Process Of Cell Spreading Research Articles

Leishmania amazonensis infection impairs VLA-4 clustering and adhesion complex assembly at the adhesion site of J774 cells.

Cutaneous leishmaniasis is an infectious disease that may lead to a single or multiple disseminated cutaneous lesions. The mechanisms involved in Leishmania dissemination to different areas of the skin and the internal organs remain poorly understood. Evidence shows that Very Late Antigen-4 (VLA-4)-dependent phagocyte adhesion is impaired by Leishmania infection, which may be related to the mechanisms of parasite dissemination. We investigated factors potentially associated with decreased VLA-4-mediated adhesion in Leishmania-infected macrophages, including lipid raft-mediated VLA-4 mobilization along the cellular membrane, integrin cluster formation at the cell base (adhesion site), and focal adhesion complex assembly. Phagocytes treated with Methyl-β-Cyclodextrin (MβCD) demonstrated reduced adhesion, similarly to Leishmania amazonensis-infected J774 cells. Infected and MβCD-treated macrophages presented decreased VLA-4 mobilization to the adhesion plane, as well as reduced integrin clustering. Leishmania amazonensis-infected cells exhibited talin depletion, as well as a decreased mobilization of adhesion complex proteins, such as talin and viculin, which were associated with lower VLA-4 concentrations at the adhesion site and limited cell-spreading. Our results suggest that Leishmania infection may modulate the firm adhesion phase of the cell-spreading process, which could contribute to the bloodstream dissemination of infected cells.

Recent advances in the application of mesoporous silica-based nanomaterials for bone tissue engineering.

Silica nanomaterials (SNMs) and their composites have recently been investigated as scaffolds for bone tissue engineering. SNM scaffolds possess the ability to encourage bone cell growth and also allow the simultaneous delivery of biologically active biomolecules that are encapsulated in the mesopores. Their high mechanical strength, low cytotoxicity, ability to stimulate both the proliferation and osteogenic differentiation of progenitor cells make the SNMs appropriate scaffolds. Their physiochemical properties facilitate the cell spreading process, allow easy access to nutrients and help the cell-cell communication process during bone tissue engineering. The ability to deliver small biomolecules, such as dexamethasone, different growth factors, vitamins and mineral ions depends on the morphology, porosity, and crystallinity of SNMs and their composites with other polymeric materials. In this review, the abilities of SNMs to perform as suitable scaffolds for bone tissue engineering are comprehensively discussed.

<p>α-Bisabolol-Loaded Cross-Linked Zein Nanofibrous 3D-Scaffolds For Accelerating Wound Healing And Tissue Regeneration In Rats</p>

ObjectivesNovel α-bisabolol (BIS)-loaded citric acid cross-linked zein nanofibrous scaffolds (C-ZNFs) were proposed to serve as safe platforms for promoting wound repair in rats.MethodsZNFs were synthesized using electrospinning technique, then NFs, with adequate water resistance, were produced using citric acid as a safe cross-linker.ResultsCompared to the uncross-linked ZNFs, cross-linking with 7% w/w citric acid decreased swelling index by 3 folds, while the tensile strength and the contact angle were enhanced to 2.5 and 3.8 folds, respectively. SEM images showed beads-free homogeneous NFs with a fully inter-connected 3D-network, where the average diameter of optimized C-ZNFs was 181.7±50 nm. After 24 h, C-ZNFs exhibited a decreased BIS release rate (45.6%), compared to uncross-linked mats (84.9%). By increasing BIS concentration, the cell adhesion (WI38 fibroblasts) was improved which can be attributed mainly to BIS activation of transforming growth factor-beta (TGF-β1). The MTT-OD obtained values indicated that all tested zein scaffolds significantly enhanced the viability of WI38 fibroblasts, compared to the control after 48h of incubation which can be referred to the proliferative potential of zein by provoking cell spreading process. The scratch wound assay demonstrated that BIS-loaded ZNF scaffolds showed accelerated migration and proliferation of fibroblasts expressed by significantly higher wound closure rates compared to the control sample. BIS-loaded-C-ZNFs prominently accelerated tissue regeneration for wound closure demonstrated by entirely grown epithelium with normal keratinization and rapid wound contraction, compared to the control. Immunohistochemical results confirmed the superiority of BIS-loaded-C-ZNFs, where the observed reduced NF-κB and the elevated cytokeratin expressions confirmed the anti-inflammatory and proliferative effects of the scaffolds, respectively.ConclusionIn-vitro, optimized C-ZNFs offered a satisfactory cytocompatibility, adhesion and healing which were consistent with the in-vivo results. BIS-loaded-C-ZNFs could be regarded as a promising and effective biomaterial for tissue regeneration and for accelerating the wound healing process.

Universal Kinetics of the Onset of Cell Spreading on Substrates of Different Stiffness

When plated onto substrates, cell morphology and even stem-cell differentiation are influenced by the stiffness of their environment. Stiffer substrates give strongly spread (eventually polarized) cells with strong focal adhesions and stress fibers; very soft substrates give a less developed cytoskeleton and much lower cell spreading. The kinetics of this process of cell spreading is studied extensively, and important universal relationships are established on how the cell area grows with time. Here, we study the population dynamics of spreading cells, investigating the characteristic processes involved in the cell response to the substrate. We show that unlike the individual cell morphology, this population dynamics does not depend on the substrate stiffness. Instead, a strong activation temperature dependence is observed. Different cell lines on different substrates all have long-time statistics controlled by the thermal activation over a single energy barrier ΔG ≈ 18 kcal/mol, whereas the early-time kinetics follows a power law ∼t5. This implies that the rate of spreading depends on an internal process of adhesion complex assembly and activation; the operational complex must have five component proteins, and the last process in the sequence (which we believe is the activation of focal adhesion kinase) is controlled by the binding energy ΔG.

Controlling Cellular Volume via Mechanical and Physical Properties of Substrate

The mechanical and physical properties of substrate play a crucial role in regulating many cell functions and behaviors. However, how these properties affect cell volume is still unclear. Here, we show that an increase in substrate stiffness, available spread area, or effective adhesion energy density results in a remarkable cell volume decrease (up to 50%), and the dynamic cell spreading process is also accompanied by dramatic cell volume decrease. Further, studies of ion channel inhibition and osmotic shock suggest that these volume decreases are due to the efflux of water and ions. We also show that disrupting cortex contractility leads to bigger cell volume. Collectively, these results reveal the “mechanism of adhesion-induced compression of cells,” i.e., stronger interaction between cell and substrate leads to higher actomyosin contractility, expels water and ions, and thus decreases cell volume.

Free energy analysis of cell spreading

In this study we present a steady-state adaptation of the thermodynamically motivated stress fiber (SF) model of Vigliotti et al. (2015). We implement this steady-state formulation in a non-local finite element setting where we also consider global conservation of the total number of cytoskeletal proteins within the cell, global conservation of the number of binding integrins on the cell membrane, and adhesion limiting ligand density on the substrate surface. We present a number of simulations of cell spreading in which we consider a limited subset of the possible deformed spread-states assumed by the cell in order to examine the hypothesis that free energy minimization drives the process of cell spreading. Simulations suggest that cell spreading can be viewed as a competition between (i) decreasing cytoskeletal free energy due to strain induced assembly of cytoskeletal proteins into contractile SFs, and (ii) increasing elastic free energy due to stretching of the mechanically passive components of the cell. The computed minimum free energy spread area is shown to be lower for a cell on a compliant substrate than on a rigid substrate. Furthermore, a low substrate ligand density is found to limit cell spreading. The predicted dependence of cell spread area on substrate stiffness and ligand density is in agreement with the experiments of Engler et al. (2003). We also simulate the experiments of Théry et al. (2006), whereby initially circular cells deform and adhere to “V-shaped” and “Y-shaped” ligand patches. Analysis of a number of different spread states reveals that deformed configurations with the lowest free energy exhibit a SF distribution that corresponds to experimental observations, i.e. a high concentration of highly aligned SFs occurs along free edges, with lower SF concentrations in the interior of the cell. In summary, the results of this study suggest that cell spreading is driven by free energy minimization based on a competition between decreasing cytoskeletal free energy and increasing passive elastic free energy.

Modeling the mechanics of cells in the cell-spreading process driven by traction forces.

Mechanical properties of cells and their mechanical interaction with the extracellular environments are main factors influencing cellular function, thus indicating the progression of cells in different disease states. By considering the mechanical interactions between cell adhesion molecules and the extracellular environment, we developed a cell mechanical model that can characterize the mechanical changes in cells during cell spreading. A cell model was established that consisted of various main subcellular components, including cortical cytoskeleton, nuclear envelope, actin filaments, intermediate filaments, and microtubules. We demonstrated the structural changes in subcellular components and the changes in spreading areas during cell spreading driven by traction forces. The simulation of nanoindentation tests was conducted by integrating the indenting force to the cell model. The force-indentation curve of the cells at different spreading states was simulated, and the results showed that cell stiffness increased with increasing traction forces, which were consistent with the experimental results. The proposed cell mechanical model provides a strategy to investigate the mechanical interactions of cells with the extracellular environments through the adhesion molecules and to reveal the cell mechanical properties at the subcellular level as cells shift from the suspended state to the adherent state.

Reproducibility of scratch assays is affected by the initial degree of confluence: Experiments, modelling and model selection

Scratch assays are difficult to reproduce. Here we identify a previously overlooked source of variability which could partially explain this difficulty. We analyse a suite of scratch assays in which we vary the initial degree of confluence (initial cell density). Our results indicate that the rate of re-colonisation is very sensitive to the initial density. To quantify the relative roles of cell migration and proliferation, we calibrate the solution of the Fisher–Kolmogorov model to cell density profiles to provide estimates of the cell diffusivity, D, and the cell proliferation rate, λ. This procedure indicates that the estimates of D and λ are very sensitive to the initial density. This dependence suggests that the Fisher–Kolmogorov model does not accurately represent the details of the collective cell spreading process, since this model assumes that D and λ are constants that ought to be independent of the initial density. Since higher initial cell density leads to enhanced spreading, we also calibrate the solution of the Porous–Fisher model to the data as this model assumes that the cell flux is an increasing function of the cell density. Estimates of D and λ associated with the Porous–Fisher model are less sensitive to the initial density, suggesting that the Porous–Fisher model provides a better description of the experiments.

Delineating the Tes Interaction Site in Zyxin and Studying Cellular Effects of Its Disruption.

Focal adhesions are integrin-based structures that link the actin cytoskeleton and the extracellular matrix. They play an important role in various cellular functions such as cell signaling, cell motility and cell shape. To ensure and fine tune these different cellular functions, adhesions are regulated by a large number of proteins. The LIM domain protein zyxin localizes to focal adhesions where it participates in the regulation of the actin cytoskeleton. Because of its interactions with a variety of binding partners, zyxin has been proposed to act as a molecular scaffold. Here, we studied the interaction of zyxin with such a partner: Tes. Similar to zyxin, Tes harbors three highly conserved LIM domains of which the LIM1 domain directly interacts with zyxin. Using different zyxin variants in pull-down assays and ectopic recruitment experiments, we identified the Tes binding site in zyxin and showed that four highly conserved amino acids are crucial for its interaction with Tes. Based upon these findings, we used a zyxin mutant defective in Tes-binding to assess the functional consequences of abrogating the zyxin-Tes interaction in focal adhesions. Performing fluorescence recovery after photobleaching, we showed that zyxin recruits Tes to focal adhesions and modulates its turnover in these structures. However, we also provide evidence for zyxin-independent localization of Tes to focal adhesions. Zyxin increases focal adhesion numbers and reduces focal adhesion lifetimes, but does so independent of Tes. Quantitative analysis showed that the loss of interaction between zyxin and Tes affects the process of cell spreading. We conclude that zyxin influences focal adhesion dynamics, that it recruits Tes and that this interaction is functional in regulating cell spreading.

Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding.

The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies.

Arp2/3 complex inhibition radically alters lamellipodial actin architecture, suspended cell shape, and the cell spreading process.

Recent studies have investigated the dendritic actin cytoskeleton of the cell edge's lamellipodial (LP) region by experimentally decreasing the activity of the actin filament nucleator and branch former, the Arp2/3 complex. Here we extend these studies via pharmacological inhibition of the Arp2/3 complex in sea urchin coelomocytes, cells that possess an unusually broad LP region and display correspondingly exaggerated centripetal flow. Using light and electron microscopy, we demonstrate that Arp2/3 complex inhibition via the drug CK666 dramatically altered LP actin architecture, slowed centripetal flow, drove a lamellipodial-to-filopodial shape change in suspended cells, and induced a novel actin structural organization during cell spreading. A general feature of the CK666 phenotype in coelomocytes was transverse actin arcs, and arc generation was arrested by a formin inhibitor. We also demonstrate that CK666 treatment produces actin arcs in other cells with broad LP regions, namely fish keratocytes and Drosophila S2 cells. We hypothesize that the actin arcs made visible by Arp2/3 complex inhibition in coelomocytes may represent an exaggerated manifestation of the elongate mother filaments that could possibly serve as the scaffold for the production of the dendritic actin network.

The structural basis of differential inhibition of human calpain by indole and phenyl α-mercaptoacrylic acids.

Excessive activity of neutrophils has been linked to many pathological conditions, including rheumatoid arthritis, cancer and Alzheimer's disease. Calpain-I is a Ca(2+)-dependent protease that plays a key role in the extravasation of neutrophils from the blood stream prior to causing damage within affected tissues. Inhibition of calpain-I with small molecule mercaptoacrylic acid derivatives slows the cell spreading process of live neutrophils and so these compounds represent promising drug leads. Here we present the 2.05 and 2.03 Å co-crystal X-ray structures of the pentaEF hand region, PEF(S), from human calpain with (Z)-3-(4-chlorophenyl)-2-mercaptoacrylic acid and (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid. In both structures, the α-mercaptoacrylic acid derivatives bind between two α-helices in a hydrophobic pocket that is also exploited by a leucine residue of the endogenous regulatory calpain inhibitor calpastatin. Hydrophobic interactions between the aromatic rings of both inhibitors and the aliphatic residues of the pocket are integral for tight binding. In the case of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid, hydrogen bonds form between the mercaptoacrylic acid substituent lying outside the pocket and the protein and the carboxylate group is coplanar with the aromatic ring system. Multiple conformations of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid were found within the pocket. The increased potency of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid relative to (Z)-3-(4-chlorophenyl)-2-mercaptoacrylic acid may be a consequence of the indole group binding more deeply in the hydrophobic pocket of PEF(S) than the phenyl ring.

Rap1 Can Bypass the FAK-Src-Paxillin Cascade to Induce Cell Spreading and Focal Adhesion Formation

We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.

Contemporaneous cell spreading and phagocytosis: Magneto-resistive real-time monitoring of membrane competing processes

Adhesion and spreading of cells strongly depend on the properties of the underlying surface, which has significant consequences in long-term cell behavior adaption. This relationship is important for the understanding of both biological functions and their bioactivity in disease-related applications. Employing our magnetic lab-on-a-chip system, we present magnetoresistive-based real-time and label-free detection of cellular phagocytosis behavior during their spreading process on particle-immobilized sensor surfaces. Cell spreading experiments carried out on particle-free and particle-modified surfaces reveal a delay in spreading rate after an elapsed time of about 2.2h for particle-modified surfaces due to contemporaneous cell membrane loss by particle phagocytosis. Our associated magnetoresistive measurements show a high uptake rate at early stages of cell spreading, which decreases steadily until it reaches saturation after an average elapsed time of about 100min. The corresponding cellular average uptake rate during the entire cell spreading process accounts for three particles per minute. This result represents a four times higher phagocytosis efficiency compared to uptake experiments carried out for confluently grown cells, in which case cell spreading is already finished and, thus, excluded. Furthermore, other dynamic cell-surface interactions at nano-scale level such as cell migration or the dynamics of cell attachment and detachment are also addressable by our magnetic lab-on-a-chip approach.

Force at Early Adhesion Formation: Integrin Activation Triggers Contractile Movement and Subsequent Outward Translocation of Integrin-RGD Clusters

Integrin-mediated adhesions play important roles in many biology events, including stem cell differentiation, cancer metastasis, and the immune response. However, The mechanism of early adhesion formation remains unclear. Using mobile RGD ligands on lipid bilayers with nano-fabricated physical barriers, we observe surprising long-range lateral movements of ligated integrins during the process of cell spreading. At first, RGD-activated integrin clusters stimulate actin polymerization that radiates from the clusters. Myosin II contraction of actin from adjacent clusters produces contractile pairs that move toward each other against barriers. Force generated by myosin II stimulates a Src kinase-dependent lamellipodial extension and outward movement of clusters. Subsequent retraction by myosin II causes inward movement of clusters. The final cell spread area increases with the density of periodic barriers. Early integrin clustering recruits adhesion proteins, talin, paxillin and focal adhesion kinase, irrespective of force generation. However, recruitment of vinculin is only observed upon contraction. Thus, we suggest that integrin activation and early clustering are independent of lateral forces. Src-dependent actin polymerization on integrin clusters and subsequent contraction stimulate active spreading with outward forces from actin polymerization followed by a second wave of contraction. These early mechanical steps provide new targets to control integrin-dependent adhesion and motility.

Early integrin binding to Arg-Gly-Asp peptide activates actin polymerization and contractile movement that stimulates outward translocation

Integrin-mediated adhesions are critical for stem cell differentiation, cancer metastasis, and the immune response [Hynes RO (2009) Science 326:1216-1219]. However, the mechanisms of early adhesion formation remain unclear, especially the effects of lateral clustering of integrins and the role of the Src family kinases. Using mobile Arg-Gly-Asp (RGD) peptide ligands on lipid bilayers with nano-fabricated physical barriers [Salaita K, et al. (2010) Science 327:1380-1385], we observe surprising long-range lateral movements of ligated integrins during the process of cell spreading. Initially, RGD-activated integrin clusters stimulate actin polymerization that radiates from the clusters. Myosin II contraction of actin from adjacent clusters produces contractile pairs that move toward each other against barriers. Force generated by myosin II stimulates a Src kinase-dependent lamellipodial extension and outward movement of clusters. Subsequent retraction by myosin II causes inward movement of clusters. The final cell spread area increases with the density of periodic barriers. Early integrin clustering recruits adhesion proteins, talin, paxillin, and FAK, irrespective of force generation. However, recruitment of vinculin is only observed upon contraction. Thus, we suggest that integrin activation and early clustering are independent of lateral forces. Clustering activates Src-dependent actin polymerization from clusters. Myosin contraction of clusters to lines stimulates active spreading with outward forces from actin polymerization followed by a second wave of contraction. Many of these early mechanical steps are not evident in cells spreading on immobilized matrices perhaps because of the low forces involved. These observations can provide new targets to control integrin-dependent adhesion and motility.

Force generated by actomyosin contraction builds bridges between adhesive contacts

Extracellular matrices in vivo are heterogeneous structures containing gaps that cells bridge with an actomyosin network. To understand the basis of bridging, we plated cells on surfaces patterned with fibronectin (FN)-coated stripes separated by non-adhesive regions. Bridges developed large tensions where concave cell edges were anchored to FN by adhesion sites. Actomyosin complexes assembled near those sites (both actin and myosin filaments) and moved towards the centre of the non-adhesive regions in a treadmilling network. Inhibition of myosin-II (MII) or Rho-kinase collapsed bridges, whereas extension continued over adhesive areas. Inhibition of actin polymerization (latrunculin-A, jasplakinolide) also collapsed the actomyosin network. We suggest that MII has distinct functions at different bridge regions: (1) at the concave edges of bridges, MIIA force stimulates actin filament assembly at adhesions and (2) in the body of bridges, myosin cross-links actin filaments and stimulates actomyosin network healing when breaks occur. Both activities ensure turnover of actin networks needed to maintain stable bridges from one adhesive region to another.

Grp78 promotes the invasion of hepatocellular carcinoma

BackgroundGlucose regulated protein 78 (Grp78) is involved in the invasion and metastasis in many human cancers including gastric cancer, breast cancer, prostate cancer. But the role of Grp78 in the invasion of human hepatocellular carcinoma has not been reported. In this article, we examined if Grp78 was associated with the invasion of hepatocellular carcinoma and explored the possible underlying mechanism.MethodsThe Grp78 and FAK expression levels in 44 patients with hepatocellular carcinoma were examined using immunohistochemistry. Grp78 overexpressing SMMC7721 cells were established by pcDNA3.1 (+)-Grp78 transfection and screened by G418. Grp78 and FAK levels in Grp78 overexpressing cells were down-regulated by siRNA transfection. The invasion status of tumor cells was evaluated by transwell assay in vitro, and chick embryo metastasis model in vivo. Cell spreading was determined by cell spreading assay, and quantitatively measured by Orisis software HUG. Grp78, pY397 FAK, pY576/577 FAK and FAK levels were detected by western blot. RhoA activity was detected by GST pulldown assay. The distribution of actin cytoskeleton was observed by fluorescent staining.ResultsGrp78 expression levels in 44 patients with hepatocellular carcinoma were negatively correlated with tumor grading, and positively correlated with portal invasion and intra-hepatic invasion. Overexpression of Grp78 in SMMC7721 cells promoted the invasion of cancer cells in vitro and in vivo, and this increase in tumor cell invasion was blocked by Grp78 siRNA knockdown. Our results also revealed that overexpression of Grp78 in SMMC7721 cells accelerated the process of cell spreading and promoted lamellipodia formation. Further analysis showed that overexpression of Grp78 in SMMC7721 cells increased pY397 and pY576/577 levels of FAK. Grp78 siRNA knockdown decreased FAK activation and activity. Our results also revealed that Grp78 overexpression in SMMC7721 cells decreased RhoA-GTP level, and Grp78 siRNA knockdown rescued RhoA-GTP level in Grp78 overexpressing cells, indicating Grp78 inhibited RhoA activity in hepatocellular carcinoma cells. Furthermore, overexpression of Grp78 in SMMC7721 cells increased phospho-p190RhoGAP level. FAK siRNA knockdown in Grp78 overexpressing cells reversed phospho-p190RhoGAP level. These data suggested that Grp78 inhibited RhoA activity by up-regulated phospho-p190RhoGAP level and Grp78 mediated p190RhoGAP phosphorylation is FAK dependent.ConclusionGrp78 promoted the invasion of hepatocellular carcinoma both in vitro and in vivo. Overexpression of Grp78 in hepatocellular carcinoma cells enhanced the activation and activity of FAK which negatively regulated Rock kinase activity by promoting the phosphorylation of p190RhoGAP.

RON-expressing MCF-10A breast epithelial cells exhibit alterations of hyaluronan expression, promoting RON-mediated early adhesion events

The receptor tyrosine kinase known as RON appears to play a role in the progression of human carcinomas, and is associated with a poor patient prognosis. Our current study demonstrates that RON expression in MCF-10A breast epithelial cells lead to an alteration of cell-surface hyaluronan compared to the parental cells. We found that hyaluronan was important for initial cell attachment to poly- d-lysine-coated coverslips, but did not contribute to the process of cell spreading. Previous data implied that the Src kinase was important for spreading but not the initial attachment of 10A cells, and here we demonstrate Src activation was also not necessary for hyaluronan production in these cells.

Computational modeling for cell spreading on a substrate mediated by specific interactions, long-range recruiting interactions, and diffusion of binders

A continuum model was proposed to study cell spreading on a flat substrate mediated by specific interaction, long-range recruiting interaction, and the diffusion of binders. Specific interactions between the mobile receptors embedded in the cell membrane and ligands coated on the substrate surface result in cell adhesion to the substrate surface. This receptor-ligand interaction was described by a chemical reaction equation. Long-range recruiting interactions between the receptors and the substrate were simplified by a traction-separation law. The governing equations and boundary conditions were formulated for the entire process of cell spreading and solved using a finite element scheme. Parametric studies were conducted to investigate the effect of system parameters on the cell spreading kinetics. It is shown that kinetic factors play an important role in cell adhesion and three regimes, that is, the binder reaction limited regime, long-range recruiting force-driven binder recruitment limited regime, and the concentration gradient-driven diffusion limited regime, were identified.