Sheet Migration Research Articles

New Opportunities for Electric Fields in Promoting Wound Healing: Collective Electrotaxis.

Significance: It has long been hypothesized that naturally occurring electric fields (EFs) aid wound healing by guiding cell migration. Consequently, the application of EFs has significant potential for promoting wound healing. However, the mechanisms underlying the cellular response to EFs remain unclear. Recent Advances: Although the directed migration of isolated single cells under EFs has been studied for decades, only recently has experimental evidence demonstrated the distinct collective migration of large sheets of keratinocytes and corneal epithelial cells in response to applied EFs. Accumulating evidence suggests that the emergent properties of cell groups in response to EF guidance offer new opportunities for EF-assisted directional migration. Critical Issues: In this review, we provide an overview of the field of collective electrotaxis, highlighting key advances made in recent years. We also discuss advanced engineering strategies utilized to manipulate collective electrotaxis. Future Directions: We outline a series of unanswered questions in this field and propose potential applications of collective electrotaxis in developing electrical stimulation technologies for wound healing.

α-catenin middle- and actin-binding domain unfolding mutants differentially impact epithelial strength and sheet migration.

α-catenin (α-cat) displays force-dependent unfolding and binding to actin filaments through direct and indirect means, but features of adherens junction structure and function most vulnerable to loss of these allosteric mechanisms have not been directly compared. By reconstituting an α-cat F-actin-binding domain unfolding mutant known to exhibit enhanced binding to actin (α-cat-H0-FABD+) into α-cat knockout Madin Darby Canine Kidney (MDCK) cells, we show that partial loss of the α-cat catch bond mechanism (via an altered H0 α-helix) leads to stronger epithelial sheet integrity with greater colocalization between the α-cat-H0-FABD+ mutant and actin. α-cat-H0-FABD+ -expressing cells are less efficient at closing scratch-wounds, suggesting reduced capacity for more dynamic cell-cell coordination. Evidence that α-cat-H0-FABD+ is equally accessible to the conformationally sensitive α18 antibody epitope as WT α-cat and shows similar vinculin recruitment suggests this mutant engages lower tension cortical actin networks, as its M-domain is not persistently open. Conversely, α-cat-M-domain salt-bridge mutants with persistent recruitment of vinculin and phosphorylated myosin light chain show only intermediate monolayer adhesive strengths, but display less directionally coordinated and thereby slower migration speeds during wound-repair. These data show α-cat M- and FABD-unfolding mutants differentially impact cell-cell cohesion and migration properties, and suggest signals favoring α-cat-cortical actin interaction without persistent M-domain opening may improve epithelial monolayer strength through enhanced coupling to lower tension actin networks.

Protease-activated receptor 2 drives migration in a colon cancer cell line but not in noncancerous human epithelial cells.

The inflamed mucosa contains a complex assortment of proteases that may participate in wound healing or the development of inflammation-associated colon cancer. We sought to determine the role of protease-activated receptor 2 (PAR2) in epithelial wound healing in both untransformed and transformed colonic epithelial cells. Monolayers of primary epithelial cells derived from organoids cultivated from patient colonic biopsies and of the T84 colon cancer cell line were grown to confluence, wounded in the presence of a selective PAR2-activating peptide, and healing was visualized by live cell microscopy. Inhibitors of various signaling molecules were used to assess the relevant pathways responsible for wound healing. Activation of PAR2 induced an enhanced wound-healing response in T84 cells but not primary cells. The PAR2-enhanced wound-healing response was associated with the development of lamellipodia in cells at the wound edge, consistent with sheet migration. The response to PAR2 activation in T84 cells was completely dependent on Src kinase activity and partially dependent on Rac1 activity. The Src-associated signaling molecules, focal adhesion kinase, and epidermal growth factor receptor, which typically mediate wound-healing responses, were not involved in the PAR2 response. Experiments repeated in the presence of the inflammatory cytokines TNF and IFNγ revealed a synergistically enhanced PAR2 wound-healing response in T84s but not primary cells. The epithelial response to proteases may be different between primary and cancer cells and is accentuated in the presence of inflammatory cytokines. Our findings have implications for understanding epithelial restitution in the context of inflammatory bowel disease (IBD) and inflammation-associated colon cancer.NEW & NOTEWORTHY Protease-activated receptor 2 enhances wound healing in the T84 colon cancer cell line, but not in primary cells derived from patient biopsies, an effect that is synergistically enhanced in the presence of the inflammatory cytokines TNF and IFNγ.

Sustained intestinal epithelial monolayer wound closure after transient application of a FAK-activating small molecule.

M64HCl, which has drug-like properties, is a water-soluble Focal Adhesion Kinase (FAK) activator that promotes murine mucosal healing after ischemic or NSAID-induced injury. Since M64HCl has a short plasma half-life in vivo (less than two hours), it has been administered as a continuous infusion with osmotic minipumps in previous animal studies. However, the effects of more transient exposure to M64HCl on monolayer wound closure remained unclear. Herein, we compared the effects of shorter M64HCl treatment in vitro to continuous treatment for 24 hours on monolayer wound closure. We then investigated how long FAK activation and downstream ERK1/2 activation persist after two hours of M64HCl treatment in Caco-2 cells. M64HCl concentrations immediately after washing measured by mass spectrometry confirmed that M64HCl had been completely removed from the medium while intracellular concentrations had been reduced by 95%. Three-hour and four-hour M64HCl (100 nM) treatment promoted epithelial sheet migration over 24 hours similar to continuous 24-hour exposure. 100nM M64HCl did not increase cell number. Exposing cells twice with 2-hr exposures of M64HCl during a 24-hour period had a similar effect. Both FAK inhibitor PF-573228 (10 μM) and ERK kinase (MEK) inhibitor PD98059 (20 μM) reduced basal wound closure in the absence of M64HCl, and each completely prevented any stimulation of wound closure by M64HCl. Rho kinase inhibitor Y-27632 (20 μM) stimulated Caco-2 monolayer wound closure but no further increase was seen with M64HCl in the presence of Y-27632. M64HCl (100 nM) treatment for 3 hours stimulated Rho kinase activity. M64HCl decreased F-actin in Caco-2 cells. Furthermore, a two-hour treatment with M64HCl (100 nM) stimulated sustained FAK activation and ERK1/2 activation for up to 16 and hours 24 hours, respectively. These results suggest that transient M64HCl treatment promotes prolonged intestinal epithelial monolayer wound closure by stimulating sustained activation of the FAK/ERK1/2 pathway. Such molecules may be useful to promote gastrointestinal mucosal repair even with a relatively short half-life.

Analysis of tsagaan lake sediment, valley of the gobi lakes, Mongolia, to determine past environmental changes and the effect of ongoing global warming

The arid region of Mongolia is sensitive to climate change. Understanding of climate variations in the past plays an important role in predicting further climate changes. In this study, we aim to investigate the relationship between the climatic factor and sedimentation history in the late Holocene. We analyzed the 18TS1 core sample (136 cm in length) from the Tsagaan lake in the Valley of the Gobi Lakes, Mongolia. The sediment core mainly consists of clay and silt sediments, occasionally interrupted by the sand-dominated layers. 210Pb dating method is applied and indicates that the upper 24 cm sediment spans the sedimentation history of the last 87 years. Along-core variations in sediment characteristics such as organic matter, carbonate, and amorphous silica abundance are indicative of the drought -inducing period based on the significant correlations between sediment characteristics and Self-calibrated Palmer Drought Severity Index. Moreover, the enrichment of carbonates is a great indicator of shallow lake conditions prior to the lake disappearance during the dry period. The chemical composition of sand-dominated units in the sediment core well agrees with that of sand sheets and sand dunes in the catchment. Modern expansion and migration of sand sheets are identified by aerial photo imagery. The insufficient sediment chronology is limiting our interpretation, though; the comparison with the other paleoclimate records helps the establishment of environmental changes in the last millennium.

Mesenchymal Stem Cell Extracellular Vesicles from Tissue-Mimetic System Enhance Epidermal Regeneration via Formation of Migratory Cell Sheets.

The secretome of adipose-derived mesenchymal stem cells (ASCs) offers a unique approach to understanding and treating wounds, including the critical process of epidermal regeneration orchestrated by keratinocytes. However, 2D culture techniques drastically alter the secretory dynamics of ASCs, which has led to ambiguity in understanding which secreted compounds (e.g., growth factors, exosomes, reactive oxygen species) may be driving epithelialization. A novel tissue-mimetic 3D hydrogel system was utilized to enhance the retainment of a moreregenerative ASC phenotype and highlight the functional secretome differences between 2D and 3D. Subsequently, the ASC-secretome was stratified by molecular weight and the presence/absence of extracellular vesicles (EVs). The ASC-secretome fractions were then evaluated to assess for the capacity to augment specific keratinocyte activities. Culture of ASCs within the tissue-mimetic system enhanced protein secretion ~ 50%, exclusively coming from the > 100kDa fraction. The ASC-secretome ability to modulate epithelialization functions, including migration, proliferation, differentiation, and morphology, resided within the "> 100kDa" fraction, with the 3D ASC-secretome providing the greatest improvement. 3D ASCEV secretion was enhanced two-fold and exhibited dose-dependent effects on epidermal regeneration. Notably, ASC-EVs induced morphological changes in keratinocytes reminiscent of native regeneration, including formation of stratified cell sheets. However, only 3D-EVs promoted collective cell sheet migration and an epithelial-to-mesenchymal-like transition in keratinocytes, whereas 2D-EVs contained an anti-migratory stimulus. This study demonstrates how critical the culture environment is on influencing ASC-secretome regenerative capabilities. Additionally, the critical role of EVs in modulating epidermal regeneration is revealed and their translatability for future clinical therapies is discussed.

CD9 negatively regulates collective electrotaxis of the epidermal monolayer by controlling and coordinating the polarization of leader cells.

Endogenous electric fields (EFs) play an essential role in guiding the coordinated collective migration of epidermal cells to the wound centre during wound healing. Although polarization of leadercells is essential for collective migration, the signal mechanisms responsible for the EF-induced polarization of leader cells under electrotactic collective migration remain unclear. This study aims to determine how the leader cells are polarized and coordinated during EF-guided collective migration of epidermal cell sheets. Collective migration of the human epidermal monolayer (human immortalized keratinocytes HaCaT) under EFs was observed via time-lapse microscopy. The involvement of tetraspanin-29 (CD9) in EF-induced fibrous actin (F-actin) polarization of leader cells as well as electrotactic migration of the epidermal monolayer was evaluated by genetic manipulation. Blocking, rescue and co-culture experiments were conducted to explore the downstream signalling of CD9. EFs guided the coordinated collective migration of the epithelial monolayer to the anode, with dynamic formation of pseudopodia in leader cells at the front edge of the monolayer along the direction of migration. F-actin polarization, as expected, played an essential role in pseudopod formation in leader cells under EFs. By confocal microscopy, we found that CD9 was colocalized with F-actin on the cell surface and was particularly downregulated in leader cells by EFs. Interestingly, genetic overexpression of CD9 abolished EF-induced F-actin polarization in leader cells as well as collective migration in the epidermal monolayer. Mechanistically, CD9 determined the polarization of F-actin in leader cells by downregulating a disintegrin and metalloprotease 17/heparin-binding epidermal growth factor-like growth factor/epidermal growth factor receptor (ADAM17/HB-EGF/EGFR) signalling. The abolished polarization of leader cells due to CD9 overexpression could be restored in a co-culture monolayer where normal cells and CD9-overexpressing cells were mixed; however, this restoration was eliminated again by the addition of the HB-EGF-neutralizing antibody. CD9 functions as a key regulator in the EF-guided collective migration of the epidermal monolayer by controlling and coordinating the polarization of leader cells through ADAM17/HB-EGF/EGFR signalling.

Propagation dynamics of electrotactic motility in large epithelial cell sheets

SummaryDirectional migration initiated at the wound edge leads epithelia to migrate in wound healing. How such coherent migration is achieved is not well understood. Here, we used electric fields to induce robust migration of sheets of human keratinocytes and developed an in silico model to characterize initiation and propagation of epithelial collective migration. Electric fields initiate an increase in migration directionality and speed at the leading edge. The increases propagate across the epithelial sheets, resulting in directional migration of cell sheets as coherent units. Both the experimental and in silico models demonstrated vector-like integration of the electric and default directional cues at free edge in space and time. The resultant collective migration is consistent in experiments and modeling, both qualitatively and quantitatively. The keratinocyte model thus faithfully reflects key features of epithelial migration as a coherent tissue in vivo, e.g. that leading cells lead, and that epithelium maintains cell-cell junction.

Investigating wound healing characteristics of gingival and skin keratinocytes in organotypic cultures

ObjectivesThe gingiva heals at an accelerated rate with reduced scarring when compared to skin. Potential well-studied factors include immune cell number, angiogenesis disparities and fibroblast gene expression. Differential keratinocyte gene expression, however, remains relatively understudied. This study explored the contrasting healing efficiencies of gingival and skin keratinocytes, alongside their differential gene expression patterns. Methods3D organotypic culture models of human gingiva and skin were developed using temporarily immortalised primary keratinocytes. Models were wounded for visualisation of re-epithelialisation and analysis of keratinocyte migration to close the wound gap. Concurrently, differentially expressed genes between primary gingival and skin keratinocytes were identified, validated, and functionally assessed. ResultsCharacterisation of the 3D cultures of gingiva and skin showed differentiation markers that recapitulated organisation of the corresponding in vivo tissue. Upon wounding, gingival models displayed a significantly higher efficiency in re-epithelialisation and stratification versus skin, repopulating the wound gap within 24 hours. This difference was likely due to distinct patterns of migration, with gingival cells demonstrating a form of sheet migration, in contrast to skin, where the leading edge was typically 1-2 cells thick. A candidate approach was used to identify several genes that were differentially expressed between gingival and skin keratinocytes. Knockdown of PITX1 resulted in reduced migration capacity of gingival cells. ConclusionGingival keratinocytes retain in vivo superior wound healing capabilities in in vitro 2D and 3D environments. Intrinsic gene expression differences could result in gingival cells being ‘primed’ for healing and play a role in faster wound resolution. Clinical Significance StatementThe successful development of organotypic models, that recapitulate re-epithelialisation, will underpin further studies to analyse the oral response to wound stimuli, and potential therapeutic interventions, in an in vitro environment.

Bladder Cancer Cells Exert Pleiotropic Effects on Human Adipose-Derived Stem Cells.

Stem cell-based therapies are considered one of the most promising disciplines in biomedicine. Bladder cancer patients could benefit from therapies directed to promote healing after invasive surgeries or to lessen urinary incontinence, a common side effect of both cancer itself and the treatment. However, the local delivery of cells producing large amounts of paracrine factors may alter interactions within the microenvironment. For this reason, reconstructive cellular therapies for patients with a history of cancer carry a potential risk of tumor reactivation. We used an indirect co-culture model to characterize the interplay between adipose-derived stem cells and bladder cancer cells. Incubation with ASCs increased MCP-1 secretion by bladder cancer cells (from 2.1-fold to 8.1-fold, depending on the cell line). Cancer cell-derived factors altered ASC morphology. Cells with atypical shapes and significantly enlarged volumes appeared within the monolayer. Incubation in a conditioned medium (CM) containing soluble mediators secreted by 5637 and HB-CLS-1 bladder cancer cell lines decreased ASC numbers by 47.5% and 45.7%. A significant increase in adhesion to ECM components, accompanied by reduced motility and sheet migration, was also observed after incubation in CM from 5637 and HB-CLS-1 cells. No differences were observed when ASCs were co-cultured with HT-1376 cells. Our previous and present results indicate that soluble mediators secreted by ASCs and bladder cancer cells induce opposite effects influencing cells that represent the non-muscle-invasive urinary bladder.

Use of sugar beet cellulose nanofibers for paper coating

Sugar beet pulp (SBP) is a widely available byproduct from sugar industry in several countries. In the current work, cellulose nanofibers (CNFs) were isolated from SBP depectinated using two different methods, namely sulfuric acid hydrolysis and hydrothermal extraction, to obtain nanofibers with different residual pectic substance. The isolated CNFs had width of ~ 5 nm and several microns in length using both methods of pectin extraction. Chemical composition was studied regarding α-cellulose, pentosans, residual pectic substance, and degree of polymerization (DP). Films cast from CNFs were characterized regarding their crystallinity, mechanical properties (tensile strength, Young's modulus, and strain), water contact angle measurement, as well as by Fourier transform infrared (FTIR). The results showed higher tensile strength properties and higher hydrophilicity for films made from CNFs isolated from the hydrothermal-depectinated SBP (HDCNF) than those from the acid-depectinated SBP (ADCNF). Both of HDCNF and ADCNF were used as coating for bagasse paper sheets; the coated paper sheets were characterized regarding their tensile strength, burst strength, water vapor permeability, air porosity, and grease-proof properties. Coating of paper sheets with CNFs resulted in remarkable increase in their mechanical properties and decrease in air porosity; higher effect was recorded in case of using the HDCNF coating. Paper sheets coated with HDCNF or ADCNF showed good grease-proof property. Finally, measuring the overall migration of the CNFs-coated paper sheets showed safety of the applied coatings for food packaging applications.

Real time monitoring of the through thickness moisture profile of thin sheets using NMR

The through-thickness distribution of liquids and liquid migration over time in thin, porous media is highly relevant for the production and product properties of materials such as textiles, membranes, paper, barrier materials, and thin films. We present a 1D NMR profiling method for the analysis of through-thickness liquid distribution during liquid absorption, migration, and drying of thin sheets. The good temporal resolution (1.3 s) and high spatial resolution (10 μm) make it possible to study changes of liquid distribution in sheets of about 100 μm thickness. A key aspect of our method is the simultaneous scanning of a liquid saturated reference sheet. Using a reference, local liquid saturation and absolute amount of liquid present over time can be evaluated. We studied the drying of water-based inkjet ink on paper substrates with differing water absorption behaviors. Differences in z-directional liquid distribution, penetration depth, and drying kinetics over time could be observed.

Lack of IRF6 Disrupts Human Epithelial Homeostasis by Altering Colony Morphology, Migration Pattern, and Differentiation Potential of Keratinocytes.

Variants within the gene encoding for the transcription factor Interferon Regulatory Factor 6 (IRF6) are associated with syndromic and non-syndromic Cleft Lip/Palate (CLP) cases. IRF6 plays a vital role in the regulation of the proliferation/differentiation balance in keratinocytes and is involved in wound healing and migration. Since a fraction of CLP patients undergoing corrective cleft surgery experience wound healing complications, IRF6 represents an interesting candidate gene linking the two processes. However, Irf6 function has been mainly studied in mice and knowledge on IRF6 in human cells remains sparse. Here, we aimed to elucidate the role of IRF6 in human postnatal skin- and oral mucosa-derived keratinocytes. To do so, we applied CRISPR/Cas9 to ablate IRF6 in two TERT-immortalized keratinocyte cultures, which we used as model cell lines. We show that IRF6 controls the appearance of single cells and colonies, with the latter being less cohesive in its absence. Consequently, IRF6 knockout keratinocytes often moved as single cells instead of a collective epithelial sheet migration but maintained their epithelial character. Lack of IRF6 triggered severe keratinocyte differentiation defects, which were already apparent in the stratum spinosum and extended to the stratum corneum in 3D organotypic skin cultures, while it did not alter their growth rate. Finally, proteomics revealed that most of the differentially expressed proteins in the absence of IRF6 could be associated with differentiation, cell-cell adhesion as well as immune response. Our data expand the knowledge on IRF6 in human postnatal keratinocytes, which will help to better understand IRF6-related pathologies.

Pannexin1: Role as a Sensor to Injury Is Attenuated in Pretype 2 Corneal Diabetic Epithelium.

Epithelial wound healing is essential to repair the corneal barrier function after injury and requires coordinated epithelial sheet movement over the wounded region. The presence and role of pannexin1 on multilayered epithelial sheet migration was examined in unwounded and wounded corneal epithelium from C57BL/6J (B6) control and diet-induced obese (DiO) mice, a pretype 2 diabetic model. We hypothesize that pannexin1 is dysregulated, and the interaction of two ion-channel proteins (P2X7 and pannexin1) is altered in pretype 2 diabetic tissue. Pannexin1 was found to be present along cell borders in unwounded tissue, and no significant difference was observed between DiO and B6 control. However, an epithelial debridement induced a striking difference in pannexin1 localization. The B6 control epithelium displayed intense staining near the leading edge, which is the region where calcium mobilization was detected, whereas the staining in the DiO corneal epithelium was diffuse and lacked distinct gradation in intensity back from the leading edge. Cells distal to the wound in the DiO tissue were irregular in shape, and the morphology was similar to that of epithelium inhibited with 10Panx, a pannexin1 inhibitor. Pannexin1 inhibition reduced mobilization of calcium between cells near the leading edge, and MATLAB scripts revealed a reduction in cell-cell communication that was also detected in cultured cells. Proximity ligation was performed to determine if P2X7 and pannexin1 interaction was a necessary component of motility and communication. While there was no significant difference in the interaction in unwounded DiO and B6 control corneal epithelium, there was significantly less interaction in the wounded DiO corneas both near the wound and back from the edge. The results demonstrate that pannexin1 contributes to the healing response, and P2X7 and pannexin1 coordination may be a required component of cell-cell communication and an underlying reason for the lack of pathologic tissue migration.

Abstract 266: Differential collective cell migratory behaviors modulated by phospholipid nanoparticles

Abstract Phospholipid nanoparticles have been actively explored for biological and biomedical applications. These nanoparticles display excellent cargo encapsulation efficiency, high water dispersibility, and outstanding biocompatibility, rendering them highly attractive for cancer nanomedicine applications. While the promising utilizations of phospholipid nanoparticles for cancer theranostic have been widely documented, the cellular biophysical responses elicited by phospholipid nanoparticles have been less investigated. Notably, the effects of phospholipid nanoparticles on collective cancer cell behaviors have not been well delineated. In fact, most of the studies examining nanoparticle-cancer interactions have focused largely on the nanoparticle toxicity on individual cells, but paid little attention to other nanoparticle effects on multicellular systems and their collective behaviors. A better appreciation of the effects of nanoparticles on multicellular system dynamics is necessary as collective cancer cell behaviors are central to the regulation of various processes, particularly cancer invasion and metastasis. Motivated by this, we sought to interrogate the effects of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) nanoparticles, which are some of the most commonly used phospholipid nanoparticles for cancer nanomedicine, on the collective behaviors of healthy and cancerous breast epithelial cell monolayers. DSPE-PEG nanoparticles with two sizes (i.e., 20 and 60 nm) and three surface functional groups (i.e., COOH, OCH3, and NH2) as well as three types of breast epithelial cell sheets with varying malignancy potential (i.e., MCF-10A, MCF-7, and MDA-MB-231) were used in this work. Utilizing a series of microscopy and molecular biology techniques and quantitative image analysis, we comprehensively examined the collective cell migratory dynamics in the absence and presence of DSPE-PEG nanoparticles. We noted that DSPE-PEG nanoparticles retarded the migration of the healthy MCF-10A epithelial cell sheets. In contrast, the cancerous MCF-7 and MDA-MB-231 cell sheets experienced accelerated collective migration in the presence of the nanoparticles. Moreover, these nanoparticles altered the migration directionality of the cancerous breast cell sheets. We ascribed the differential nanoparticle-modulated collective cell migratory behaviors to changes in the stiffness of the cell nuclei, cytoplasm, and cell-cell junctions, as well as the reorganization of the cellular actin filament networks after the nanoparticle-cell interactions. We anticipate that this study will provide a deeper insight into the nanomaterial-cancer interactions and aid the formulation of phospholipid nanoparticles for more effective and safer cancer theranostic and nanomedicine applications. Citation Format: Kenry, Bin Liu. Differential collective cell migratory behaviors modulated by phospholipid nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 266.

The Kunitz-type serine protease inhibitor Spint2 is required for cellular cohesion, coordinated cell migration and cell survival during zebrafish hatching gland development

We have previously shown that the Kunitz-type serine protease inhibitor Spint1a, also named Hai1a, is required in the zebrafish embryonic epidermis to restrict the activity of the type II transmembrane serine protease (TTSP) Matriptase1a/St14a, thereby ensuring epidermal homeostasis. A closely related Kunitz-type inhibitor is Spint2/Hai2, which in mammals plays multiple developmental roles that are either redundant or non-redundant with those of Spint1. However, the molecular bases for these non-redundancies are not fully understood. Here, we study spint2 during zebrafish development. It is co-expressed with spint1a in multiple embryonic epithelia, including the outer/peridermal layer of the epidermis. However, unlike spint1a, spint2 expression is absent from the basal epidermal layer but present in hatching gland cells. Hatching gland cells derive from the mesendodermal prechordal plate, from where they undergo a thus far undescribed transit into, and coordinated sheet migration within, the interspace between the outer and basal layer of the epidermis to reach their final destination on the yolk sac. Hatching gland cells usually survive their degranulation that drives embryo hatching but die several days later. In spint2 mutants, cohesion among hatching gland cells and their collective intra-epidermal migration are disturbed, leading to a discontinuous organization of the gland. In addition, cells undergo precocious cell death before degranulation, so that embryos fail to hatch. Chimera analyses show that Spint2 is required in hatching gland cells, but not in the overlying periderm, their potential migration and adhesion substrate. Spint2 acts independently of all tested Matriptases, Prostasins and other described Spint1 and Spint2 mediators. However, it displays a tight genetic interaction with and acts at least partly via the cell-cell adhesion protein E-cadherin, promoting both hatching gland cell cohesiveness and survival, in line with formerly reported effects of E-cadherin during morphogenesis and cell death suppression. In contrast, no such genetic interaction was observed between Spint2 and the cell-cell adhesion molecule EpCAM, which instead interacts with Spint1a. Our data shed new light onto the mechanisms of hatching gland morphogenesis and hatching gland cell survival. In addition, they reveal developmental roles of Spint2 that are strikingly different from those of Spint1, most likely due to differences in the expression patterns and relevant target proteins.

RhoGEF17-An Essential Regulator of Endothelial Cell Death and Growth.

The Rho guanine nucleotide exchange factor RhoGEF17 was described to reside in adherens junctions (AJ) in endothelial cells (EC) and to play a critical role in the regulation of cell adhesion and barrier function. The purpose of this study was to analyze signal cascades and processes occurring subsequent to AJ disruption induced by RhoGEF17 knockdown. Primary human and immortalized rat EC were used to demonstrate that an adenoviral-mediated knockdown of RhoGEF17 resulted in cell rounding and an impairment in spheroid formation due to an enhanced proteasomal degradation of AJ components. In contrast, β-catenin degradation was impaired, which resulted in an induction of the β-catenin-target genes cyclin D1 and survivin. RhoGEF17 depletion additionally inhibited cell adhesion and sheet migration. The RhoGEF17 knockdown prevented the cells with impeded cell–cell and cell–matrix contacts from apoptosis, which was in line with a reduction in pro-caspase 3 expression and an increase in Akt phosphorylation. Nevertheless, the cells were not able to proliferate as a cell cycle block occurred. In summary, we demonstrate that a loss of RhoGEF17 disturbs cell–cell and cell–substrate interaction in EC. Moreover, it prevents the EC from cell death and blocks cell proliferation. Non-canonical β-catenin signaling and Akt activation could be identified as a potential mechanism.

Discovery of Novel Small-Molecule FAK Activators Promoting Mucosal Healing.

Gastrointestinal mucosal wounds are common to patients injured by factors as diverse as drugs, inflammatory bowel disease, peptic ulcers, and necrotizing enterocolitis. However, although many drugs are used to ameliorate injurious factors, there is no drug available to actually stimulate mucosal wound healing. Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase, induces epithelial sheet migration and wound healing, making FAK a potential pharmacological target in this regard. In our previous research, we found a lead compound with drug-like properties, ZINC40099027, which promotes FAK phosphorylation, inducing mucosal healing in murine models. Herein we describe the design and optimization of a small library of novel FAK activators based on ZINC40099027 and their applications toward human intestinal epithelial wound closure and mouse ulcer healing.

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

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

Engineering slit-like channels for studying the growth of epithelial tissues in 3D-confined spaces.

The development of epithelial lumens in ducts is essential to the functioning of various organs and in organogenesis. Ductal elongation requires the collective migration of cell cohorts in three-dimensional (3D) confined spaces, while maintaining their epithelial integrity. Epithelial lumens generally adopt circular morphologies, however abnormalities in complex physiological environments can lead to the narrowing of glandular spaces that adopt elongated and slit-like morphologies. Here, we describe a simple method to form epithelial tissues in microchannels of various widths (100-300 µm) with a constant height of 25 µm that mimic elongated geometries of glandular spaces. The significance of this biomimetic platform has been evidenced by studying the migration of epithelial cell sheets inside these narrow slits of varying dimensions. We show that the growth of epithelial tissues in 3D-confined slits leads to a gradient of cell density along the slit axis and that the migration cell velocity depends on the extent of the spatial confinement. Our findings indicate that nuclear orientation is higher for leader cells and depends on the slit width, whereas YAP protein was predominantly localized in the nucleus of leader cells. This method will pave the way to studies aiming at understanding how 3D-confined spaces, which are reminiscent of in vivo pathological conditions, can affect the growth and the homeostasis of epithelial tissues.