Fibronectin Matrix Research Articles

Coronin-1C Protein and Caveolin Protein Provide Constitutive and Inducible Mechanisms of Rac1 Protein Trafficking

Sustained directional fibroblast migration requires both polarized activation of the protrusive signal, Rac1, and redistribution of inactive Rac1 from the rear of the cell so that it can be redistributed or degraded. In this work, we determine how alternative endocytic mechanisms dictate the fate of Rac1 in response to the extracellular matrix environment. We discover that both coronin-1C and caveolin retrieve Rac1 from similar locations at the rear and sides of the cell. We find that coronin-1C-mediated extraction, which is responsible for Rac1 recycling, is a constitutive process that maintains Rac1 protein levels within the cell. In the absence of coronin-1C, the effect of caveolin-mediated endocytosis, which targets Rac1 for proteasomal degradation, becomes apparent. Unlike constitutive coronin-1C-mediated trafficking, caveolin-mediated Rac1 endocytosis is induced by engagement of the fibronectin receptor syndecan-4. Such an inducible endocytic/degradation mechanism would predict that, in the presence of fibronectin, caveolin defines regions of the cell that are resistant to Rac1 activation but, in the absence of fibronectin leaves more of the membrane susceptible to Rac1 activation and protrusion. Indeed, we demonstrate that fibronectin-stimulated activation of Rac1 is accelerated in the absence of caveolin and that, when caveolin is knocked down, polarization of active Rac1 is lost in FRET experiments and culminates in shunting migration in a fibrous fibronectin matrix. Although the concept of polarized Rac1 activity in response to chemoattractants has always been apparent, our understanding of the balance between recycling and degradation explains how polarity can be maintained when the chemotactic gradient has faded.

Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

Pluripotent embryonic stem cells (ESCs) exert low-traction forces on their niche in vitro whereas specification to definitive endoderm in vivo coincides with force-mediated motility, suggesting a differentiation-mediated switch. However, the onset of contractility and extent to which force-mediated integrin signaling regulates fate choices is not understood. To address the requirement of tractions forces for differentiation, we examined mouse embryonic stem cell (ESC) specification towards definitive endoderm on fibrillar fibronectin containing a deformation-sensitive FRET probe. Inhibiting contractility resulted in an increase in the observed fibronectin FRET intensity ratio but also decreased the amount of phosphorylated nuclear SMAD2, leading to reduced expression of the definitive endoderm marker SOX17. By contrast ESCs maintained in pluripotency medium did not exert significant tractions against the fibronectin matrix. When laminin-111 was added to fibrillar matrices to improve the efficiency of definitive endoderm induction, ESCs decreased their fibronectin traction forces in a laminin-dependent manner; blocking the laminin-binding α3-integrin restored fibronectin matrix deformation and reduced SOX17 expression and SMAD2 phosphorylation, probably because of compensation of inhibitory signaling from SMAD7 after 5 days in culture. These data imply that traction forces and integrin signaling are important regulators of early fate decisions in ESCs.

Increased expression of Solute carrier family 12 member 5 via gene amplification contributes to tumour progression and metastasis and associates with poor survival in colorectal cancer

ObjectiveUsing whole genome sequencing, we identified gene amplification of solute carrier family 12 member 5 (SLC12A5) located at 20q13.12 in colorectal cancer (CRC). We analysed its amplification, overexpression, biological effects...

You-gui Pill ameliorates renal tubulointerstitial fibrosis via inhibition of TGF-β/Smad signaling pathway

Ethnopharmacological relevanceYou-gui Pill (YGP), a traditional Chinese medicinal prescription, was widely used to warm and recuperate “kidney-yang” clinically for hundreds of years in China. Recent studies found that YGP had a potential benefit for renoprotection. Aim of the studyThe present study aimed to elucidate the in vivo and in vitro efficacy of YGP on renal tubulointerstitial fibrosis, and the molecular mechanism is also investigated. Materials and methodsRat renal tubulointerstitial fibrosis model was elicited by unilateral ureteral obstruction (UUO). Sprague–Dawley rats underwent UUO and were studied after 14 days. Animals were randomly subjected to six groups: sham, UUO, UUO/YGP (0.14, 0.42, 1.26g/kg/d), and UUO/enalapril (10mg/kg/d). HE, Masson and ELISA were used for evaluate renal injury and function. Immunohistochemical analysis and western blot were used to detect the expressions of α-SMA, fibronectin, collagen matrix and Smads. In vitro studies were investigated in TGF-β1-stiumlated NRK-49F cell line. ResultsOral administration of YGP significantly decreased UUO-induced inflammatory cell infiltration, tubular atrophy and interstitial fibrosis, and there was no significant difference between YGP at 1.26g/kg and enalapril at 10mg/kg treatment (P>0.05). Meanwhile, serum creatinine and blood urea nitrogen levels were reduced dramatically (P<0.01). In coincide with the decreased of TGF-β1, α-SMA, fibronectin and collagen matrix expressions were also declined with YGP treatment in both UUO kidneys and TGF-β1-stimulated NRK-49F cell line. Additionally, nuclear translocation of p-Smad2/3 was markedly down-regulated by YGP (P<0.001), with a relative mild up-regulated expression of Smad7 (P<0.05). ConclusionsOur findings demonstrate that YGP had a renoprotective effect in ameliorating renal tubulointerstitial fibrosis, and this activity possibly via suppression of the TGF-β and its downstream regulatory signaling pathway, including Smad2/3.

Molecular differences between stromal cell populations from deciduous and permanent human teeth.

IntroductionDeciduous and permanent human teeth represent an excellent model system to study aging of stromal populations. Aging is tightly connected to self-renewal and proliferation and thus, mapping potential molecular differences in these characteristics between populations constitutes an important task.MethodsUsing specifically designed microarray panels, Real-Time Quantitative Polymerase Chain Reaction (RT q-PCR), Western blot, immunohistochemistry and siRNA-mediated knock down experiments, we have detected a number of molecules that were differentially expressed in dental pulp from deciduous and permanent teeth extracted from young children and adults, respectively.ResultsAmong the differentially regulated genes, high-mobility group AT-hook 2 (HMGA2), a stem cell-associated marker, stood out as a remarkable example with a robust expression in deciduous pulp cells. siRNA-mediated knock down of HMGA2 expression in cultured deciduous pulp cells caused a down-regulated expression of the pluripotency marker NANOG. This finding indicates that HMGA2 is a pulpal stem cell regulatory factor. In addition to this, we discovered that several proliferation-related genes, including CDC2A and CDK4, were up-regulated in deciduous pulp cells, while matrix genes COL1A1, fibronectin and several signaling molecules, such as VEGF, FGFr-1 and IGFr-1 were up-regulated in the pulp cells from permanent teeth.ConclusionsTaken together, our data suggest that deciduous pulp cells are more robust in self- renewal and proliferation, whereas adult dental pulp cells are more capable of signaling and matrix synthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0056-7) contains supplementary material, which is available to authorized users.

Design of an osteoinductive extracellular fibronectin matrix protein for bone tissue engineering.

Integrin-mediated cell-matrix interactions play an important role in osteogenesis. Here, we constructed a novel osteoinductive fibronectin matrix protein (oFN) for bone tissue engineering, designed to combine the integrin-binding modules from fibronectin (iFN) and a strong osteoinductive growth factor, bone morphogenetic protein-2. Compared with iFN, the purified oFN matrix protein caused a significant increase in cell adhesion and osteogenic differentiation of pre-osteoblast MC3T3-E1 cells (p < 0.05).

MAP1S‐enhanced Autophagy Alleviates Tissue Fibrosis and Sustains Mouse Survival under Stress Imposed by LC3‐induced Overexpression of Fibronectin

Autophagy is a cellular process that controls and executes the turnover of misfolded and abnormally aggregated proteins. Microtubule‐associated protein 1S (MAP1S) interacts with autophagy marker light chain 3 (LC3) and positively regulates autophagy flux. LC3 also binds with fibronectin mRNA and facilitates its sorting along microtubules onto rough endoplasmic reticulum where it is translated into fibronectin protein. Fibronectin is exported to surface of plasma membrane to initiate the assembly of fibronectin extracellular matrix and other extracellular matrix proteins. Fibronectin matrix is degraded in lysosomes after it is engulfed into cytosol via endocytosis. Here we show that overexpression of LC3 in wildtype mice promoted the synthesis and degradation of fibronectin and consequently resulted in a balanced levels of fibronectin; therefore, the GFP‐LC3 transgenic mice had a normal length of lifespan as the wild‐type mice did. In GFP‐LC3 transgenic mice with MAP1S deleted, LC3 overexpression enhanced synthesis of fibronectin while MAP1S‐deficiency‐caused autophagy defect impaired the degradation of fibronectin. The accumulation of fibronectin protein led to enhanced levels of oxidative stress, genomic instability and tissue fibrosis, and to a dramatic 35% reduction in mouse lifespan. Therefore, autophagy may play an important role in maintaining mouse lifespan via regulating the homeostasis of fibronectin extracellular matrix.This work was supported by a NIH R01CA142862 to Leyuan Liu.

β1‐Integrin, KCa3.1 and TRPC4 Channels Play Complementary Roles in Alveolar Epithelial Repair

Extensive alveolar epithelial injury and remodelling are typical features of acute respiratory distress syndrome (ARDS). It has been established that epithelial regeneration, and secondary lung oedema resorption, are crucial for the resolution of ARDS parameters. Interestingly, many evidences indicate that K+ channels are regulating epithelial repair processes; however the role of Ca2+‐activated KCa3.1 channels in alveolar repair has never been investigated before.Wound healing assays first demonstrated that the repair rates of primary rat alveolar cell monolayers were increased on a fibronectin matrix compared to non‐coated supports. KCa3.1 inhibition impaired the fibronectin‐stimulated wound‐healing rates, as well as cell migration and proliferation, but was without effect in absence of coating. We then evaluated a putative relationship between KCa3.1 and the β1‐integrin, which is activated by fibronectin. Immunofluorescence and co‐immunoprecipitation assays indicated a cellular co‐distribution and a physical link between the two proteins. In addition, KCa3.1 and β1‐integrin membrane expressions were both increased on a fibronectin matrix. We also showed increased [Ca2+]i as well as enhanced expression of TRPC4, a non‐voltage gated Ca2+ channel of the large TRP channel family, in the presence of fibronectin. Finally, wound healing assays showed that TRPC4 function is also involved in alveolar repair.Altogether, our data demonstrate for the first time a complementary role of β1‐integrin, KCa3.1 and TRPC4 channels in the regulation of alveolar repair processes.Funded by CIHR, FRQS and NSERC.

Combined effect of insulin-like growth factor-1 and CC chemokine ligand 2 on angiogenic events in endothelial cells.

Therapeutic angiogenesis may be applied in medical conditions to promote stimulation of angiogenesis. Angiogenesis is a multistep process, which includes endothelial cell proliferation, migration, and tube formation, which is mediated by various angiogenic polypeptides. Thus, studies that elucidate the cellular mechanisms involved in these processes are necessary to develop novel therapeutic strategies. This study investigated the in vitro effects of the pro-angiogenic factors, insulin-like growth factor-1 (IGF-1) and/or chemokine (CC motif) ligand 2 (CCL2), on endothelial cells. Flow cytometry analysis showed that IGF-1 and CCL2 treatment did not interfere with IGF-1 receptor (IGF-1R) expression, but CCL2 treatment increased CCL2 receptor (CCR2) expression. Immunofluorescence analysis revealed that the IGF-1/CCL2 combination induced a greater increase in fibronectin deposition, but the treatments did not alter the expression of the fibronectin receptors, CD49e and CD44. The interaction of fibronectin with cytokines demonstrated that IGF-1/CCL2 promoted changes in intermediate F-actin remodeling that may result in increased endothelial cell adhesion and cell migration mediated by fibronectin. Furthermore, IGF-1/CCL2 stimulated endothelial cells, grown on fibronectin, to form capillary-like structures and intercellular lumina with greater luminal area. These data suggest that IGF-1/CCL2 combination and a fibronectin matrix may contribute to the angiogenesis process to stimulate adhesion, migration, and tube formation by endothelial cells as a result of F-actin remodeling.

Extracellular Matrix Fibronectin Attenuates PDGF‐Induced Intracellular Calcium Release

The extracellular matrix (ECM) has structural and signaling roles within tissues. Fibronectin is an ECM protein that is secreted by fibroblasts in a protomeric form and assembled into a fibrillar matrix by a cell‐mediated process termed fibronectin matrix assembly. ECM proteins affect growth factor signaling. Platelet derived growth factor (PDGF) is a major mitogen for fibroblasts that promotes proliferation and migration. The objective of this study was to determine the effect of ECM fibronectin on the response of fibroblasts to PDGF. The response of cells to PDGF was quantified by measuring intracellular calcium release in Fluo‐4 loaded cells. Calcium release was measured in fibronectin‐null mouse embryonic fibroblasts, allowing for control over the amount of fibronectin to which cells were exposed and the initiation of fibronectin matrix assembly. Results showed that in the absence of fibronectin, addition of 50 ng/mL PDGF to cells transiently increased intracellular calcium concentration. In contrast, incubation of cells with 25 nM fibronectin for 20 hr significantly attenuated the PDGF‐induced increase in calcium release. Fibronectin‐mediated attenuation of PDGF‐induced calcium release was blocked by an inhibitor of fibronectin matrix assembly, indicating that the ECM form of fibronectin is required for fibronectin‐mediated attenuation of PDGF signaling. A fibronectin fragment engineered to mimic the ECM form of fibronectin also attenuated PDGF‐induced calcium release. Attenuation of PDGF‐induced calcium release was not due to downregulation of PDGF receptor β expression. This study demonstrates that fibronectin matrix assembly attenuates PDGF‐induced intracellular calcium release by an as‐yet unidentified mechanism.Supported by: NIH/NHLBI: T32 HL066988

Megakaryoblastic leukemia-1 is required for the development of bleomycin-induced pulmonary fibrosis.

BackgroundFibrosing disorders of the lung, such as idiopathic pulmonary fibrosis, are characterized by progressive extracellular matrix accumulation that is driven by myofibroblasts. The transcription factor megakaryoblastic leukemia-1 (MKL1) mediates myofibroblast differentiation in response to several profibrotic stimuli, but the role it plays in mediating pulmonary fibrosis has not been fully elucidated. In this study, we utilized mice that had a germline deletion of MKL1 (MKL1 (−,-)) to determine the role that MKL1 plays in the development of bleomycin-induced pulmonary fibrosis.MethodsBleomycin or normal saline were intratracheally delivered to 9 to 12 week old female MKL1 (+,+) and MKL1 (−,-) mice. Mice were assessed for weight loss and survival to 28 days. Inflammatory responses were assessed through bronchoalveolar lavage at days 3 and 7 post-treatment. The development of pulmonary fibrosis was characterized using hydroxyproline assay and histological staining. MKL1 (+,+) and MKL1 (−,-) mouse lung fibroblasts were isolated to compare morphologic, gene expression and functional differences.ResultsMKL1 (−,-) mice demonstrated increased survival, attenuated weight loss, and decreased collagen accumulation compared to wild-type animals 28-days after intratracheal instillation of bleomycin. Histological analysis demonstrated decreased trichrome, smooth muscle α-actin, and fibronectin staining in MKL1(−,-) mice compared to MKL1 (+,+) controls. Differential cell counts from bronchoalveolar lavage demonstrated that there was attenuated neutrophilia 3 days after bleomycin administration, but no difference at day 7. Isolated mouse lung fibroblasts from MKL1 (−,-) mice had decreased contractility and deposited less fibronectin matrix compared to wild-type controls, suggesting a defect in key remodeling functions.ConclusionsAltogether, these data demonstrate that MKL1 plays a significant role in mediating the fibrotic response to bleomycin injury. Loss of MKL1 attenuated early neutrophil influx, as well as myofibroblast-mediated remodeling. Targeting MKL1 activity may therefore be a useful strategy in treating pulmonary fibrosis.

Fibronectin Matrix Assembly after Spinal Cord Injury.

After spinal cord injury (SCI), a fibrotic scar forms at the injury site that is best characterized by the accumulation of perivascular fibroblasts and deposition of the extracellular matrix protein fibronectin. While fibronectin is a growth-permissive substrate for axons, the fibrotic scar is inhibitory to axon regeneration. The mechanism behind how fibronectin contributes to the inhibitory environment and how the fibronectin matrix is assembled in the fibrotic scar is unknown. By deleting fibronectin in myeloid cells, we demonstrate that fibroblasts are most likely the major source of fibronectin in the fibrotic scar. In addition, we demonstrate that fibronectin is initially present in a soluble form and is assembled into a matrix at 7 d post-SCI. Assembly of the fibronectin matrix may be mediated by the canonical fibronectin receptor, integrin α5β1, which is primarily expressed by activated macrophages/microglia in the fibrotic scar. Despite the pronounced cavitation after rat SCI, fibrotic scar also is observed in a rat SCI model, which is considered to be more similar to human pathology. Taken together, our study provides insight into the mechanism of fibrotic scar formation after spinal cord injury.

Myofibroblasts Exhibit Enhanced Fibronectin Assembly That Is Intrinsic to Their Contractile Phenotype

Myofibroblasts have increased expression of contractile proteins and display augmented contractility. It is not known if the augmented contractile gene expression characterizing the myofibroblast phenotype impacts its intrinsic ability to assemble fibronectin (FN) and extracellular matrix. In this study we investigated whether myofibroblasts displayed increased rates of FN fibril assembly when compared with their undifferentiated counterparts. Freshly plated myofibroblasts assemble exogenous FN (488-FN) into a fibrillar matrix more rapidly than fibroblasts that have not undergone myofibroblast differentiation. The augmented rate of FN matrix formation by myofibroblasts was dependent on intact Rho/Rho kinase (ROCK) and myosin signals inasmuch as treatment with Y27632 or blebbistatin attenuated 488-FN assembly. Inhibiting contractile gene expression by pharmacologic disruption of the transcription factors megakaryoblastic leukemia-1 (MKL1)/serum response factor (SRF) during myofibroblast differentiation resulted in decreased contractile force generation and attenuated 488-FN incorporation although not FN expression. Furthermore, disruption of the MKL1/SRF target gene, smooth muscle α-actin (α-SMA) via siRNA knockdown resulted in attenuation of 488-FN assembly. In conclusion, this study demonstrates a linkage between increased contractile gene expression, most importantly α-SMA, and the intrinsic capacity of myofibroblasts to assemble exogenous FN into fibrillar extracellular matrix.

Molecular pathways: linking tumor microenvironment to epithelial-mesenchymal transition in metastasis.

During tumor development, tumor cells constantly communicate with the surrounding microenvironment through both biochemical and biophysical cues. In particular, the tumor microenvironment can instruct carcinoma cells to undergo a morphogenesis program termed epithelial-to-mesenchymal transition (EMT) to facilitate local invasion and metastatic dissemination. Growing evidence uncovered a plethora of microenvironmental factors in promoting EMT, including proinflammatory cytokines secreted by locally activated stromal cells, hypoxia conditions, extracellular matrix components, and mechanical properties. Here, we review various biochemical and biophysical factors in the tumor microenvironment that directly impinge upon the EMT program. Specifically, cytokines such as TGFβ, TNFα, and IL6 and hypoxia are capable of inducing EMT in various tumors. Several extracellular matrix (ECM) proteins, including collagen-I, fibronectin, and hyaluronan, and ECM remodeling via extracellular lysyl oxidase are also implicated in regulating EMT. In preclinical studies and ongoing clinical trials, targeting these tumor microenvironmental signals has shown promises in halting tumor progression in various human cancers.

Development of a biological scaffold engineered using the extracellular matrix secreted by skeletal muscle cells

The performance of implantable biomaterials derived from decellularized tissue, including encouraging results with skeletal muscle, suggests that the extracellular matrix (ECM) derived from native tissue has promising regenerative potential. Yet, the supply of biomaterials derived from donated tissue will always be limited, which is why the in-vitro fabrication of ECM biomaterials that mimic the properties of tissue is an attractive alternative. Towards this end, our group has utilized a novel method to collect the ECM that skeletal muscle myoblasts secrete and form it into implantable scaffolds. The cell derived ECM contained several matrix constituents, including collagen and fibronectin that were also identified within skeletal muscle samples. The ECM was organized into a porous network that could be formed with the elongated and aligned architecture observed within muscle samples. The ECM material supported the attachment and in-vitro proliferation of cells, suggesting effectiveness for cell transplantation, and was well tolerated by the host when examined in-vivo. The results suggest that the ECM collection approach can be used to produce biomaterials with compositions and structures that are similar to muscle samples, and while the physical properties may not yet match muscle values, the in-vitro and in-vivo results indicate it may be a suitable first generation alternative to tissue derived biomaterials.

Relocation of CLIC1 promotes tumor cell invasion and colonization of fibrin.

Chloride intracellular channel 1 (CLIC1) has been shown to be upregulated in various malignancies but its exact function remains unclear. Here, it is revealed that CLIC1 is critical for the stability of invadopodia in endothelial and tumor cells embedded in a 3-dimensional (3D) matrix of fibrin. Invadopodia stability was associated with the capacity of CLIC1 to induce stress fiber and fibronectin matrix formation following its β3 integrin (ITGB3)-mediated recruitment into invadopodia. This pathway, in turn, was relevant for fibrin colonization as well as slug (SNAI2) expression and correlated with a significant role of CLIC1 in metastasis in vivo. Mechanistically, a reduction of myosin light chain kinase (MYLK) in CLIC1-depleted as well as β3 integrin-depleted cells suggests an important role of CLIC1 for integrin-mediated actomyosin dynamics in cells embedded in fibrin. Overall, these results indicate that CLIC1 is an important contributor to tumor invasion, metastasis, and angiogenesis. This study uncovers an important new function of CLIC1 in the regulation of cell-extracellular matrix interactions and ability of tumor cells to metastasize to distant organs.

Role of tumor cell surface lysosome-associated membrane protein-1 (LAMP1) and its associated carbohydrates in lung metastasis.

Expression of lysosome-associated membrane protein-1 (LAMP1) on the surface correlates with metastatic potential of B16 melanoma cells. Downregulation of their expression in high metastatic (B16F10) cells reduced their surface expression and metastatic potential. Present investigations explore if overexpression of LAMP1 on the surface of low metastatic (B16F1) cells augment their metastatic ability, and if so, how? B16F1 cells were transduced with lentiviral vector carrying mutant-LAMP1 (Y386A) (mutLAMP1). Surface expression of LAMP1 and carbohydrates was analyzed by flow cytometry, immunofluorescence and/or immunoprecipitation and Western blotting. Cell spreading and motility were assessed on components of extracellular matrix (ECM) (fibronectin) and basement membrane (BM) (matrigel), and galectin-3-coated coverslips/plates. Metastatic potential was assessed using experimental metastasis assay. Pre-incubation with anti-LAMP1 antibodies significantly reduced lung metastasis of B16F10 cells. Overexpression of mutLAMP1 significantly increased its surface expression on B16F1 cells, resulting in increased cellular spreading and motility on fibronectin and matrigel. LAMP1 is the major carrier of poly-N-acetyllactosamine (polyLacNAc) on B16F10 cells. However, significantly higher expression of mutLAMP1 had no effect on galectin-3 binding on cell surface or on spreading or motility of cells on galectin-3-coated coverslips/plates. These cells also failed to show any gain in metastatic ability. This could be because LAMP1 from these cells carried significantly lower levels of polyLacNAc in comparison with B16F10 cells. PolyLacNAc on B16F10 cells and galectin-3 on lungs are the major participants in melanoma metastasis. Although surface LAMP1 promotes interactions with organ ECM and BM, carbohydrates on LAMP1 play a decisive role in dictating lung metastasis.

Inflammatory cell infiltration in experimental liver fibrosis is affected by inhibition of fibronectin matrix assembly

Background and Aims: The accumulation of inflammatory cells is an important step in liver fibrosis. Fibronectin has been shown to enhance tissue infiltration of various cell types including immune cells. Our aim was to determine how the loss of fibronectin in the liver might affect the inflammatory response to liver injury. In prior work by our group, deletion of fibronectin production by the myofibroblasts resulted in an increase in available TGF-β and hence an increase in fibrotic signals within the liver precluding an examination of the direct effect of fibronectin on inflammation in the response to fibrosis induction. We therefore instead inhibited fibronectin fibril formation pharmacologically and evaluated the various inflammatory cells in a toxic liver injury model.

PDGF‑stimulated dispersal of cell clusters and disruption of fibronectin matrix on three-dimensional collagen matrices requires matrix metalloproteinase-2.

Formation of cell clusters is a common morphogenic cell behavior observed during tissue and organ development and homeostasis, as well as during pathological disorders. Dynamic regulation of cell clustering depends on the balance between contraction of cells into clusters and migration of cells as dispersed individuals. Previously we reported that under procontractile culture conditions, fibronectin fibrillar matrix assembly by human fibroblasts functioned as a nucleation center for cell clustering on three-dimensional collagen matrices. Here we report that switching preformed cell clusters from procontractile to promigratory culture conditions results in cell dispersal out of clusters and disruption of FN matrix. Experiments using small interfering RNA silencing and pharmacological inhibition demonstrated that matrix metalloproteinase activity involving MMP-2 was necessary for fibronectin matrix disruption and dispersal of cell clusters.

Increasing throughput of AFM-based single cell adhesion measurements through multisubstrate surfaces.

Mammalian cells regulate adhesion by expressing and regulating a diverse array of cell adhesion molecules on their cell surfaces. Since different cell types express distinct sets of cell adhesion molecules, substrate-specific adhesion is cell type- and condition-dependent. Single-cell force spectroscopy is used to quantify the contribution of cell adhesion molecules to adhesion of cells to specific substrates at both the cell and single molecule level. However, the low throughput of single-cell adhesion experiments greatly limits the number of substrates that can be examined. In order to overcome this limitation, segmented polydimethylsiloxane (PDMS) masks were developed, allowing the measurement of cell adhesion to multiple substrates. To verify the utility of the masks, the adhesion of four different cell lines, HeLa (Kyoto), prostate cancer (PC), mouse kidney fibroblast and MDCK, to three extracellular matrix proteins, fibronectin, collagen I and laminin 332, was examined. The adhesion of each cell line to different matrix proteins was found to be distinct; no two cell lines adhered equally to each of the proteins. The PDMS masks improved the throughput limitation of single-cell force spectroscopy and allowed for experiments that previously were not feasible. Since the masks are economical and versatile, they can aid in the improvement of various assays.