Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia
2008; Springer Nature; Volume: 27; Issue: 21 Linguagem: Inglês
10.1038/emboj.2008.206
ISSN1460-2075
AutoresJordi Alcaraz, Ren Xu, Hidetoshi Mori, Celeste M. Nelson, Rana Mroue, Virginia A. Spencer, Douglas Brownfield, Derek C. Radisky, Carlos Bustamante, Mina J. Bissell,
Tópico(s)Cancer Cells and Metastasis
ResumoArticle9 October 2008Open Access Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia Jordi Alcaraz Corresponding Author Jordi Alcaraz Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Present address: Unitat de Biofísica i Bioenginyeria, Universitat de Barcelona, Barcelona, 08036 Spain Search for more papers by this author Ren Xu Ren Xu Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Hidetoshi Mori Hidetoshi Mori Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Celeste M Nelson Celeste M Nelson Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Rana Mroue Rana Mroue Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Virginia A Spencer Virginia A Spencer Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Doug Brownfield Doug Brownfield Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Derek C Radisky Derek C Radisky Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Present address: Mayo Clinic Cancer Center, Jacksonville, FL 32224 USA Search for more papers by this author Carlos Bustamante Carlos Bustamante Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Department of Physics, University of California, Berkeley, CA, USA Howard Hughes Medical Institute, University of California, Berkeley, CA, USA Search for more papers by this author Mina J Bissell Corresponding Author Mina J Bissell Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Jordi Alcaraz Corresponding Author Jordi Alcaraz Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Present address: Unitat de Biofísica i Bioenginyeria, Universitat de Barcelona, Barcelona, 08036 Spain Search for more papers by this author Ren Xu Ren Xu Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Hidetoshi Mori Hidetoshi Mori Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Celeste M Nelson Celeste M Nelson Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Rana Mroue Rana Mroue Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Virginia A Spencer Virginia A Spencer Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Doug Brownfield Doug Brownfield Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Derek C Radisky Derek C Radisky Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Present address: Mayo Clinic Cancer Center, Jacksonville, FL 32224 USA Search for more papers by this author Carlos Bustamante Carlos Bustamante Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Department of Physics, University of California, Berkeley, CA, USA Howard Hughes Medical Institute, University of California, Berkeley, CA, USA Search for more papers by this author Mina J Bissell Corresponding Author Mina J Bissell Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Author Information Jordi Alcaraz 1,2,5, Ren Xu1, Hidetoshi Mori1, Celeste M Nelson1, Rana Mroue1, Virginia A Spencer1, Doug Brownfield1, Derek C Radisky1,6, Carlos Bustamante2,3,4 and Mina J Bissell 1 1Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA 2Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA 3Department of Physics, University of California, Berkeley, CA, USA 4Howard Hughes Medical Institute, University of California, Berkeley, CA, USA 5Present address: Unitat de Biofísica i Bioenginyeria, Universitat de Barcelona, Barcelona, 08036 Spain 6Present address: Mayo Clinic Cancer Center, Jacksonville, FL 32224 USA *Corresponding authors: Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road MS 977R225A, Berkeley, CA 94720, USA. Tel.: + 510 4864365; Fax: + 510 4865586; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2008)27:2829-2838https://doi.org/10.1038/emboj.2008.206 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In the mammary gland, epithelial cells are embedded in a ‘soft’ environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for β-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of β-casein expression required both laminin signalling and a ‘soft’ extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of β-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues’ unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis. Introduction Signals from the microenvironment are essential to direct normal tissue development and, in the adult organism, to maintain tissue-specific functions (Nelson and Bissell, 2006). Studies in three-dimensional (3D) cultures have identified key biochemical microenvironmental cues underlying mammary-specific structure and function. The extracellular matrix (ECM) component, laminin-111 (LM1), is necessary to induce polarity and acinar morphogenesis (Gudjonsson et al, 2002) and, together with lactogenic hormones, is required for β-casein expression in mammary epithelial cells (MECs) (Streuli et al, 1991; Xu et al, 2007). In addition to a unique biochemical milieu, different tissue microenvironments exhibit distinct mechanical properties (Discher et al, 2005). There is growing evidence that, rather than being a passive property of the tissue, the mechanical properties of the microenvironment have a direct impact on tissue-specific morphogenetic programmes in MECs and other cell types (Wozniak et al, 2003; Paszek et al, 2005; Engler et al, 2006). Furthermore, because abnormally high stiffness and loss of tissue function are hallmarks of solid tumours (Paszek et al, 2005), and increased mammographic density is a risk factor for breast cancer (Boyd et al, 1998), it has been suggested that aberrant tissue stiffness may facilitate the acquisition of a malignant phenotype (Wozniak et al, 2003; Paszek et al, 2005; Provenzano et al, 2006). Tissue elasticity is thought to be maintained by a mechanical homeostatic mechanism largely determined by the reciprocal mechanochemical interactions between cells and their surrounding ECM (Bissell et al, 1982; Discher et al, 2005; Paszek et al, 2005). Previous studies have examined the effects of either the biochemical composition or the elasticity of the substrata on functional differentiation or cell mechanics individually; however, a comprehensive approach aiming to dissect how these signals modulate each other and how these signals integrate to control tissue-specific gene expression have been lacking (Alcaraz et al, 2004). To determine how the mechanochemistry of the cellular microenvironment affects tissue-specific gene expression, we used two mammary epithelial cell lines (SCp2 and EpH4) that in culture can be induced to functionally differentiate (defined here as expression of an abundant mouse milk protein, β-casein) in the presence of appropriate ECM. The fact that, in the presence of a LM1-containing gels, cell—cell contact is not required for β-casein expression (Streuli et al, 1991) (Supplementary Figure 1) allowed us to examine single cells cultured on top of gels under these conditions. This ‘single cell on top’ assay allowed us to use atomic force microscopy (AFM) to assess the elasticity or stiffness (defined by the Young's elastic modulus E) of single cells and their underlying substrata independently. Using this quantitative and comprehensive approach, we unraveled the intimate interrelationship among LM1 binding, ECM stiffness, cell shape and cell stiffness, as well as the synergistic effects of these mechanochemical properties on tissue-specific gene expression. Furthermore, we show that variations of these properties from biomimetic values, that is, those comparable with normal physiological conditions, may be linked potentially to dedifferentiation that occurs in tumours. Results Single cells exhibit elastic moduli comparable with cells within primary organoids when exposed to LM1 and biomimetic extracellular elasticity Previous studies have reported that normal mouse mammary tissue is very soft (E∼100 Pa) (Table I) (Paszek et al, 2005) and have suggested that this low stiffness is caused mainly by the ECM. However, the actual elastic modulus of epithelial cells within the mammary gland has not been measured. To estimate the elasticity of fully differentiated MECs in vivo, we isolated milk-synthesizing primary mammary organoids from early pregnant mice and cultured them on top of Matrigel in differentiation medium for 24 h. Matrigel is a suitable substratum because it is rich in LM1 and exhibits biomimetic elastic modulus, as measured by AFM and rheometry (Table I). A schematic representation of this experimental setup is shown in Figure 1A. Mammary organoids in culture are often heterogeneous in size. To account for this heterogeneity, we measured the elasticity of both small- and medium-sized organoids (Figure 1B), which typically contain about a dozen or several dozen cells, respectively. We found that irrespective of the size, cells within the organoids exhibited comparable average elastic moduli between 400 and 800 Pa (Figure 1C). We used this range as a reference for biomimetic cellular elasticity throughout this work. Figure 1.The ‘physiological’ or ‘biomimetic’ elasticity of MECs can be defined in culture. (A) Schematic representation of the experimental setup: either mammary organoids (MO) or single MECs (SCp2 or EpH4) were plated on top of an ECM gel. An AFM probe was used to quantify the elasticity of either the cell or the underlying ECM gel. (B) Morphology of primary mammary organoids isolated from mice in early pregnancy and cultured on top of a LM1-rich ECM gel (Matrigel) in differentiation medium for 24 h. The elastic modulus of the substrata as measured by AFM is indicated at the bottom of each image. Most mammary organoids had either small or medium sizes, corresponding to roughly a dozen or a few dozen cells, respectively. (C) Elastic moduli of cells within either small- or medium-sized primary mammary organoids cultured as in (B). Dashed horizontal lines correspond to the lower and upper values defined by the elasticity of cells within mammary organoids, used as a reference for biomimetic cell elastic moduli throughout this study. All scale bars correspond to 20 μm throughout the figures unless otherwise indicated. Download figure Download PowerPoint Table 1. Summary of mechanical parameters: comparison between rodent mammary tissue, different biological substrata and single MECs cultured on top of these substrata Sample Tissue or substratum Single cellsa EAFM (Pa)b Ebulk (Pa)c EAFM (Pa)b Normal mammary tissue n.a. 170±30d Average mammary tumour n.a. 4050±940d Mammary organoids on Matrigel 120±20 n.a. 600±200 Matrigel 120±20 220±10 700±100 Laminin-111 110±30 n.a. 730±110 Laminin-111+collagen I (2 g/l) (40:60% v/v) 72±8 150±40 600±90 Collagen I (2 g/l) 290±100 240±40 400±100 PolyHEMA n.a. n.a. 1700±300 Borosilicate glass (2D) n.a. 63 × 109e 1300±200 Data are mean±s.e.; n.a., not available. a Mean of all cell lines. b Young's modulus measured by AFM. c Young's modulus measured with a rheometer. d From Paszek et al (2005). e According to the manufacturer's instructions. The two mammary cell lines, SCp2 and EpH4, have been used extensively to study functional differentiation of MECs in culture (Desprez et al, 1993; Pujuguet et al, 2001). The elastic moduli of either single SCp2 or EpH4 cells cultured on top of Matrigel for 24 h (Figure 2A, bottom panel) fell within the biomimetic range defined by cells in mammary organoids (parallel dashed lines) (Figure 2C). As MECs exhibit a round morphology both in vivo and when exposed to LM1 in culture (Roskelley et al, 1994), it is possible that their similar elastic moduli is due to the round shape per se (Le Beyec et al, 2007). To address this question, we measured the elasticity of the two cell lines rounded in the absence of LM1 signalling by culturing them on top of a substratum coated with the non-adhesive poly-(2-hydroxyethyl methacrylate) (poly-HEMA) (Figure 2A, top panel). Unlike cells on Matrigel, cells cultured on poly-HEMA were significantly stiffer (Figure 2C) and did not express β-casein, as assessed by quantitative RT—PCR (Figure 2B). The stiffening of MECs on poly-HEMA was not due to increased cell death (Muschler et al, 1999). These data confirm that LM1 signalling is necessary for functional differentiation of MECs and indicate that biomimetic cellular elasticity in MECs is cell line independent and downstream of cell—ECM rather than cell—cell adhesion or cell rounding per se. Figure 2.Laminin-111 signalling and biomimetic extracellular elasticity, but not cell shape per se, induce robust β-casein expression and a cellular elasticity comparable with cells within mammary organoids. Two MECs (SCp2 and EpH4) were cultured in the presence (Matrigel) or absence (poly-HEMA) of ECM signalling. (A–C) Both culture conditions produced a similarly round morphology in SCp2 (A) and EpH4 cells (data not shown), but differed dramatically in their effects on β-casein, as measured by quantitative RT—PCR (B) and cellular elasticity (C). Note that fold β-casein/18S rRNA data are plotted on a logarithmic scale throughout the figures. **P<0.05 and ***P<0.01 were determined by two-tailed Student's t-test with respect to (w.r.t.) Matrigel or the corresponding control throughout this work unless otherwise indicated. Download figure Download PowerPoint Increasing extracellular elasticity beyond normal mammary tissue values promotes spreading and stiffening and inhibits -casein expression in MECs As normal mammary tissue is soft (Table I), we hypothesized that strong functional differentiation in culture would be achieved only by using substrata where the elasticity mimics normal tissue. Accordingly, we used two culture assays that allow increasing extracellular stiffness beyond biomimetic values, while maintaining biochemical signalling constant (Alcaraz et al, 2004). In the first assay (Figure 3A–C), EpH4 cells were cultured on top of gels containing LM1 mixed with collagen type I (COL-I) (3 g/l) (40:60% v/v). Four hours after plating, differentiation media was added and half of the gels were gently detached from their container along the gel's edges and rendered floating in the medium (Michalopoulos and Pitot, 1975). As AFM requires samples to be somewhat anchored, caution was taken to avoid complete gel detachment by leaving the bottom-center of the gel attached to the underlying glass surface. AFM measurements revealed that the average floating gel elasticity was close to that of bulk mammary tissue, whereas the attached gel was three-fold stiffer (Figure 3A, bottom image). Such gel stiffening was sufficient to dramatically downregulate β-casein (Figure 3B) and increase the elastic modulus of the cells (Figure 3C). In the second assay (Figure 3D–H), EpH4 cells were plated on top of polyacrylamide gels exhibiting elastic moduli either close to mammary tissue (referred to as ‘soft’), comparable with or even higher than mammary tumours (referred to as ‘stiff’). Only 24 h after plating on the stiffer substrata, cells displayed a spread morphology (Figure 3D) and non-biomimetic elasticity (Figure 3F). To induce β-casein, cells were overlaid with 2% Matrigel diluted in differentiation medium. In agreement with the first assay, stiffer substrata downregulated β-casein transcription, measured both by quantitative RT—PCR (Figure 3E) and by the fluorescence of cells transfected with a construct containing 16 concatenated copies of the mouse β-casein gene promoter driving cyan fluorescent protein (CFP) expression (Figure 3G and H). These findings support our hypothesis and indicate that LM1-dependent functional differentiation is modulated by the extracellular elasticity. Figure 3.Increasing extracellular elasticity beyond normal mammary tissue values inhibits β-casein expression and promotes spreading and stiffening in MECs. We used two independent culture assays to increase extracellular elasticity beyond normal mammary tissue values while maintaining biochemical composition constant. The elastic modulus of the substrata as measured by AFM is indicated at the bottom of each image. (A—C) Floating gel assay: EpH4 cells were cultured on top of LM1 mixed with COL-I (3 g/l) (40:60% v/v). Four hours after plating, differentiation media was added and half of the gels were gently detached from the container. The elastic modulus of the floating gel was comparable to normal tissue (A, top image), whereas that of the attached gel was three-fold stiffer (A, bottom image). Corresponding β-casein expression (B) and cell stiffness (C) in these culture conditions. (D—H) Polyacrylamide gel assay: EpH4 cells were cultured on top of gels coated with equal fibronectin concentration but exhibiting a stiffness comparable with normal tissue (referred to as ‘soft’) or within the range of mammary tumours (referred to as ‘stiff’). Cell morphology (D) and elasticity (F) 24 h after plating on either soft or stiff polyacrylamide gels. (E) β-Casein expression 48 h after overlaying cells with 2% Matrigel diluted in differentiation media. (G) Phase contrast (top) and corresponding CFP fluorescence intensity images (bottom panels) of EpH4 cells stably transfected with 16 concatenated copies of the β-casein promoter fused to CFP cultured as in (E). (H) Corresponding quantification of CFP intensity of cells cultured as in (E). The two assays consistently reported a downregulation of β-casein expression as well as non-biomimetic cell shape and elasticity in gels with non-biomimetic elastic moduli (for more details see the Discussion). *P<0.1 and ***P<0.01 were determined by two-tailed Student's t-test. Download figure Download PowerPoint Loss of LM1 signalling induces non-biomimetic cellular elasticity and/or morphology and downregulates -casein expression Mammary epithelial cells in vivo are in contact with a basement membrane, a specialized ECM rich in LM1 that physically separates mammary epithelium from the stroma; the latter is rich in COL-I (Provenzano et al, 2006) and contains much less LM1 (Klinowska et al, 1999). During tumour cell invasion, the integrity of the basement membrane is often compromised (Wetzels et al, 1989) and MECs can contact the stroma directly. To investigate how this loss of LM1 signalling affects functional differentiation and the mechanical properties of MECs, we examined SCp2 and EpH4 cells plated on top of LM1 gels mixed with increasing ratio of COL-I (2 g/l). We found that as little as 10% LM1 was sufficient to maintain β-casein expression and biomimetic extra- and intracellular elasticity (Figure 4A–E). Interestingly, reducing LM1 concentration below 10% led to a dramatic downregulation of β-casein expression (Figure 4A), a decrease in β-casein promoter activity (Figure 4B and C), non-biomimetic cellular elasticity (Figure 4D) and a sudden increase in the gel's elastic moduli (Figure 4E). Both SCp2 and EpH4 cells exhibited a similar relative downmodulation of β-casein as the COL-I/LM1 ratio increased. However, unlike SCp2 cells, loss of LM1 signalling in EpH4 cells induced stiffening (Figure 4D) and spreading comparable with that found on glass substrata (Supplementary Figure 2). The differences between SCp2 and EpH4 on these gels most probably arise from the distinct expression profiles of LM1 and COL-I integrin receptors (Figure 4G). SCp2 cells appeared stiffer on softer gels (Figure 4F), thereby revealing a strong non-linear relationship between extra- and intracellular elasticity in these cells, and confirming that our methodology to probe cell mechanics with AFM was not biased by the stiffness of the underlying substrata (further discussion on the lack of contribution by the underlying substrata elasticity on cell mechanical measurements is presented in Supplementary data). These experiments reveal that at a concentration of 10% or above, LM1 signalling is dominant over COL-I signalling, and that COL-I-dependent loss of β-casein expression is associated with non-biomimetic extra- and intercellular elastic moduli and changes in cell shape. Figure 4.Loss of LM1 signalling downregulates β-casein expression and induces non-biomimetic cellular elasticity and/or morphology in SCp2 and EpH4 MECs. (A) β-Casein expression of MECs cultured on top of COL-I (2 g/l) gels mixed with decreasing concentrations of LM1. (B and C) Visualization (C) and corresponding quantification (B) of the activity of the β-casein promoter in EpH4 cells cultured as in (A). (D) Elastic moduli of MECs cultured as in (A). Although both cell lines exhibited non-biomimetic elasticity for LM1 concentrations below 10%, the elastic moduli of SCp2 was lower than the physiological range (dashed lines), whereas that of EpH4 was higher. (E) Elasticity of LM1—COL-I mixed gels. (F) Elasticity of SCp2 cells as a function of gel elasticity. Note the non-linear relationship between these two properties. (G) Messenger RNA levels of LM1- and COL-I-specific integrin receptors in SCp2 and EpH4 MECs cultured in 2D assessed by RT—PCR. Download figure Download PowerPoint Receptors involved in LM1-dependent biomimetic cellular elasticity To start defining the molecular mechanisms underlying LM1-dependent biomimetic cellular elasticity, we examined the function of laminin-specific receptors, β1- and α6-integrins (Muschler et al, 1999) and dystroglycan (DG) (Weir et al, 2006), shown previously to be necessary for β-casein expression in culture and in vivo (Muschler et al, 1999; Naylor et al, 2005; Weir et al, 2006). To inhibit integrin receptors, we used function-blocking antibodies against either β1- or α6-integrins as described previously (Muschler et al, 1999). Blocking β1-integrin in SCp2 dramatically decreased their elastic moduli (Figure 5A), whereas blocking α6-integrin had only a weak effect (Figure 5B). Unlike β1-integrin blocking experiments, we did not observe a statistically significant difference between the elasticity of DG negative (DG−/−) and DG expressing (DG+/+) cells (kindly provided by Dr John Muschler at the California Pacific Medical Center) (P=0.14) (Figure 5C). These data suggest that a β1-integrin other than α6β1 mediates LM1-dependent biomimetic cellular elasticity. Figure 5.Receptors containing the β1-integrin subunit are major candidates for mediating LM1-induced cell biomimetic elasticity in MECs. (A and B) Elastic moduli of SCp2 cells cultured on top of Matrigel in the presence of function blocking antibodies against β1- (A) and α6-integrins (B) or corresponding isotype controls. (C) Elasticity of dystroglycan-expressing cells (DG+/+) or DG-negative (DG−/−). Both cell types remained fairly round under all conditions. Download figure Download PowerPoint Biomimetic cellular elasticity is associated with low non-muscle myosin II activity and low actin polymerization The actin—myosin cytoskeleton is the major contributor to cellular elasticity (Wakatsuki et al, 2003; Roca-Cusachs et al, 2008). When SCp2 cells cultured on two-dimensional (2D) glass substrata for 24 h were treated with specific inhibitors of actin polymerization (latrunculin B), myosin II ATPase activity (blebbistatin) and its upstream effector Rho kinase (ROCK) (Y27632), cellular elasticity markedly decreased towards the biomimetic range, although the difference was significantly smaller than cells cultured on top of Matrigel (Figure 6A). In contrast, two different inhibitors of microtubule polymerization essentially had no effect on cellular elasticity (Supplementary Figure 3). Treating SCp2 cells cultured on Matrigel with the same concentration of the inhibitors against the actin—myosin cytoskeleton revealed that actin polymerization still contributed to cellular elasticity. However, unlike 2D conditions, we did not observe any further reduction in cellular elasticity upon inhibition of ROCK or myosin II activities in Matrigel (Figure 6B). Confocal visualization in SCp2 cells of F-actin and phosphorylated myosin II light chain (MLC-II) fluorescence staining at Thr18 and Ser19, the latter being indicative of the specific activity of myosin II, revealed that cells on Matrigel were round, F-actin was mostly cortical and diphosphorylated myosin was scarce. In contrast, MECs on 2D spread and the corresponding F-actin and active myosin II were abundant both at the cell cortex and throughout the cytoplasm. (Figure 6C). Increased spreading and F-actin in 2D conditions were confirmed by quantitative analysis of the cell-projected area (Figure 6D) and of the average fluorescence intensity of phalloidin staining per cell (Figure 6E), respectively. Similar findings were obtained in EpH4 cells (data not shown). In agreement with the confocal images, immunoblot analysis of EpH4 cells showed that diphosphorylation of myosin II was significantly lower on Matrigel than on 2D tissue culture plastic (Figure 6F and G). These results indicate that LM1-dependent cellular biomimetic elastic modulus is mediated, at least in part, by targeting the actin—myosin cytoskeleton through downregulation of actin polymerization and myosin II activity. Figure 6.Laminin-111-induced cell biomimetic elasticity is partially mediated through downregulation of both actin polymerization and myosin II activity. (A, B) Comparison of the elasticity of SCp2 cells cultured for 24 h on 2D (A) or on top of LM1-rich gel (Matrigel) (B) after 30 min treatment with either vehicle (DMSO) or inhibitors of actin polymerization (latrunculin B), ROCK (Y26732) and myosin II ATPase (blebbistatin) activity using concentrations described in Materials and methods. *P<0.1, **P<0.05 and ***P<0.01 were determined by two-tailed Student's t-test w.r.t. untreated cells. (C) F-actin (red) and diphosphorylated MLC-II (green) organization in SCp2 cells spread on 2D substrata or rounded on top of Matrigel studied with confocal microscopy. Images correspond to the maximum intensity value projected on either the x—y plane (top images) or the z—x plane (bottom images). Both horizontal and vertical scale bars indicate 5 μm. (D) Box plot of cell spreading of SCp2 cells cultured as in (C). **P<0.05 was determined using Mann—Whitney rank sum test. (E) Quantification of the average fluorescence intensity of F-actin phalloidin staining per cell. (F) Immunoblot of total and diphosphorylated MLC-II in MECs cultured as in (C); (G) corresponding quantification by densitometry analysis of the ratio between diphosphorylated and total MLC-II. 2D substrata corresponds to either untreated borosilicate glass or tissue culture polystyrene dish. Download figure Download PowerPoint Discussion In vivo, signals from the microenvironment are essential for normal development and organ homoeostasis, and abnormalities in these signals contribute to various pathologies including tumorigenesis (Ingber, 2003; Nelson and Bissell, 2006). Nevertheless, the detailed mechanochemical signalling by which the microenvironment controls these processes are still ill-defined (Alcaraz et al, 2004). Previous studies using cultured cells have examined the effects of extracellular biochemical or biophysical cues on differentiation (Engler et al, 2004b; Le Beyec et al, 2007), morphology (Engler et al, 2004a) and mechanics (Solon et al, 2007), each in isolation. Here, we used a c
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