Promoted Cell Detachment Research Articles

Bacteria Tune a Trade-off between Adhesion and Migration to Colonize Surfaces under Flow

The bacterial colonization of surfaces is a ubiquitous process that shapes nature and profoundly affects human health. While much is known about the biology of this process, the pivotal interplay between physical environment and active bacterial micromechanics remains poorly understood. In fact, strong adhesion and high motility, both of which are essential for surface colonization, are two apparently contradictory goals, as they mutually obstruct each other. Here, we investigate how the human pathogen optimizes its behavior for colonization of surfaces under flow. From the analysis of the dynamics of fluorescently labeled type-IV pili, we construct a mathematical model that quantitatively connects individual motor dynamics with whole-cell motility and migration. The data analysis also reveals that cells upregulate the number of visible pili on surface contact, although individual pili do not display a measurable sensory response to surfaces. When applying shear flow, we unexpectedly find that robust sticking to a surface requires passive surface adhesion rather than pilus activity. Instead, pilus activity actually promotes cell detachment while enabling migration. Using genetic perturbations of the pilus apparatus, it is shown that wild-type cells achieve a trade-off between adhesion and migration by limiting the number of pili. Simulations reveal a generic underlying trait space, where, depending on the interplay of active and passive forces, adhesion and migration are either compatible or a trade-off is required for efficient bacterial surface colonization. The discovered adhesion-migration problem is paradigmatic of a broad class of piliated bacteria and may also have implications for other cells. Published by the American Physical Society 2024

Terminal cationization of poly(N-isopropylacrylamide) brush surfaces facilitates efficient thermoresponsive control of cell adhesion and detachment

ABSTRACT A variety of poly(N-isopropylacrylamide) (PIPAAm)-grafted surfaces have been reported for temperature-controlled cell adhesion/detachment. However, the surfaces reported to date need further improvement to achieve good outcomes for both cell adhesion and detachment, which are inherently contradictory behaviors. This study investigated the effects of terminal cationization and length of grafted PIPAAm chains on temperature-dependent cell behavior. PIPAAm brushes with three chain lengths were constructed on glass coverslips via surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization. Terminal substitution of the grafted PIPAAm chains with either monocationic trimethylammonium or nonionic isopropyl moieties was performed through the reduction of terminal RAFT-related groups and subsequent thiol-ene reaction with the corresponding acrylamide derivatives. Although the thermoresponsive properties of the PIPAAm brush surfaces were scarcely affected by the terminal functional moiety, the zeta potentials of the cationized PIPAAm surfaces were higher than those of the nonionized ones, both below and above the phase transition temperature of PIPAAm (30°C). When bovine endothelial cells were cultured on each surface at 37°C, the number of adherent cells decreased with longer PIPAAm. Notably, cell adhesion on the cationized PIPAAm surfaces was higher than that on the nonionized surfaces. This terminal effect on cell adhesion gradually weakened with increasing PIPAAm length. In particular, long-chain PIPAAm brushes virtually showed cell repellency even at 37°C, regardless of the termini. Interestingly, moderately long-chain PIPAAm brushes promoted cell detachment at 20°C, with negligible terminal electrostatic interruption. Consequently, both cell adhesion and detachment were successfully improved by choosing an appropriate PIPAAm length with terminal cationization.

Real-Time Wireless Monitoring of Cell Proliferation and Detachment Based on pH-Responsive Conductive Polymer Dots.

In situ wireless monitoring for cell proliferation and detachment kinetics was conducted using pH-responsive zwitterionic polymer dots (Z-PDs), based on changes in electrochemical signals derived from Z-PD-coated substrates via the interaction of charges transferred between Z-PDs and cells. Z-PD-coated substrates were found to be a potent means to monitor and manipulate cell adhesion and detachment because of their high sensitivity over a wide range of pH conditions, and modification of the coated substrates was confirmed using a wireless system. At neutral pH, Z-PD-coated wireless sensors exhibited π-π stacking involving aromatic rings with hydrophobic interactions, thereby promoting cell proliferation; consequently, an increase in the measured resistance was observed. In contrast, Z-PD-coated substrates triggered by acidic and basic conditions promoted cell detachment, which induced an increase in the resistance compared with Z-PD substrates at pH 6.8, as a result of charges transferred to support Z-PD internalization through cell membranes after detachment. Therefore, as a wireless biosensor with excellent pH responsiveness that facilitates cell proliferation and detachment and whose electrochemical signals could be additionally acquired via a smartphone, Z-PD biosensors demonstrated a more favorable approach for monitoring cell-surface interactions than conventional optically based methods.

Influence of poly(N-isopropylacrylamide) (PIPAAm) graft density on properties of PIPAAm grafted poly(dimethylsiloxane) surfaces and their stability

A previous report shows that poly(N-isopropylacrylamide) (PIPAAm) gel grafted onto poly(dimethylsiloxane) (PDMS) (PI-PDMS) surfaces with large PIPAAm graft density (Lar-PI-PDMS), is prepared by using electron beam irradiation, demonstrating that applied mechanical stretching affects properties of the Lar-PI-PDMS surface. However, the influence of PIPAAm graft density on the properties of PI-PDMS surfaces and their stability are not understood. To provide insight into these points, the properties of PI-PDMS surfaces with low PIPAAm graft density (Low-PI-PDMS) surfaces with stretched (stretch ratio = 20%) and unstretched states were examined as stretchable temperature-responsive cell culture surface using contact angle measurement and cell attachment/detachment assays, compared to those with Lar-PI-PDMS, as previously reported. Long-term contact angle measurements (61 days) for unstretched Low-PI-PDMS and Lar-PI-PDMS surfaces indicated that the cross-linked structure of the grafted PIPAAm gel suppressed hydrophobic recovery of the basal PDMS surface. The cell attachment assay revealed that the stretched Low-PI-PDMS surface was less cell adhesive than that of the unstretched Low-PI-PDMS surface despite of a larger amount of adsorbed fibronectin (FN). The lower cell adhesiveness was possibly explained by denaturation of adsorbed FN, which was induced by the strong hydrophobic property of the stretched Low-PI-PDMS surface. The cell detachment assay revealed that dual stimuli, low temperature treatment and mechanical shrinking stress applied to the stretched Low-PI-PDMS surface promoted cell detachment compared to a single stimulus, low temperature treatment or mechanical shrinking stress. These results suggested that the PIPAAm gelgrafted PDMS surface was chemically stable and did not suffer from hydrophobic recovery. External mechanical stretching stress not only strongly dehydrated grafted PIPAAm chains, but also denatured the adsorbed FN when the grafted PIPAAm layer was extremely thin, as in Low-PI-PDMS surfaces. Thus, PI-PDMS may be utilized as a stretchable temperature-responsive cell culture surface without significant hydrophobic recovery.

Extracellular Vesicle Integrins Distinguish Unique Cancers.

The proteomic profile of extracellular vesicles (EVs) has been of increasing interest, particularly in understanding cancer growth, drug resistance, and metastatic behavior. Emerging data suggest that cancer-derived EVs carry an array of oncogenic cargo, including certain integrin proteins that may, in turn, promote cell detachment, migration, and selection of future metastatic sites. We previously reported a large comparison of secreted vesicle protein cargo across sixty diverse human cancer cell lines. Here, we analyze the distinct integrin profiles of these cancer EVs. We further demonstrate the enrichment of integrin receptors in cancer EVs compared to vesicles secreted from benign epithelial cells. The total EV integrin levels, including the quantity of integrins α6, αv, and β1 correlate with tumor stage across a variety of epithelial cancer cells. In particular, integrin α6 also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumors. This study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumor stage. Differential expression of integrins across cancer cells and selective packaging of integrins into EVs may contribute to further understanding the development and progression of tumor growth and metastasis across a variety of cancer types.

Inhibition of cell migration by focal adhesion kinase: Time-dependent difference in integrin-induced signaling between endothelial and hepatoblastoma cells.

Angiogenesis plays an important role in the development and progression of tumors, and it involves a series of signaling pathways contributing to the migration of endothelial cells for vascularization and to the invasion of cancer cells for secondary tumor formation. Among these pathways, the focal adhesion kinase (FAK) signaling cascade has been implicated in a variety of human cancers in connection with cell adhesion and migration events leading to tumor angiogenesis, metastasis and invasion. Therefore, the inhibition of FAK in endothelial and/or cancer cells is a potential target for anti-angiogenic therapy. In the present study, a small-molecule FAK inhibitor, 1,2,4,5-benzenetetramine tetrahydrochloride (Y15), was used to study the effects of FAK inhibition on the adhesion and migration behaviors of vascular endothelial cells (VECs) and human hepatoblastoma cells. Furthermore, the time-dependent differences in proteins associated with the integrin-mediated FAK/Rho GTPases signaling pathway within 2 h were examined. The results indicated that the inhibition of FAK significantly decreased the migration ability of VECs and human hepatoblastoma cells in a dose-dependent manner. Inhibition of FAK promoted cell detachment by decreasing the expression of focal adhesion components, and blocked cell motility by reducing the level of Rho GTPases. However, the expression of crucial proteins involved in integrin-induced signaling in two cell lines exhibited a time-dependent difference with increased duration of FAK inhibitor treatment, suggesting different mechanisms of FAK-mediated cell migration behavior. These results suggest that the mechanism underlying FAK-mediated adhesion and migration behavior differs among various cells, which is expected to provide evidence for future FAK therapy targeted against tumor angiogenesis.

Orexin receptor agonist Yan 7874 is a weak agonist of orexin/hypocretin receptors and shows orexin receptor-independent cytotoxicity.

Two promising lead structures of small molecular orexin receptor agonist have been reported, but without detailed analyses of the pharmacological properties. One of them, 1-(3,4-dichlorophenyl)-2-[2-imino-3-(4-methylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl]ethan-1-ol (Yan 7874), is commercially available, and we set out to analyze its properties. As test system we utilized human OX1 and OX2 orexin receptor-expressing Chinese hamster ovary (CHO) K1 cells as well as control CHO-K1 and neuro-2a neuroblastoma cells. Gq-coupling was assessed by measurement of intracellular Ca2+ and phospholipase C activity, and the coupling to Gi and Gs by adenylyl cyclase inhibition and stimulation, respectively. At concentrations above 1 μM, strong Ca2+ and low phospholipase C responses to Yan 7874 were observed in both OX1- and OX2-expressing cells. However, a major fraction of the response was not mediated by orexin receptors, as determined utilizing the non-selective orexin receptor antagonist N-biphenyl-2-yl-1-{[(1-methyl-1H-benzimidazol-2-yl)sulfanyl]acetyl}-L-prolinamide (TCS 1102) as well as control CHO-K1 cells. Yan 7874 did not produce any specific adenylyl cyclase response. Some experiments suggested an effect on cell viability by Yan 7874, and we thus analyzed this. Within a few hours of exposure, Yan 7874 markedly changed cell morphology (shrunken, rich in vacuoles), reduced growth, promoted cell detachment, and induced necrotic cell death. The effect was equal in cells expressing orexin receptors or not. Thus, Yan 7874 is a weak partial agonist of orexin receptors. It also displays strong off-target effects in the same concentration range, culminating in necrotic cell demise. This makes Yan 7874 unsuitable as orexin receptor agonist.

Chronic stress induces CD99, suppresses autophagy, and affects spontaneous adipogenesis in human bone marrow stromal cells

BackgroundBone marrow-derived mesenchymal stromal cells (MSCs) are multipotent cells with a high constitutive level of autophagy and low expression of CD99. Under certain conditions, MSCs may develop tumorigenic properties. However, these transformation-induced conditions are largely unknown. Recently, we have identified an association between Hsp70, a main participant in cellular stress response and tumorigenesis, and CD99. Preliminary observations had revealed upregulation of both proteins in stressed long-term cultured MSCs. And so we hypothesized that CD99 is implicated in stress-induced mechanisms of cellular transformation in MSCs. Hence, we investigated the effects of prolonged stress on MSCs and the role of CD99 and autophagy in their survival.MethodsHuman telomerase reverse transcriptase (hTERT) overexpressing immortalized MSCs and primary bone marrow stromal cells were used to investigate the influence of long-term serum deprivation and hypoxia on growth and differentiation of MSCs. Cell proliferation and apoptosis were evaluated using flow cytometry, differentiation capabilities of MSCs were assessed by immunohistochemical staining followed by microscopic examination. CD99, Hsp70 expression were analyzed using flow cytometry, western blotting, and reverse transcriptase polymerase chain reaction. Autophagy was explored with specific inhibitors using cell morphology examination and western blotting.ResultsChronic stress factors are able to change the morphology of MSCs and to inhibit spontaneous differentiation into adipocyte lineage. Furthermore, CD99 elevation and downregulation of p53 and p21 accompanied defective autophagy, which is usually associated with tumor formation. We found that inhibition of autophagy by chloroquine promoted cell detachment and modulated CD99 expression level whereas incorporation of CD99 recombinant protein into the cells suppressed autophagy.ConclusionsObtained results provide a model for chronic stress-induced transformation of MSCs via CD99 and may therefore be highly relevant to mesenchymal tumorigenesis.

Cbl-b promotes cell detachment via ubiquitination of focal adhesion kinase.

Cancer cell detachment from the primary tumor site represents the first stage of metastasis. Previous studies have identified that cell detachment is triggered by cytoskeletal disruption, which may induce a wide variety of cellular changes. Focal adhesion kinase (FAK) exhibits crucial cellular functions, including regulation of the cytoskeleton. These observations have provided exciting insights into the effect of FAK in cell detachment; however, the involvement of FAK in cell detachment remains controversial. The aim of the present study was to evaluate the effect of FAK and its function in the process of cell detachment. The results revealed that FAK expression was downregulated following trypsin treatment in human gastric, lung, colon and breast cancer cell lines, as well as a human gastric epithelial cell line. Knockdown of FAK enhanced cell detachment in gastric cancer MGC803 cells, indicating that FAK inhibits cell detachment. Further investigation revealed that trypsin induced monoubiquitination of FAK. In addition, the lysosome inhibitor, NH4Cl, decreased trypsin-induced degradation of FAK. Casitas B-lineage lymphoma-b (Cbl-b), an E3 ubiquitin ligase, was involved in this process, which interacted with FAK, as demonstrated by co-precipitation experiments, and promoted trypsin-induced ubiquitin-lysosome degradation of FAK. These results indicate that Cbl-b promotes cell detachment via ubiquitination of FAK. These findings provide novel insights regarding the effect of FAK and Cbl-b in the process of cancer cell detachment.

Generation of a monoclonal antibody recognizing the CEACAM glycan structure and inhibiting adhesion using cancer tissue-originated spheroid as an antigen.

Spheroids cultured directly from tumours can better reflect in vivo tumour characteristics than two-dimensional monolayer culture or three-dimensional culture of established cell lines. In this study, we generated antibodies by directly immunizing mice with primary-cultured living spheroids from human colorectal cancer. We performed phenotypic screening via recognition of the surface of the spheroids and inhibition of their adhesion to extracellular matrices to identify a monoclonal antibody, clone 5G2. The antibody inhibited cell migration in two-dimensional culture and promoted cell detachment. Western blotting and immunohistochemistry detected the 5G2 signal in many colorectal cancer spheroids, as well as patient tumours, but failed to detect in various cell lines examined. We found that 5G2 recognized the Lea and Lec on N-glycan, and their major carrier proteins were CEACAM5 and CEACAM6. Pre-incubation of the spheroids with 5G2 impaired translocation of integrin β4 from the lateral membrane to the contact interface between the extracellular matrix when embedded in it. As we successfully obtained a functional antibody, which antigen was glycan structures and lost in cell lines, cancer tissue-originated spheroids can be a useful antigen for generating novel anti-cancer antibodies.

Cbl-b accelerates trypsin-induced cell detachment through ubiquitination and degradation of proline-rich tyrosine kinase 2.

Trypsin is a digestive enzyme that is widely used for cell detachment, which is the first stage of tumor metastasis. Recent studies show that adhesion-related kinases are involved in cell detachment. Proline-rich tyrosine kinase 2 (Pyk2) is a crucial kinase in the regulation of cell adhesion and detachment. However, the effect of Pyk2 on cell detachment is controversial. In the present study, we found that Pyk2 expression was rapidly decreased after trypsin treatment in gastric cancer, breast cancer, colon cancer, lung cancer, and human gastric epithelial cells. Knockdown of Pyk2 accelerated cell detachment. Furthermore, lysosome inhibitor NH4CL suppressed cell detachment and increased ubiquitination of Pyk2. Cbl-b is a type of E3 ubiquitin ligase that interacted with Pyk2, reduced the expression of Pyk2, and promoted trypsin-induced degradation of Pyk2. These findings suggest that Cbl-b promoted cell detachment through mono-ubiquitination of Pyk2. Our data provide a new insight into the role of Cbl-b in cell detachment.

Hyperoxia increases the elastic modulus of alveolar epithelial cells through Rho kinase

Patients with acute lung injury are administered high concentrations of oxygen during mechanical ventilation, and while both hyperoxia and mechanical ventilation are necessary, each can independently cause additional injury. However, the precise mechanisms that lead to injury are not well understood. We hypothesized that alveolar epithelial cells may be more susceptible to injury caused by mechanical ventilation because hyperoxia causes cells to be stiffer due to increased filamentous actin (f-actin) formation via the GTPase RhoA and its effecter Rho kinase (ROCK). We examined cytoskeletal structures in cultured murine lung alveolar epithelial cells (MLE-12) under normoxic and hyperoxic (48 h) conditions. We also measured cell elasticity (E) using an atomic force microscope in the indenter mode. Hyperoxia caused increased f-actin stress fibers and bundle formation, an increase in g- and f-actin, an increase in nuclear area and a decrease in nuclear height, and cells became stiffer (higher E). Treatment with an inhibitor (Y-27632) of ROCK significantly decreased E and prevented the cytoskeletal changes, while it did not influence the nuclear height and area. Pre-exposure of cells to hyperoxia promoted detachment when cells were subsequently stretched cyclically, but the ROCK inhibitor prevented this effect. Hyperoxia caused thickening of vinculin focal adhesion plaques, and inhibition of ROCK reduced the formation of distinct focal adhesion plaques. Phosphorylation of focal adhesion kinase was significantly reduced by both hyperoxia and treatment with Y-27632. Hyperoxia caused increased cell stiffness and promoted cell detachment during stretch. These effects were ameliorated by inhibition of ROCK.

Hyperoxia Increases Elastic Modulus of Alveolar Epithelial Cells Through Rho Kinase

Patients with acute lung injury are administered high concentrations of oxygen during mechanical ventilation, and both hyperoxia and mechanical ventilation can independently cause injury. However, the precise mechanisms that lead to epithelial injury are not well understood. We hypothesized that alveolar epithelial cells may be more susceptible to injury caused by mechanical ventilation because hyperoxia causes cells to be more stiff due to increased f‐action formation via activation of the GTPase RhoA and its downstream effecter Rho kinase (ROCK). We used fluorescence microscopy to examine cytoskeletal structures in murine lung alveolar epithelial cells. We measured cell elasticity (E) using atomic force microscopy in the indenter mode. Hyperoxia caused increased f‐actin stress fibers formation, and cells became stiffer (higher E). Treatment with an inhibitor of ROCK significantly decreased E and prevented the cytoskeletal changes. Pre‐exposure of cells to hyperoxia promoted detachment when cells were subsequently stretched using the Flexercell device, but the ROCK inhibitor prevented this effect. Loss of cell adhesion would result in decreased barrier function. In summary, hyperoxia caused cytoskeletal remodeling that resulted in increased cell stiffness and promoted cell detachment during stretch. These effects were ameliorated by inhibition of ROCK. Supported by NIH grant HL094366 (CMW).

Thermosensitive polymeric matrices for three-dimensional cell culture strategies

A completely new strategy for cell culture focusing on the design of three-dimensional (3D) smart surfaces by supercritical fluid technology has been developed. This approach might overcome the limitations on cell expansion and proliferation of currently existing techniques. An alternative technology, based on supercritical carbon dioxide, was used to polymerize poly(N-isopropylacrylamide) (PNIPAAm) and to foam poly( d, l-lactic acid) (P D,LLA), creating a thermosensitive 3D structure which has proven to have potential as a substrate for cell growth and expansion. We demonstrated that the thermosensitive matrices promoted cell detachment, thus P D,LLA scaffolds have the potential to be used as substrates for cell growth and expansion avoiding enzymatic and mechanical methods of cell harvesting. The harvested cells were replated to evaluate their viability, which was not compromised. A major advantage of this technology is the fact that the prepared materials can be recovered and reused. Therefore, the same substrate can be recycled and reused for different batches. An indirect impact of the technology developed is related to the field of biotechnology, as this novel technology for cell expansion can be applied to any adherent cell cultures.

CRTAM: A molecule involved in epithelial cell adhesion

Class I-restricted T cell associated molecule (CRTAM) is a member of the immunoglobulin superfamily that complies with the structural characteristics of the JAM family of proteins and is phylogenetically more closely related to nectin-like proteins. Here we demonstrate for the first time, that CRTAM is expressed in epithelial cells along the lateral membrane and is important for early cell-cell contacts and cell-substrate interactions. CRTAM is sensitive to intermediate filament disruption and treatment of monolayers with soluble CRTAM enhances cell-cell dissociation and lowers transepithelial electrical resistance. Incubation of newly plated cells with anti-CRTAM antibody decreases the formation of cell aggregates and promotes cell detachment. Co-cultures of epithelial cells and fibroblasts that lack CRTAM expression and in vitro binding assays, demonstrate the participation of CRTAM in homotypic and heterotypic trans-interactions. Hence we conclude that CRTAM is a molecule involved in epithelial cell adhesion.

New Roles for the LKB1-NUAK Pathway in Controlling Myosin Phosphatase Complexes and Cell Adhesion

The AMPK-related kinases NUAK1 and NUAK2 are activated by the tumor suppressor LKB1. We found that NUAK1 interacts with several myosin phosphatases, including the myosin phosphatase targeting-1 (MYPT1)-protein phosphatase-1beta (PP1beta) complex, through conserved Gly-Ile-Leu-Lys motifs that are direct binding sites for PP1beta. Phosphorylation of Ser(445), Ser(472), and Ser(910) of MYPT1 by NUAK1 promoted the interaction of MYPT1 with 14-3-3 adaptor proteins, thereby suppressing phosphatase activity. Cell detachment induced phosphorylation of endogenous MYPT1 by NUAK1, resulting in 14-3-3 binding to MYPT1 and enhanced phosphorylation of myosin light chain-2. Inhibition of the LKB1-NUAK1 pathway impaired cell detachment. Our data indicate that NUAK1 controls cell adhesion and functions as a regulator of myosin phosphatase complexes. Thus, LKB1 can influence the phosphorylation of targets not only through the AMPK family of kinases but also by controlling phosphatase complexes.

A Dual Role for Oncostatin M Signaling in the Differentiation and Death of Mammary Epithelial Cells in Vivo

Recent studies in breast cancer cell lines have shown that oncostatin M (OSM) not only inhibits proliferation but also promotes cell detachment and enhances cell motility. In this study, we have looked at the role of OSM signaling in nontransformed mouse mammary epithelial cells in vitro using the KIM-2 mammary epithelial cell line and in vivo using OSM receptor (OSMR)-deficient mice. OSM and its receptor were up-regulated approximately 2 d after the onset of postlactational mammary regression, in response to leukemia inhibitory factor (LIF)-induced signal transducer and activator of transcription-3 (STAT3). This resulted in sustained STAT3 activity, increased epithelial apoptosis, and enhanced clearance of epithelial structures during the remodeling phase of mammary involution. Concurrently, OSM signaling precipitated the dephosphorylation of STAT5 and repressed expression of the milk protein genes beta-casein and whey acidic protein (WAP). Similarly, during pregnancy, OSM signaling suppressed beta-casein and WAP gene expression. In vitro, OSM but not LIF persistently down-regulated phosphorylated (p)-STAT5, even in the continued presence of prolactin. OSM also promoted the expression of metalloproteinases MMP3, MMP12, and MMP14, which, in vitro, were responsible for OSM-specific apoptosis. Thus, the sequential activation of IL-6-related cytokines during mammary involution culminates in an OSM-dependent repression of epithelial-specific gene expression and the potentiation of epithelial cell extinction mediated, at least in part, by the reciprocal regulation of p-STAT5 and p-STAT3.

Fibronectin Unfolding Revisited: Modeling Cell Traction-Mediated Unfolding of the Tenth Type-III Repeat

Fibronectin polymerization is essential for the development and repair of the extracellular matrix. Consequently, deciphering the mechanism of fibronectin fibril formation is of immense interest. Fibronectin fibrillogenesis is driven by cell-traction forces that mechanically unfold particular modules within fibronectin. Previously, mechanical unfolding of fibronectin has been modeled by applying tensile forces at the N- and C-termini of fibronectin domains; however, physiological loading is likely focused on the solvent-exposed RGD loop in the 10th type-III repeat of fibronectin (10FNIII), which mediates binding to cell-surface integrin receptors. In this work we used steered molecular dynamics to study the mechanical unfolding of 10FNIII under tensile force applied at this RGD site. We demonstrate that mechanically unfolding 10FNIII by pulling at the RGD site requires less work than unfolding by pulling at the N- and C- termini. Moreover, pulling at the N- and C-termini leads to 10FNIII unfolding along several pathways while pulling on the RGD site leads to a single exclusive unfolding pathway that includes a partially unfolded intermediate with exposed hydrophobic N-terminal β-strands – residues that may facilitate fibronectin self-association. Additional mechanical unfolding triggers an essential arginine residue, which is required for high affinity binding to integrins, to move to a position far from the integrin binding site. This cell traction-induced conformational change may promote cell detachment after important partially unfolded kinetic intermediates are formed. These data suggest a novel mechanism that explains how cell-mediated forces promote fibronectin fibrillogenesis and how cell surface integrins detach from newly forming fibrils. This process enables cells to bind and unfold additional fibronectin modules – a method that propagates matrix assembly.

Inhibition of microparticle release triggers endothelial cell apoptosis and detachment

Endothelial cell cultures contain caspase 3-containing microparticles (EMP), which are reported to form during or after cell detachment. We hypothesize that also adherent endothelial cells release EMP, thus protecting these cells from caspase 3 accumulation, detachment and apoptosis. Human umbilical vein endothelial cells (HUVEC) were incubated with and without inhibitors of microparticle release (Y-27632, calpeptin), both in the absence or presence of additional "external stress", i.e. the apoptotic agent staurosporin (200 nM) or the activating cytokine interleukin (IL)-1alpha (5 ng/ml). Control cultures contained mainly viable adherent cells and minor fractions of apoptotic detached cells and microparticles in the absence of inhibitors. In the presence of inhibitors, caspase 3 accumulated in adherent cells and detachment tended to increase. During incubation with either staurosporin or IL-1alpha in the absence of inhibitors of microparticle release, adherent cells remained viable, and detachment and EMP release increased slightly. In the presence of inhibitors, dramatic changes occurred in staurosporin-treated cultures. Caspase 3 accumulated in adherent cells and >90% of the cells detached within 48 hours. In IL-1alpha-treated cultures no accumulation of caspase 3 was observed in adherent cells, although detachment increased. Scanning electron microscopy studies confirmed the presence of EMP on both adherent and detached cells. Prolonged culture of detached cells indicated a rapid EMP formation as well as some EMP formation at longer culture periods. Inhibition of EMP release causes accumulation of caspase 3 and promotes cell detachment, although the extent depends on the kind of "external stress". Thus, the release of caspase 3-containing microparticles may contribute to endothelial cell survival.

EphB–ephrinB bi-directional endocytosis terminates adhesion allowing contact mediated repulsion

Eph receptors and their membrane-associated ephrin ligands mediate cell-cell repulsion to guide migrating cells and axons. Repulsion requires that the ligand-receptor complex be removed from the cell surface, for example by proteolytic processing of the ephrin ectodomain. Here we show that cell contact-induced EphB-ephrinB complexes are rapidly endocytosed during the retraction of cells and neuronal growth cones. Endocytosis occurs in a bi-directional manner that comprises of full-length receptor and ligand complexes. Endocytosis is sufficient to promote cell detachment and seems necessary for axon withdrawal during growth cone collapse. Here, we show a mechanism for the termination of adhesion and the promotion of cell repulsion after intercellular (trans) interaction between two transmembrane proteins.