The cell signaling adaptor protein EPS-8 is essential for C. elegans epidermal elongation and interacts with the ankyrin repeat protein VAB-19

Background

The epidermal cells of the C. elegans embryo undergo coordinated cell shape changes that result in the morphogenetic process of elongation. The cytoskeletal ankyrin repeat protein VAB-19 is required for cell shape changes and localizes to cell-matrix attachment structures. The molecular functions of VAB-19 in this process are obscure, as no previous interactors for VAB-19 have been described.

Methodology/Principal Findings

In screens for VAB-19 binding proteins we identified the signaling adaptor EPS-8. Within C. elegans epidermal cells, EPS-8 and VAB-19 colocalize at cell-matrix attachment structures. The central domain of EPS-8 is necessary and sufficient for its interaction with VAB-19. eps-8 null mutants, like vab-19 mutants, are defective in epidermal elongation and in epidermal-muscle attachment. The eps-8 locus encodes two isoforms, EPS-8A and EPS-8B, that appear to act redundantly in epidermal elongation. The function of EPS-8 in epidermal development involves its N-terminal PTB and central domains, and is independent of its C-terminal SH3 and actin-binding domains. VAB-19 appears to act earlier in the biogenesis of attachment structures and may recruit EPS-8 to these structures.

Conclusions/Significance

EPS-8 and VAB-19 define a novel pathway acting at cell-matrix attachments to regulate epithelial cell shape. This is the first report of a role for EPS-8 proteins in cell-matrix attachments. The existence of EPS-8B-like isoforms in Drosophila suggests this function of EPS-8 proteins could be conserved among other organisms.     

Regulated Expression of Human Filaggrin in Keratinocytes Results in Cytoskeletal Disruption, Loss of Cell–Cell Adhesion, and Cell Cycle Arrest

Filaggrin is an intermediate filament (IF)-associated protein that aggregates keratin IFs in vitro and is thought to perform a similar function during the terminal differentiation of epidermal keratinocytes. To further explore the role of filaggrin in the cytoskeletal rearrangement that accompanies epidermal differentiation, we generated keratinocyte cell lines that express human filaggrin using a tetracycline-inducible promoter system. Filaggrin expression resulted in reduced keratinocyte proliferation and caused an alteration in cell cycle distribution consistent with a post-G1 phase arrest. Keratin filament distribution was disrupted in filaggrin-expressing lines, while the organization of actin microfilaments and microtubules was more mildly affected. Evidence for direct interaction of filaggrin and keratin IFs was seen by overlay assays of GFP-filaggrin with keratin proteins in vitro and by filamentous filaggrin distribution in cells with low levels of expression. Cells expressing moderate to high levels of filaggrin showed a rounded cell morphology, loss of cell-cell adhesion, and compacted cytoplasm. There was also partial or complete loss of the desmosomal proteins desmoplakin, plakoglobin, and desmogleins from cell-cell borders, while the distribution of the adherens junction protein E-cadherin was not affected. No alterations in keratin cytoskeleton, desmosomal protein distribution, or cell shape were observed in control cell lines expressing beta-galactosidase. Filaggrin altered the cell shape and disrupted the actin filament distribution in IF-deficient SW13 cells, demonstrating that filaggrin can affect cell morphology independent of the presence of a cytoplasmic IF network. These studies demonstrate that filaggrin, in addition to its known effects on IF organization, can affect the distribution of other cytoskeletal elements including actin microfilaments, which can occur in the absence of a cytoplasmic IF network. Further, filaggrin can disrupt the distribution of desmosome proteins, suggesting an additional role(s) for this protein in the cytoskeletal and desmosomal reorganization that occurs at the granular to cornified cell transition during terminal differentiation of epidermal keratinocytes.     

Cancer invasion and the microenvironment: plasticity and reciprocity

Cancer invasion is a cell- and tissue-driven process for which the physical, cellular, and molecular determinants adapt and react throughout the progression of the disease. Cancer invasion is initiated and maintained by signaling pathways that control cytoskeletal dynamics in tumor cells and the turnover of cell-matrix and cell-cell junctions, followed by cell migration into the adjacent tissue. Here, we describe the cell-matrix and cell-cell adhesion, protease, and cytokine systems that underlie tissue invasion by cancer cells. We explain how the reciprocal reprogramming of both the tumor cells and the surrounding tissue structures not only guides invasion, but also generates diverse modes of dissemination. The resulting "plasticity" contributes to the generation of diverse cancer invasion routes and programs, enhanced tumor heterogeneity, and ultimately sustained metastatic dissemination.     

Cell-matrix adhesion

The complex interactions of cells with extracellular matrix (ECM) play crucial roles in mediating and regulating many processes, including cell adhesion, migration, and signaling during morphogenesis, tissue homeostasis, wound healing, and tumorigenesis. Many of these interactions involve transmembrane integrin receptors. Integrins cluster in specific cell-matrix adhesions to provide dynamic links between extracellular and intracellular environments by bi-directional signaling and by organizing the ECM and intracellular cytoskeletal and signaling molecules. This mini review discusses these interconnections, including the roles of matrix properties such as composition, three-dimensionality, and porosity, the bi-directional functions of cellular contractility and matrix rigidity, and cell signaling. The review concludes by speculating on the application of this knowledge of cell-matrix interactions in the formation of cell adhesions, assembly of matrix, migration, and tumorigenesis to potential future therapeutic approaches.     

Epidermal growth factor induces an increase in cell adhesion and an arrangement of actin skeleton in stress fibres in pituitary cultured cells from infantile rats but not adult rats

The rat anterior pituitary gland undergoes changes in its cyto-architecture during the second and third weeks of postnatal life. However, little is known about the factors that regulate these tissue conformational changes. The epidermal growth factor (EGF) is one of the growth factors that are synthesized by the pituitary gland, and almost all of the pituitary cells have EGF receptors (EGFR). In addition to the effects of the EGF on mitosis and differentiation, this growth factor can modulate cell adhesion, cell migration, and cytoskeletal organization. In this study we focussed our attention in examining the effects of EGF on the adhesion of cells to the extracellular matrix and on the actin cytoskeletal arrangement of pituitary cells from infantile and adult rats. Our results show that in infantile cells the EGF induces cell adhesion with increase in cell surface area. The arrangement of actin-F in infantile EGF-treated cells was in stress fibers and vinculin acquired a striped shape at the membrane border, suggesting the assembly of focal adhesion contacts. In contrast, in adult pituitary cells EGF does not induce any change in cell adhesion, and the cells maintain a rounded shape with an arrangement of actin-F in thin cortical bands even though, immuno-localization of the EGFR was observed in adult cells cultured in defined medium. We also looked for the EGFR in membrane preparations from infantile and adult pituitaries, and a marked difference in membrane EGFR was observed between them, the infantile pituitaries showing a significantly higher amount. Our results suggest that in infantile cells EGF induces the assembly of focal adhesion contacts, and that in adult cells the receptor of this growth factor is uncoupled of the signaling pathway by which a rearrangement of actin cytoskeleton occurs.     

Epithelial-mesenchymal interactions in tooth morphogenesis: the roles of extracellular matrix, growth factors, and cell surface receptors.

Morphogenesis and cell differentiation in the developing tooth are controlled by a series of reciprocal interactions between the epithelial and mesenchymal tissues. The exact molecular mechanisms operating in these interactions are unknown at present, but both structural components of the extracellular matrix (ECM) and diffusible growth factors have been suggested to be involved. In this review article we summarize our findings on the distribution patterns of three ECM molecules and two cell surface receptors during tooth morphogenesis through bud, cap, and bell stages of development. The examined molecules include fibronectin, type III collagen, and tenascin, which all represent components of the mesenchymal ECM, the cell surface proteoglycan, syndecan, which functions as a receptor for interstitial matrix, and the cell surface receptor for epidermal growth factor. Based on the observed changes in distribution patterns and on experimental evidence, roles are suggested for these molecules in epithelial-mesenchymal interactions during tooth development. Fibronectin is suggested to be involved in the cell-matrix interaction that controls odontoblast differentiation. Epidermal growth factor and its receptors are suggested to be involved in a paracrine fashion in the epithelial-mesenchymal interactions regulating morphogenesis of bud- and cap-stage teeth. Tenascin and syndecan are accumulated in the dental mesenchyme during the bud stage of development, and it is suggested that they represent a couple of a cell surface receptor and its matrix ligand and that they are involved in mesenchymal cell condensation during the earliest stages of tooth morphogenesis.     

Cardiac myocyte remodeling mediated by N-cadherin-dependent mechanosensing

Cell-to-cell adhesions are crucial in maintaining the structural and functional integrity of cardiac cells. Little is known about the mechanosensitivity and mechanotransduction of cell-to-cell interactions. Most studies of cardiac mechanotransduction and myofibrillogenesis have focused on cell-extracellular matrix (ECM)-specific interactions. This study assesses the direct role of intercellular adhesion, specifically that of N-cadherin-mediated mechanotransduction, on the morphology and internal organization of neonatal ventricular cardiac myocytes. The results show that cadherin-mediated cell attachments are capable of eliciting a cytoskeletal network response similar to that of integrin-mediated force response and transmission, affecting myofibrillar organization, myocyte shape, and cortical stiffness. Traction forces mediated by N-cadherin were shown to be comparable to those sustained by ECM. The directional changes in predicted traction forces as a function of imposed loads (gel stiffness) provide the added evidence that N-cadherin is a mechanoresponsive adhesion receptor. Strikingly, the mechanical sensitivity response (gain) in terms of the measured cell-spread area as a function of imposed load (adhesive substrate rigidity) was consistently higher for N-cadherin-coated surfaces compared with ECM protein-coated surfaces. In addition, the cytoskeletal architecture of myocytes on an N-cadherin adhesive microenvironment was characteristically different from that on an ECM environment, suggesting that the two mechanotransductive cell adhesion systems may play both independent and complementary roles in myocyte cytoskeletal spatial organization. These results indicate that cell-to-cell-mediated force perception and transmission are involved in the organization and development of cardiac structure and function.     

Reciprocal integrin/integrin antagonism through kindlin-2 and Rho GTPases regulates cell cohesion and collective migration

Collective cell behaviour during embryogenesis and tissue repair requires the coordination of intercellular junctions, cytoskeleton-dependent shape changes controlled by Rho GTPases, and integrin-dependent cell-matrix adhesion. Many different integrins are simultaneously expressed during wound healing, embryonic development, and sprouting angiogenesis, suggesting that there is extensive integrin/integrin cross-talk to regulate cell behaviour. Here, we show that fibronectin-binding β1 and β3 integrins do not act synergistically, but rather antagonize each other during collective cell processes in neuro-epithelial cells, placental trophoblasts, and endothelial cells. Reciprocal β1/β3 antagonism controls RhoA activity in a kindlin-2-dependent manner, balancing cell spreading, contractility, and intercellular adhesion. In this way, reciprocal β1/β3 antagonism controls cell cohesion and cellular plasticity to switch between extreme and opposing states, including epithelial versus mesenchymal-like phenotypes and collective versus individual cell migration. We propose that integrin/integrin antagonism is a universal mechanism to effectuate social cellular interactions, important for tissue morphogenesis, endothelial barrier function, trophoblast invasion, and sprouting angiogenesis.     

Cytoskeletal network in colon cancer: from genes to clinical application

Colorectal cancer arises from well-defined sequential steps characterised by distinct genetic events. Abnormalities in the expression and functional activity of cell adhesion molecules are implicated in the development and progression of the majority of colorectal cancers. Intercellular (e.g. E-cadherin/catenin complex) and cell-matrix (e.g. integrins) adhesion molecules are more than just cementing substances but regulate cell polarity, differentiation, proliferation, migration and invasion. Many of these cellular events are mediated through their intimate association with the actin cytoskeletal network. A dynamic actin cytoskeleton characterises normal epithelial cells and polymerisation and depolymerisation of actin filaments enables cell shape to change during migration and mitosis. In colorectal cancer, cells lose actin cytoskeletal organisation and normal cell adhesion when they become invasive. Future investigations should allow the unravelling of new cytoskeletal network functions in tumour biology and may lead to the development of novel therapeutic strategies based on the manipulation of its associated molecules.     

Shaping up for shipping out: PLCgamma signaling of morphology changes in EGF-stimulated fibroblast migration

For effective migration, cells must establish an asymmetry in cell/substratum biophysical interactions permitting cellular protrusive and contractile motive forces to produce net cell body translocation; often this is superficially manifested as a polarized cell shape. This change is most easily noted for epithelial cells, which typically undergo a mesenchymal transition prior to rapid motility, and for hematopoietic cells, which must transition from non-adherent to adherent states. These two situations entail dramatic changes that also involve cell-cell contact and differentiation-related changes, and thus introduce confounding events and signals in defining control elements. Hence, a simpler biochemical and biophysical model system may be useful for gaining fundamental insights into the underlying mechanisms. Fortunately, even relatively "uniform" fibroblasts also undergo an initial shape change to commence locomotion. Investigators have recently begun to probe underlying signals that contribute to the reorganization of the actin cytoskeleton. We describe here a model for fibroblast shape changes involved in epidermal growth factor (EGF) stimulation of motility, focusing on signals through EGF receptor (EGFR) -mediated pathways influencing cytoskeletal organization and cell/substratum adhesion. We present new data addressing specifically phospholipase C-gamma (PLCgamma) pathway activation of actin-modifying proteins, including gelsolin, that contributes to these changes and promotes cell migration by increasing the fraction of cells in a motility-permissive morphology and the time spent in such a state.     

Drosophila alpha-actinin in ovarian follicle cells is regulated by EGFR and Dpp signalling and required for cytoskeletal remodelling

alpha-Actinin is an evolutionarily conserved actin filament crosslinking protein with functions in both muscle and non-muscle cells. In non-muscle cells, interactions between alpha-actinin and its many binding partners regulate cell adhesion and motility. In Drosophila, one non-muscle and two muscle-specific alpha-actinin isoforms are produced by alternative splicing of a single gene. In wild-type ovaries, alpha-actinin is ubiquitously expressed. The non-muscle alpha-actinin mutant Actn(Delta233), which is viable and fertile, lacks alpha-actinin expression in ovarian germline cells, while somatic follicle cells express alpha-actinin at late oogenesis. Here we show that this latter population of alpha-actinin, termed FC-alpha-actinin, is absent from the dorsoanterior follicle cells, and we present evidence that this is the result of a negative regulation by combined Epidermal growth factor receptor (EGFR) and Decapentaplegic signalling. Furthermore, EGFR signalling increased the F-actin bundling activity of ectopically expressed muscle-specific alpha-actinin. We also describe a novel morphogenetic event in the follicle cells that occurs during egg elongation. This event involves a transient repolarisation of the basal actin fibres and the assembly of a posterior beta-integrin-dependent adhesion site accumulating alpha-actinin and Enabled. Clonal analysis using Actn null alleles demonstrated that although alpha-actinin was not necessary for actin fibre formation or maintenance, the cytoskeletal remodelling was perturbed, and Enabled did not localise in the posterior adhesion site. Nevertheless, epithelial morphogenesis proceeded normally. This work provides the first evidence that alpha-actinin is involved in the organisation of the cytoskeleton in a non-muscle tissue in Drosophila.     

Molecular mechanisms of cell shape changes that contribute to vertebrate neural tube closure

During early development of the central nervous system, the neuroepithelial cells undergo dynamic changes in shape, cumulative action of which cause the neural plate to bend mediolaterally to form the neural tube. The apicobasal elongation changes the cuboidal cells into columnar ones, whereas apical constriction minimizes the cell apices, causing them to adopt wedge-like shapes. To achieve the morphological changes required for the formation of a hollow structure, these cellular changes must be controlled in time and space. To date, it is widely accepted that spatial and temporal changes of the cytoskeletal organization are fundamental to epithelial cell shape changes, and that noncetrosomal microtubules assembled along apicobasal axis and actin filaments and non-muscle myosin II at the apical side are central machineries of cell elongation and apical constriction, respectively. Hence, especially in the last decade, intracellular mechanisms regulating these cytoskeletons have been extensively investigated at the molecular level. As a result, several actin-binding proteins, Rho/ROCK pathway, and cell-cell adhesion molecules have been proven to be the central regulators of apical constriction, while the regulatory mechanisms of cell elongation remain obscure. In this review, we first describe the distribution and role of cytoskeleton in cell shape changes during neural tube closure, and then summarize the current knowledge about the intracellular proteins that directly modulate the cytoskeletal organization and thus the neural tube closure.     

Behavior of a cell line derived from normal human hepatocytes on non-physiological and physiological-type substrates: Evidence for enhancement of secretion of liver-specific proteins by a three-dimensional growth pattern

Summary The behavior of a recently described cell line, HH25, derived from normal human hepatocytes, has been investigated on several different substrates—tissue-culture plastic, glass, a thin layer of rat-tail collagen I, and thin layers or thick gels of extracellular matrix derived from the Engelbreth-Holm-Swarm murine sarcoma (EHS matrix). Cellular morphology, proliferation, and secretion of three hepatocyte-specific proteins (albumin, α₁ acid glycoprotein, and α₁ antitrypsin) have been examined. There were no differences in morphology, proliferation, or differentiated function in the cells on either plastic, glass, collagen, I, or a thin layer of EHS matrix, but on a thick EHS matrix gel the cells altered their morphology (forming three-dimensional colonies with canalicular-like structures) and their production of albumin and α₁ acid glycoprotein was enhanced. This suggests that the enhanced differentiated function is associated with the morphological change (occurring only on the thick EHS gel) rather than with receptor-mediated cell-matrix interactions (which can also occur on the thin layer of EHS matrix). This cell line is therefore a good in vitro cellular model for the investigation of the roles of morphological changes and of cell-cell and cell-matrix interactions in the control of human hepatocyte behavior without the need for an extensive source of primary tissue.     

Coronin 1C negatively regulates cell-matrix adhesion and motility of intestinal epithelial cells

Coronins, WD-repeat actin-binding proteins, are known to regulate cell motility by coordinating actin filament turnover in lamellipodia of migrating cell. Here we report a novel mechanism of Coronin 1C-mediated cell motility that involves regulation of cell-matrix adhesion. RNAi silencing of Coronin 1C in intestinal epithelial cells enhanced cell migration and modulated lamellipodia dynamics by increasing the persistence of lamellipodial protrusion. Coronin 1C-depleted cells showed increased cell-matrix adhesions and enhanced cell spreading compared to control cells, while over-expression of Coronin 1C antagonized cell adhesion and spreading. Enhanced cell-matrix adhesion of coronin-deficient cells correlated with hyperphosphorylation of focal adhesion kinase (FAK) and paxillin, and an increase in number of focal adhesions and their redistribution at the cell periphery. siRNA depletion of FAK in coronin-deficient cells rescued the effects of Coronin 1C depletion on motility, cell-matrix adhesion, and spreading. Thus, our findings provide the first evidence that Coronin 1C negatively regulates epithelial cell migration via FAK-mediated inhibition of cell-matrix adhesion.     

Overexpression of c-Src enhances cell-matrix adhesion and cell migration in PDGF-stimulated NIH3T3 fibroblasts

c-Src is normally associated with the plasma membrane, but upon activation by tyrosine kinase receptors it translocates to the cytoskeleton. Activation of c-Src alters its conformation and induces the association of c-Src with cytoskeletal proteins. c-Src is implicated in tyrosine phosphorylation of cytoskeletal proteins, which might affect the cytoskeletal architecture. Rearrangements of the cytoskeleton affect cell-matrix adhesion and cell migration. In this study NIH3T3 fibroblasts, that overexpress c-Src, were used to analyze the effect of c-Src on both cell-matrix adhesion and cell migration. Upon PDGF stimulation translocation of c-Src to the cytoskeleton was detected. PDGF treatment also increased cell-matrix adhesion and cell migration. The cell line with the highest c-Src expression showed the largest increases in both phenomena. These findings suggest that translocation of c-Src to the cytoskeleton results in enhanced cell-matrix adhesion and cell migration.     

Vascular endothelial-cadherin regulates cytoskeletal tension, cell spreading, and focal adhesions by stimulating RhoA

Changes in vascular endothelial (VE)-cadherin-mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion coordinate to affect the physical and mechanical rearrangements of the endothelium, although the mechanisms for such cross talk remain undefined. Herein, we describe the regulation of focal adhesion formation and cytoskeletal tension by intercellular VE-cadherin engagement, and the molecular mechanism by which this occurs. Increasing the density of endothelial cells to increase cell-cell contact decreased focal adhesions by decreasing cell spreading. This contact inhibition of cell spreading was blocked by disrupting VE-cadherin engagement with an adenovirus encoding dominant negative VE-cadherin. When changes in cell spreading were prevented by culturing cells on a micropatterned substrate, VE-cadherin-mediated cell-cell contact paradoxically increased focal adhesion formation. We show that VE-cadherin engagement mediates each of these effects by inducing both a transient and sustained activation of RhoA. Both the increase and decrease in cell-matrix adhesion were blocked by disrupting intracellular tension and signaling through the Rho-ROCK pathway. In all, these findings demonstrate that VE-cadherin signals through RhoA and the actin cytoskeleton to cross talk with cell-matrix adhesion and thereby define a novel pathway by which cell-cell contact alters the global mechanical and functional state of cells.     

Functional role for stable microtubules in lens fiber cell elongation

The process of tissue morphogenesis, especially for tissues reliant on the establishment of a specific cytoarchitecture for their functionality, depends a balanced interplay between cytoskeletal elements and their interactions with cell adhesion molecules. The microtubule cytoskeleton, which has many roles in the cell, is a determinant of directional cell migration, a process that underlies many aspects of development. We investigated the role of microtubules in development of the lens, a tissue where cell elongation underlies morphogenesis. Our studies with the microtubule depolymerizing agent nocodazole revealed an essential function for the acetylated population of stable microtubules in the elongation of lens fiber cells, which was linked to their regulation of the activation state of myosin. Suppressing myosin activation with the inhibitor blebbistatin could attenuate the loss of acetylated microtubules by nocodazole and rescue the effect of this microtubule depolymerization agent on both fiber cell elongation and lens integrity. Our results also suggest that acetylated microtubules impact lens morphogenesis through their interaction with N-cadherin junctions, with which they specifically associate in the region where lens fiber cell elongate. Disruption of the stable microtubule network increased N-cadherin junctional organization along lateral borders of differentiating lens fiber cells, which was prevented by suppression of myosin activity. These results reveal a role for the stable microtubule population in lens fiber cell elongation, acting in tandem with N-cadherin cell-cell junctions and the actomyosin network, giving insight into the cooperative role these systems play in tissue morphogenesis.     

Interchangeable functions of Arabidopsis PIROGI and the human WAVE complex subunit SRA1 during leaf epidermal development

The WAVE complex is an essential regulator of actin-related protein (ARP) 2/3-dependent actin filament nucleation and cell shape change in migrating cells. Although the composition of the WAVE complex is well characterized, the cellular mechanisms that control its activity and localization are not well known. The 'distorted group' defines a set of Arabidopsis genes that are required to remodel the actin cytoskeleton and maintain the polarized elongation of branched, hair-like cells termed trichomes. Several loci within this group encode homologs of ARP2/3 subunits. In addition to trichome distortion, ARP2/3 subunit mutants have reduced shoot fresh weight and widespread defects in epidermal cell-cell adhesion. The precise cellular function of plant ARP2/3, and the means by which it is regulated, is not known. In this paper, we report that the 'distorted group' gene PIROGI encodes a homolog of the WAVE complex subunit SRA1. The similar cell shape and actin phenotypes of pir and ARP2/3 complex subunit mutants suggest that PIROGI positively regulates ARP2/3. PIROGI directly interacts with the small GTPase ATROP2 with isoform specificity and with selectivity for active forms of the protein. PIROGI shares only 30% amino acid identity with its human homolog. However, both WAVE subunit homologs are functionally interchangeable and display identical physical interactions with RHO family GTPases and the Arabidopsis homolog of the WAVE complex subunit NAP125. These results demonstrate the utility of the 'distorted group' mutants to study ARP2/3 complex functions from signaling input to cell shape output.     

Diaphanous regulates myosin and adherens junctions to control cell contractility and protrusive behavior during morphogenesis

Formins are key regulators of actin nucleation and elongation. Diaphanous-related formins, the best-known subclass, are activated by Rho and play essential roles in cytokinesis. In cultured cells, Diaphanous-related formins also regulate cell adhesion, polarity and microtubules, suggesting that they may be key regulators of cell shape change and migration during development. However, their essential roles in cytokinesis hamper our ability to test this hypothesis. We used loss- and gain-of-function approaches to examine the role of Diaphanous in Drosophila morphogenesis. We found that Diaphanous has a dynamic expression pattern consistent with a role in regulating cell shape change. We used constitutively active Diaphanous to examine its roles in morphogenesis and its mechanisms of action. This revealed an unexpected role in regulating myosin levels and activity at adherens junctions during cell shape change, suggesting that Diaphanous helps coordinate adhesion and contractility of the underlying actomyosin ring. We tested this hypothesis by reducing Diaphanous function, revealing striking roles in stabilizing adherens junctions and inhibiting cell protrusiveness. These effects also are mediated through coordinated effects on myosin activity and adhesion, suggesting a common mechanism for Diaphanous action during morphogenesis.     

Cdc42 regulates extracellular matrix remodeling in three dimensions

Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce nonspecific effects. Here, we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of matrix metalloproteinase 9 (MMP9) and altered fibronectin deposition patterning. A Cdc42 mutant rescue experiment found that downstream of Cdc42, p21-activated kinase (PAK), but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation, and vesicle trafficking, Cdc42 is essential in ECM remodeling in three dimensions.