Substrate engagement of integrins α5 β1 and αv β3 is necessary, but not sufficient, for high directional persistence in migration on fibronectin

The interplay between specific integrin-mediated matrix adhesion and directional persistence in cell migration is not well understood. Here, we characterized fibroblast adhesion and migration on the extracellular matrix glycoproteins fibronectin and vitronectin, focusing on the role of α5 β1 and αv β3 integrins. Fibroblasts manifested high directional persistence in migration on fibronectin-, but not vitronectin-coated substrates, in a ligand density-dependent manner. Fibronectin stimulated α5 β1-dependent organization of the actin cytoskeleton into oriented, ventral stress fibers, and assembly of dynamic, polarized protrusions, characterized as regions free of stress fibers and rich in nascent adhesions at their edge. Such protrusions correlated with persistent, local leading edge advancement, but were not sufficient, nor necessary for directional migration over longer times. Selective blocking of αv β3 or α5 β1 integrins using small molecule integrin antagonists reduced directional persistence on fibronectin, indicating integrin cooperativity in maintaining directionality. On the other hand, patterned substrates, designed to selectively engage either integrin, or their combination, were not sufficient to establish directional migration. Overall, our study demonstrates adhesive coating-dependent regulation of directional persistence in fibroblast migration and challenges the generality of the previously suggested role of β1 and β3 integrins in directional migration.Instituto de Investigaciones Fisicoquímicas Teóricas y AplicadasConsejo Nacional de Investigaciones Científicas y Técnica

Substrate engagement of integrins α5 β1 and αv β3 is necessary, but not sufficient, for high directional persistence in migration on fibronectin

The interplay between specific integrin-mediated matrix adhesion and directional persistence in cell migration is not well understood. Here, we characterized fibroblast adhesion and migration on the extracellular matrix glycoproteins fibronectin and vitronectin, focusing on the role of α5 β1 and αv β3 integrins. Fibroblasts manifested high directional persistence in migration on fibronectin-, but not vitronectin-coated substrates, in a ligand density-dependent manner. Fibronectin stimulated α5 β1-dependent organization of the actin cytoskeleton into oriented, ventral stress fibers, and assembly of dynamic, polarized protrusions, characterized as regions free of stress fibers and rich in nascent adhesions at their edge. Such protrusions correlated with persistent, local leading edge advancement, but were not sufficient, nor necessary for directional migration over longer times. Selective blocking of αv β3 or α5 β1 integrins using small molecule integrin antagonists reduced directional persistence on fibronectin, indicating integrin cooperativity in maintaining directionality. On the other hand, patterned substrates, designed to selectively engage either integrin, or their combination, were not sufficient to establish directional migration. Overall, our study demonstrates adhesive coating-dependent regulation of directional persistence in fibroblast migration and challenges the generality of the previously suggested role of β1 and β3 integrins in directional migration.Instituto de Investigaciones Fisicoquímicas Teóricas y AplicadasConsejo Nacional de Investigaciones Científicas y Técnica

The Effect of Substrate Elasticity and Actomyosin Contractility on Different Forms of Endocytosis

<div><p>Substrate mechanical properties have emerged as potent determinants of cell functions and fate. We here tested the hypothesis that different forms of endocytosis are regulated by the elasticity of the synthetic hydrogels cells are cultured on. Towards this objective, we quantified cell-associated fluorescence of the established endocytosis markers transferrin (Tf) and cholera toxin subunit B (CTb) using a flow-cytometry based protocol, and imaged marker internalization using microscopy techniques. Our results demonstrated that clathrin-mediated endocytosis of Tf following a 10-minute incubation with a fibroblast cell line was lower on the softer substrates studied (5 kPa) compared to those with elasticities of 40 and 85 kPa. This effect was cancelled after 1-hour incubation revealing that intracellular accumulation of Tf at this time point did not depend on substrate elasticity. Lipid-raft mediated endocytosis of CTb, on the other hand, was not affected by substrate elasticity in the studied range of time and substrate elasticity. The use of pharmacologic contractility inhibitors revealed inhibition of endocytosis for both Tf and CTb after a 10-minute incubation and a dissimilar effect after 1 hour depending on the inhibitor type. Further, the internalization of fluorescent NPs, used as model drug delivery systems, showed a dependence on substrate elasticity, while transfection efficiency was unaffected by it. Finally, an independence on substrate elasticity of Tf and CTb association with HeLa cells indicated that there are cell-type differences in this respect. Overall, our results suggest that clathrin-mediated but not lipid-raft mediated endocytosis is potentially influenced by substrate mechanics at the cellular level, while intracellular trafficking and accumulation show a more complex dependence. Our findings are discussed in the context of previous work on how substrate mechanics affect the fundamental process of endocytosis and highlight important considerations for future studies.</p></div

Association of latex nanoparticles but not of high MW dextran with REF52 cells depends on substrate elasticity.

<p>Normalized MFI values of REF52 cells incubated for 1(A) dextran (MW = 70,000) or (B) carboxylate-modified, polystyrene nanoparticles, 100 nm in diameter, as a function of substrate elasticity. Dot plots and mean values are presented with each dot representing an independent experiment.</p

Association of Tf and CTb with REF52 cells is not correlated with cell size.

<p>(A) Different sized polystyrene beads were used to derive a linear relationship between bead diameter and the FSC detector signal. (B, C) Typical dot plots and correlation coefficients (Pearson's) for association of Tf (B) or CTb (C) and apparent cell diameter of live cells. (D) Results of Pearson's correlation coefficients for 5 different flow cytometry analyses are presented as dot plots and mean values. The low values (<0.3) indicate very low to no correlation between cell size and amounts of Tf or CTb associated with cells.</p

Substrate elasticity significantly affects association of Tf with REF52 cells after a 10-minute but not 1-hour incubation but does not have a significant effect on CTb association.

<p>(A) Schematic representation of experimental setup used to quantify cell association of fluorescent markers. Cells on PEG hydrogels of variable elasticity were incubated for a specified duration with fluorescent markers, washed to remove non-associated markers, detached using trypsin and analyzed using flow cytometry. Histograms of cell-associated fluorescence were acquired after gating live cells. (B) Histograms of a typical experiment showing efficient internalization of Tf by cells on hydrogels compared to negative controls and exhibiting a narrow Gaussian distribution. Tf association with REF52 cells was similar on soft, intermediate and stiff substrates after 1 hour incubation (C) but showed a significant lower normalized MFI on softer gels at the shorter time point of 10 minutes (D). Dot plots and mean values are presented, with each dot representing an independent experiment. (E) Epifluorescence microscopy images of REF52 cells on substrates with intermediate stiffness incubated 1 hour with Alexa Fluor 568-labeled Tf and stained for plasma membrane (WGA) confirmed that uptake of Tf by REF52 cells was uniform among the cell population and further showed that Tf localized in a perinuclear region of the cells. Scale bar: 100 µm (F) Histograms of a typical experiment of CTb association with REF52 cells showing a broad distribution of intensity value/cell. Normalized MFI values of CTb association were similar on soft, intermediate and stiff substrates after both 1 hour (G) or 10 minute (H) incubation with CTb. Dot plots and mean values are presented with each dot representing an independent experiment. (I) Epifluorescence microscopy images of REF52 cells on substrates with intermediate stiffness incubated 1 hour with Alexa Fluor 568-labeled CTb and stained for plasma membrane (WGA) showed a heterogeneous population of cells in respect to CTb internalization. Scale bar: 100 µm.</p

Inhibition of actomyosin contractility has a major effect on cell morphology and F-actin cytoskeleton but only a moderate, time-dependent influence on Tf and CTb association.

<p>(A) Epifluorescence microscopy images of REF52 cells 6 hours after seeding on FN-coated glass and stained for plasma membrane (WGA) and F-actin cytoskeleton (Phalloidin-TRITC). Cells were treated for 1 hour with 10 µM Y27632 or 50 µM blebbistatin, or left untreated as controls. Scale bars: 10 µm. Normalized MFI values for Tf (B) or CTb (C) association in presence or absence of 10 µΜ Y27632 or Blebbistatin, after a 10-minute or 1-hour incubation. Dot plots and mean values are presented. Normalized FSC (D) and SSC (E) intensity values of REF52 cells in presence or absence of 10 µΜ Y27632 or Blebbistatin as determined by flow cytometry analysis. Dot plots and mean values are presented with each dot representing an independent experiment.</p

In vitro uptake of amphiphilic, hydrogel nanoparticles by J774A.1 cells

We here report improved synthesis and in vitro interactions of amphiphilic hydrogel nanoparticles with the macrophage cell line J774A.1. Nanoparticles comprising dispersed hydrophobic nanodomains of poly(propylene glycol) within a continuous phase of hydrophilic poly (ethylene glycol) (PEG) were prepared via inverse emulsion crosslinking polymerization, using acrylated PEG and Pluronic" F127 as macromonomer blocks. Functionality and fluorescent labeling were achieved through incorporation of reactive comonomers and a posteriori reaction with fluorescein, respectively. When introduced to a static cell culture of adhered J774A.1 macrophages, the cells internalized these hydrogel nanoparticles in a dose- and time-dependent manner through clathrin-mediated and other pathways. Amphiphilic nanoparticle uptake was however dramatically lower than that of a model system (Fluospheres (R)) and similar to PEG-coated colloids reported in the literature, which are considered "stealth." Our findings support the potential of the nanoparticles presented here as long-circulating drug carriers. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res 93A: 1557-1565, 201

Combined Effects of PEG Hydrogel Elasticity and Cell-Adhesive Coating on Fibroblast Adhesion and Persistent Migration

The development and use of synthetic, cross-linked, macromolecular substrates with tunable elasticity has been instrumental in revealing the mechanisms by which cells sense and respond to their mechanical microenvironment. We here describe a hydrogel based on radical-free, cross-linked poly­(ethylene glycol) to study the effects of both substrate elasticity and type of adhesive coating on fibroblast adhesion and migration. Hydrogel elasticity was controlled through the structure and concentration of branched precursors, which efficiently react via Michael-type addition to produce the polymer network. We found that cell spreading and focal adhesion characteristics are dependent on elasticity for all types of coatings (RGD peptide, fibronectin, vitronectin), albeit with significant differences in magnitude. Importantly, fibroblasts migrated slower but more persistently on stiffer hydrogels, with the effects being more pronounced on fibronectin-coated substrates. Therefore, our results validate the hydrogels presented in this study as suitable for future mechanosensing studies and indicate that cell adhesion, polarity, and associated migration persistence are tuned by substrate elasticity and biochemical properties