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Control of the proliferation/differentiation balance in skeletal myoblasts by integrin and syndecan targeting peptides Varvara Gribova, Isabelle Paintrand, Laure Fourel, Rachel Auzély-Velty, Corinne Albige-Rizo, Cécile Gauthier-Rouvière, and Catherine Picart ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00557 • Publication Date (Web): 28 Jan 2016 Downloaded from http://pubs.acs.org on February 10, 2016

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ACS Biomaterials Science & Engineering

Control of the proliferation/differentiation balance in skeletal myoblasts by integrin and syndecan targeting peptides Varvara Gribova1, 2, 3, 4†, Isabelle Paintrand1, 2, Laure Fourel5, Rachel Auzely-Velty3, 4, Corinne Albigès-Rizo5, Cécile Gauthier-Rouvière6,7 Catherine Picart 1, 2*

1

. Univ. Grenoble Alpes, LMGP, F-38016 Grenoble, France

2

. CNRS, LMGP, F-38016 Grenoble, France

3

. Centre de Recherches sur les Macromolécules Végétales (CERMAV, CNRS UPR 5301),

affiliated with Université Joseph Fourier, and member of the Institut de Chimie Moléculaire de Grenoble, 601 rue de la Chimie, Domaine Universitaire de Grenoble-St Martin d'Hères, France 4

. CNRS, CERMAV, F-38016 Grenoble, France

5

. INSERM U823, ERL CNRS5284, Université Joseph Fourier, Institut Albert Bonniot, Site

Santé, BP170, 38042 Grenoble cedex 9, France 6

. Universités Montpellier 2 et 1, CRBM, F-34293 Montpellier, France

7.

CNRS, CRBM, F-34293 Montpellier, France

Keywords Extracellular matrix, polyelectrolyte multilayer films peptides, integrins, syndecan, layer-bylayer, myogenesis

ACS Paragon Plus Environment

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Abstract Controlling the different steps of cell differentiation in vitro using bioactive surfaces may be useful in view of future cell therapies. Substrates presenting peptides, which are minimal fragments of extracellular matrix (ECM) proteins may be used for this purpose. In this work, we used polyelectrolyte multilayer films presenting two peptides derived from different muscle ECM proteins to target syndecan or/and integrin receptors. We showed that the presence of laminin-derived peptide to target syndecan-1 promotes lamellipodia formation, increases migration speed, directionality and cell proliferation but impaired myotube formation. The cellular effects of L2synd are under the control of Rac1 and Cdc42 activities and involved β1 integrin in contrast to RGD-containing peptide, which enabled adhesion via β3 integrins and muscle cell differentiation. Our

results

show

that

peptides

grafted

onto

multilayered

films

can

guide

the

proliferation/differentiation balance and reveal crosstalk between different adhesion receptors.

Introduction Skeletal muscle tissue engineering holds promise for the treatment of muscle diseases, for drug screening and for studying the functional effects of patient-specific mutations. In this context, controlling the proliferation and differentiation of muscle precursor cells, which are able to regenerate the injured muscle fibers1, may contribute to propose new therapies for cell amplification prior to in vivo engraftment. In muscle fibers, satellite cells reside between the sarcolemma and the basal lamina, a thin sheet of the extracellular matrix (ECM) proteins laminins and collagens

2,3

, which is connected to the fibronectin-rich reticular lamina4.


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Fibronectin may also be transiently expressed and localized to the basal lamina during muscle regeneration 5. It was shown recently that fibronectin is a critical component for the maintenance of the satellite cell pool 6. Laminins are already known to affect myoblast cell shape, migration, proliferation and differentiation, both in vitro and in vivo

7-10

. Being the predominant laminin α

chain expressed in skeletal muscle, the laminin-α2 chain (a component of laminin-211 (α2/β1/γ1) and laminin-221 (α2/β2/γ1) is involved in anchoring myofibers to the basement membrane, promoting muscle cell integrity and survival

11-13

. Genetic mutation of this protein results in

muscle dystrophy 14. Laminins and fibronectin have the ability to interact with the cellular receptors integrins and with syndecans. Both beta 1 (β1) integrin, a major laminin and fibronectin receptor, and beta 3 (β3) integrin, which interacts with fibronectin via the RGD tripeptide sequence, are playing an important role during skeletal muscle development and function receptors for cell adhesion and muscle function

19 20

15-18

. Syndecans are also key

. Whereas syndecan-3 and syndecan-4 are

implicated in the quiescence and activation of satellite cells

21

, syndecan-1 (SDC-1) is down-

regulated during myoblast differentiation 22. Its overexpression inhibited cell differentiation

23,24

while promoting cell proliferation 25. In addition, Adams et al. showed that SDC-1 was involved in C2C12 myoblast adhesion and directional migration on laminin-coated plates 26. Interestingly, syndecan-1 (SDC-1) is known to interact with the globular domain of laminin-α2 27. Altogether, these data demonstrate an important role of syndecan-1 in muscle regeneration likely through its binding to laminin-α2 as well as an important role for β1 and β3 integrin receptors via binding to fibronectin. Interestingly, previous studies have demonstrated a cooperation between syndecan-1 and β1 and β3 integrin receptors in the control of cell adhesion, although precise mechanisms of their crosstalk remain unclear

28-30

. Materials presenting peptide sequences are


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interesting in that they provide adhesive signals in a minimal system

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31

, thus simplifying the

complexity of full-length proteins. The IKVAV peptide derived from laminin has recently being studied in the context of myogenesis 32. The aim of this study was to activate SDC-1 and integrins in skeletal myoblasts and to study their individual or combined effects in cell fate. To this end, we created a SDC-1-, integrin- and SDC-1/integrin-targeting surface by using polypeptide layer-by-layer films with two different peptides targeting SDC-1 and integrins. The first peptide is a syndecan targeting peptide, named hereafter L2synd, derived from the LG4 globular domain of laminin-α2 shown to bind to SDC-1

27

33

, which has been

. The second peptide is a RGD peptide targeting β1 and β3 integrins,

which was recently shown to promote myogenesis34. We studied the effects of L2synd and RGD peptides alone or in combination on the sequential steps of myogenesis, including adhesion, migration, polarity and differentiation, using C2C12 myoblasts as a cellular model. We showed that adhesion to L2synd through SDC-1 also involved β1 integrins and resulted in cell polarization, proliferation and absence of myogenesis, while adhesion to RGD via β3 integrins enabled muscle cell differentiation.

Materials and methods PEM film buildup Poly(L-lysine) (PLL, P2636, Sigma) and poly(L-glutamic) acid (PGA, P-4886, Sigma) were dissolved at 0.5 mg/mL in a HEPES-NaCl buffer (150 mM NaCl and 20 mM HEPES at pH 7.4). For all experiments, films were deposited manually in 96-well plates starting with a first layer of poly(ethyleneimine) (PEI) at 5 mg/ml. To deposit the subsequent polyelectrolyte layers, 50 µL of


4

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the polyelectrolyte solution was deposited in each well and let adsorbed for 8 min before being rinsed twice for 30 sec and 5 min, respectively, with 100 µL of 150 mM NaCl (pH 6.5). This sequence was repeated until the buildup of a (PGA/PLL)6 film was achieved.

Polyelectrolyte film functionalization by adhesion peptides The laminin alpha2 chain derived peptide L2synd was chosen according to a published sequence that was shown to interact with the transmembrane proteoglycan syndecan-1 containing the RGD central sequence differentiation

34

36

27,33,35

. A peptide

was used as a control peptide promoting myogenic

. A scrambled L2synd peptide was designed using a tool available at the

following website: http://www.mimotopes.com. The RGD (CGPKGDRGDAGPKGA), L2synd (CGKNRLTIELEVRT) and scrambled L2synd (CGERRTETLVKNIL) peptides were purchased from GeneCust (Dudelange, Luxembourg). The protocol for peptide grafting was adapted from a previously published protocol

36

. Briefly,

the first step consisted in grafting maleimide groups onto PGA. To this end, 60 mg of PGA were dissolved in 3 mL of a solution containing 10 mM HEPES buffer (pH 6.5), 20 mg of 1-ethyl-3-(3dimethylamino-propyl) carbodiimide (EDC), and 3 mg of N-hydroxysulfo succinimide (S-NHS) in an

inert atmosphere (nitrogen gas) under magnetic stirring. Then, 24 mg of N-(2-aminoethyl) maleimide trifluoroacetate was added. The reaction was allowed to proceed at room temperature (RT) for 24 h. After removal of the byproducts via dialysis against water, the PGA–maleimide was freeze-dried. The average number of maleimide groups bound to PGA was equal to 16% (i.e. in average 16 maleimide groups every one hundred PGA repeat unit), as determined via 1H NMR analysis. In the second step, the PGA-maleimide was adsorbed on the top of PLL-ending films and reacted with 100 µL of 60 µg/mL L2synd peptide (dissolved in water), 50 µg/mL of


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RGD peptide or 1:1 (v/v) mix of both. The grafting was carried out overnight at RT under agitation and the films were then rinsed twice with MilliQ water to remove the unbound peptide. The final architecture of the film is thus PEI-(PGA/PLL)6 –PGA-peptide or PGA-maleimide in case no peptide is grafted.

Cell culture C2C12 cells (from ATCC, used at passages 5 to 15) were maintained in petri dishes in an incubator at 37° C and 5% CO2 and cultured in growth medium (GM) composed of Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (1:1; Gibco, Invitrogen, Cergy-Pontoise, France) supplemented with 10% fetal bovine serum (PAA Laboratories, Les Mureaux, France) containing 10 U/mL of penicillin G and 10 µg/mL of streptomycin (Gibco, Invitrogen, CergyPontoise, France). Cells were subcultured prior to reaching 60–70% confluence (approximately every 2 days). For all experiments, C2C12 cells were first allowed to adhere in a serum-free medium composed of DMEM/F12 1:1 and supplemented with antibiotics. After 4 h of adhesion, the cells were fixed or the medium was replaced by the GM, depending on the type of experiment (see below). Cell were differentiated in a differentiation medium (DM) composed of DMEM/F12 (1:1) supplemented with 2% horse serum (PAA Laboratories, Les Mureaux, France) and antibiotics.

Cell adhesion assays For cell adhesion tests, C2C12 cells were seeded at 15 000 cells/cm2 in 96-well plates and allowed to adhere in serum free medium for 4 h, then fixed in 3.7% formaldehyde. For the adhesion specificity test using scrambled peptide, the films were grafted with either original or


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scrambled L2synd peptide, and the cells were allowed to adhere on L2synd-grafted films for 4 h in serum-free medium. For adhesion assays in presence of Rac1 and Cdc42 inhibitors, the cells were allowed to adhere for 4h in absence or in presence of 50 µg/ml of NSC23766 (Rac1 inhibitor) or 10 µM of ML141 (Cdc42 inhibitor) in serum free medium. Dimethylsulfoxide (DMSO) was used as a control for ML141 which is dissolved in DMSO. For cell morphological measurements, experiments were done in duplicate and at least 50 cells from 2 different wells were analyzed in each experimental condition for each experiment.

Immunostaining Cells were first rinsed in phosphate buffer saline (PBS) and fixed in 3.7% formaldehyde for 30 min at RT before being permeabilized in 0.5% Triton X-100 for 4 min. After rinsing with PBS, samples were incubated for 1 h in 0.1 % BSA in TRIS-buffered saline (TBS, 50 mM TRIS at pH 7.4, 150 mM NaCl, 0.1% NaN3). F-actin was labeled with phalloidin-TRITC (1:800, Sigma) for 30 min. Cell nuclei were stained with Hoechst 33342 (Invitrogen) at 5 µg/ml for 10 min. After the incubations with the primary antibodies (diluted in 0.2% TBS-gelatin) for 30 min at RT, cells were washed 3 times in TBS and incubated for 30 min with the secondary antibodies. Mouse anti-β-tubulin antibody (1:200, Sigma), rabbit anti-FAK pY397 antibody (1:200, Invitrogen), rabbit anti-myogenin antibody (1:30, Tebu-Bio), mouse anti-troponin T (1:100, Sigma), mouse skeletal myosin (fast, 1:500, Sigma), mouse N-cadherin antibody (1:200, BD Biosciences), were used as primary antibodies. Alexa-Fluor 488, 594 and 647-conjugated antibodies (Invitrogen) were used at 1:1000 as secondary antibodies.


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For Syndecan-1 immunostaining, a protocol adapted from Garcia and coworkers was applied 37. Briefly, cells were rinsed in PBS and incubated in ice-cold 1 mM 3,3-dithiobis(sulfosuccinimidyl)propionate (DTSSP), Calbiochem-Merck, Merck Chemicals, Nottingham, UK) in PBS for 30 min. Unreacted cross-linker was quenched with 50 mM Tris for 15 min and bulk cellular components were extracted in 0.1% SDS in PBS. The slides were then blocked in BSA (0.1% in TBS). After this, Syndecan-1 was immunostained with 281-2 mouse anti Syndecan-1 (CD138) antibody conjugated to biotin (1:100, BD Pharmingen, BD Biosciences) and FITC-Streptavidin (1:500, BD Pharmingen, BD Biosciences) was used for visualization.

Image acquisition and quantification Images were taken with the Zeiss LSM 700 confocal microscope equipped with 20x and 63x objectives. To quantify cell morphology, cell area and aspect ratio (ratio of the length of the Major Axis to the Minor Axis of the particle’s fitted ellipse) images were analyzed using ImageJ (v 1.44p, NIH, Bethesda). 3D confocal images were used to measure the cell height.

Transfection by siRNA Cells were transfected with siRNA against β1 or β3 integrins (ON-TARGET plus SMARTpool, respectively Mouse ITGB1 and Mouse ITGB3, Thermo Scientific Dharmacon) as described in our previous work

34

, or against SDC-1, Rac1 and Cdc42 (ON-TARGET plus SMARTpool,

respectively Mouse Sdc1, Mouse Rac1 and Mouse Cdc42 siRNA, Thermo Scientific Dharmacon). All the siRNA were designed by Dharmacon.


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Cell proliferation, differentiation and migration Cell proliferation was quantified by the 5-ethynyl-2'-deoxyuridine (EdU) assay (Click-iT EdU Imaging Kit, Invitrogen). For quantification of cell proliferation in GM, cells were seeded at 15 000 cells/cm2 and allowed to adhere for 1 h in serum free medium, then cultured in GM for 24 h, 48 h or 72 h. For proliferation in DM, the cells were seeded at 30 000 cells/cm2 and allowed to adhere for 1 h in serum-free medium. Cells were then grown for 1 day in GM and then for 1 day in DM. At the chosen time point the cells were incubated with EdU diluted at 1/1000 in cell culture medium for 1 h at 37°C. The detection was carried out following the manufacturer instructions. At the end, nuclei were counter-stained with Hoechst 33432 (Invitrogen). The images of EdU and Hoechst-labeled nuclei were taken using a Zeiss LSM 700 confocal microscope. To calculate the ratio of proliferating cells, 10 images taken at 20x magnification were analysed. To follow cell migration, C2C12 cells were seeded at 15 000 cells/cm2 in 96-well plates. Images were taken every 10 min during 10 h. For analysis, at least 20 cells were tracked using ImageJ (v1.45d, NIH, Bethesda) using the chemotaxis tool to get quantitative measurement of cell velocity (µm/h) and directionality. The directionality is defined as the ratio of displacement d over total path length of the cell D. For differentiation assays, cells were seeded at 30 000 cells/cm2 and allowed to adhere for 1 h in serum-free medium. Cells were then grown for 1 day in GM and then switched to DM. The medium was changed every 3 days.


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Scanning Electron Microscopy To image the C2C12 cells on the films by scanning electron microscopy (SEM), the polyelectrolyte films were deposited on 1 x 1 cm silicon wafers. The cells were seeded at 15 000 cells/cm2 and allowed to adhere for 4 h before fixing them in 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2 for 30 min. After rinsing with 0.1 M cacodylate buffer pH 7.2, the samples were dehydrated as following: 10 min in 70% ethanol, 10 min in 95% ethanol and 10 min in 100% ethanol. After drying, the samples were imaged by SEM using a FEI-Quanta 250 SEM-FEG (1 kV).

Statistics Data are reported as means ± standard deviation. Box plot representations indicate the 25%, 50% and 75% of the values with lower and upper boundaries being 10 and 90%. The 5% and 95 % upper limits are indicated with closed circles. Experiments were reproduced in duplicate or triplicate. Statistical comparisons were performed using SigmaPlot Version 11.0 software and based on an analysis of variance (ANOVA) followed by an appropriate pairwise comparison or comparison versus control group procedure. (P < 0.05) was considered significant. Statistically different values are reported on the figures.

Results Myoblasts adopted an elongated morphology on L2synd-grafted films as compared to RGD-grafted films To activate syndecan and integrin receptors, we grafted the L2synd or RGD peptides to polyelectrolyte films as shown in Fig. 1A. The film buildup was followed in situ by quartz


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crystal microbalance with dissipation monitoring (QCM-D) (Fig. S1), which allowed to measure film thickness using the Voigt model 38 and to calculate the deposited mass of each peptide. Film thickness was ∼ 70 nm for a film made of 6 layer pairs, and the deposited amount of peptide was 480 ng/cm2. The (PLL/PGA) films can be considered as soft, as the Young’s modulus of the films deposited on a thick polyelectrolyte cushion has previously been measured at 51 ± 17 kPa 39

.

First, we studied the effect of L2synd peptide on C2C12 myoblast cell adhesion at early time (Fig. 1B and C, 4 h in serum free medium) in comparison to RGD that promoted cell adhesion and differentiation

34

. In addition, mixed L2synd/RGD-grafted films were prepared to study

possible interplay between the signals provided by the two peptides. (PLL/PGA) films without grafted peptides were used as negative controls. As few cells adhered onto these films, as previously shown

34

, these films were discarded from the subsequent experiments. Cells were

elongated on L2synd films while they were spread on RGD (Fig 1B). Cell area was about 1.5 to 2 fold lower on L2synd only films in comparison to RGD peptide alone or a mix of L2synd/RGD (Fig. 1C). Conversely, the aspect ratio was higher for films containing L2synd peptide as compared to RGD films (3.2 ± 1.8 versus 2.2 ± 0.9). Thus, each peptide can induce cell elongation (L2synd) or spreading (RGD) and can act in synergy to induce both cell spreading and elongation.

Myoblasts interaction with peptide-grafted films To further assess myoblast interaction with L2synd peptide, cell adhesion on L2synd-grafted films was compared to adhesion on a scramble peptide, which was grafted using the same protocol (Fig. 2A). The number of adherent cells was ∼ 4 times lower on scrambled peptide as


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compared to L2synd peptide. SDC-1 labeling (green) was detected at cell extremities close to actin (red) bundles on L2synd films (Fig. 2B, white arrows) but was not visible on RGD films. We next investigated the role of SDC-1 or integrins (β1 or β3) in myoblast adhesion to L2synd grafted films (Fig 2C,D) as these integrins are known to be important in C2C12 myoblasts {Lan, 2005 #3941}. The receptors were knock-down using specific silencing RNA (siRNA) and cell area and aspect ratio were quantified. Neither β1 nor β3 silencing had an effect on the cell spreading area while silencing SDC-1 lead to increased cell spreading. In contrast, cell rounding was notable after knock-down of SDC-1 and β1 integrins, with a 2-fold decrease in the aspect ratio (Fig. 2D). In contrast, β3 integrin knock-down has no effect on the cell response to SDC-1, while we previously showed that β3 integrins, but not β1, are involved in C2C12 myoblast adhesion to RGD peptide-functionalized surface 34 (Fig S2). These findings revealed that, on L2synd films, neither β1 nor β3 integrins are involved in cell spreading but β1 played a role in maintaining an elongated cell shape similarly to SDC-1. On RGD ending films, β3 has a major role in cell spreading while β1 has a minor role in cell shape.

L2synd and RGD films induce different adhesion mode and cytoskeletal organization Cell adhesion and cytoskeleton organization on L2synd films were analyzed in more details in comparison to RGD (Fig 3). On L2synd films, cells presented front-rear polarity morphology with bulgy nucleus and large and flat lamellipodium at the leading edge, while on RGD films, cells were well spread and flattened (Fig. 3A). The visualization of focal adhesions by labeling phosphorylated focal adhesion kinase (pFAK) (Fig. 3A), an important component of mature focal adhesions, revealed the presence small complexes on L2synd films at the leading edge of the lamelipodium in contrast to robust focal adhesions on RGD films. By SEM, a large flat


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lamellipodia containing filopodia that orthogonally encounter the leading edge was visible on L2synd films, whereas the extremities of actin stress fiber were visible at the cell periphery on RGD films (Fig. 3B). The 3D reconstruction of confocal images exhibited a bulgy nucleus on L2synd films with an “egg-on-a-plate” shape while they presented a flattened nucleus on RGD peptide (Fig. 3C). Vertical cross-sections also revealed a difference in cell height (Fig. 3D,D’), with respectively 9.6 ± 1.5 µm and 8.1 ± 0.9 µm on L2synd and RGD films. The cells seeded on mixed L2synd/RGD film spread similarly to RGD film. Because cooperation between actin cytoskeleton and microtubules is known to be crucial for cell polarization and lamellipodia formation

40

, we also observed and quantified the organization of

microtubules (Fig S3). On L2synd films, the angular distribution was rather homogenous without particular orientation. 33% of microtubules were oriented at < 30° and 30% at > 60°, whereas the corresponding percentage were of 63% and 15%, respectively on RGD films (Fig. S3B,C). In this later condition, microtubules were essentially radial microtubules, emerged from the centrosome and extended toward the cell periphery (Fig. S3A). Besides, on L2synd films, the centrosomes were localized between the nucleus and the lamellipodium at the front edge of the cell, indicating that, in addition to front-rear polarity, a centrosomal polarity was present. Thus, our data revealed completely different myoblast adhesion modes on L2synd versus RGD films, with a polarized lamellipodial morphology on L2synd films associated with noncentrosomal microtubules.


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Cell polarity and cytoskeletal organization on L2synd films is associated with a directionally persistent migration Cell polarity and movement are determined by cooperation between the actin and microtubule cytoskeletons

40

and the balance between MT minus-end capture and release from the

centrosome was shown to be critical for efficient cell migration

41

. To investigate if the

difference in cell morphology, polarity and microtubule orientation was associated with differences in cell migration, we recorded cell migration during 10 h on the different films (Fig. 4A) and quantified both cell migration speed (Fig. 4B) and directionality (Fig. 4C). Cell migration speed and directionality were significantly higher on L2synd as compared to RGD films. The migration velocity was also higher on L2synd/RGD films but directionality was close to RGD films. These results showed that cell polarity and cytoskeletal organization on L2synd films is associated with a faster and directionally persistent cell migration.

L2synd-induced cell proliferation and decrease of cell-cell adhesions We next assessed whether the L2synd peptide played a role on myoblast cell proliferation and cell-cell contacts (Fig 5). The percentage of cell proliferation in GM and DM was assessed by quantitative analysis of EdU staining (Fig 5A and S4). Cell proliferation was lower on the L2synd films in comparison to RGD after 24h in GM. After 1 day in DM, proliferation remained significantly higher than for the RGD and RGD/L2synd, suggesting the absence of cell cycle exit. In contrast, proliferation significantly dropped on RGD films. Cell-cell contact mediated by cadherin receptors is important in growth and maintenance of muscle cell interactions. To visualize the confluency of the cell layer, we labeled N-cadherin, which is involved in myogenesis and in contact-dependent growth inhibition 42,43 (Fig. 5B). Cells


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formed confluent layers on all three film types and the level of N-cadherin expression. However, the distribution of N-cadherin at cell-cell contacts was not identical, with a disorganized cell monolayer on L2synd films and no clear accumulation of N-cadherin at cell-cell contacts. In contrast, N-cadherin accumulated at cell-cell contacts (indicated with arrows) on RGD peptides. On L2synd/RGD films, the organization was intermediate. All together, these data indicated that the L2synd peptide impacts the organization of the cell layers and the localization of N-cadherin.

L2synd peptide-induced impairment of cell differentiation. The C2C12 myoblasts are a well-known model for the in vitro study of myogenic differentiation 44 45

, which is a highly regulated and temporal process. To monitor myogenic differentiation, we

quantified the expression of the transcription factor myogenin at early times of the differentiation process (Day 1) (Fig. 6A) and myoblast fusion during 5 days in DM (Fig. 6B,C). On L2synd films, the number of myogenin-positive cells was significantly lower than on RGD. In addition, cells formed few or no myotubes at all on L2synd films as they formed aggregates, which often detached after 2 days in DM, while cells on RGD films formed myotubes as previously observed 34

.

These data showed that L2synd-grafted films allowed myoblast proliferation but not myogenic differentiation, in contrast to the RGD peptide 34.

Rac1 and Cdc42 are involved in cell polarization, proliferation and impaired differentiation on L2synd films The Rho GTPases Rac1 and Cdc42 are implicated in the formation of focal complexes, cell polarization and directed cell movement46,47. To investigate if cell polarity and differentiation on


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L2synd films were related to Rac1 and Cdc42 signaling, we analyzed the cell spreading area, aspect ratio as well as markers of differentiation (Fig. 7). To this end, we used two complimentary approaches that consisted in knocking-down of CdC42 and Rac1 using silencing RNA or using inhibitors (NSC23766 for Rac1 and ML141 for Cdc42). On L2synd, nor the Rac1 neither Cdc42 inhibitors affected the cell spreading area, while inhibition of Rac1 using siRNA significantly decreased cell spreading (Fig. 7A, B). In contrast, the aspect ratio significantly decreased after inhibition or specific knock-down of Rac1 and Cdc42. This indicated that Rac1/Cdc42 pathway regulates front-rear cell polarization. Interestingly, such cell response to Rac1 and Cdc42 inhibition on L2synd-containing films was similar to the effect of β1 integrin knock-down (Fig. 2D,E). Both Rac1 and Cdc42 inhibition also increased the percentage of myogenin-positive nuclei on L2synd films (Fig. 7C). However, myoblast fusion was still impaired (Fig. 7D).

Discussion In this study, we analyzed the response of C2C12 myoblasts after targeting of SDC-1 and/or integrins, using two peptides from different ECM proteins: a 12 amino-acid long peptide derived from laminin-α2 chain

33

to target SDC-1 and a 15 amino-acid long peptide derived from

collagen to target integrins

34,36

. Our aim was to study the specific effect of each of these

peptides, individually or in combination, on the sequential events leading to myogenesis, especially on adhesion, migration, polarity and differentiation. The adhesion assays revealed that the L2synd peptide was responsible for elongated cell morphology, cell polarization with lamellipodia at the leading edge and increased migration (Fig 1-4), while the RGD-peptide induced cell spreading with no particular polarization. Interestingly,


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the cells seeded on mixed L2synd/RGD film presented both of these features. Using a minimal system consisting of a small peptide derived from a laminin chain, we were able to mimic the cellular effects (i.e. elongated phenotype, lamellipodia formation and directional migration) that were previously observed for myoblasts cultured on full length laminin-coated or anti-SDC-1 antibody-coated substrates

7,26,48

. Besides, the peptides can act simultaneously and induce the

combined effects of each of them. Our data are in agreement with previous studies on different cell types suggesting the additive effect of different peptides 29,33,49,50. Interestingly, directional cell migration is usually induced by environmental cues such as growth factor or chemokine gradients

51,52

. Our data demonstrated that an adhesive ligand presented

from the basal side could promote cell directional migration (Fig. 4) even in the absence of classic directionality-inducing factors. Microtubule growth and organization are linked to the formation of lamellipodia and cell polarization 40. The Rac1 and Cdc42 small G proteins, which are known to promote cell polarization and directional migration, were shown to be associated to microtubules through a downstream protein linker at the leading edge

53,54

. Here, polarized cell

morphology on L2synd films was associated with the presence of non-centrosomal microtubules, faster migration and increased directionality (Fig. 4 and SI3). Moreover, Rac1 and Cdc42 inhibition or silencing impaired the L2synd-dependent polarized morphology (Fig. 7). Indeed, both modifications of SDC-1 expression and altered Rac1 activity are observed in many cancer types, leading to increased cell proliferation, loss of polarity and increased motility 46. The role and function of non-centrosomal microtubules are not fully understood yet, and their formation process seems to be dependent on cell type

55

. We suggest that non-centrosomal microtubules

may be involved in lamellipodia formation on L2synd peptide and play a role in stabilizing and maintaining cell polarity.


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We found that cells on L2synd peptide remained in a proliferative state, that the localization of N-cadherin at cell-cell contact sites was perturbed and their differentiation impaired (Fig 5, 6). N-cadherin, a member of the Ca2+-dependent cell–cell adhesion molecule family

56

, plays a

major role in cell cycle exit and in the induction of the skeletal muscle differentiation program 20,42,57

. It was previously described that N-cadherin triggers signaling events that promote the

commitment to myogenesis through RhoA-positive and Rac1-negative regulation42. In C2C12 cells, Rac1 activation impaired the accumulation of N-cadherin at cell-cell contacts consequently affected contact inhibition

42,57

58

, which

. Moreover, L6 myoblasts cell cycle exit was

impaired upon expression of activated Rac1 and Cdc42

59,60

. Thus, Rac1 activation on L2synd

films may be the consequence of a direct signaling of syndecan cell receptors in response to L2synd peptide or of the perturbation of N-cadherin-mediated intercellular junction formation.

Synergistic control of cell adhesion by integrins and syndecans as well as a their cooperation to regulate Rac1 have already been described in several studies

28

, although the mechanisms and

precise roles of this interplay remain unclear. Here, we observed an effect of β1 integrin knockdown on the cell morphology induced by L2synd peptide, whereas β3 integrin knock-down strongly impacted the cell response to the RGD-ending film (Fig. 2 and SI2). Thus, our data support a crosstalk between β1 integrin and syndecans in myoblast response to L2synd-grafted films to regulate cell polarity and directionality, whereas the RGD peptide signals mostly via β3 integrins in myoblasts

34

to regulate myogenic differentiation. Indeed, SDC-1 was previously

shown to support α2β1-integrin mediated adhesion to collagen in Chinese hamster ovary cells

61

and two peptides (SDC-1/4 and α2β1 integrin binding peptides) derived from laminin-α1 were shown to synergistically accelerate adhesion of human dermal fibroblasts

29

. In contrast,


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adhesion of human submandibular gland cells to L2synd peptide was not affected by anti-β1 integrin antibodies 27. Such difference in different experimental studies may be explained by the use of different cell types and/or different peptide presentation modes (coating vs grafting). It is interesting to note the myoblast behavior on the L2synd peptide is reminiscent of the cell behavior on stiff polyelectrolyte films (i.e. chemically crosslinked polyelectrolyte films), where cells remained in a high proliferative state without myogenic differentiation

34

. On these stiff

films, adhesion involved β1 integrins in addition to β3 integrins and promoted ROCK activation. In Fig. 8, we summarize the cellular processes occurring following C2C12 myoblast interaction with the L2synd or RGD peptides presented by the soft films to activate integrin or syndecan receptors. Cell interaction with the L2synd peptide via syndecan involves a crosstalk with β1integrin and front-rear polarization, increases migration speed, directionality and prevents Ncadherin accumulation at cell-cell contacts. It also maintains the cells in proliferative state and inhibits myogenic differentiation, presumably via an activation of the Rac1/ Cdc42 signaling pathway. In contrast, cell interaction with RGD peptide induces integrin activation mostly via β3 integrins, leads to cell cycle exit and promotes myogenesis.

Conclusions This work revealed an important role of syndecan targeting peptides in the promotion of myoblast proliferation and impairment of myogenesis. We showed that L2synd peptide induced a cell elongation with lamellipodia at the leading edge and an increase in cell height while RGD promoted cell spreading and flattening. Different integrins were involved in the cell response: β1 being responsible for maintaining the elongated cell shape and polarized morphology on L2synd films while β3 had a major role in cell spreading on RGD ending films. We also revealed that


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L2synd presenting films increased migration speed, directionality and were favorable for cell proliferation, but were inappropriate for myogenic differentiation, in contrast to RGD presenting films, which enabled myotube formation. We showed that the polarized cell morphology on L2synd films was associated with the presence of non-centrosomal microtubules and required Rac1 and Cdc42 activities. Peptide-grafted surfaces enable to study the roles of adhesion receptors without changing their normal expression level by transfection or knock-down as is usually done in cell biology. Two or more ligands targeting different receptors may be combined in order to investigate their synergistic effects. Moreover, these biochemical functionalities may be further combined to additional stimuli, such as stiffness modulation or spatial patterning. These thin films could be further used to study the molecular mechanisms of cytoskeleton remodeling/cell migration and the underlying signaling pathways. We also envision their use to control the expansion of stem cells prior to their injection in vivo for the purpose of cell therapy.


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Supporting Information. The following files are available free of charge. Supplementary Information (PDF). Figure S1. Film growth followed by quartz crystal microbalance. Figure S2. Silencing RNA approach on RGD-ending films. Figure S3. Microtubule organization. Figure S4. EdU staining on peptidegrafted films.

Corresponding Author * Prof. C. Picart. Univ Grenoble Alpes, CNRS-UMR 5628, LMGP, 3 parvis L. Néel, F-38016 Grenoble, France E-mail: [email protected] Phone : (33)-(0)4 56 52 93 11 ; Fax : (33)-(0)4 56 52 93 01

Present Addresses †V. Gribova Institute of Condensed Matter and Nanosciences (Bio- and Soft Matter), Université catholique de Louvain, Croix du Sud 1, box L7.04.02, B-1348 Louvain-la-Neuve, Belgium E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


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Acknowledgments This research was supported by Region Rhônes-Alpes via a PhD fellowship to VG. CP wishes to thank the European Commission for support in the framework of FP7 via an ERC Starting grant 2010 (GA 259370, BIOMIM) and the Association Française contre les Myopathies (AFM, grant Microtiss 16530). The authors thank Manuel Théry for his precious advices, Quentin Lubart for help in the film characterization by quartz crystal microbalance, Marylin Vantard for providing antibodies, Claire Monge for useful comments on the manuscript, Anne Valat and Mélanie Arboléas for technical help with the qPCR experiments.

Abbreviations ANOVA, analysis of variance; BSA, bovine serum albumin; DM, differentiation medium; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; DTSSP, 3,3dithiobis-(sulfosuccinimidyl)propionate;

ECM,

extracellular

matrix;

EDC,

1-ethyl-3-(3-

dimethylamino-propyl) carbodiimide; EdU, 5-ethynyl-2'-deoxyuridine; GM, growth medium; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MT, microtubules; PBS, phosphate buffer saline; pFAK, phosphorylated focal adhesion kinase; PEI, poly(ethyleneimine); PGA, poly(L-glutamic) acid; PLL, poly(L-lysine); RT, room temperature; SDC-1, syndecan-1; SEM, scanning electron microscopy; S-NHS: N-hydroxysulfo succinimide; TBS: TRIS-Buffered Saline; TRIS, 2-amino-2-hydroxymethyl-propane-1,3-diol; TRITC, tetramethylrhodamine.


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FIGURE LEGEND Figure 1. Cell adhesion and morphology on peptide-grafted films. (A) Design and characterization of peptide-grafted films. 1- A polyelectrolyte multilayer film is built onto a substrate by alternating deposits of PLL and of PGA. 2- PGA-maleimide is added as last layer of the film. 3- Biochemical functionality is provided by adding peptides that covalently bind to the maleimide group. 4- C2C12 myoblasts are cultured on peptide-grafted films. (B) Actin (red) and nuclei (blue) staining of C2C12 cells to visualize cell adhesion and spreading after 4 h of culture on L2synd-, L2synd/RGD and RGD-grafted films. Scale bar: 50 µm. (C) Quantification of cell spreading area and aspect ratio (length L over width W). Data are from 3 independent experiments (150 cells for each condition). * p < 0.05.

Figure 2. Role of β chain integrins and SDC-1 in myoblast adhesion and spreading on L2synd-grafted films. (A) Adhesion on a scramble peptide-grafted film in comparison to L2synd; error bars correspond to SD. (B) Expression of Syndecan-1 (green) on L2synd- and RGD-grafted films; F-actin is labeled in red. Scale bar: 5 µm. (C) SDC-1, β1 and β3 integrins were knocked-down using specific siRNA. Actin labeling after 4 h of adhesion, scale bar 50 µm and (D) corresponding quantification of the cell area and aspect ratio (the mean value being set to 1 for the scramble siRNA). Data are pooled from 3 independent experiments (total of 150 cells analyzed for each condition). * p < 0.05 compared to control (scrambled siRNA).

Figure 3. Lamellipodia formation and cytoskeleton organization on peptide-presenting films. C2C12 myoblasts were seeded onto RGD- or L2synd-grafted films, fixed after 4 h and analyzed as described. (A) Focal adhesion and cytoskeleton staining: phosphorylated focal


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adhesion kinase (pFAK Y397, green), F-actin (red) and nuclei (blue). Scale bar: 10 µm. (B) Scanning electron microscopy images. Scale bar: 10 µm. (C) 3D confocal images of cells. Scale bar: 20 µm. (D) Confocal cross-sections showing β-tubulin (green), actin (red) and nuclei (blue). Scale bar: 10 µm. (D’) Quantification of cell height. * p < 0.05. Data from 20 cells.

Figure 4. Cell migration on peptide-grafted films. (A) Migration was measured for 10 h after 4 h of adhesion. Migration paths are plotted for approximately 40 cells, scale is given in µm. Quantification of (B) cell velocity, * p < 0.05 compared to L2synd and (C) directionality, defined as displacement d / total path length of the cell D; * p < 0.05 compared to L2synd. Data were collected from 3 independent experiments (65 cells analyzed in total per experimental condition).

Figure 5. Cell proliferation on peptide-grafted films. (A). (B) Percentage of EdU-positive nuclei after 24 h culture in GM and at Day 1 of differentiation (24 h in GM then 24 h in DM), Data are from 3 independent experiments * p < 0.05 compared to L2synd. (C) Staining of Ncadherin (green), F-actin (red) and nuclei (blue) after 72 h of culture in GM. Scale bar: 20 µm. Arrows indicate cell-cell contacts.

Figure 6. Myoblast differentiation on peptide grafted films (A) Quantification of the percentage of myogenin-positive nuclei at Day 1 in DM. Data are from three independent experiments; * p < 0.05. (B) Fluorescent labeling of troponin T (green), F-actin (red) and nuclei (blue) at Day 5 of differentiation Scale bar: 50 µm. (C) Higher magnification of skeletal myosin staining reveals myotube striation on RGD-grafted films. Scale bar: 20 µm.


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Figure 7. Involvement of Rac1 and Cdc42 in cell response to L2synd films. (A) Cell adhesion after 4 h of Rac1 inhibitor (NSC23766) or Cdc42 inhibitor (ML141) as compared to control (no inhibitor) and corresponding quantification of cell area and aspect ratio. * p < 0.05 compared to control (no inhibitor) (data are pooled from 2 independent experiments, 100 cells analyzed per condition). Scare bar 20 µm. (B) Knock-down of Rac1 and Cdc42 using siRNA approach and corresponding quantification of cell area and aspect ratio. * p < 0.05 compared to control (scrambled siRNA) (data are pooled from 3 independent experiments, 150 cells analyzed per condition). Scare bar 50 µm. (C) Quantification of the percentage of myogenin-positive nuclei in presence of Rac1 or Cdc42 inhibitors as compared to control at Day 1 in DM. Data are from 2 independent experiments; * p < 0.05 compared to control. (D) Fluorescent labeling of troponin T (green), F-actin (red) and nuclei (blue) at Day 5 of differentiation in presence of Rac1 or Cdc42 inhibitor as compared to control. Scale bar: 50 µm.

Figure 8. Schematic for C2C12 myoblast interaction with syndecan and integrin targeting peptides. The cell interaction with L2synd peptide induces SDC-1 activation and cell directionality while maintaining cell proliferation high. This activation involves the Rac1/ Cdc42 signalling pathway and is associated with β1-integrins while cell interaction with the RGD containing peptide preferentially targets β3-integrins, leading to inhibition of proliferation and myogenic commitment.


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(53) Fukata, M.; Watanabe, T.; Noritake, J.; Nakagawa, M.; Yamaga, M.; Kuroda, S.; Matsuura, Y.; Iwamatsu, A.; Perez, F.; Kaibuchi, K. Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170. Cell 2002, 109, 873-885. (54) Watanabe, T.; Wang, S.; Noritake, J.; Sato, K.; Fukata, M.; Takefuji, M.; Nakagawa, M.; Izumi, N.; Akiyama, T.; Kaibuchi, K. Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration. Dev Cell 2004, 7, 871883. (55) Bartolini, F.; Gundersen, G. G. Generation of noncentrosomal microtubule arrays. J Cell Sci 2006, 119, 4155-4163. (56) Angst, B. D.; Marcozzi, C.; Magee, A. I. The cadherin superfamily: diversity in form and function. J Cell Sci 2001, 114, 629-641. (57) Lassar, A. B.; Skapek, S. X.; Novitch, B. Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal. Curr Opin Cell Biol 1994, 6, 788-794. (58) Comunale, F.; Causeret, M.; Favard, C.; Cau, J.; Taulet, N.; Charrasse, S.; Gauthier-Rouviere, C. Rac1 and RhoA GTPases have antagonistic functions during N-cadherindependent cell-cell contact formation in C2C12 myoblasts. Biol Cell 2007, 99, 503-517. (59) Meriane, M.; Roux, P.; Primig, M.; Fort, P.; Gauthier-Rouviere, C. Critical activities of Rac1 and Cdc42Hs in skeletal myogenesis: antagonistic effects of JNK and p38 pathways. Mol Biol Cell 2000, 11, 2513-2528. (60) Meriane, M.; Charrasse, S.; Comunale, F.; Mery, A.; Fort, P.; Roux, P.; GauthierRouviere, C. Participation of small GTPases Rac1 and Cdc42Hs in myoblast transformation. Oncogene 2002, 21, 2901-2907. (61) Vuoriluoto, K.; Jokinen, J.; Kallio, K.; Salmivirta, M.; Heino, J.; Ivaska, J. Syndecan-1 supports integrin alpha2beta1-mediated adhesion to collagen. Exp Cell Res 2008, 314, 3369-3381.


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TOC Graphic


30

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Figure 1 1. Construction of (PLL/PGA)5-PLL film

B

2. Addition of PGA-maleimide

L2synd

C

4500 4000 2

3500

3. Grafting of peptides 4. Culture of C2C12 to maleimide group myoblasts

RGD

L2synd/RGD

* * *

10

* ** *

Aspect ratio

A

Cell area (µm )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3000 2500 2000 1500 1000 500

1

L2synd L2synd/ RGD RGD




Figure 2 2

A

Cell number (/mm )

200

*

B

150 100 50 0

scramble L2synd

Ctrl

C

Ctrl siRNA (Scr)

siRNA β1

siRNA β3

2

*

D 2

Aspect ratio

Relative cell area

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1

0

cr

Scr S

-1 C β11 β33 SDC-1 SD

**

siRNA SDC-1

*

1

0

1

ScrScr β1 1 β3 3 SDC-1 DC


S

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Figure 3

A

D L2synd

L2synd

RGD RGD

L2synd/RGD

10 µm

B

RGD L2synd

RGD

D’ 12

C

L2synd/ RGD L2synd

RGD

Cell height (µm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


*

*

10

8

6




Figure 4

A

40

*

30

C 1,0

Directionality

B Migration speed (µm/h)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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20

*

**

0,8 0,6 0,4

10 0,2 0

0,0




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Figure 5 80

Edu+ cells (%)

A

80

24H

60

60

* 40

40

20

20

B Actin + N-cadherin + Nuclei

DM1

*

*

0

0

N-cadherin

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




L2synd

L2synd/RGD

RGD

L2synd

L2synd/RGD

RGD



Figure 6

A

A’

Day 1, DM

4500

* ** * *

20

RGD/L2synd

RGD

2

L2synd

Myogenin-"+" cells (%)

4000

Cell area (µm )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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3500 15

3000 2500

10

2000 1500

5

1000 500

0


B L2synd

C L2synd/RGD

RGD


RGD

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Figure 7

*

1,5 1,5

Ctrl

NSC

ML

1,0 1,0

0,5 0,5

2,0

Aspect ratio

A

Relative cell area area Relative cell

0,5

Ctrl

siRac1

siCdc42

Relative cell Relative cellarea area

2,0

Ctrl

1,5 1,5 1,0 1,0 0,5 0,5

12

Myogenin + cells (%)

C

1,0

Ctrl SiRac1 SiCdc42

B

* *

1,5

0,0

0,0 0,0

Aspect Aspect ratio ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


*

10

*

0,0 0,0

D Ctrl

8

Ctrl

NSC

6 4 2 0

Ctrl

NSC

ML


NSC ML

SiRac1 SiCdc42

* *

1,5 1,5 1,0 1,0 0,5 0,5 0,0 0,0

ML

Ctrl

NSC ML


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8

L2synd peptide

RGD peptide C2C12 myoblasts

β1

β3

SDC-1

L2synd

RGD

Interaction invovling β1-integrins and SDC-1, Rac1 and Cdc42 activity

Interaction invovling β3-integrins

Cell spreading Cell elongation and polarity

Directional migration Cell proliferation

Cell differentiation


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Differentiation Balance ... - ACS Publications

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