2696 Research Article -1 microfibril deposition is dependent on fibronectin assembly

Rachel Kinsey1, Matthew R. Williamson1, Shazia Chaudhry1, Kieran T. Mellody1, Amanda McGovern1, Seiichiro Takahashi2, C. Adrian Shuttleworth1 and Cay M. Kielty1,* 1Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Science, Michael Smith Building, Oxford Road, University of Manchester, Manchester M13 9PT, UK 2Max Planck Institute of Biochemistry, Department of Molecular Medicine, Am Klopferspitz 18, 82152 Martinsried, Germany *Author for correspondence (e-mail: [email protected])

Accepted 3 June 2008 Journal of Cell Science 121, 2696-2704 Published by The Company of Biologists 2008 doi:10.1242/jcs.029819

Summary Newly deposited microfibrils strongly colocalise with fibronectin microfibril assembly. In fibronectinRGE/RGE mutant mouse in primary fibroblasts. Microfibril formation is grossly inhibited fibroblast cultures, which do not engage α5β1 integrin, by fibronectin depletion, but rescued by supplementation with extracellular assembly of both fibronectin and microfibrils is exogenous cellular fibronectin. As integrin receptors are key markedly reduced. Thus, pericellular microfibril assembly is determinants of fibronectin assembly, we investigated whether regulated by fibronectin fibrillogenesis. they also influenced microfibril deposition. Analysis of β1- integrin-receptor-null fibroblasts, blockage of cell surface Supplementary material available online at integrin receptors that regulate fibronectin assembly and http://jcs.biologists.org/cgi/content/full/121/16/2696/DC1 disruption of Rho kinase all result in suppressed deposition of both fibronectin and microfibrils. Antibody activation of β1 Key words: Fibrillin-1, Microfibrils, Fibronectin, Integrin, Rho integrins in fibronectin-depleted cultures is insufficient to rescue kinase.

Introduction to exert force on matrix leads to exposure of cryptic self-association Fibrillin-1 (official protein symbol FBN1) is a large glycoprotein sites that support higher-order fibronectin assembly (Wu et al., 1995; that assembles pericellularly into microfibrils that have a complex Zhong et al., 1998; Baneyx et al., 2002; Ilic et al., 2004; Wang et structural organisation and are widespread in elastic tissues such as al., 2005; Féral et al., 2007; Somanath et al., 2007; Somers and

Journal of Cell Science , lung, arteries and ligaments (Hubmacher et al., 2006; Kielty, Mosher, 1993; Yoneda et al., 2007). Fibronectin can also assemble 2006). It contains 47 epidermal growth factor-like domains, 43 of without RGD-dependent α5β1 integrin engagement, in this case which bind calcium, which are interspersed with eight cysteine- forming short thick pericellular filaments through αvβ3 integrin containing transforming growth factor-β (TGF-β) binding-protein- ligation of an N-terminal NGR motif (Takahashi et al., 2007). like (TB) modules (Pereira et al., 1993). The process of fibrillin-1 We tested the hypothesis that fibronectin plays a central role in microfibril assembly remains poorly understood; however, as with fibrillin microfibril assembly. Here we show that the deposition of other major (ECM) assemblies such as extensive microfibril arrays is regulated by α5β1-integrin-mediated fibronectin and I , assembly might be driven by both fibronectin fibrillogenesis. self-assembly and cell-directed mechanisms. Fibrillin-1 N- and C- terminal interactions are thought to support linear assembly (Trask Results et al., 1999; Lin et al., 2002; Marson et al., 2005). Cellular control Fibronectin is required for fibrillin-1 microfibril assembly of assembly is apparent at the level of furin processing at N- and Confocal microscopy of primary human dermal fibroblasts (HDFs) C-termini, which may be a prerequisite for interactions between was employed to determine whether fibronectin is an orchestrator fibrillin-1 termini during assembly (Wallis et al., 2003). However, of fibrillin-1 microfibril assembly (Fig. 1). In 24 hour cultures, the potential contribution of cell surface receptors to the process of fibronectin had formed short extracellular fibrils, whereas fibrillin- microfibril assembly is unknown. 1 was mainly cell-associated with some pericellular staining. The Evidence is emerging that fibronectin orchestrates the assembly 5 day cultures had assembled extensive organised, directionally of ECM (Velling et al., 2002; Dallas et al., 2005; Dallas et al., 2006; aligned fibrillin-1 microfibrils and fibronectin arrays that strongly Sottile et al., 2007). Fibronectin fibrillogenesis occurs pericellularly colocalised. RNAi was then used in HDFs to determine whether and is dependent upon cell-surface-receptor engagement. Assembly fibronectin influences fibrillin-1 microfibril formation. is initiated by N-terminal fibronectin interactions with the cell surface, which induces α5β1 integrin clustering and translocation Fibronectin knockdown disrupts fibrillin-1 microfibril from focal contacts into fibrillar adhesions, followed by fibronectin assembly self-association (Wierzbicka-Patynowski et al., 2003; Mao and Fibronectin was depleted in HDFs using either of two siRNA Schwarzbauer, 2005; Pankov et al., 2000; Tomasini-Johansson et oligonucleotide sequences, designated FN1 and FN2 (Fig. 2). Control al., 2006). Integrin-activated RhoA/ROCK, FAK, Akt and protein HDFs were transfected with a scrambled version of the siRNA kinase C pathways regulate cytoskeletal changes, and cellular ability oligonucleotide FN2, or were untransfected. In all experiments, cells Fibrillin-1 microfibril assembly 2697

or control cultures (not shown). Confocal microscopy of the fibronectin-depleted HDF cultures at 5 days showed limited rescue of fibronectin and microfibril assembly by plasma fibronectin (not shown) but strong rescue and colocalisation of both assemblies by cellular fibronectin (Fig. 2D). These knockdown and rescue experiments show that fibronectin is required for fibrillin-1 microfibril assembly.

Microfibril assembly also requires α5β1-integrin- mediated fibronectin assembly We investigated whether integrin α5β1 and αvβ3, which are key regulators of extracellular fibronectin assembly (Mao and Schwarzbauer, 2005; Takahashi et al., 2007), also influence fibrillin-1 assembly. Flow cytometry confirmed the expression by HDFs of both α5β1 and αvβ3 integrin (Fig. 3A).

Blocking integrin α5β1 disrupts fibronectin and microfibril assembly Immunoblotting of cell lysates and conditioned medium from the 24 hour and 5 day cultures confirmed Fig. 1. Fibrillin-1 microfibril assembly by human dermal fibroblasts. (A) Representative confocal microscopy images of the assembly of microfibrils (using rabbit anti-proline-rich that protein levels of fibrillin-1 were similar in the region pAb; red) and fibronectin (FN) (using FN-3E2 mAb to cellular fibronectin; green) by presence or absence of integrin-blocking antibodies HDFs at 24 hours and 5 days. Cell nuclei were stained with DAPI (blue). Scale bars: 50 μm. (Fig. 3B). Confocal microscopy of 24 hour and 5 day (B) Enlarged images from A. These experiments were repeated several times with similar cultures, treated with an anti-β1-integrin function- results. blocking antibody (mAb13) and an anti-α5-integrin function-blocking antibody (mAb16), showed severely were cultured in medium containing serum from which plasma ablated fibronectin and microfibril deposition compared with fibronectin had been stripped. Fibronectin knockdown by FN1 or untreated cultures (Fig. 3C) or cultures treated with the non- FN2 oligonucleotides, assessed at the mRNA level by RT-PCR, was function-blocking α5-integrin mAb11 (not shown). In the cultures significant compared with levels in control cells (P=0.0002). mRNA treated with blocking antibodies, fibrillin-1 still colocalised with expression levels of fibrillin-1, and also collagen I as a control ECM fibronectin, although not all fibronectin was associated with fibrillin- molecule, were not significantly altered by fibronectin RNAi (Fig. 1. Thus, inhibiting integrin α5β1 grossly disrupted the extracellular

Journal of Cell Science 2A). Western blotting showed drastically reduced levels of fibronectin assembly of both fibronectin and fibrillin-1 microfibrils. in the cell lysates and conditioned medium of 24 hour and 5 day FN1 and FN2 siRNA cultures compared with control cells (Fig. 2B). Integrin-β1-null murine embryonic fibroblasts disrupt However, fibrillin-1 protein levels were similar in control and fibronectin and microfibril assembly fibronectin siRNA cultures (Fig. 2B). Subsequent knockdown To confirm that β1 integrins are needed for fibronectin and experiments utilised FN1 oligonucleotides. microfibril assembly, wild-type and β1-integrin-null mouse Confocal microscopy of HDF cultures from 24 hours to 5 days embryonic fibroblasts (MEFs) were analysed at 24 hours and 5 days. after fibronectin knockdown revealed that, as expected, fibronectin Flow cytometry confirmed that integrin subunits β1 and α5 were deposition was greatly reduced in the fibronectin-depleted cultures. expressed by the wild-type MEFs but not by β1-integrin-knockout From 24 hours to 3 days in the siRNA cultures, there was very little MEFs (Fig. 4A). Confocal microscopy revealed that the wild-type fibronectin staining, but by 5-7 days, a few fibrils were detected cells began to assemble extracellular fibronectin fibrils by 24 hours, (Fig. 2C). Fibronectin RNAi also profoundly disrupted fibrillin-1 whereas fibrillin-1 staining was mostly cell-associated (not shown). microfibril assembly. At 24 hours, fibrillin-1 staining was mostly By 3 days, wild-type MEFs had deposited dense colocalising cell-associated, as seen in control cells (see Fig. 1); however, in the networks of fibronectin and fibrillin-1 (Fig. 4B). By contrast, the 5 day fibronectin siRNA cells, fibronectin and microfibril deposition β1-integrin-knockout MEFs had deposited markedly reduced was severely ablated compared with that in control cells (Fig. 2D). fibronectin and fibrillin-1 microfibrils by 3 days, although colocalisation was still apparent (Fig. 4B). These data confirm that Exogenous fibronectin rescues microfibril assembly in β1 integrin plays a critical role in fibronectin assembly, and indicate fibronectin siRNA cultures that the deposition of microfibrils follows fibronectin assembly (Fig. To investigate whether exogenous fibronectin rescues fibrillin-1 4B). As β1-integrin-null murine cells express αvβ3 (Retta et al., microfibril assembly, fibronectin siRNA cultures were supplemented 2001), the low levels of fibronectin might have assembled through with human plasma or cellular fibronectin. RT-PCR confirmed that αvβ3 integrin (Takahashi et al., 2007). addition of exogenous fibronectin to the fibronectin siRNA cultures did not alter the depleted fibronectin mRNA levels or the fibrillin- Blocking integrin-induced cytoskeletal signals disrupts 1 and collagen I control mRNA levels (not shown). Western blotting fibronectin and microfibril assembly also confirmed that addition of fibronectin did not alter fibrillin-1 Integrins link the ECM to the actin cytoskeleton, regulating cell levels in the lysates or conditioned medium of fibronectin siRNA contractility and generating tension that allows fibronectin self- 2698 Journal of Cell Science 121 (16)

Fig. 2. Fibronectin RNAi disrupts fibrillin-1 microfibril assembly by human dermal fibroblasts. (A) RT-PCR analysis of RNAi knockdown of fibronectin in HDFs. FN1 and FN2 are knockdowns using different oligonucleotides (see Materials and Methods). FN2 scr, scrambled oligonucleotide control; Col I, collagen I; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B) Immunoblotting of fibrillin-1 [using rabbit anti-proline-rich region (PRO) pAb] and fibronectin (using FN-3E2 mAb to cellular fibronectin) Journal of Cell Science on lysates and medium from knockdown and control cultures, as outlined in A, with β-actin loading controls (using mAb AC-74) for the cell lysates. (C) Representative confocal microscopy images of fibronectin assembly (using FN-3E2 mAb to cellular fibronectin) in the RNAi and control cultures, from 1-7 days. (D) Representative confocal microscopy images of microfibril assembly (using rabbit anti-proline- rich region (PRO) pAb; red) and fibronectin (using FN-3E2 mAb to cellular fibronectin; green) in control cultures, fibronectin RNAi cultures, and fibronectin RNAi cultures supplemented with cellular fibronectin (10 μg/ml), all at 5 days. Microfibril assembly was grossly disrupted in the fibronectin siRNA cells, and partially rescued by cellular fibronectin. Scale bars: 50 μm. (E) Enlarged images from D. All experiments were repeated at least three times.

association. Integrin α5β1 activates RhoA and downstream Rho pathway is also necessary for fibrillin-1 microfibril deposition. The kinase (ROCKI and ROCKII) (Yoneda et al., 2007), and Rho kinase selective ROCK inhibitors, H1152 and Y27632, at concentrations activity is necessary for fibronectin assembly (Baneyx et al., 2002; of 10 μM and 30 μM, respectively, caused grossly different fibrillar Wu et al., 1995). We therefore investigated whether this signalling organisation and reduced deposition of both fibronectin and fibrillin- Fibrillin-1 microfibril assembly 2699

Fig. 3. Blocking α5β1 integrins disrupts fibrillin-1 microfibril assembly by human dermal fibroblasts. (A) FACS analysis of HDFs, confirming expression of α5β1 and αvβ3 integrins. The antibodies used were mAb13 (β1 integrin), mAb16 (α5 integrin), 17E6 (αv integrin) and 23C6 (αvβ3 integrin). (B) HDFs were incubated for 24 hours or 5 days in the presence of integrin-blocking antibodies mAb13 (β1) and mAb16 (α5) or a rabbit IgG control (all at 10 μg/ml). Medium and cell lysates were immunoblotted for fibrillin-1 [using the rabbit anti- proline-rich region (PRO) pAb] or fibronectin (using FN-3E2 mAb), with β-actin (using mAb AC-74) loading controls for the cell lysates. Molecular masses are indicated (in kDa). Quantitative analysis on the media

Journal of Cell Science was done by densitometry as a measure of pixel density. Cell lysates were quantified using densitometry with data normalised against β-actin. All data are represented as the mean ± s.d. of two repeated experiments. There was no statistical difference between treated samples and the control when using the Student’s t-test. (C) Representative confocal microscopy images of the assembly of microfibrils (using PRO pAb; red) and fibronectin (using FN-3E2 mAb to cellular fibronectin; green), in the presence or absence of mAb13 (blocks β1 integrins) and mAb16 (blocks α5 integrin), at 5 days. Cell nuclei were stained with DAPI (blue). Microfibril assembly was grossly disrupted in the antibody-treated cells. Scale bars: 50 μm. Enlarged images are also shown. All experiments were repeated at least three times.

1, compared with control cells either supplemented with 0.2% (v/v) specific cyclic RGD peptide inhibitor of αvβ3 integrin, did not DMSO or unsupplemented cultures (Fig. 5). Immunoblotting of cell disrupt either fibronectin or microfibril assembly (Mitjans et al., lysates and conditioned medium at 5 days confirmed that fibronectin 1995) (not shown). Cells incubated with the αvβ3-integrin function- and fibrillin-1 protein levels were unaffected by ROCK inhibition blocking antibody LM609 also assembled well-organised networks (not shown). Thus, integrin-regulated cytoskeletal changes suppress of both molecules (not shown). Incubation with mAb 17E6, which fibronectin and microfibril assembly. inhibits and internalises αv integrins (Castell et al., 2001), also did not ablate either fibronectin or microfibril assembly (not shown). Integrin αvβ3 is not essential for microfibril assembly Immunoblotting of cell lysates and medium from the LM609 Although integrin α5β1 is a primary regulator of extracellular antibody experiments showed that there was no alteration in mRNA fibronectin assembly (Mao and Schwarzbauer, 2005), αvβ3 integrin or protein levels of either molecule (not shown). Together, these also supports the assembly of short pericellular fibronectin fibrils data show that αvβ3 integrin is not a major regulator of fibronectin (Takahashi et al., 2007). Incubation with cilengitide (0.1 μg/ml), a or microfibril assembly in primary fibroblasts. 2700 Journal of Cell Science 121 (16)

Fig. 4. Fibrillin-1 microfibril assembly is disrupted in β1-null murine embryonic fibroblasts. (A) FACS analysis detected α5 (5H10 mAb) or β1 (KM16 mAb) integrin subunits in wild-type but not β1-integrin-null MEFs. (B) Representative confocal microscopy images of microfibril assembly (using PRO pAb; red) and fibronectin (using FN-3E2 mAb to cellular fibronectin; green) in wild-type and β1-integrin-null MEFs, at 3 days. Cell nuclei were stained with DAPI (blue). fibronectin and microfibril assembly was markedly reduced in the β1-integrin-null cells. Scale bars: 50 μm. Enlarged images are also shown. These experiments were repeated three times.

Activation of β1 integrins in fibronectin-depleted cultures lysates and media showed that TS2/16 supplementation had no does not rescue microfibril assembly effect on protein levels of fibronectin or fibrillin-1 (not shown). Having established that the assembly of extensive microfibril arrays Confocal microscopy of fibronectin-depleted or control cell cultures is suppressed whenever α5β1-integrin-mediated fibronectin at 24 hours and 5 days showed that microfibril assembly was not fibrillogenesis is disrupted, we investigated whether fibronectin- rescued by TS2/16 (not shown). Thus, activation of β1 integrins independent activation of β1 integrins can directly rescue in fibronectin-depleted cells is not sufficient to support microfibril microfibril assembly, using fibronectin-depleted cultures incubated assembly. β Journal of Cell Science with the integrin- 1-activating antibody TS2/16 at a concentration (10 μg/ml) that activates β1- integrin-mediated signalling (Lomas et al., 2007), or with mouse IgG as a control. RT-PCR results indicated that fibronectin was significantly reduced by RNAi (P=0.0027) and neither TS2/16 nor a mouse IgG control stimulated fibronectin expression (not shown). mRNA levels of fibrillin- 1 and a control matrix molecule, collagen type I, were not affected by fibronectin RNAi or by supplementation with either antibody (not shown). Immunoblotting of the 24 hour and 5 day cell

Fig. 5. Rho kinase inhibitors disrupt fibrillin-1 microfibril assembly by human dermal fibroblasts. (A) Representative confocal microscopy images of microfibril assembly (using PRO pAb; red) and fibronectin (using FN-3E2 mAb to cellular fibronectin; green) in the presence or absence of either of two ROCK inhibitors (H1152 or Y27632) or DMSO (dimethylsulphoxide) control, at 5 days. Cell nuclei were stained with DAPI (blue). Microfibril assembly was grossly disrupted in the ROCK-inhibitor-treated cells. Scale bars: 50 μm. (B) Representative confocal microscopy images of F-actin stress fibres, stained with phalloidin, in the presence or absence of either H1152 or Y27632 or DMSO control, at 5 days. Stress fibres were markedly reduced in the inhibitor-treated cultures. These experiments were repeated three times. Fibrillin-1 microfibril assembly 2701

Fig. 6. Fibrillin-1 microfibril assembly is disrupted in murine embryonic fibroblasts expressing mutant fibronectinRGE/RGE. Representative confocal microscopy images of microfibril assembly [using Santa Cruz N-19 anti-fibrillin-1 pAb (SC-7541); green, FITC] and fibronectin assembly (using FN-3E2 mAb to cellular fibronectin; Texas Red) by nonpermeabilised wild-type or fibronectinRGE/RGE mutant MEFs, at 14 days. Cell nuclei were stained with DAPI (blue). Mutant cells deposited reduced fibronectin and very little extracellular fibrillin-1. Scale bars: 50 μm. Enlarged images are also shown. These experiments were repeated three times.

α5β1-integrin-mediated fibronectin assembly is required for Immunofluorescence microscopy showed that extracellular deposition of extensive microfibril arrays fibronectin and fibrillin-1 microfibrils strongly colocalise (although Studies on fibronectinRGE/RGE mice and cells have shown that, in fibronectin is more abundant, and not all fibronectin is associated the absence of RGD-dependent α5β1 integrin interactions, with microfibrils), and that siRNA depletion of fibronectin grossly fibronectin can assemble into short thick pericellular fibrils through reduced both fibronectin and fibrillin-1 microfibril deposition. Thus, αvβ3 integrin (Takahashi et al., 2007). We used confocal there is a critical assembly relationship between these ECM polymers.

Journal of Cell Science immunomicroscopy of nonpermeabilised cells (N-19 pAb) to The deposition of fibronectin and microfibrils was more strongly examine microfibril deposition in wild-type and fibronectinRGE/RGE rescued by cellular fibronectin, which contains EDA and EDB murine cells, in order to establish whether microfibril deposition is domains flanking the central cell-binding region (Pankov and Yamada, dependent on α5β1-integrin-mediated fibronectin assembly. In the 2002), than by soluble plasma fibronectin. Thus, fibronectin isoforms wild-type MEFs, abundant extracellular microfibril networks were may differentially influence microfibril assembly in vivo. deposited and strongly colocalised with fibronectin (Fig. 6). In the Fibronectin fibrillogenesis generally requires α5β1 integrin fibronectinRGE/RGE cultures, markedly reduced levels of extracellular (Clark et al., 2005), although αvβ3 integrin can support reduced fibronectin were detected and there were very few extracellular assembly of short thick pericellular fibronectin fibrils (Takahashi microfibrils (Fig. 6). These experiments show that the deposition et al., 2007). Using integrin antibody-blocking and inhibitor of extensive microfibril arrays requires α5β1-integrin-mediated experiments, and β1-integrin-null murine cells, it was clear that fibronectin fibrillogenesis. α5β1 integrin is also critical for the deposition of fibrillin-1 microfibril arrays because both fibronectin and microfibril Discussion deposition is greatly reduced in these conditions. The cosuppression Despite intense research, fibrillin microfibril assembly remains a of fibronectin and fibrillin-1 assembly in the antibody-blocking and poorly understood process. It is widely considered that microfibrils mouse mutant cultures supports a model in which microfibril assemble pericellularly, following furin processing, through high- assembly is dependent upon fibronectin assembly. Furthermore, affinity N- and C-terminal interactions (Hubmacher et al., 2006; since antibody activation of β1 integrins failed to rescue microfibril Kielty, 2006). However, assembly of other ECM polymers, such deposition in fibronectin-depleted cells, activated integrin alone is as fibronectin and collagen fibrils, are driven by both cell-directed insufficient to drive microfibril assembly. These data, which and self-assembly processes (Mao and Schwarzbauer, 2005; Velling indicated that microfibril assembly is dependent on fibronectin et al., 2002; Dallas et al., 2006; Sottile et al., 2007). In this study, formation through α5β1 integrin, were reinforced using we have investigated how the cell-matrix interface influences fibronectinRGE/RGE cultures, which cannot assemble fibronectin fibrillin-1 assembly, by testing the hypothesis that integrin-mediated through α5β1 integrin (Takahashi et al., 2007), because almost no fibronectin assembly is critical for fibrillin-1 microfibril assembly extracellular fibrillin-1 microfibrils were apparent. In addition, (Fig. 7). Our novel findings are that the deposition of microfibrillar although HDFs can adhere to fibrillin-1 molecules and microfibrils arrays is dependent upon the assembly of fibronectin through α5β1 in an RGD-dependent manner through α5β1 integrin (Bax et al., integrin. 2003; Bax et al., 2007), we found that supplementation with fibrillin- 2702 Journal of Cell Science 121 (16)

Fig. 7. Model of pericellular fibronectin and fibrillin- 1 assembly. Diagram illustrating how fibronectin and α5β1 integrin may contribute to fibrillin-1 microfibril assembly. The model predicts that activated α5β1 integrin and associated cytoskeletal changes drive fibronectin fibrillogenesis, which in turn facilitates the deposition of extensive microfibril arrays. Although fibrillin-1 may associate with pericellular fibronectin even when fibronectin cannot assemble through α5β1 (Takahashi et al., 2007), in these conditions there is little microfibril assembly (see Figs 4, 6).

1 RGD fragments or a fibrillin-1-specific RGD antibody (Lee et glycoproteins and interacts directly with the N-terminal region of al., 2004) has no disruptive effect on microfibril assembly (our fibrillin-1 (Isogai et al., 2003; Chaudhry et al., 2007). It is of note unpublished data), so microfibril assembly does not require direct that, although the matrix deposition of LTBP-1 requires fibronectin α5β1 integrin interactions. Fibrillin-1 interactions with cells may (Dallas et al., 2005), LTBP1 binds fibrillin-1 but does not interact be physiologically important in the form of assembled microfibrils, directly with fibronectin, so its deposition might in fact be dependent as seen in blood vessels (Bunton et al., 2001). on fibronectin-mediated microfibril assembly. In summary, we have Integrin α5β1 ligation of fibronectin at the cell surface activates shown that the pericellular assembly of fibrillin-1 microfibrils is cell-signalling pathways that regulate the cytoskeleton, including dependent on α5β1-integrin-mediated fibronectin assembly. RhoA/ROCK (Yoneda et al., 2007; Féral et al., 2007), and tensional forces may then expose cryptic self-assembly sites (Zhong et al., 1998). We confirmed that cytoskeletal disruption due to ROCK Materials and Methods Reagents inhibition suppresses fibronectin assembly, and the concurrent Primary antibodies used were: rabbit anti-human fibronectin polyclonal antibody disruption to microfibril deposition supports the dependence of (pAb) (plasma and cellular) (Sigma-Aldrich); fibronectin-3E2 mouse anti-cellular fibrillin-1 assembly on α5β1-integrin-mediated fibronectin fibronectin monoclonal antibody (mAb) (Sigma-Aldrich); MAB1919/clone 11C1.3 α β mouse anti-fibrillin-1 mAb (Chemicon); rabbit anti-fibrillin-1 proline-rich region Journal of Cell Science assembly. It seems likely that the fibronectin interaction with 5 1 (raised to a recombinant peptide) (here designated ‘PRO’) pAb (gift from P. A. integrin, which induces exposure of cryptic fibronectin self- Handford, Oxford, UK); rabbit anti-fibrillin-1 proline-rich region (raised to a association sites, results in a pericellular form of fibronectin that synthetic peptide) pAb (raised to a synthetic peptide by Eurogentec); rabbit pAb raised acts as a suitable template to support the assembly of extensive (Bethyl Laboratories) to the fibrillin-1 RGD and flanking sequence (CYLDIRPRGDNGTA), as previously reported (Sakamoto et al., 1996); goat anti- microfibril arrays. Although fibrillin-1 can colocalise with fibrillin-1 N-terminal region (N-19) pAb (Santa Cruz, sc-7541), anti-human integrin fibronectin that is not assembled through α5β1 integrin, microfibril α5 mAbs (blocking,mAb16; non-inhibitory, mAb11); mouse anti-human integrin α5 deposition is greatly reduced in such conditions (see Fig. 4B, Fig. mAb (12G10) and rat anti-mouse integrin α5 mAb (5H10) (gifts from M. J. Humphries, Manchester, UK); TS2/16 mouse anti-human integrin β1 mAb (Pierce 6). Using in vitro binding assays, we detected molecular interactions Biotechnology); KMI6 rat anti-mouse integrin β1 mAb (BD Biosciences); LM609 between fibrillin-1 and specific recombinant fragments of mouse anti-human integrin αVβ3 mAb (Chemicon); 17E6 mouse anti-human integrin fibronectin, including the N-terminal region of fibronectin that is αV blocking mAb (kindly supplied by S. Goodman, Merck KGaA, Germany); 23C6 particularly important in assembly (supplementary material Fig. S1); mouse anti-human αvβ3 (Abcam); AC-74 mouse anti-β-actin (Sigma-Aldrich). Secondary antibodies were donkey anti-mouse (FITC), donkey anti-rabbit (FITC), these interactions may support transient cell surface associations. donkey anti-rabbit (TRITC), donkey anti-rat (FITC), donkey anti-rat (TRITC), donkey Only weak interactions were detected between plasma fibronectin anti-sheep (FITC) and donkey anti-sheep Cy3 (Jackson ImmunoResearch and fibrillin-1, implicating cryptic fibronectin sites that may be Laboratories); rabbit anti-mouse HRP, goat anti-rabbit HRP, and goat anti-sheep HRP (DAKO). Control immunoglobulins (IgGs) from murine serum, rabbit serum, rat exposed through integrin interactions. It is also possible that cell serum and sheep serum were from Sigma. F-actin was stained using Texas Red-X surface heparan sulphate, which binds both molecules, could phalloidin (Molecular Probes). A cyclic RGD peptide inhibitor of αv integrin mediate their association. (cilengitide) was kindly provided by S. Goodman, Merck KGaA (Germany) and used μ The dependence of fibrillin-1 assembly on fibronectin and α5β1 at 10 M concentration. α β integrin has pathological implications. 5 1-integrin-null mice are Cells and cell culture embryonic lethal (Bouvard et al., 2001), as are fibronectin-null mice, HDFs (Invitrogen) were cultured in Modified Eagle’s Medium (MEM) plus Earle’s which suffer from severe cardiovascular defects (George et al., plus L-glutamine (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS; BioWhittaker), 1 mM sodium pyruvate, non-essential amino acids (100ϫ), MEM 1997); these pathologies may thus reflect loss of fibronectin and vitamins (100ϫ), 2 mM L-glutamine, 10 μg/ml penicillin, 5 μg/ml streptomycin also consequent disruption to the assembly of fibrillin-1. Fibronectin sulphate (all from Gibco) at 37°C, 5% CO2 up to passage 8. FBS was stripped of also regulates the assembly of other ECM molecules, including the plasma fibronectin by passing the serum over a gelatin SepharoseTM 4B column latent TGFβ binding protein (LTBP1), which controls TGFβ (Amersham Biosciences), as described (McKeown-Longo et al., 1985). Where appropriate, medium was supplemented with either 20 μg/ml human plasma bioavailability. LTBP1, which also contains cbEGF and TB fibronectin (Chemicon) or with 10 μg/ml cellular fibronectin isolated from human domains, is a member of the fibrillin superfamily of ECM foreskin fibroblasts (Sigma). In some experiments, medium was supplemented with Journal of Cell Science 121 () Fibrillin-1 microfibril assembly 2703

inhibitory or stimulatory antibodies or control immunoglobulins (IgGs), at a concentration of 2 M. After washing twice with PBS, cells were lysed with 25 mM concentration of 10 μg/ml, or with either of two well-characterised inhibitors of Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS ROCKI and ROCKII, H1152 and Y-27632 (Calbiochem), respectively, at working for 30 minutes, in the presence of a protease inhibitor cocktail (1:2000), and scraped concentrations of 10 μM and 30 μM, or with recombinant fibrillin-1 RGD fragments from the tissue culture flask. Debris was pelleted at 20,000 g for 5 minutes. (Bax et al., 2007). Supernatants were removed and analysed by SDS-PAGE and immunoblotting. Wild-type and fibronectinRGE/RGE mutant mouse embryonic fibroblasts (MEFs), a gift from Reinhardt Fässler (Munich, Germany) (Takahashi et al., 2007), were cultured SDS-PAGE for up to 14 days in Medium 231 with serum-free growth supplement (Cascade Samples were mixed with 4ϫ NuPAGE lithium dodecyl sulphate (LDS) sample buffer Biologics) at 37°C, 5% CO2. Immortalised wild-type and β1-integrin-knockout MEFs (Invitrogen) for analysis by SDS-PAGE. Where appropriate, samples were reduced (from M. J. Humphries, Manchester, UK) were cultured at 33°C in Dulbecco’s MEM in 10ϫ NuPAGE reducing agent (Invitrogen) at 57°C for 30 minutes. Samples were supplemented with L-glutamine (BioWhittaker), 10% (v/v) FBS (BioWhittaker) run on SDS-PAGE gels in reducing conditions using the NuPAGE system (Invitrogen). precleared of fibronectin, 1 mM sodium pyruvate (Gibco), 2 mM L-glutamine (Gibco) Gels were silver stained or immunoblotted. For the immunoblotting, transferred and 20 U/ml interferon gamma (Sigma). membranes were blocked in 5% (w/v) dried milk in TTBS (0.15 M NaCl, 25 mM Tris-HCl, 0.5% (v/v) Tween-20, pH 8.0) for 1 hour at room temperature before Flow cytometry incubation with primary antibodies diluted in 2% (w/v) milk in TTBS for 1 hour at Cell surface receptors were analysed by flow cytometry, using established protocols. 20°C. Membranes were washed in TTBS before incubation with HRP-conjugated Briefly, 100 μl of each cell suspension (1ϫ106 cells) was mixed with 100 μl primary secondary antibodies diluted in 2% (w/v) milk in TTBS for 1 hour at 20°C. Membranes antibody (for MEFs: mAbs 5H10, KM16; for HDFs: mAbs mAb13, mAb16, 17E6, were washed in TTBS prior to a final wash in TBS (0.15 M NaCl, 25 mM Tris-HCl, 23C6) diluted to 20 μg/ml in phosphate-buffered saline (PBS), and incubated on ice pH 8.0). Membranes were incubated with a 1:1 mixture of stable peroxide: enhancer for 45 minutes. Cells were harvested by centrifugation for 5 minutes at 800 g and ECL reagents (Amersham Biosciences) for 1 minute at 20°C then exposed to Kodak washed three times in 600 μl PBS containing 1% (v/v) FBS. Cell pellets were Biomax MR film. The nitrocellulose membrane was reduced in 4 M urea, 10 mM resuspended in 50 μl FITC-conjugated secondary antibody diluted 1:50 in PBS Tris-HCl pH 8.0, 10 mM DTT for 30 minutes at 20°C. Membranes were then blocked containing 10% (v/v) human serum (Sigma-Aldrich) and incubated on ice for 45 and probed, as described above. Fibrillin-1 was detected using the rabbit anti-fibrillin- minutes in the dark. Cells were collected by spinning for 5 minutes at 800 g and 1 proline-rich region pAb, and fibronectin using FN-3E2 mouse anti-cellular washed twice in 600 μl PBS containing 1% (v/v) FBS. Cells were washed in 300 μl fibronectin, in all blots. PBS and resuspended in 400 μl PBS for analysis. For each sample, 20,000 cells were counted using a Cyan flow cytometer (Dako) at a flow rate of less than 200 Reverse transcriptase polymerase chain reaction (RT-PCR) events/second and an excitation wavelength of 488 nm. RNA was isolated from cells using an SV Total RNA Isolation Kit (Promega Corporation). For first strand cDNA synthesis, 200 ng random primers (Promega Immunofluorescence microscopy Corporation), 500 ng polyA+ RNA and DNase, RNase-free UltraPURE water (Gibco) Cells were immunostained using established protocols. Briefly, cells were cultured up to a volume of 22.5 μl were mixed in an RNase-free tube and incubated at 70°C for various lengths of time before being fixed with 3% (v/v) formaldehyde in PBS for 10 minutes. Tubes were chilled on ice, and 8 μl of 5ϫ reverse transcriptase reaction for 20 minutes at room temperature. Cells were washed three times with PBS and buffer (Roche), 4 μl 0.1M dithiothreitol (DTT; Promega Corporation), 4 μl of 10 formaldehyde groups were quenched with 0.2 M glycine in PBS for 20 minutes at mM dNTPs (Bioline) and 0.5 μl of 40 U/μl RNase inhibitor (Roche) were added. room temperature. Cells were washed three times with PBS before being permeabilised Mixtures were incubated at 37°C for 2 minutes prior to the addition of 1 μl AMV in 0.5% (v/v) Triton X-100 in PBS for 4 minutes at 20°C. For the ‘nonpermeabilised’ reverse transcriptase (25 U/μl; Roche), then incubated at 37°C for a further hour. fibronectinRGE/RGE MEF experiments, cells were washed in PBS but were not treated Reactions were stopped by incubation at 95°C for 5 minutes. with Triton X-100. After further PBS washes, cells were blocked with 3% (w/v) PCR was performed using 2.5 μl first strand cDNA, mixed with 2.5 μl 10ϫ PCR bovine serum albumin (BSA) for 1 hour at room temperature. Cells were incubated reaction buffer with MgCl2 (Roche), 0.25 μl of 25 mM dNTPs (Bioline), forward and with primary antibodies diluted in 3% (w/v) BSA for 1 hour at room temperature, reverse primers to a final concentration of 0.5 μM, 1.25 U Taq DNA polymerase and were washed three times with PBS before incubation with fluorophore-conjugated (Roche) and DNase, RNase-free UltraPURE water (Gibco) to a final volume of 25 secondary antibody (Jackson ImmunoResearch Laboratories) diluted in 3% BSA, for μl. Reaction mixtures were incubated for 2 minutes at 94°C, followed by 30 seconds 45 minutes at room temperature in the dark. For HDFs, the primary antibodies were at 94°C, 30 seconds at 55°C and 30 seconds at 72°C for 25 cycles, then a 10 minute anti-fibrillin-1 PRO pAb (1:200) or anti-fibronectin (FN-3E2) mAb (1:200). For the incubation at 72°C. PCR was performed using a GeneAmp PCR system (Applied Journal of Cell Science β1-integrin-null and wild-type MEFs, the primary antibodies were anti-fibrillin-1 PRO Biosystems). Oligonucleotide primers for PCR were designed using Primer3 software RGE/RGE pAb (1:200) or anti-fibronectin (FN-3E2) mAb (1:200). For the fibronectin (Rozen and Skaletsky, 2000), using the same parameters resulting in similar Tm values and wild-type MEFs, the primary antibodies were anti-fibrillin-1 N-19 pAb (1:50) and product lengths as shown. Primers used (MWG Biotech or Eurogentec) were: or anti-fibronectin (FN-3E2) mAb (1:200). Cells were washed three times with PBS Fibrillin-1, 100 bp; forward primer 5Ј-CCTGTTCCGCTGTGAGTG-3Ј,Tm 58.90; Ј before incubating with 300 nM 4 ,6-diamidino-2-phenylindole, dihydrochloride reverse primer, 5Ј-ACTGATGCACGTGGTTGG-3Ј, Tm 59.04 (Ball et al., 2004). (DAPI; Molecular Probes) for five minutes at room temperature in the dark. Collagen type I α1 chain, 86bp; forward primer, 5Ј-GGATTGACCCCAACCAAG- ® Coverslips were mounted onto slides using Prolong antifade mounting medium 3Ј, Tm 58.69; reverse primer, 5Ј-AGTGGGGTACACGCAGGT-3Ј, Tm 58.97 (Ball et (Molecular Probes) and sealed with nail varnish. Slides were stored in the dark at al., 2004). Fibronectin EDa splice variant, 82bp; forward primer, 5Ј-ACTCGAGCC- 4°C until examination. Slides were examined either by conventional epifluorescence CTGAGGATG-3Ј, Tm 58.86; reverse primer, 5Ј-CTGAGGCCTTGCAGCTCT-3Ј, Tm microscopy using an Olympus BX51 microscope and a Photometrics Coolsnap HQ 59.39 (Ball et al., 2004). GAPDH, 93bp; forward primer, 5Ј-GAAGGCTGGGGCT- camera driven by Metamorph software, or by confocal microscopy using a Leica CATTT-3Ј, Tm 60.16; reverse primer, 5Ј-TGGTTCACACCCATGACG-3Ј, Tm 59.92. SP2 AOBS (acusto-optical beam splitter) confocal microscope. Images were compiled PCR products were resolved by 2.5% UltraPURE agarose gels (Gibco) run in TAE and analysed using ImageJ software (National Institute of Health). Imaging of FITC buffer (40 mM Tris-acetate, 1 mM EDTA), with either a 1 kb DNA ladder or TRITC was always conducted at nonoverlapping wavelengths. (NewEngland Biolabs) or hyperladder V (Bioline Ltd). DNA was visualised under UV after by staining with 0.5 μg/ml ethidium bromide in TAE for 30 minutes. RNA interference RNA interference was performed with siRNA oligonucleotides that were synthesised, Fibrillin-1 interaction with fibronectin purified and duplexed by Dharmacon RNA Technologies. siRNA duplexes were Plasma fibronectin and recombinant fibronectin fragments (N-terminal fragments 30 designed against the target sequence using Dharmacon siDESIGN® Center (two kDa and 45 kDa) were purchased from the Sigma Chemical Company (Poole, Dorset, fibronectin sequences targeted: FN1, 5Ј-AACAAATCTCCTGCCTGGTAC-3Ј; FN2, UK). The cell binding region 50 kDa, and the H120 fragment (Schofield et al., 1998) 5Ј-AAGTGGTCCTGTCGAAGTATT-3Ј. The order of nucleotides in the second were obtained from M. J. Humphries (Manchester, UK). The recombinant fibrillin- fibronectin sequence targeted was scrambled to generate a control sequence: FN2 1 fragments were generated as previously described (Rock et al., 2004; Marson et scrambled, 5Ј-AACTAGGCGATAACACTCAAC-3Ј). HDFs were transfected with al., 2005; Bax et al., 2003; Bax et al., 2007). Domain structures of these fragments 100 nM siRNA duplex using an Amaxa NucleofectorTM device and NucleofectorTM and an SDS-PAGE gel of the FN fragments are shown in supplementary material kit optimised for HDFs (Amaxa Biosystems) according to the manufacturer’s Fig. S1A. instructions. Cells were cultured for up to 7 days, and analysed by RT-PCR, Plasma fibronectin or recombinant fibronectin fragments were immobilised onto immunofluorescence and western blotting of culture medium and cell lysates. RNAi 96-well plates (Immulon 4) at 10 µg/ml (100 µl/well) overnight at 4°C. Unbound knockdown efficacy was analysed using unpaired Student’s t-tests (GraphPad Prism FN was removed and the wells blocked with 200 µl 5% BSA (w/v) in Tris-buffered 2.0). Results were statistically significant when P<0.05 (*P<0.05, **P<0.001 and saline (TBS) for 2-3 hours at 20°C. Wells were washed three times with 200 μl wash ***P<0.0001). solution (TBS + 0.1% BSA), then 100 µl of each recombinant fibrillin-1 fragment (His6-tagged) was added to the wells and incubated overnight at 4°C. Unbound Cell layer extractions fibrillin-1 fragments were removed, and the wells washed three times with 200 μl Cells were cultured in six-well plates until confluent. The medium was removed and wash solution. Bound ligand was detected by incubation with anti-His6 antibody protease inhibitor cocktail (Sigma), diluted 1:2000 was added with urea, to a final diluted 1:1000 in wash buffer for 3 hours, followed by anti-mouse horse radish 2704 Journal of Cell Science 121 (16)

peroxidase (HRP) secondary antibody diluted 1:2000 in wash buffer for 1 hour. Wells Lee, S. S., Knott, V., Jovanovic, J., Harlos, K., Grimes, J. M., Choulier, L., Mardon, were washed four times with 200 μl wash solution then 100 μl detection reagent (11 H. J., Stuart, D. I. and Handford, P. A. (2004). Structure of the integrin binding mg 2,2Ј-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) dissolved in 0.5 fragment from fibrillin-1 gives new insights into microfibril organization. Structure 12, ml H2O + 10 ml NaOAc (0.1 M)/NaH2PO4 (0.05 M) pH 5.0 +1 μl 30% H2O2) was 717-729. added to the wells. Absorbance was read at 405 nm using a Dynex plate reader. Lin, G., Tiedemann, K., Vollbrandt, T., Peters, H., Batge, B., Brinckmann, J. and Binding was analysed using unpaired Student’s t-tests (GraphPad Prism 2.0). Results Reinhardt, D. P. (2002). Homo- and heterotypic fibrillin-1 and –2 interactions constitute J. Biol. Chem. were statistically significant when P<0.05 (*P<0.05, **P<0.001 and ***P<0.0001). the basis for the assembly of microfibrils. 277, 50795-50804. To establish whether fibronectin could act as a direct template for cell surface Lomas, A. C., Mellody, K. T., Freeman, L. J., Bax, D. V., Shuttleworth, C. A. and Kielty, C. M. (2007). Fibulin-5 binds human smooth-muscle cells through alpha5beta1 fibrillin-1 assembly, we investigated potential molecular interactions using and alpha4beta1 integrins, but does not support receptor activation. Biochem. J. 405, recombinant fibrillin-1 and fibronectin fragments in solid-phase binding assays. Only 417-428. weak interactions were detected between plasma fibronectin and fibrillin-1 (not Mao, Y. and Schwarzbauer, J. E. (2005). Fibronectin fibrillogenesis, a cell-mediated matrix shown). However, several significant interactions were detected between specific assembly process. Matrix Biol. 24, 389-399. recombinant human fibrillin-1 and FN fragments (supplementary material Fig. S1B). Marson, A., Rock, M. J., Cain, S. A., Freeman, L. J., Morgan, A., Mellody, K., Fibrillin-1 overlapping fragments PF5 and PF7 bound to the fibronectin 30 kDa N- Shuttleworth, C. A., Baldock, C. and Kielty, C. M. (2005). Homotypic fibrillin-1 terminal fragment but not to the adjacent fibronectin 45 kDa fragment. PF5 and PF7 interactions in microfibril assembly. J. Biol. Chem. 280, 5013-5021. also bound the FN H120 fragment. N-terminal overlapping fibrillin-1 fragments PF1 McKeown-Longo, P. J. and Mosher, D. F. (1985). Interaction of the 70,000-mol-wt amino- and PF2 both bound to the FN 50 kDa central cell-binding region. These interactions terminal fragment of fibronectin with the matrix-assembly receptor of fibroblasts. J. Cell may align fibrillin-1 molecules transiently on fibronectin during assembly. Biol. 100, 364-374. Mitjans, F., Sander, D., Adán, J., Sutter, A., Martinez, J. M., Jäggle, C. S., Moyano, J. M., Kreysch, H. G., Piulats, J. and Goodman, S. L. (1995). An anti-alpha v-integrin This work was funded by the Wellcome Trust (R.K.), the Medical antibody that blocks integrin function inhibits the development of a human melanoma Research Council (G0200246) and the European Union (LSHM-CT- in nude mice. J. Cell Sci. 108, 2825-2838. 2005-018960). C.M.K. is a Royal Society Wolfson Research Merit Pankov, R. and Yamada, K. M. (2002). Fibronectin at a glance. J. Cell Sci. 115, 3861- Award holder. We thank Reinhardt Fässler (Max-Planck Institute of 3863. Pankov, R., Cukierman, E., Katz, B. Z., Matsumoto, K., Lin, D. C., Lin, S., Hahn, C. Biochemistry, Department of Molecular Medicine, Martinsried, and Yamada, K. M. (2000). Integrin dynamics and matrix assembly: tensin-dependent Germany) for kindly supplying the fibronectin RGE mutant cells. translocation of alpha(5)beta(1) integrins promotes early fibronectin fibrillogenesis. J. Cell Biol. 148, 1075-1090. Pereira, L., D’Alessio, M., Ramirez, F., Lynch, J. R., Sykes, B., Pangilinan, T. and References Bonadio, J. (1993). Genomic organization of the sequence coding for fibrillin, the Ball, S. G., Shuttleworth, C. A. and Kielty, C. M. (2004). Direct cell contact influences defective gene product in . Hum. Mol. Genet. 2, 961-968. Int. J. Biochem. Cell Biol. bone marrow mesenchymal stem cell fate. 36, 714-727. Retta, S. F., Cassarà, G., D’Amato, M., Alessandro, R., Pellegrino, M., Degani, S., De Baneyx, G., Baugh, L. and Vogel, V. (2002). Fibronectin extension and unfolding within Leo, G., Silengo, L. and Tarone, G. (2001). Cross talk between beta(1) and alpha(V) Proc. Natl. Acad. Sci. USA cell matrix fibrils controlled by cytoskeletal tension. 99, integrins: beta(1) affects beta(3) mRNA stability. Mol. Biol. Cell 12, 3126-3138. 5139-5143. Rock, M. J., Cain, S. A., Freeman, L. J., Morgan, A., Mellody, K., Marson, A., Bax, D. V., Bernard, S. E., Lomas, A., Morgan, A., Humphries, J., Shuttleworth, C. Shuttleworth, C. A., Weiss, A. S. and Kielty, C. M. (2004). Molecular basis of elastic A., Humphries, M. J. and Kielty, C. M. (2003). Cell adhesion to fibrillin-1 molecules fiber formation. Critical interactions and a tropoelastin-fibrillin-1 cross-link. J. Biol. J. Biol. and microfibrils is mediated by alpha 5 beta 1 and alpha v beta 3 integrins. Chem. 279, 23748-23758. Chem. 278, 34605-34616. Rozen, S. and Skaletsky, H. (2000). Primer3 on the WWW for general users and for Bax, D. V., Mahalingam, Y., Cain, S. A., Mellody, K., Freeman, L., Younger, K., biologist programmers. Methods Mol. Biol. 132, 365-386. Shuttleworth, C. A., Humphries, M. J., Couchman, J. R. and Kielty, C. M. (2007). Sakamoto, H., Broekelmann, T., Cheresh, D. A., Ramirez, F., Rosenbloom, J. and Cell adhesion to fibrillin-1: identification of an Arg-Gly-Asp-dependent synergy region Mecham, R. P. (1996). Cell-type specific recognition of RGD- and non-RGD-containing and a heparin-binding site that regulates focal adhesion formation. J. Cell Sci. 120, 1383- cell binding domains in fibrillin-1. J. Biol. Chem. 271, 4916-4922. 1392. Schofield, K. P., Humphries, M. J., de Wynter, E., Testa, N. and Gallagher, J. T. (1998). Bouvard, D., Brakebusch, C., Gustafsson, E., Aszódi, A., Bengtsson, T., Berna, A. and The effect of alpha4 beta1-integrin binding sequences of fibronectin on growth of cells Fässler, R. (2001). Functional consequences of integrin gene mutations in mice. Circ. from human hematopoietic progenitors. Blood 91, 3230-3238. Res. Journal of Cell Science 89, 211-223. Somanath, P. R., Kandel, E. S., Hay, N. and Byzova, T. V. (2007). Akt1 signaling regulates Bunton, T. E., Biery, N. J., Myers, L., Gayraud, B., Ramirez, F. and Dietz, H. C. (2001). integrin activation, matrix recognition, and fibronectin assembly. J. Biol. Chem. 282, Phenotypic alteration of vascular smooth muscle cells precedes elastolysis in a mouse 22964-22976. model of Marfan syndrome. Circ. Res. 88, 37-43. Somers, C. E. and Mosher, D. F. (1993). Protein kinase C modulation of fibronectin matrix Castell, S., Roser Pagan, R., Mitjans, F., Piulats, J., Goodman, S., Jonczyk, A., Huber, assembly. J. Biol. Chem. 268, 22277-22280. F., Vilaró, S. and Reina, M. (2001). RGD peptides and monoclonal antibodies, Sottile, J., Shi, F., Rublyevska, I., Chiang, H. Y., Lust, J. and Chandler, J. (2007). antagonists of v-integrin, enter the cells by independent endocytic pathways. Lab. Invest. Fibronectin-dependent collagen I deposition modulates the cell response to fibronectin. 81, 1615-1626. Am. J. Physiol., Cell. Physiol. 293, C1934-C1946. Chaudhry, S. S., Cain, S. A., Morgan, A., Dallas, S. L., Shuttleworth, C. A. and Kielty, Takahashi, S., Leiss, M., Moser, M., Ohashi, T., Kitao, T., Heckmann, D., Pfeifer, A., C. M. (2007). Fibrillin-1 regulates the bioavailability of TGFbeta1. J. Cell Biol. 176, Kessler, H., Takagi, J., Erickson, H. P. et al. (2007). The RGD motif in fibronectin is 355-367. essential for development but dispensable for fibril assembly. J. Cell Biol. 178, 167-178. Clark, K., Pankov, R., Travis, M. A., Askari, J. A., Mould, A. P., Craig, S. E., Newham, Tomasini-Johansson, B. R., Annis, D. S. and Mosher, D. F. (2006). The N-terminal 70- P., Yamada, K. M. and Humphries, M. J. (2005). A specific alpha5beta1-integrin kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence conformation promotes directional integrin translocation and fibronectin matrix of intact fibronectin. Matrix Biol. 25, 282-293. formation. J. Cell Sci. 118, 291-300. Trask, T. M., Ritty, T. M., Broekelmann, T., Tisdale, C. and Mecham, R. P. (1999). N- Dallas, S. L., Sivakumar, P., Jones, C. J., Chen, Q., Peters, D. M., Mosher, D. F., terminal domains of fibrillin 1 and fibrillin 2 direct the formation of homodimers: a Humphries, M. J. and Kielty, C. M. (2005). Fibronectin regulates latent transforming possible first step in microfibril assembly. Biochem. J. 340, 693-701. growth factor-beta (TGF beta) by controlling matrix assembly of latent TGF beta-binding Velling, T., Risteli, J., Wennerberg, K., Mosher, D. F. and Johansson, S. (2002). protein-1. J. Biol. Chem. 280, 18871-18880. Polymerization of type I and III is dependent on fibronectin and enhanced by Dallas, S. L., Chen, Q. and Sivakumar, P. (2006). Dynamics of assembly and integrins alpha 11beta 1 and alpha 2beta 1. J. Biol. Chem. 277, 37377-37381. reorganization of extracellular matrix proteins. Curr. Top. Dev. Biol. 75, 1-24. Wallis, D. D., Putnam, E. A., Cretoiu, J. S., Carmical, S. G., Cao, S. N., Thomas, G. Féral, C. C., Zijlstra, A., Tkachenko, E., Prager, G., Gardel, M. L., Slepak, M. and and Milewicz, D. M. (2003). Profibrillin-1 maturation by human dermal fibroblasts: Ginsberg, M. H. (2007). CD98hc (SLC3A2) participates in fibronectin matrix assembly proteolytic processing and molecular chaperones. J. Cell. Biochem. 90, 641-652. by mediating integrin signalling. J. Cell Biol. 178, 701-711. Wang, R., Clark, R. A., Mosher, D. F. and Ren, X. D. (2005). Fibronectin’s central cell- George, E. L., Baldwin, H. S. and Hynes, R. O. (1997). Fibronectins are essential for binding domain supports focal adhesion formation and Rho signal transduction. J. Biol. heart and blood vessel morphogenesis but are dispensable for initial specification of Chem. 280, 28803-28810. precursor cells. Blood 90, 3073-3081. Wierzbicka-Patynowski, I. and Schwarzbauer, J. E. (2003). The ins and outs of Hubmacher, D., Tiedemann, K. and Reinhardt, D. P. (2006). : from biogenesis fibronectin matrix assembly. J. Cell Sci. 116, 3269-3276. of microfibrils to ignalling functions. Curr. Top. Dev. Biol. 75, 93-123. Wu, C., Keivens, V. M., O’Toole, T. E., McDonald, J. A. and Ginsberg, M. H. (1995). Ilic, D., Kovacic, B., Johkura, K., Schlaepfer. D. D., Tomasevic, N., Han, Q., Kim, J. Integrin activation and cytoskeletal interaction are essential for the assembly of a B., Howerton, K., Baumbusch, C., Ogiwara, N. et al. (2004). FAK promotes fibronectin matrix. Cell 83, 715-724. organization of fibronectin matrix and fibrillar adhesions. J. Cell Sci. 117, 177-187. Yoneda, A., Ushakov, D., Multhaupt, H. A. and Couchman, J. R. (2007). Fibronectin Isogai, Z., Ono, R. N., Ushiro, S., Keene, D. R., Chen, Y., Mazzieri, R., Charbonneau, matrix assembly requires distinct contributions from Rho kinases I and -II. Mol. Biol. N. L., Reinhardt, D. P., Rifkin, D. B. and Sakai, L. Y. (2003). Latent transforming Cell 18, 66-75. growth factor beta-binding protein 1 interacts with fibrillin and is a microfibril-associated Zhong, C., Chrzanowska-Wodnicka, M., Brown, J., Shaub, A., Belkin, A. M. and protein. J. Biol. Chem. 278, 2750-2757. Burridge, K. (1998). Rho-mediated contractility exposes a cryptic site in fibronectin Kielty, C. M. (2006). Elastic fibres in health and disease. Expert Rev. Mol. Med. 8, 1-23. and induces fibronectin matrix assembly. J. Cell Biol. 141, 539-551.