Fibronectin Matrix Assembly Research Articles

MYPT1 regulates contractility and microtubule acetylation to modulate integrin adhesions and matrix assembly

Although much is known about how individual cytoskeletal systems contribute to physiological processes such as cell migration and branching morphogenesis, little is known about how these different systems actively coordinate their functions after polymerization. Here we show that both fibroblasts and developing glands reciprocally coordinate levels of cellular contractility and microtubule acetylation. We find that this balance is achieved by interaction of the myosin phosphatase target subunit of myosin phosphatase with either myosin light chain or HDAC6, a microtubule deacetylase. This balance of contractility and microtubule acetylation controls progression of adhesion maturation by regulating surface density of α5β1 integrin and fibronectin. Thus, we propose that a homeostatic balance between contractility and microtubule acetylation is mediated by myosin phosphatase via controlled activation and deactivation of myosin II and HDAC6. This regulates the surface density of α5β1 integrin to modulate fibronectin matrix assembly and governs rates of cell migration and branching morphogenesis.

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

Extracellular matrix fibronectin fibrils serve as passive structural supports for the organization of cells into tissues, yet can also actively stimulate a variety of cell and tissue functions, including cell proliferation. Factors that control and coordinate the functional activities of fibronectin fibrils are not known. Here, we compared effects of cell adhesion to vitronectin versus type I collagen on the assembly of and response to, extracellular matrix fibronectin fibrils. The amount of insoluble fibronectin matrix fibrils assembled by fibronectin-null mouse embryonic fibroblasts adherent to collagen- or vitronectin-coated substrates was not significantly different 20h after fibronectin addition. However, the fibronectin matrix produced by vitronectin-adherent cells was ~10-fold less effective at enhancing cell proliferation than that of collagen-adherent cells. Increasing insoluble fibronectin levels with the fibronectin fragment, anastellin did not increase cell proliferation. Rather, native fibronectin fibrils polymerized by collagen- and vitronectin-adherent cells exhibited conformational differences in the growth-promoting, III-1 region of fibronectin, with collagen-adherent cells producing fibronectin fibrils in a more extended conformation. Fibronectin matrix assembly on either substrate was mediated by α5β1 integrins. However, on vitronectin-adherent cells, α5β1 integrins functioned in a lower activation state, characterized by reduced 9EG7 binding and decreased talin association. The inhibitory effect of vitronectin on fibronectin-mediated cell proliferation was localized to the cell-binding domain, but was not a general property of αvβ3 integrin-binding substrates. These data suggest that adhesion to vitronectin allows for the uncoupling of fibronectin fibril formation from downstream signaling events by reducing α5β1 integrin activation and fibronectin fibril extension.

Hsp90 Binds Directly to Fibronectin (FN) and Inhibition Reduces the Extracellular Fibronectin Matrix in Breast Cancer Cells

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis.

Fibronectin matrix assembly is essential for cell condensation during chondrogenesis.

Mesenchymal cell condensation is the initiating event in endochondral bone formation. Cell condensation is followed by differentiation into chondrocytes, which is accompanied by induction of chondrogenic gene expression. Gene mutations involved in chondrogenesis cause chondrodysplasias and other skeletal defects. Using mesenchymal stem cells (MSCs) in an in vitro chondrogenesis assay, we found that knockdown of the diastrophic dysplasia (DTD) sulfate transporter (DTDST, also known as SLC26A2), which is required for normal cartilage development, blocked cell condensation and caused a significant reduction in fibronectin matrix. Knockdown of fibronectin with small interfering RNAs (siRNAs) also blocked condensation. Fibrillar fibronectin matrix was detected prior to cell condensation, and its levels increased during and after condensation. Inhibition of fibronectin matrix assembly by use of the functional upstream domain (FUD) of adhesin F1 from Streptococcus pyogenes prevented cell condensation by MSCs and also by the chondrogenic cell line ATDC5. Our data show that cell condensation and induction of chondrogenesis depend on fibronectin matrix assembly and DTDST, and indicate that this transporter is required earlier in chondrogenesis than previously appreciated. They also raise the possibility that certain of the skeletal defects in DTD patients might derive from the link between DTDST, fibronectin matrix and condensation.

Abstract PR11: Ovarian cancer cells activate the mesothelium in the peritoneal microenvironment promoting fibronectin production and the early steps of metastasis

Abstract The main site of ovarian cancer (OvCa) metastasis is the omentum, which is covered by a single layer of mesothelial cells overlaying an extracellular matrix interspersed with fibroblasts (mesothelium). A 3D model of the human peritoneal surface (in vitro), fragments of human omentum (ex vivo), and a mouse xenograft model (in vivo) of OvCa were used to investigate the early effects of cancer cells on the host stromal microenvironment. Our results reveal that fibronectin is overexpressed in the stroma of more than 93% of OvCa omental metastases. OvCa cells induce fibronectin matrix assembly in omental stromal cells, and stimulate fibronectin expression in mesothelial cells. The fibronectin produced by human mesothelial stromal cells promotes OvCa cell adhesion, invasion and proliferation. Inhibition of fibronectin production in mesothelial cells or omental surface cells using a fibronectin specific siRNA decreases OvCa cell adhesion, invasion and proliferation. Genetic knock-down of fibronectin in vitro and in vivo, using fibronectinfl/fl mice, also impaired OvCa cell adhesion/invasion and metastasis. Re-analysis of an Australian OvCa study gene array data set suggested that OvCa, which highly express fibronectin, show co-activation of TGFβ1/Rac1 dependent signaling pathways. Indeed, OvCa cells increase phosphorylation of Smad2/3 in mesothelial cells upon co-culture. Inhibition of TGFβ1 in OvCa cells or inhibition of Smad 3, as well as TGFβRI or TGFβRII, in mesothelial cells resulted in decreased fibronectin production. Moreover, Rac 1 activity was increased upon the co-culture of OvCa and mesothelial cells. Subsequent inhibition of Rac 1 in mesothelial cells led to a decrease in fibronectin secretion. Our data suggest that OvCa cells regulate fibronectin expression in mesothelial cells through a TGFβ1/Rac1 dependent pathway. In vitro and in vivo therapy with a fibronectin inhibitor, ATN161, a peptide derived from the synergy region of fibronectin, reduced OvCa invasion, proliferation and metastasis. In summary, we show that abdominally metastasizing cancer cells induce fibronectin production in the peritoneal and omental microenvironment and that inhibition of this fibronectin-response prevents the first steps of peritoneal metastasis. By understanding the mechanism of OvCa metastasis we will be better able to target cells in the tumor microenvironment (i.e. mesothelial cells, fibroblasts and macrophages) during early ovarian cancer metastasis. This abstract is also presented as Poster A60. Citation Format: Hilary A. Kenny, Chun-Yi Chiang, Andras Ladanyi, Joshy George, David D. Bowtell, Katja Gwin, Andrew Mazar, Ernst Lengyel. Ovarian cancer cells activate the mesothelium in the peritoneal microenvironment promoting fibronectin production and the early steps of metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR11.

A role for fibronectin matricryptic site signaling in short‐term responses of endothelium to flow

Endothelial cell (EC) responses to mechanical forces exerted by flowing blood are well established; a principal response is realignment of the EC cytoskeleton in the direction of flow. The existing paradigm holds that this realignment is signaled directly from changes in perceived wall shear stress by mechanosensors (e.g. integrins and the glycocalyx) on the EC surface. We sought to determine whether short‐term responses of the EC cytoskeleton to flow have an ECM fibronectin (FN) ‐dependent component. Human umbilical vein endothelial cells (HUVEC) were cultured in perfusable microslides and subjected to flow (10 dyn/cm2 for 4 hrs) in the presence or absence of 9D2 (an anti‐FNIII‐1 mAb that blocks FN matrix assembly). HUVECs were then fixed and labeled for FN and F‐Actin and immunofluorescence images from 20 sites in 4 microslides per condition were obtained. In ECs incubated with 9D2 for 2 hrs prior to flow, there was a decrease in F‐Actin alignment compared to non‐treated controls (42.04 ± 0.37° vs 35.02 ± 0.38°, p< 0.01). This finding demonstrates that when FN signaling is blocked by 9D2, realignment of F‐ Actin stress fibers is significantly attenuated. Therefore, these data suggest that ECM FN plays a role in regulating F‐Actin realignment of endothelial cells due to flow. Supported by NIH HL105909

Segmental Assembly of Fibronectin Matrix Requires rap1b and integrin α5

During segmentation of the zebrafish embryo, inside-out signaling activates Integrin α5, which is necessary for somite border morphogenesis. The direct activator of Integrin α5 during this process is unknown. One candidate is Rap1b, a small monomeric GTPase implicated in Integrin activation in the immune system. Knockdown of rap1b, or overexpression of a dominant negative rap1b, causes a mild axis elongation defect in zebrafish. However, disruption of rap1b function in integrin α5(-/-) mutants results in a strong reduction in Fibronectin (FN) matrix assembly in the paraxial mesoderm and a failure in somite border morphogenesis along the entire anterior-posterior axis. Somite patterning appears unaffected, as her1 oscillations are maintained in single and double morphants/mutants, but somite polarity is gradually lost in itgα5(-/-) ; rap1b MO embryos. In itgα5(-) (/) (-) mutants, rap1b is required for proper somite border morphogenesis in zebrafish. The loss of somite borders is not a result of aberrant segmental patterning. Rather, somite boundary formation initiates but is not completed, due to the failure to assemble FN matrix along the nascent boundary. We propose a model in which Rap1b activates Integrin/Fibronectin receptors as part of an "inside-out" signaling pathway that promotes Integrin binding to FN, FN matrix assembly, and subsequent stabilization of morphological somite boundaries.

RSK2 Protein Suppresses Integrin Activation and Fibronectin Matrix Assembly and Promotes Cell Migration

Modulation of integrin activation is important in many cellular functions including adhesion, migration, and assembly of the extracellular matrix. RSK2 functions downstream of Ras/Raf and promotes tumor cell motility and metastasis. We therefore investigated whether RSK2 affects integrin function. We report that RSK2 mediates Ras/Raf inactivation of integrins. As a result, we find that RSK2 impairs cell adhesion and integrin-mediated matrix assembly and promotes cell motility. Active RSK2 appears to affect integrins by reducing actin stress fibers and disrupting focal adhesions. Moreover, RSK2 co-localizes with the integrin activator talin and is present at integrin cytoplasmic tails. It is thereby in a position to modulate integrin activation and integrin-mediated migration. Activation of RSK2 promotes filamin phosphorylation and binding to integrins. We also find that RSK2 is activated in response to integrin ligation to fibronectin. Thus, RSK2 could participate in a feedback loop controlling integrin function. These results reveal RSK2 as a key regulator of integrin activity and provide a novel mechanism by which it may promote cell migration and cancer metastasis.

Fibroblast cluster formation on 3D collagen matrices requires cell contraction dependent fibronectin matrix organization

Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin (FN) fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell–matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy.

Paracrine role for TGF-β-induced CTGF and VEGF in mesangial matrix expansion in progressive glomerular disease.

Transforming growth factor-β (TGF-β) is a key regulator of extracellular matrix (ECM), and may mediate the development of glomerulosclerosis with accumulation of mesangial matrix. Mesangial cells secrete TGF-β in response to common in vitro fibrogenic stimuli. Yet mesangial immunostaining for active TGF-β1 is frequently negative in chronic glomerular disease. TGF-β is rather expressed and/or activated by podocytes in both mesangial and podocyte diseases. Activated TGF-β/Smad signaling by podocytes may induce connective tissue growth factor (CTGF or CCN2) and vascular endothelial growth factor (VEGF) expression. Podocyte CTGF seems to have paracrine effects on mesangial cells to stimulate CTGF expression. CTGF appears to stimulate the fibronectin-matrix assembly via enhanced cell-surface expression of α5β1 integrin in the mesangium of diseased glomeruli. Podocyte VEGF-A overexpression also seems to play a paracrine role on mesangial cells to upregulate VEGF/VEGF receptor systems and to overproduce matrix proteins. Thus, paracrine CTGF and VEGF may contribute to mesangial matrix accumulation in chronic glomerular disease, culminating in the development of glomerulosclerosis. Together, these data bring new mechanistic insights into our understanding of the pathogenic role of TGF-β-induced CTGF and VEGF in mesangial matrix expansion in chronic progressive glomerular disease.

Abstract 306: Atheroprone Hemodynamics Regulate Smooth Muscle Cell Phenotype Through Endothelial PECAM and Fibronectin

Introduction: Atherosclerosis is an inflammatory disease that develops preferentially in regions of disturbed hemodynamic shear stress. The extracellular matrix protein fibronectin (FN) is deposited in the sub-endothelial layer of pre-atherosclerotic and advanced lesions. Atheroprone shear stress promotes FN deposition and inflammatory signaling pathways in endothelial cells (ECs). Platelet endothelial cell adhesion molecule (PECAM), a mechanosensory protein, is necessary for the production, secretion, and assembly of FN matrix by ECs. Similar to ECs, vascular smooth muscle cells (SMCs) also display a pro-inflammatory phenotype in regions of atherogenesis, and this phenotype is key to the progression of atherosclerosis. Hypothesis: We hypothesize that endothelial PECAM and FN signaling promotes a pro-inflammatory smooth muscle cell phenotype in response to atheroprone shear stress patterns. Methods: An in vitro cone-and-plate viscometer model was used to apply human-derived atheroprone or atheroprotective shear stress patterns to human umbilical vein ECs co-cultured with underlying human umbilical vein SMCs. Results: Atheroprone flow increased SMC NFkB activity compared to atheroprotective flow (1.94±0.35 fold, p<0.05). This increase of SMC NFkB activity under atheroprone flow was attenuated by siRNA knockdown of either endothelial PECAM (0.56±0.08 fold, p<0.01) or FN (0.50±0.10 fold, p<0.01). Endothelial PECAM knockdown also reduced genes marking the SMC inflammatory phenotype, including smooth muscle alpha actin, vascular cell adhesion molecule 1, and cyclooxygenase-2. Conclusion: These results suggest that SMCs switch to a pro-inflammatory phenotype when overlying ECs are exposed to atheroprone shear stress, and that PECAM and FN signaling in the ECs regulate this phenotype.

A Collapsin Response Mediator Protein 2 Isoform Controls Myosin II-Mediated Cell Migration and Matrix Assembly by Trapping ROCK II

Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two binding domains but also trapped and inhibited the kinase. CRMP-2L protein levels profoundly affected haptotactic migration and the actin-myosin cytoskeleton of carcinoma cells as well as nontransformed epithelial cell migration in a ROCK activity-dependent manner. Moreover, the ectopic expression of CRMP-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions through the inhibition of ROCK II in nonneuronal cells.

Abstract 5171: Restoration of fibronectin matrix assembly impedes invasion and reduces tumor-stromal cell affinity in prostate cancer

Abstract Cell-cell and cell-ECM interactions are important regulators of cohesion, a property previously demonstrated to mediate malignant invasion. Intermixing of the epithelial and stromal compartments is often associated with increased invasive capacity and poor prognosis. The studies reported here propose a novel role for α5β1 integrin, the principle mediator of fibronectin matrix assembly (FNMA), as an invasion suppressor in prostate cancer cells. Employing a combination of biophysical and cell biological techniques we explore the relationship between cohesion, invasion, FNMA and tumor-stromal interactions, using a well-characterized prostate cancer model. We show that cohesion is inversely proportional to invasive capacity. We also show that the most invasive cells are deficient in FNMA and express reduced levels of α5β1 integrin. We generated cells over-expressing either wild-type α5 integrin or a non-functional α5 in which the cytoplasmic domain was replaced with that of α2. We show that only wild-type α5 was able to increase cohesion and reduce the invasiveness of aggressive cells. In order to further explore the influence of FNMA on tumor-stromal cell interaction we employed a liquid miscibility model in which interactions between tumor and stroma are rooted in the same thermodynamic principles that govern the mixing of simple fluids. In this model, whether tumor and stromal cells segregate or intermix depends on the ratio of the strength of self-cohesion to cross-adhesion; a high ratio corresponds with segregation, whereas a lower ratio denotes intermixing. We hypothesized that segregation between tumor and stroma can be promoted by increasing the strength of tumor cell cohesion. To test this hypothesis, we first showed that aggressive prostate cancer cells intermix with prostate fibroblasts. We then treated the cancer cells with MEK inhibitors (MEKi) and showed that treatment restored FNMA, leading to increased cell-cell cohesion. When mixed with prostate stromal cells, MEKi-treated cells segregated, suggesting that increasing cohesion between tumor cells is sufficient to reduce their affinity for stromal cells. These data suggest that by increasing tumor cell cohesion through a fibronectin matrix mediated adhesion mechanism, it may be possible to effectively reduce affinity of tumor cells for stroma, thus inhibiting invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5171. doi:1538-7445.AM2012-5171

Epidermal growth factor receptor transactivation and fibronectin matrix assembly by the G‐protein coupled receptor, GPER, requires a transmembrane signaling complex consisting of PTPN12, integrin α5β1, and MMP‐3

G‐protein estrogen receptor (GPER) positively associates with breast tumor progression, including tumor size and extramammary metastases. Via activation of integrin α5β1, GPER coordinates two cellular activities that facilitate breast tumor cell survival, fibronectin matrix assembly and the release of membrane‐tethered proHB‐EGF. RNAi knockdown of either MMP‐3 or PTPN12 inhibits both of these cellular processes regulated by effectors on opposing sides of the plasma membrane. Moreover, PTPN12 knockdown also negatively influences the ability of GPER to promote the centripetal movement of integrin α5β1 from focal to fibrillar adhesions, the site of fibronectin fibril formation, and results in the accumulation of the Shc adaptor protein on the cytoplasmic tail of integrin α5β1. In support of the concept that PTPN12 is necessary for activated integrin α5β1, SKBR3 cells stably expressing PTPN12 shRNA showed reduced formation of anchorage independent colonies. Efforts to further understand the influence of PTPN12 knockdown on “inside‐out” integrin signaling events required for EGFR transactivation are underway with particular interest in understanding its impact on MMP‐3 activation and HB‐EGF release. A better understand of the molecular mechanism of GPER induced EGFR transactivation may lead to the development of future therapeutic strategies that target this receptor and prevent metastatic disease.

Fibronectin matrix-mediated cohesion suppresses invasion of prostate cancer cells

BackgroundInvasion is an important early step in the metastatic cascade and is the primary cause of death of prostate cancer patients. In order to invade, cells must detach from the primary tumor. Cell-cell and cell-ECM interactions are important regulators of cohesion - a property previously demonstrated to mediate cell detachment and invasion. The studies reported here propose a novel role for α5β1 integrin - the principle mediator of fibronectin matrix assembly (FNMA) - as an invasion suppressor of prostate cancer cells.MethodsUsing a combination of biophysical and cell biological methods, and well-characterized prostate cancer cell lines of varying invasiveness, we explore the relationship between cohesion, invasiveness, and FNMA.ResultsWe show that cohesion is inversely proportional to invasive capacity. We also show that more invasive cells express lower levels of α5β1 integrin and lack the capacity for FNMA. Cells were generated to over-express either wild-type α5 integrin or an integrin in which the cytoplasmic domain of α5 was replaced with that of α2. The α2 construct does not promote FNMA. We show that only wild-type α5 integrin promotes aggregate compaction, increases cohesion, and reduces invasion of the more aggressive cells, and that these effects can be blocked by the 70-kDa fibronectin fragment.ConclusionsWe propose that restoring capacity for FNMA in deficient cells can increase tumor intercellular cohesion to a point that significantly reduces cell detachment and subsequent invasion. In prostate cancer, this could be of therapeutic benefit by blocking an early key step in the metastatic cascade.

The Fate of Internalized Alpha-5 Integrin is Regulated by Fibronectin Matrix Assembly

The Fate of Internalized Alpha-5 Integrin is Regulated by Fibronectin Matrix Assembly

Fibronectin Matrix Assembly Regulates Shear Stress-Induced Structural Remodeling and Motility

Complex spatial profiles of shear stress have been implicated in focal development of atherosclerotic lesions by mechanisms involving force transmission through endothelial cell (EC) focal adhesions to cytoskeleton. Fibronectin is a primary component of the provisional extracellular matrix (ECM) deposited in atherosclerotic lesions. Integrin-mediated mechanosignaling pathways in ECs are required for both adaptation to shear stress and fibronectin fibrillogenesis, but how fibronectin assembly state modulates EC responses to shear stress remains unknown. To investigate this question, focal adhesion displacement, cytoskeletal reorganization, and migration were measured before and after a step increase from 0 to 12 dyn/cm2 steady unidirectional shear stress acting on ECs interacting with either assembled, fibrillar fibronectin matrix or unassembled, fragmented fibronectin on glass. Shear stress onset induced the arrest of focal adhesion displacement in ECs on unassembled fibronectin but not in cells on assembled fibronectin. In subconfluent layers, ECs on unassembled fibronectin migrated downstream after shear stress onset, but cells on assembled fibronectin migrated in random directions associated with local fibril orientations. In confluent monolayers, ECs interacting with unassembled fibronectin aligned in the flow direction faster than in monolayers on fibrillar fibronectin matrix, suggesting that fibronectin assembly regulated shear stress-induced cytoskeletal remodeling. Since cytoskeletal remodeling and focal adhesion displacement reflect early mechanosensing events after the onset of shear stress, these data suggest that fibronectin assembly regulates mechanosensitivity at the cell-matrix interface, which leads to shear stress-induced adaptation of cell motility and alignment.

Surface derivatization strategy for combinatorial analysis of cell response to mixtures of protein domains.

We report a robust strategy for conjugating mixtures of two or more protein domains to nonfouling polyurethane surfaces. In our strategy, the carbamate groups of polyurethane are reacted with zirconium alkoxide from the vapor phase to give a surface-bound oxide that serves as a chemical layer that can be used to bond organics to the polymer substrate. A hydroxyalkylphosphonate monolayer was synthesized on this layer, which was then used to covalently bind primary amine groups in protein domains using chloroformate-derived cross-linking. The effectiveness of this synthesis strategy was gauged by using an ELISA to measure competitive, covalent bonding of cell-binding (III(9-10)) and fibronectin-binding (III(1-2)) domains of the cell adhesion protein fibronectin. Cell adhesion, spreading, and fibronectin matrix assembly were examined on surfaces conjugated with single domains, a 1:1 surface mixture of III(1-2) and III(9-10), and a recombinant protein "duplex" containing both domains in one fusion protein. The mixture performed as well as or better than the other surfaces in these assays. Our surface activation strategy is amenable to a wide range of polymer substrates and free amino group-containing protein fragments. As such, this technique may be used to create biologically specific materials through the immobilization of specific protein groups or mixtures thereof on a substrate surface.

Fibronectin Aggregation and Assembly: THE UNFOLDING OF THE SECOND FIBRONECTIN TYPE III DOMAIN

The mechanism of fibronectin (FN) assembly and the self-association sites are still unclear and contradictory, although the N-terminal 70-kDa region ((I)1-9) is commonly accepted as one of the assembly sites. We previously found that (I)1-9 binds to superfibronectin, which is an artificial FN aggregate induced by anastellin. In the present study, we found that (I)1-9 bound to the aggregate formed by anastellin and a small FN fragment, (III)1-2. An engineered disulfide bond in (III)2, which stabilizes folding, inhibited aggregation, but a disulfide bond in (III)1 did not. A gelatin precipitation assay showed that (I)1-9 did not interact with anastellin, (III)1, (III)2, (III)1-2, or several (III)1-2 mutants including (III)1-2KADA. (In contrast to previous studies, we found that the (III)1-2KADA mutant was identical in conformation to wild-type (III)1-2.) Because (I)1-9 only bound to the aggregate and the unfolding of (III)2 played a role in aggregation, we generated a (III)2 domain that was destabilized by deletion of the G strand. This mutant bound (I)1-9 as shown by the gelatin precipitation assay and fluorescence resonance energy transfer analysis, and it inhibited FN matrix assembly when added to cell culture. Next, we introduced disulfide mutations into full-length FN. Three disulfide locks in (III)2, (III)3, and (III)11 were required to dramatically reduce anastellin-induced aggregation. When we tested the disulfide mutants in cell culture, only the disulfide bond in (III)2 reduced the FN matrix. These results suggest that the unfolding of (III)2 is one of the key factors for FN aggregation and assembly.

Membrane-type 1 matrix metalloproteinase regulates fibronectin assembly to promote cell motility

Fibronectin (FN) matrix assembly is an essential process in normal vertebrate development, which is frequently lost in tumor cells. Here we show that membrane-type 1 matrix metalloproteinase (MT1-MMP) regulates FN matrix assembly. MT1-MMP knockdown induced FN assembly in breast carcinoma cells. Ectopic expression of MT1-MMP reduced specifically the assembled FN matrix level without affecting whole FN production in fibroblasts. Treatment of fibrosarcoma HT1080 cells with dexamethasone (DEX) enhanced FN synthesis, resulting in short fibrils but not dense matrix formation. Combined treatment of DEX and MT1-MMP inhibitor accelerated FN matrix assembly, which mediated cellular adhesion and reduced cell migration and invasion. These results indicate that MT1-MMP stimulates cell migration and invasion by negatively regulating FN assembly. Structured summary of protein interactionsFN and Integrin Beta 1colocalize by fluorescence microscopy (View interaction)FN and Paxillincolocalize by fluorescence microscopy (View interaction)