Myosin-mediated cytoskeleton contraction and Rho GTPases regulate laminin-5 matrix assembly

Laminin-5 is a major structural element of epithelial tissue basement membranes. In the matrix of cultured epithelial cells, laminin-5 is arranged into intricate patterns. Here we tested a hypothesis that myosin II-mediated actin contraction is necessary for the proper assembly of a laminin-5 matrix by cultured SCC12 epithelial cells. To do so, the cells were treated with ML-7, a myosin II light chain kinase inhibitor, or Y-27632, an inhibitor of Rho-kinase (ROCK), both of which block actomyosin contraction. Under these conditions, laminin-5 shows an aberrant localization in dense patches at the cell periphery. Since ROCK activity is regulated by the small GTPase Rho, this suggests that members of the Rho family of GTPases may also be important for laminin-5 matrix assembly by SCC12 cells. We confirmed this hypothesis since SCC12 cells expressing mutant proteins that inhibit RhoA, Rac, and Cdc42 assemble the same aberrant laminin-5 protein arrays as drug-treated cells. We have also evaluated the organization of the laminin-5 receptors alpha3beta1 and alpha6beta4 integrin and hemidesmosome proteins in ML-7- and Y-27632-treated cells or in cells in which RhoA, Rac, and Cdc42 activity were inhibited. In all instances, alpha3beta1 and alpha6beta4 integrin heterodimers, as well as hemidesmosome proteins, localize precisely with laminin-5 in the matrix of the cells. In summary, our results provide evidence that myosin II-mediated actin contraction and the activity of Rho GTPases are necessary for the proper organization of a laminin-5 matrix and localization of hemidesmosome protein arrays in epithelial cells.     

Integrin beta4 regulates migratory behavior of keratinocytes by determining laminin-332 organization

Whether alpha6beta4 integrin regulates migration remains controversial. beta4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express beta4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type beta4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with alpha6beta4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the alpha6beta4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells.     

Integrin engagement differentially modulates epithelial cell motility by RhoA/ROCK and PAK1

Integrin-ligand binding regulates tumor cell motility and invasion. Cell migration also involves the Rho GTPases that control the interplay between adhesion receptors and the cytoskeleton. We evaluated how specific extracellular matrix ligands modulate Rho GTPases and control motility of human squamous cell carcinoma cells. On laminin-5 substrates, the epithelial cells rapidly spread and migrated, but on type I collagen the cells spread slowly and showed reduced motility. We found that RhoA activity was suppressed in cells attached to laminin-5 through the alpha3 integrin receptor. In contrast, RhoA was strongly activated in cells bound to type I collagen and this was mediated by the alpha2 integrin. Inhibiting the RhoA pathway by expression of a dominant-negative RhoA mutant or by directly inhibiting ROCK, reduced focal adhesion formation and enhanced cell migration on type I collagen. Cdc42 and Rac and their downstream target PAK1 were activated following adhesion to laminin-5. PAK1 activation induced by laminin-5 was suppressed by expression of a dominant-negative Cdc42. Moreover, constitutively active PAK1 stimulated migration on collagen I substrates. Our results indicate that in squamous epithelial cells, collagen-alpha2beta1 integrin binding activates RhoA, slowing cell locomotion, whereas laminin-5-alpha3beta1 integrin interaction inhibits RhoA and activates PAK1, stimulating cell migration. The data demonstrate that specific ligand-integrin pairs regulate cell motility differentially by selectively modulating activities of Rho GTPases and their effectors.     

Regulation of the Type II Hemidesmosomal Plaque Assembly in Intestinal Epithelial Cells

Hemidesmosomes (HDs) are cellular junctions that anchor epithelial cells to the extracellular matrix (ECM) and are associated morphologically with the cytoskeleton. Hemidesmosomal molecular components include two proteins involved in linking intermediate filaments, HD1/plectin and BP230, and two transmembrane proteins, BP180 and the alpha6beta4 integrin, a laminin receptor. In cells lacking BP230 and BP180, HD1/plectin still associates with alpha6beta4 integrin, forming HD-like structures, called type II HDs. In the present study, we used an intestinal epithelial cell line that expresses HD1/plectin and the alpha6beta4 integrin to investigate the regulation of assembly of these proteins in type II HDs. These compounds were found to be clustered at sites of cell-ECM contact and their polarized localization was influenced by either cell confluency or extracellular matrix deposition. Conventional and immunoelectron microscopy showed that HD1/plectin and the beta4 integrin subunit are colocalized in an adhesion structure. Using cytoskeleton-disrupting drugs and confocal microscopy, we demonstrated that type II HDs are made up of numerous individual plaques whose assembly into a cluster requires actin filaments, but not microtubules.     

Crucial role of the specificity-determining loop of the integrin beta4 subunit in the binding of cells to laminin-5 and outside-in signal transduction

Within each hemidesmosome, alpha6beta4 integrin plays a crucial role in hemidesmosome assembly by binding to laminin-5 in the basement membrane zone of epithelial tissue. Recent analyses have implicated "specificity-determining loops" (SDLs) in the I-like domain of beta integrin in regulating ligand binding. Here, we investigated the function of an SDL-like motif within the extracellular I-like domain of beta4 integrin. We generated point mutations within the SDL of beta4 integrin tagged with green fluorescent protein (GFP-beta4K150A and GFP-beta4Q155L). We also generated a mutation within the I-like domain of the beta4 integrin, lying outside the SDL region (GFP-beta4V284E). We transfected constructs encoding the mutated beta4 integrins and a GFP-conjugated wild type beta4 integrin (GFP-beta4WT) into 804G cells, which assemble hemidesmosomes, and human endothelial cells, which express little endogenous beta4 integrin. In transfected 804G cells, GFP-beta4WT and GFP-beta4V284E colocalize with hemidesmosome proteins, whereas hemidesmosomal components in cells expressing GFP-beta4K150A and GFP-beta4Q155L are aberrantly localized. In endothelial cells, GFP-beta4WT and mutant proteins are co-expressed at the cell surface with alpha6 integrin. When transfected endothelial cells are plated onto laminin-5 matrix, GFP-beta4WT and GFP-beta4V284E localize with laminin-5, whereas GFP-beta4K150A and GFP-beta4Q155L do not. GFP-beta4WT and GFP-beta4V284E expressed in endothelial cells associate with the adaptor protein Shc when the cells are stimulated with laminin-5. However, GFP-beta4K150A and GFP-beta4Q155L fail to associate with Shc even when laminin-5 is present, thus impacting downstream signaling. These results provide evidence that the SDL segment of the beta4 integrin subunit is required for ligand binding and is involved in outside-in signaling.     

Laminin-11.

Laminins are a family of glycoproteins which are ubiquitous components of basement membranes and play key structural and functional roles. Eleven isoforms have been identified to date; each is an alpha beta gamma heterotrimer assembled from a repertoire of five alpha, three beta and two gamma chains. Studies of laminin-11 (alpha 5 beta 2 gamma 1) illustrate the unique expression patterns and distinct functions that can be displayed by laminin isoforms. Laminin-11 is found in the glomerular basement membrane in kidney, in the neuromuscular synaptic cleft in skeletal muscle and in other tissues such as placenta and lung. Mice lacking laminin-11 exhibit defective glomerular filtration and developmental defects in neuromuscular synapse formation, with Schwann cells invading the synaptic cleft. Consistent with these observations, both motoneurons and Schwann cells distinguish laminin-11 from other isoforms in vitro. These results suggest that laminin-11 is a structural component of the basement membrane which influences cell behavior in physiologically relevant ways. A greater understanding of laminin-11 assembly and basement membrane incorporation could provide a logical basis for therapy in merosin-deficient congenital muscular dystrophy.     

Processing of Laminin-5 and Its Functional Consequences: Role of Plasmin and Tissue-type Plasminogen Activator

The laminin-5 component of the extracellular matrices of certain cultured cells such as the rat epithelial cell line 804G and the human breast epithelial cell MCF-10A is capable of nucleating assembly of cell– matrix adhesive devices called hemidesmosomes when other cells are plated upon them. These matrices also impede cell motility. In contrast, cells plated onto the laminin-5–rich matrices of pp126 epithelial cells fail to assemble hemidesmosomes and are motile. To understand these contradictory phenomena, we have compared the forms of heterotrimeric laminin-5 secreted by 804G and MCF-10A cells with those secreted by pp126 cells, using a panel of laminin-5 subunit-specific antibodies. The α3 subunit of laminin-5 secreted by pp126 cells migrates at 190 kD, whereas that secreted by 804G and MCF-10A cells migrates at 160 kD. The pp126 cell 190-kD α3 chain of laminin-5 can be specifically proteolyzed by plasmin to a 160-kD species at enzyme concentrations that do not apparently effect the laminin-5 β and γ chains. After plasmin treatment, pp126 cell laminin-5 not only impedes cell motility but also becomes competent to nucleate assembly of hemidesmosomes. The possibility that plasmin may play an important role in processing laminin-5 subunits is supported by immunofluorescence analyses that demonstrate colocalization of laminin-5 and plasminogen in the extracellular matrix of MCF-10A and pp126 cells. Whereas tissue-type plasminogen activator (tPA), which converts plasminogen to plasmin, codistributes with laminin-5 in MCF-10A matrix, tPA is not present in pp126 extracellular matrix. Treatment of pp126 laminin-5–rich extracellular matrix with exogenous tPA results in proteolysis of the laminin-5 α3 chain from 190 to 160 kD. In addition, plasminogen and tPA bind laminin-5 in vitro. In summary, we provide evidence that laminin-5 is a multifunctional protein that can act under certain circumstances as a motility and at other times as an adhesive factor. In cells in culture, this functional conversion appears dependent upon and is regulated by tPA and plasminogen.     

The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes

Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the incorporation of laminin-5 into its proper higher-order structure within the extracellular matrix of keratinocytes and (2) that the organizational state of laminin-5 has an influence on laminin-5 matrix function.     

Bone marrow-derived cells as progenitors of lung alveolar epithelium.

We assessed the capacity of plastic-adherent cultured bone marrow cells to serve as precursors of differentiated parenchymal cells of the lung. By intravenously delivering lacZ-labeled cells into wild-type recipient mice after bleomycin-induced lung injury, we detected marrow-derived cells engrafted in recipient lung parenchyma as cells with the morphological and molecular phenotype of type I pneumocytes of the alveolar epithelium. At no time after marrow cell injection, did we detect any engraftment as type II pneumocytes. In addition, we found that cultured and fresh aspirates of bone marrow cells can express the type I pneumocyte markers, T1alpha and aquaporin-5. These observations challenge the current belief that adult alveolar type I epithelial cells invariably arise from local precursor cells and raise the possibility of using injected marrow-derived cells for therapy of lung diseases characterized by extensive alveolar damage.     

The slingshot family of phosphatases mediates Rac1 regulation of cofilin phosphorylation, laminin-332 organization, and motility behavior of keratinocytes

The motility of keratinocytes is an essential component of wound closure and the development of epidermal tumors. In vitro, the specific motile behavior of keratinocytes is dictated by the assembly of laminin-332 tracks, a process that is dependent upon alpha6beta4 integrin signaling to Rac1 and the actin-severing protein cofilin. Here we have analyzed how cofilin phosphorylation is regulated by phosphatases (slingshot (SSH) or chronophin (CIN)) downstream of signaling by alpha6beta4 integrin/Rac1 in human keratinocytes. Keratinocytes express all members of the SSH family (SSH1, SSH2, and SSH3) and CIN. However, expression of phosphatase-dead versions of all three SSH proteins, but not dominant inactive CIN, results in phosphorylation/inactivation of cofilin, changes in actin cytoskeleton organization, loss of cell polarity, and assembly of aberrant arrays of laminin-332 in human keratinocytes. SSH activity is regulated by 14-3-3 protein binding, and intriguingly, 14-3-3/alpha6beta4 integrin protein interaction is required for keratinocyte migration. We wondered whether 14-3-3 proteins function as regulators of Rac1-mediated keratinocyte migration patterns. In support of this hypothesis, inhibition of Rac1 results in an increase in 14-3-3 protein association with SSH. Thus, we propose a novel mechanism in which alpha6beta4 integrin signaling via Rac1, 14-3-3 proteins, and SSH family members regulates cofilin activation, cell polarity, and matrix assembly, leading to specific epidermal cell migration behavior.     

Integrins beta1, alpha6, and alpha3 contribute to mechanical strain-induced differentiation of fetal lung type II epithelial cells via distinct mechanisms

Mechanical forces regulate lung maturation in the fetus by promoting type II epithelial differentiation. However, the cell surface receptors that transduce these mechanical cues into cellular responses remain largely unknown. When distal lung type II epithelial cells isolated from embryonic day 19 rat fetuses were cultured on flexible plates coated with laminin, fibronectin, vitronectin, collagen, or elastin and exposed to a level of mechanical strain (5%) similar to that observed in utero, transmembrane signaling responses were induced under all conditions, as measured by ERK activation. However, mechanical stress maximally increased expression of the type II cell differentiation marker surfactant protein C when cells were cultured on laminin substrates. Strain-induced alveolar epithelial differentiation was inhibited by interfering with cell binding to laminin using soluble laminin peptides (IKVIV or YIGSR) or blocking antibodies against integrin beta1, alpha3, or alpha6. Additional studies were carried out with substrates coated directly with different nonactivating anti-integrin antibodies. Blocking integrin beta1 and alpha6 binding sites inhibited both cell adhesion and differentiation, whereas inhibition of alpha3 prevented differentiation without altering cell attachment. These data demonstrate that various integrins contribute to mechanical control of type II lung epithelial cell differentiation on laminin substrates. However, they may act via distinct mechanisms, including some that are independent of their cell anchoring role.     

Heterocellular gap junctional communication between alveolar epithelial cells

We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6 alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.     

The Integrin α6β4 Functions in Carcinoma Cell Migration on Laminin-1 by Mediating the Formation and Stabilization of Actin-containing Motility Structures

Functional studies on the α6β4 integrin have focused primarily on its role in the organization of hemidesmosomes, stable adhesive structures that associate with the intermediate filament cytoskeleton. In this study, we examined the function of the α6β4 integrin in clone A cells, a colon carcinoma cell line that expresses α6β4 but no α6β1 integrin and exhibits dynamic adhesion and motility on laminin-1. Time-lapse videomicroscopy of clone A cells on laminin-1 revealed that their migration is characterized by filopodial extension and stabilization followed by lamellae that extend in the direction of stabilized filopodia. A function-blocking mAb specific for the α6β4 integrin inhibited clone A migration on laminin-1. This mAb also inhibited filopodial formation and stabilization and lamella formation. Indirect immunofluorescence microscopy revealed that the α6β4 integrin is localized as discrete clusters in filopodia, lamellae, and retraction fibers. Although β1 integrins were also localized in the same structures, a spatial separation of these two integrin populations was evident. In filopodia and lamellae, a striking colocalization of the α6β4 integrin and F-actin was seen. An association between α6β4 and F-actin is supported by the fact that α6β4 integrin and actin were released from clone A cells by treatment with the F-actin– severing protein gelsolin and that α6β4 immunostaining at the marginal edges of clone A cells on laminin-1 was resistant to solubilization with Triton X-100. Cytokeratins were not observed in filopodia and lamellipodia. Moreover, α6β4 was extracted from these marginal edges with a Tween-40/deoxycholate buffer that solubilizes the actin cytoskeleton but not cytokeratins. Three other carcinoma cell lines (MIP-101, CCL-228, and MDA-MB-231) exhibited α6β4 colocalized with actin in filopodia and lamellae. Formation of lamellae in these cells was inhibited with an α6-specific antibody. Together, these results indicate that the α6β4 integrin functions in carcinoma migration on laminin-1 through its ability to promote the formation and stabilization of actin-containing motility structures.     

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.     

Monoclonal antibodies specific to apical surfaces of rat alveolar type I cells bind to surfaces of cultured, but not freshly isolated, type II cells

The alveolar surface of the lung is lined by two classes of epithelial cells, type I and type II cells. Type I cells cover more than 97% of the alveolar surface. Although this cell type is felt to be essential for normal gas exchange, neither unique identifying characteristics nor functions have been described for the type I cell. We have produced monoclonal antibodies to (a) component(s) of molecular weight 40,000 and 42,000 of the apical surface of rat alveolar type I cells. The antibodies are specific to the lung in Western blots of organ homogenates. In immunocytochemical studies of frozen lung at the level of both light and electron microscopy, the monoclonal antibodies appear to react specifically with the apical plasma membrane of type I cells. Airway, vascular, interstitial cells, type II cells and macrophages are not immunoreactive. Western blots of isolated type I cells (approx. 70% pure) also demonstrate immunoreactivity at molecular weights of 40,000 and 42,000. When the lung is injured, type I cells may be damaged and sloughed from the alveolar surface. Alveolar repair occurs when the second type of alveolar cell, the type II cell, divides. Cell progeny may retain type II cell morphology or may differentiate into type I cells. Western blots of freshly isolated type II cells (approx. 85% pure) do not display immunoreactivity with our monoclonal antibodies. However, type II cells maintained in culture acquire immunoreactivity to monoclonal antibodies, demonstrating that type II cells in vitro have the capacity to develop a characteristic associated with type I cells in situ. The availability of markers for a specific membrane component of type I cells should facilitate the study of many questions on alveolar functions, development and response to injury.     

T1alpha,a lung type I cell differentiation gene,is required for normal lung cell proliferation and alveolus formation at birth

T1alpha, a differentiation gene of lung alveolar epithelial type I cells, is developmentally regulated and encodes an apical membrane protein of unknown function. Morphological differentiation of type I cells to form the air-blood barrier starts in the last few days of gestation and continues postnatally. Although T1alpha is expressed in the foregut endoderm before the lung buds, T1alpha mRNA and protein levels increase substantially in late fetuses when expression is restricted to alveolar type I cells. We generated T1alpha null mutant mice to study the role of T1alpha in lung development and differentiation and to gain insight into its potential function. Homozygous null mice die at birth of respiratory failure, and their lungs cannot be inflated to normal volumes. Distal lung morphology is altered. In the absence of T1alpha protein, type I cell differentiation is blocked, as indicated by smaller airspaces, many fewer attenuated type I cells, and reduced levels of aquaporin-5 mRNA and protein, a type I cell water channel. Abundant secreted surfactant in the narrowed airspaces, normal levels of surfactant protein mRNAs, and normal patterns and numbers of cells expressing surfactant protein-B suggest that differentiation of type II cells, also alveolar epithelial cells, is normal. Anomalous proliferation of the mesenchyme and epithelium at birth with unchanged numbers of apoptotic cells suggests that loss of T1alpha and/or abnormal morphogenesis of type I cells alter the proliferation rate of distal lung cells, probably by disruption of epithelial-mesenchymal signaling.     

Atomic force microscope elastography reveals phenotypic differences in alveolar cell stiffness.

To understand the connection between alveolar mechanics and key biochemical events such as surfactant secretion, one first needs to characterize the underlying mechanical properties of the lung parenchyma and its cellular constituents. In this study, the mechanics of three major cell types from the neonatal rat lung were studied; primary alveolar type I (AT1) and type II (AT2) epithelial cells and lung fibroblasts were isolated using enzymatic digestion. Atomic force microscopy indentation was used to map the three-dimensional distribution of apparent depth-dependent pointwise elastic modulus. Histograms of apparent modulus data from all three cell types indicated non-Gaussian distributions that were highly skewed and appeared multimodal for AT2 cells and fibroblasts. Nuclear stiffness in all three cell types was similar (2.5+/-1.0 kPa in AT1 vs. 3.1+/-1.5 kPa in AT2 vs. 3.3+/-0.8 kPa in fibroblasts; n=10 each), whereas cytoplasmic moduli were significantly higher in fibroblasts and AT2 cells (6.0+/-2.3 and 4.7+/-2.9 kPa vs. 2.5+/-1.2 kPa). In both epithelial cell types, actin was arranged in sparse clusters, whereas prominent actin stress fibers were observed in lung fibroblasts. No systematic difference in actin or microtubule organization was noted between AT1 and AT2 cells. Atomic force microscope elastography, combined with live-cell fluorescence imaging, revealed that the stiffer measurements in AT2 cells often colocalized with lamellar bodies. These findings partially explain reported heterogeneity of alveolar cell deformation during in situ lung inflation and provide needed data for better understanding of how mechanical stretch influences surfactant release.     

Morphogenetic Effects of Soluble Laminin-5 on Cultured Epithelial Cells and Tissue Explants

The rat cell line 804G assembles an extracellular matrix which induces not only the rapid adhesion and spreading of epithelial cells but also the assembly of a cell–matrix attachment device called the hemidesmosome. The major component of this matrix is laminin-5. We have purified rat laminin-5 from medium conditioned by 804G cells. Epithelial cells which are co-incubated with medium supplemented with soluble laminin-5 adhere and spread rapidly. Furthermore, human carcinoma cells undergo a dramatic morphologic change in the presence of laminin-5 and form orderly arrays resembling epithelial sheets. Soluble rat laminin-5 is selectively incorporated into an insoluble matrix of epithelial cellsin vitro,since rat-specific laminin-5 antibodies stain cell–substrate contacts. Addition of medium containing soluble laminin-5 to explanted, human corneal rims induces assembly of hemidesmosomes, important cell–matrix attachment devices. Furthermore, rat-specific laminin-5 antibodies stain areas of contact between corneal epithelium and basement membrane, indicating that rat laminin-5 from the medium is incorporated into basement membrane. We discuss the use of laminin-5 as a medium supplement for the culture of both epithelial cells and epithelial tissue explants.