor array (MFSA), which consists of an array of vertically standing poly(dimethylsiloxane) (PDMS) microposts, 2 microm in diameter and 6 microm in height, with a center-to-center distance of 4 microm. In combination with new image analysis algorithms, the MFSA can achieve a spatial resolution of 40 nm and a force sensitivity of 0.5 nN. Culture experiments with various types of cells showed that this MFSA technology can effectively determine CTFs of cells with different sizes and traction force magnitudes.     

Vertex Models of Epithelial Morphogenesis

The dynamic behavior of epithelial cell sheets plays a central role during numerous developmental processes. Genetic and imaging studies of epithelial morphogenesis in a wide range of organisms have led to increasingly detailed mechanisms of cell sheet dynamics. Computational models offer a useful means by which to investigate and test these mechanisms, and have played a key role in the study of cell-cell interactions. A variety of modeling approaches can be used to simulate the balance of forces within an epithelial sheet. Vertex models are a class of such models that consider cells as individual objects, approximated by two-dimensional polygons representing cellular interfaces, in which each vertex moves in response to forces due to growth, interfacial tension, and pressure within each cell. Vertex models are used to study cellular processes within epithelia, including cell motility, adhesion, mitosis, and delamination. This review summarizes how vertex models have been used to provide insight into developmental processes and highlights current challenges in this area, including progressing these models from two to three dimensions and developing new tools for model validation.