Sheet Migration Research Articles

E-cadherin Is Critical for Collective Sheet Migration and Is Regulated by the Chemokine CXCL12 Protein During Restitution

Chemokines and other immune mediators enhance epithelial barrier repair. The intestinal barrier is established by highly regulated cell-cell contacts between epithelial cells. The goal of these studies was to define the role for the chemokine CXCL12 in regulating E-cadherin during collective sheet migration during epithelial restitution. Mechanisms regulating E-cadherin were investigated using Caco2(BBE) and IEC-6 model epithelia. Genetic knockdown confirmed a critical role for E-cadherin in in vitro restitution and in vivo wound repair. During restitution, both CXCL12 and TGF-β1 tightened the monolayer by decreasing the paracellular space between migrating epithelial cells. However, CXCL12 differed from TGF-β1 by stimulating the significant increase in E-cadherin membrane localization during restitution. Chemokine-stimulated relocalization of E-cadherin was paralleled by an increase in barrier integrity of polarized epithelium during restitution. CXCL12 activation of its cognate receptor CXCR4 stimulated E-cadherin localization and monolayer tightening through Rho-associated protein kinase activation and F-actin reorganization. These data demonstrate a key role for E-cadherin in intestinal epithelial restitution.

E-cadherin plays an essential role in collective directional migration of large epithelial sheets

In wound healing and development, large epithelial sheets migrate collectively, in defined directions, and maintain tight cell-cell adhesion. This type of movement ensures an essential function of epithelia, a barrier, which is lost when cells lose connection and move in isolation. Unless wounded, epithelial sheets in cultures normally do not have overall directional migration. Cell migration is mostly studied when cells are in isolation and in the absence of mature cell-cell adhesion; the mechanisms of the migration of epithelial sheets are less well understood. We used small electric fields (EFs) as a directional cue to instigate and guide migration of epithelial sheets. Significantly, cells in monolayer migrated far more efficiently and directionally than cells in isolation or smaller cell clusters. We demonstrated for the first time the group size-dependent directional migratory response in several types of epithelial cells. Gap junctions made a minimal contribution to the directional collective migration. Breaking down calcium-dependent cell-cell adhesion significantly reduced directional sheet migration. Furthermore, E-cadherin blocking antibodies abolished migration of cell sheets. Traction force analysis revealed an important role of forces that cells in the leading rows exert on the substratum. With EF, the traction forces of the leading edge cells coordinated in directional re-orientation. Our study thus identifies a novel mechanism--E-cadherin dependence and coordinated traction forces of leading cells in collective directional migration of large epithelial sheets.

Requirement for Pak3 in Rac1-induced organization of actin and myosin during Drosophila larval wound healing

Rho-family small GTPases regulate epithelial cell sheet migration by organizing actin and myosin during wound healing. Here, we report that Pak3, but not Pak1, is a downstream target protein for Rac1 in wound closure of the Drosophila larval epidermis. Pak3-deficient larvae failed to close a wound hole and this defect was not rescued by Pak1 expression, indicating differential functions of the two proteins. Pak3 localized to the wound margin, which selectively required Rac1. Pak3-deficient larvae showed severe defects in actin-myosin organization at the wound margin and in submarginal cells, which was reminiscent of the phenotypes of Rac1-deficient larvae. These results suggest that Pak3 specifically mediates Rac1 signaling in organizing actin and myosin during Drosophila epidermal wound healing.

Graphene quantum resistive sensing skin for the detection of alteration biomarkers

Sensing skins made of reduced graphene oxide (RGO) based quantum resistive vapour sensors (vQRS) have been developed by combining two original processes, i.e., the synthesis of phase transferable graphene sheets using ionic liquid polymers (PIL) and the structuring of 3D conducting architectures by the spray layer by layer (sLbL) technique. Many advantages can be derived from this new technology, such as versatility of fabrication (sprayability, no need for a clean room), flexibility, potential transparency and low cost, making vQRS skins very attractive to develop the next generation e-noses with quick response time (less than 3 s), room temperature operability, high sensitivity and adjustable selectivity. This can open the door to a wide range of applications, in particular smart packaging, making the monitoring of the quality/safety of food possible by following VOC biomarkers emitted during its alteration. RGO based QRS are also expected to be biocompatible, exempt from cytotoxicity and the risks of migration of RGO sheets in food must be very limited, thanks to their very large surface/thickness ratio and to their embedment into a polymer matrix. Comparing pristine RGO, RGO–PIL and RGO–PIL/PEDOT QRS undoubtedly establishes the superiority of the latter in terms of sensitivity and selectivity for the detection of volatile organic solvents (VOCs) released from food during its degradation. The reason for this can be found in the unique architecture of the transducer, optimizing functionalization in solution by the combined action of PIL and PEDOT and structuring in the solid state by the step by step assembly in 3D by sLbL.

Controlling Embryonic Cell Sheet Migration using Microfluidics

Embryonic development consists of a complex series of cell signaling, cell migration and cell differentiation events whereas morphogenesis is the process that controls the organized spatiotemporal distributions of these events. Cell sheet migration is central to embryonic development, complex organs system and diseases, therefore studying cell migration within sheets is important. Embryos from the African Claw-toed frog, Xenopus laevis, are used to elucidate genes important in moving cells. However, little is known about the underlying mechanism by which cells in epithelial sheets coordinate their responses to growth factors, directional signals, and motility cues to direct sheet movement in vivo. One reason for this knowledge gap is the lack of technologies to control of the chemical microenvironment surrounding multicellular tissues. Here, we manipulate the chemical microenvironment with precise spatiotemporal delivery of cytoskeletal inhibitors and contraction-stimulating compounds using laminar flow interfaces with microfluidics. We use this approach to study motility of the cells within the sheets to these localized environments and understand how their coordinated behavior works with spatiotemporal control.Microfluidics provides an opportunity to control chemical stimulation of biological systems using laminar flow interfaces with common flow modulation methods. To deliver precise chemical stimulation to a multicellular tissue, we have developed a system for controlling the inlet pressures to a microfluidic device by modulating fluidic resistance and capacitance. We employed this system to deliver chemicals over tissue explants from Xenopus embryos with a spatiotemporal control to study mechanical patterning and local control of cell sheet migration during epibolic-type morphogenetic movements.We believe that patterning cell mechanics and controlling cell motility provide a means to initiate synthetic morphogenetic programs. In addition, the ability to control the form of multicellular tissues potentially has high impact in tissue engineering and regeneration applications in bioengineering and medicine.

TGFβ (transforming growth factor β) and keratocyte motility in 24 h zebrafish explant cultures

Fish keratocytes are used as a model system for the study of the mechanics of cell motility because of their characteristic rapid, smooth gliding motion, but little work has been done on the regulation of fish keratocyte movement. As TGFβ (transforming growth factor β) plays multiple roles in primary human keratinocyte cell migration, we investigated the possible involvement of TGFβ in fish keratocyte migration. Studying the involvement of TGFβ1 in 24 h keratocyte explant allows the examination of the cells before alterations in cellular physiology occur due to extended culture times. During this initial period, TGFβ levels increase 6.2-fold in SFM (serum-free medium) and 2.4-fold in SFM+2% FBS (fetal bovine serum), while TGFβ1 and TGFβRII (TGFβ receptor II) mRNA levels increase ∼3- and ∼5-fold respectively in each culture condition. Two measures of motility, cell sheet area and migration distance, vary with the amount of exogenous TGFβ1 and culture media. The addition of 100 ng/ml exogenous TGFβ1 in SFM increases both measures [3.3-fold (P = 4.5×10-5) and 26% (P = 2.1×10-2) respectively]. In contrast, 100 ng/ml of exogenous TGFβ1 in medium containing 2% FBS decreases migration distance by 2.1-fold (P = 1.7×10-7), but does not affect sheet area. TGFβ1 (10 ng/ml) has little effect on cell sheet area in SFM cultures, but leads to a 1.8-fold increase (P = 1.5×10-2) with 2% FBS. The variable response to TGFβ1 may be, at least in part, explained by the effect of 2% FBS on cell morphology, mode of motility and expression of endogenous TGFβ1 and TGFβRII. Together, these results suggest that expression of TGFβ and its receptor are up-regulated during zebrafish keratocyte explant culture and that TGFβ promotes fish keratocyte migration.

Tension, Free Space, and Cell Damage in a Microfluidic Wound Healing Assay

We use a novel, microfluidics-based technique to deconstruct the classical wound healing scratch assay, decoupling the contribution of free space and cell damage on the migratory dynamics of an epithelial sheet. This method utilizes multiple laminar flows to selectively cleave cells enzymatically, and allows us to present a ‘damage free’ denudation. We therefore isolate the influence of free space on the onset of sheet migration. First, we observe denudation directly to measure the retraction in the cell sheet that occurs after cell-cell contact is broken, providing direct and quantitative evidence of strong tension within the sheet. We further probe the mechanical integrity of the sheet without denudation, instead using laminar flows to selectively inactivate actomyosin contractility. In both cases, retraction is observed over many cell diameters. We then extend this method and complement the enzymatic denudation with analogies to wounding, including gradients in signals associated with cell damage, such as reactive oxygen species, suspected to play a role in the induction of movement after wounding. These chemical factors are evaluated in combination with the enzymatic cleavage of cells, and are assessed for their influence on the collective migration of a non-abrasively denuded epithelial sheet. We conclude that free space alone is sufficient to induce movement, but this movement is predominantly limited to the leading edge, leaving cells further from the edge less able to move towards the wound. Surprisingly, when coupled with a gradient in ROS to simulate the chemical effects of abrasion however, motility was not restored, but further inhibited.

Application of Small Organic Molecules Reveals Cooperative TGFβ and BMP Regulation of Mesothelial Cell Behaviors

Epicardial development is a process during which epithelial sheet movement, single cell migration, and differentiation are coordinated to generate coronary arteries. Signaling cascades regulate the concurrent and complex nature of these three events. Through simple and highly reproducible assays, we identified small organic molecules that impact signaling pathways regulating these epicardial behaviors. Subsequent biochemical analyses confirmed the specificity of these reagents and revealed novel targets for the widely used dorsomorphin (DM) and LDN-193189 molecules. Using these newly characterized reagents, we show the broad regulation of epicardial cell differentiation, sheet movement, and single cell migration by Transforming Growth Factor β (TGFβ). With the DM analogue DMH1, a highly specific Bone Morphogenetic Protein (BMP) inhibitor, we demonstrate the cooperative yet exclusive role for BMP signaling in regulation of sheet migration. The action of DMH1 reveals that small organic molecules (SOM) can intervene on a single epicardial behavior while leaving other concurrent behaviors intact. All SOM data were confirmed by reciprocal experiments using growth factor addition and/or application of established non-SOM inhibitors. These compounds can be applied to cell lines or native proepicardial tissue. Taken together, these data establish the efficacy of chemical intervention for analysis of epicardial behaviors and provide novel reagents for analysis of epicardial development and repair.

Tumor suppressor Lzap regulates cell cycle progression, doming, and zebrafish epiboly

Initial stages of embryonic development rely on rapid, synchronized cell divisions of the fertilized egg followed by a set of morphogenetic movements collectively called epiboly and gastrulation. Lzap is a putative tumor suppressor whose expression is lost in 30% of head and neck squamous cell carcinomas. Lzap activities include regulation of cell cycle progression and response to therapeutic agents. Here, we explore developmental roles of the lzap gene during zebrafish morphogenesis. Lzap is highly conserved among vertebrates and is maternally deposited. Expression is initially ubiquitous during gastrulation, and later becomes more prominent in the pharyngeal arches, digestive tract, and brain. Antisense morpholino-mediated depletion of Lzap resulted in delayed cell divisions and apoptosis during blastomere formation, resulting in fewer, larger cells. Cell cycle analysis suggested that Lzap loss in early embryonic cells resulted in a G2/M arrest. Furthermore, the Lzap-deficient embryos failed to initiate epiboly--the earliest morphogenetic movement in animal development--which has been shown to be dependent on cell adhesion and migration of epithelial sheets. Our results strongly implicate Lzap in regulation of cell cycle progression, adhesion and migratory activity of epithelial cell sheets during early development. These functions provide further insight into Lzap activity that may contribute not only to development, but also to tumor formation.

Partial Restraint Stress Induces Increased Permeability and Low Grade Inflammation in the Rat Ileum, but Not in the Jejunum

Mixed-lineage kinase-3 (MLK3) activates multiple MAPK pathways and has been shown to initiate apoptosis, proliferation, migration or differentiation in several cell types. MLK3 signaling is also required for breast cancer cell invasion. However, the role of MLK3 signaling in regulation of intestinal epithelial cell sheet migration In Vivo and In Vitro is not known. Given its role in cancer cell migration, we sought to investigate whether MLK3 signaling is important in intestinal mucosal healing and epithelial cell motility. We compared the healing of jejunal mucosal ulcers induced by topical serosal acetic acid in different age groups of MLK3 null mice with that in age-matched wild type mice and assessed the role of MLK3 signaling in human Caco-2 intestinal epithelial migration across type I collagen substrates. In older mice (30-42 weeks of age), ulcer healing at 5 days after the initial insult was reduced by 47% (p<0.05) in MLK3 null and by 29% in MLK3 heterozygous mice when compared with wild-type littermate controls. In younger animals (8-12 weeks), ulcer healing in MLK3 null mice was 25% less at day 3 (n=7, p<0.05) and 23% less at day 5 (n=7, p<0.05) than in wild-type mice. In Vitro, an MLK3 inhibitor significantly decreased closure of circular wounds in Caco-2 cell monolayers after 24 hours compared with to vehicle-treated control by 16.3% and 20.1 % respectively (p<0.05). Proliferative blockade using hydroxyurea (30mM) did not abolish the effect of MLK3 inhibitor on Caco-2 wound closure, suggesting a true effect on cell motility rather than an effect on cell proliferation. The reduction in cell motility by MLK3 inhibitor reduced level of pERK by 79% (p<0.05) and pJNK by 12% (p<0.05) revealing suppression of ERK and JNK signaling. Total MLK3 was also compared in confluent Caco-2 cells to cell that have been migrating for 96 hours. MLK3 was 53% (p<0.05) higher in migrating vs confluent cells further indicating the importance of MLK3 signaling in migrating intestinal epithelial cells. These studies reveal a novel role for MLK3 signaling in the regulation of intestinal epithelial migration and suggest that MLK3 may be an important target to influence intestinal mucosal healing.

Abstract 2183: Tumor suppressor LZAP regulates cell cycle progression and doming during zebrafish epiboly

Abstract Initial stages of embryonic development rely on rapid, synchronized cell divisions of the fertilized egg followed by a set of morphogenetic movements collectively called epiboly and gastrulation. Lzap is a putative tumor suppressor whose expression is lost in 30% of head and neck squamous cell carcinomas. Lzap activities include regulation of cell cycle progression and response to therapeutic agents. Here we explore developmental roles of the lzap gene during zebrafish morphogenesis. Lzap is highly conserved among vertebrates and is maternally deposited. Expression is initially ubiquitous during gastrulation, and later becomes more prominent in the pharyngeal arches, digestive tract and brain. Antisense morpholino-mediated depletion of Lzap resulted in delayed cell divisions during blastomere formation, resulting in fewer, larger cells. Furthermore, the Lzap-deficient embryos failed to initiate epiboly – the earliest morphogenetic movement in animal development – which has been shown to be dependent on cell adhesion and migration of epithelial sheets. Our results strongly implicate Lzap in regulation of cell cycle progression, adhesion and migratory activity of epithelial cell sheets during early development. These functions provide further insight into Lzap activity that may contribute not only to development, but also to tumor formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2183. doi:10.1158/1538-7445.AM2011-2183

A Steering Model of Endothelial Sheet Migration Recapitulates Monolayer Integrity and Directed Collective Migration

Cells in endothelial cell monolayers maintain a tight barrier between blood and tissue, but it is not well understood how endothelial cells move within monolayers, pass each other, migrate when stimulated with growth factor, and also retain monolayer integrity. Here, we develop a quantitative steering model based on functional classes of genes identified previously in a small interfering RNA (siRNA) screen to explain how cells locally coordinate their movement to maintain monolayer integrity and collectively migrate in response to growth factor. In the model, cells autonomously migrate within the monolayer and turn in response to mechanical cues resulting from adhesive, drag, repulsive, and directed steering interactions with neighboring cells. We show that lateral-drag steering explains the local coordination of cell movement and the maintenance of monolayer integrity by allowing closure of small lesions. We further demonstrate that directional steering of cells at monolayer boundaries, combined with adhesive steering of cells behind, can explain growth factor-triggered collective migration into open space. Together, this model provides a mechanistic explanation for the observed genetic modularity and a conceptual framework for how cells can dynamically maintain sheet integrity and undergo collective directed migration.

A Targeted UAS-RNAi Screen in Drosophila Larvae Identifies Wound Closure Genes Regulating Distinct Cellular Processes

Robust mechanisms for tissue repair are critical for survival of multicellular organisms. Efficient cutaneous wound repair requires the migration of cells at the wound edge and farther back within the epidermal sheet, but the genes that control and coordinate these migrations remain obscure. This is in part because a systematic screening approach for in vivo identification and classification of postembryonic wound closure genes has yet to be developed. Here, we performed a proof-of-principle reporter-based in vivo RNAi screen in the Drosophila melanogaster larval epidermis to identify genes required for normal wound closure. Among the candidate genes tested were kinases and transcriptional mediators of the Jun N-terminal kinase (JNK) signaling pathway shown to be required for epithelial sheet migration during development. Also targeted were genes involved in actin cytoskeletal remodeling. Importantly, RNAi knockdown of both canonical and noncanonical members of the JNK pathway caused open wounds, as did several genes involved in actin cytoskeletal remodeling. Our analysis of JNK pathway components reveals redundancy among the upstream activating kinases and distinct roles for the downstream transcription factors DJun and DFos. Quantitative and qualitative morphological classification of the open wound phenotypes and evaluation of JNK activation suggest that multiple cellular processes are required in the migrating epidermal cells, including functions specific to cells at the wound edge and others specific to cells farther back within the epidermal sheet. Together, our results identify a new set of conserved wound closure genes, determine putative functional roles for these genes within the migrating epidermal sheet, and provide a template for a broader in vivo RNAi screen to discover the full complement of genes required for wound closure during larval epidermal wound healing.

The leading edge during dorsal closure as a model for epithelial plasticity: Pak is required for recruitment of the Scribble complex and septate junction formation

Dorsal closure (DC) of the Drosophila embryo is a model for the study of wound healing and developmental epithelial fusions, and involves the sealing of a hole in the epidermis through the migration of the epidermal flanks over the tissue occupying the hole, the amnioserosa. During DC, the cells at the edge of the migrating epidermis extend Rac- and Cdc42-dependent actin-based lamellipodia and filopodia from their leading edge (LE), which exhibits a breakdown in apicobasal polarity as adhesions are severed with the neighbouring amnioserosa cells. Studies using mammalian cells have demonstrated that Scribble (Scrib), an important determinant of apicobasal polarity that functions in a protein complex, controls polarized cell migration through recruitment of Rac, Cdc42 and the serine/threonine kinase Pak, an effector for Rac and Cdc42, to the LE. We have used DC and the follicular epithelium to study the relationship between Pak and the Scrib complex at epithelial membranes undergoing changes in apicobasal polarity and adhesion during development. We propose that, during DC, the LE membrane undergoes an epithelial-to-mesenchymal-like transition to initiate epithelial sheet migration, followed by a mesenchymal-to-epithelial-like transition as the epithelial sheets meet up and restore cell-cell adhesion. This latter event requires integrin-localized Pak, which recruits the Scrib complex in septate junction formation. We conclude that there are bidirectional interactions between Pak and the Scrib complex modulating epithelial plasticity. Scrib can recruit Pak to the LE for polarized cell migration but, as migratory cells meet up, Pak can recruit the Scrib complex to restore apicobasal polarity and cell-cell adhesion.

663 Age dependence of T-cell lymphoma induction by radiation exposure in Mlh1-deficient mice

Ezrin/radixin/moesin (ERM) proteins are membrane-cytoskeleton linkers that also have roles in signal transduction. Here we show that G protein-coupled receptor kinase 2 (GRK2) regulates membrane protrusion and cell migration during wound closure in Madin-Darby canine kidney (MDCK) epithelial cell monolayers at least partly through activating phosphorylation of radixin on a conserved, regulatory C-terminal Thr residue. GRK2 phosphorylated radixin exclusively on Thr 564 in vitro. Expression of a phosphomimetic (Thr-564-to-Asp) mutant of radixin resulted in increased Rac1 activity, membrane protrusion and cell motility in MDCK cells, suggesting that radixin functions “upstream” of Rac1, presumably as a scaffolding protein. Phosphorylation of ERM proteins was highest during the most active phase of epithelial cell sheet migration over the course of wound closure. In view of these results, we explored the mode of action of quinocarmycin/quinocarcin analog DX-52-1, an inhibitor of cell migration and radixin function with considerable selectivity for radixin over the other ERM proteins, finding that its mechanism of inhibition of radixin does not appear to involve binding and antagonism at the site of regulatory phosphorylation.

The leading edge during dorsal closure as a model for epithelial plasticity: Pak is required for recruitment of the Scribble complex and septate junction formation

Dorsal closure (DC) of the Drosophila embryo is a model for the study of wound healing and developmental epithelial fusions, and involves the sealing of a hole in the epidermis through the migration of the epidermal flanks over the tissue occupying the hole, the amnioserosa. During DC, the cells at the edge of the migrating epidermis extend Rac- and Cdc42-dependent actin-based lamellipodia and filopodia from their leading edge (LE), which exhibits a breakdown in apicobasal polarity as adhesions are severed with the neighbouring amnioserosa cells. Studies using mammalian cells have demonstrated that Scribble (Scrib), an important determinant of apicobasal polarity that functions in a protein complex, controls polarized cell migration through recruitment of Rac, Cdc42 and the serine/threonine kinase Pak, an effector for Rac and Cdc42, to the LE. We have used DC and the follicular epithelium to study the relationship between Pak and the Scrib complex at epithelial membranes undergoing changes in apicobasal polarity and adhesion during development. We propose that, during DC, the LE membrane undergoes an epithelial-to-mesenchymal-like transition to initiate epithelial sheet migration, followed by a mesenchymal-to-epithelial-like transition as the epithelial sheets meet up and restore cell-cell adhesion. This latter event requires integrin-localized Pak, which recruits the Scrib complex in septate junction formation. We conclude that there are bidirectional interactions between Pak and the Scrib complex modulating epithelial plasticity. Scrib can recruit Pak to the LE for polarized cell migration but, as migratory cells meet up, Pak can recruit the Scrib complex to restore apicobasal polarity and cell-cell adhesion.

Electrical estimulation of retinal pigment epithelial cells

We investigated and characterized the effect of externally applied electric fields (EF) on retinal pigment epithelial (RPE) cells by exposing primary cultures of human RPE cells (hRPE) and those from the ARPE19 immortalized cell line to various strengths of EF (EF-treated cells) or to no EF (control cells) under different conditions including presence or absence of serum and gelatin and following wounding. We evaluated changes in RPE cell behavior in response to EF by using a computer based image capture and analysis system (Metamorph). We found that RPE cells responded to externally applied EFs by preferential orientation perpendicular to the EF vector, directed migration towards the anode, and faster translocation rate than control, untreated cells. These responses were voltage-dependent. Responses were observed even at low voltages, of 50–300 mV. Furthermore, the migration of hRPE cell sheets generated by wounding of confluent monolayers of cells at early and late confluence could be manipulated by the application of EF, with directed migration towards the anode observed at both sides of the wounded hRPE. In conclusion, RPE cell behaviour can be controlled by an externally applied EF. The potential for externally applied EF to be used as a therapeutic strategy in the management of selected retinal diseases warrants further investigation.

Silurian-Devonian age and tectonic setting of the Connecticut Valley-Gaspe trough in Vermont based on U-Pb SHRIMP analyses of detrital zircons

U-Pb SHRIMP ages of detrital zircons from metasedimentary rocks of the Connecticut Valley-Gaspé trough in Vermont corroborate a Silurian-Devonian age of deposition for these strata and constrain their provenances. Ages of randomly selected detrital zircons obtained from quartzites within the Waits River and Gile Mountain Formations range from Archean to Devonian with Mesoproterozoic, Neoproterozoic, Ordovician, and Silurian age populations suggesting both eastern and western sources of the sediments. The two youngest single-grain detrital zircon ages from samples collected in the Waits River Formation are 418 ± 7 and 415 ± 2 Ma. The youngest single-grain detrital zircon age from the eastern part of the Gile Mountain Formation is 411 ± 8. The youngest detrital zircons from the western portion of the Gile Mountain Formation comprise an age population with a weighted average of 409 ± 5 Ma. These ∼409 Ma zircons are likely of volcanic origin, perhaps derived from the Piscataquis magmatic belt to the east. The absence of younger volcanic zircons in the coarser-grained eastern facies of the Gile Mountain Formation suggests the eastern sediments are older and were buried during Piscataquis volcanism and deposition in the west. The shift in protoliths from calcareous silts and muds of the Waits River Formation to quartzo-feldspathic sands of the Gile Mountain Formation implies a change from a continental slope-like depositional environment to a near-shore or terrestrial environment of deposition. This change supports a transition in the nature of the basin from an intercontinental back-arc extensional setting to a foreland basin setting. Maximum depositional ages of sediments above and below this facies boundary constrain the timing of transition in basin style between about 415 and 411 Ma. Given the timing of the approaching Acadian wedge, this shift in basin style likely reflects westward migration of thrust sheets during the Acadian orogeny. The fine-grained nature of the youngest silts, muds and turbidites suggests that sedimentation occurred in increasingly deeper water. The implied basin subsidence was likely caused by lithospheric flexure as the Acadian wedge approached from the east. The timing of this subsidence is constrained to be younger than the youngest zircons at about 409 Ma.

Abstract 4811: Compression-induced cell distension stimulates coordinated migration of mammary carcinoma cells

Abstract Uncontrolled cell proliferation of a solid tumor in a confined space not only creates oxidative stress (hypoxia), but also generates mechanical compressive stress, which can influence the tumor cells and modify their interactions with neighboring cells and the extracellular matrix. Little is known about how such compressive stress affects cancer progression. Here we show, for the first time, that externally-applied compressive stress results in faster migration of mammary carcinoma cells. Compression induces migration of mammary carcinoma cells in a coordinated sheet, initiated by “leader cells” - single cells at the leading edge of the sheet, extending long filopodia. The formation of leader cells is dependent on the geometry of association with neighboring cells, but the frequency of leader-cell formation - and sheet migration speed - is increased by applying compression perpendicular to the sheet. Accompanied by redistribution of fibronectin deposition, applied compression enhances cell-matrix adhesion and stabilizes cell distension independent of actomyosin contractile machinery. Our results suggest that mechanical stress accumulated during tumor growth is sufficient to enhance cell-substrate adhesion and stimulate cancer cell migration. This mechanism opens the door to a new class of targets for blocking mechanical stress pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4811.

Upregulation proliferation and senescence markers in hyperplasia induced in mouse skin by sulfur mustard exposure

Chemical vesicants such as sulfur mustard (SM) can produce severe tissue damage. A time‐course study using mouse ear skin exposed to a low dose (0.08 mg) of SM resulted in excessive keratinocyte hyperplasia about three days after exposure. The abnormal cell proliferation was reminiscent of that observed in several types of cancer and potential oncogenic hyperproliferative signal molecules were examined to determine if the two systems were comparable. Immunohistochemical, Western blotting and RT‐PCR studies were performed and markers of skin injury and repair were examined for various time‐points. The protein, p16‐INK4a, which triggers either growth arrest or apoptosis was hyperexpressed in the hyperplasia areas of injured skin. The p16‐INK4a co‐expressed with laminin γ2, a polypeptide that promotes epithelial sheet migration over a wound bed. The endoplasmic reticulum stress‐regulated gene, GADD153/CHOP had a similar expression pattern to that of p16‐INK4a. In contrast, both keratin 5, an intermediate filament protein of basal epithelial cells and Ki67, a marker of cells in the growth stage of the cell cycle had a much more restricted distribution. Understanding the basic mechanism of action of vesicant‐induced skin injury and repair should help to identify new targets of potential medical countermeasures against chemical vesicants. This work is supported by ES005022, EY09056, and AR055073.Grant Funding Source: ES005022, EY09056, and AR055073.