Bulk and Micropatterned Conjugation of Extracellular Matrix Proteins to Characterized Polyacrylamide Substrates for Cell Mechanotransduction Assays

Damljanovic, Vesna; Lagerholm, B. Christoffer; Jacobson, Ken

doi:10.2144/000112026

Cited by 132 publications

(132 citation statements)

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“…To examine how spatial confinement of FAs resulted in migration bias, we used Traction Force Microscopy (TFM) to measure the direction of traction forces exerted by cells on a polyacrylamide gel surface functionalized with stripes of collagenIV to spatially confine FAs (23,24). Cells exerted traction stresses exclusively on collagenIV stripes at the cell periphery ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Fluorescence signal from the cells and the micropatterns was used to assess protein recruitment, cell-contour fluctuations at short time scales for measurement of lamellipodia activity, and cell position at long time scales for measurement of cell migration. TFM was performed as previously described (24) on polyacrylamide gel sheets functionalized with micropatterns of ECM (23).…”

Section: Methodsmentioning

confidence: 99%

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Regulation of cell motile behavior by crosstalk between cadherin- and integrin-mediated adhesions

Borghi¹,

Lowndes²,

Maruthamuthu

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

206

169

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During normal development and in disease, cohesive tissues undergo rearrangements that require integration of signals from cell adhesions to neighboring cells and to the extracellular matrix (ECM). How a range of cell behaviors is coordinated by these different adhesion complexes is unknown. To analyze epithelial cell motile behavior in response to combinations of cell-ECM and cell-cell adhesion cues, we took a reductionist approach at the single-cell scale by using unique, functionalized micropatterned surfaces comprising alternating stripes of ECM (collagenIV) and adjustable amounts of E-cadherin-Fc (EcadFc). On these surfaces, individual cells spatially segregated integrin-and cadherin-based complexes between collagenIV and EcadFc surfaces, respectively. Cell migration required collagenIV and did not occur on surfaces functionalized with only EcadFc. However, E-cadherin adhesion dampened lamellipodia activity on both collagenIV and EcadFc surfaces and biased the direction of cell migration without affecting the migration rate, all in an EcadFc concentration-dependent manner. Traction force microscopy showed that spatial confinement of integrin-based adhesions to collagenIV stripes induced anisotropic cell traction on collagenIV and migration directional bias. Selective depletion of different pools of αE-catenin, an E-cadherin and actin binding protein, identified a membrane-associated pool required for E-cadherin-mediated adhesion and down-regulation of lamellipodia activity and a cytosolic pool that down-regulated the migration rate in an E-cadherin adhesion-independent manner. These results demonstrate that there is crosstalk between E-cadherin-and integrin-based adhesion complexes and that E-cadherin regulates lamellipodia activity and cell migration directionality, but not cell migration rate.D uring development, cohesive tissues exhibit extensive rearrangements that range from en masse migration, such as in wound healing (1), to complex local cell rearrangements, such as cell intercalation (2). In extreme cases in development (3) and in diseases such as metastatic cancers (4), tissue cohesion is lost and single-cell migration enabled, which results in cells populating distant sites. These morphogenetic processes reveal the importance of a fine coregulation, or crosstalk, between tissue cohesion (cadherin-based cell-cell adhesion) and cell migration [integrinbased extracellular matrix (ECM) adhesion] in the maintenance of tissue integrity and function.Interest in the crosstalk between cell-cell adhesion and cell migration dates back to the pioneering studies of Abercrombie and Heaysman in the 1950s (5, 6) and even earlier (7). Abercrombie coined the term "contact inhibition" to describe how cell-cell interactions between fibroblasts initially inhibited and then redirected their migration. Whether cell-cell contact inhibition of cell migration results from cell-cell contact-dependent spatial redistribution or down-regulation of the cell migration machinery, or both remains unknown.A major component of inte...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Regulation of cell motile behavior by crosstalk between cadherin- and integrin-mediated adhesions

Borghi¹,

Lowndes²,

Maruthamuthu

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

206

169

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“…To pattern FN, the human FN (50 mg/mL; BD Bioscience) solution was added onto each stamp for 1 h and then blow-dried with nitrogen gas. To pattern collagen, collagen from rat tail (COLI, 200 mg/mL; Invitrogen) was oxidized with 4 mg/mL sodium periodate in 50 mM sodium acetate buffer for 30 min according to the method described before [25]. Afterward, the COLI solution was added onto stamps for 1 h followed by blowing dry with nitrogen gas.…”

Section: Stamp Fabrication and Microcontact Printingmentioning

confidence: 99%

Insights into the Role of Focal Adhesion Modulation in Myogenic Differentiation of Human Mesenchymal Stem Cells

Lui

Xiong

et al. 2013

Stem Cells and Development

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We report the establishment of a novel platform to induce myogenic differentiation of human mesenchymal stem cells (hMSCs) via focal adhesion (FA) modulation, giving insights into the role of FA on stem cell differentiation. Micropatterning of collagen type I on a polyacrylamide gel with a stiffness of 10.2 kPa efficiently modulated elongated FA. This elongated FA profile preferentially recruited the b 3 integrin cluster and induced specific myogenic differentiation at both transcription and translation levels with expression of myosin heavy chain and a-sarcomeric actin. This was initiated with elongation of FA complexes that triggered the RhoA downstream signaling toward a myogenic lineage commitment. This study also illustrates how one could partially control myogenic differentiation outcomes of similar-shaped hMSCs by modulating FA morphology and distribution. This technology increases our toolkit choice for controlled differentiation in muscle engineering.

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“…12 There have been at least two other published reports of extraction of PA gel elastic moduli from sphere indentation measurements. 7,13 These studies infer indentation depths by measuring z positions of embedded fluorescent fiduciary markers before and after removing the sphere from the surface, which can be difficult to accurately track and localize and require the use of sophisticated marker selection software. 13 The act of removing the sphere also sometimes damages the gel, complicating marker tracking.…”

mentioning

confidence: 99%

“…7,13 These studies infer indentation depths by measuring z positions of embedded fluorescent fiduciary markers before and after removing the sphere from the surface, which can be difficult to accurately track and localize and require the use of sophisticated marker selection software. 13 The act of removing the sphere also sometimes damages the gel, complicating marker tracking. These studies also do not offer generalized expressions that explicitly incorporate the buoyancy of the sphere, which may vary with media conditions and strongly influences measurement accuracy.…”

mentioning

confidence: 99%

High-throughput indentational elasticity measurements of hydrogel extracellular matrix substrates

D’Sa¹,

De‐Juan‐Pardo²,

Astiz³

et al. 2009

Applied Physics Letters

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Much interest surrounds the effect of extracellular matrix ͑ECM͒ elasticity on cell behavior. Here we present a rapid method for measuring the elasticity of synthetic ECM substrates based on indentation of the substrate with a ferromagnetic sphere and optical tracking of the resulting deformation. We find that this method yields order-of-magnitude agreement with atomic force microscopy elasticity measurements, but that the degree of this agreement depends strongly on sphere density and gel elasticity. In its regime of greatest accuracy, we envision that this method may be used for high-throughput characterization of ECM substrates in cell biological studies. © 2009 American Institute of Physics. ͓DOI: 10.1063/1.3197013͔Over the past two decades, cell biologists have begun to recognize that cells are exquisitely sensitive to mechanical inputs from their surroundings, including the extracellular matrix ͑ECM͒ and the solid-state biopolymeric scaffold to which cells adhere in tissues. It has become especially clear that the elasticity ͑Young's modulus͒ of the ECM can powerfully regulate many cellular properties, including structure, adhesion, migration, proliferation, and death. 1,2 For stem and progenitor cells, ECM elasticity can guide self-renewal and differentiation into specific lineages, 3,4 and for tumor cells, it can guide the initial stages of tissue dysplasia 5 as well as later proliferation and invasion. 6 This surge of interest in "cellular mechanobiology" has in turn given rise to material platforms that enable one to culture living cells on synthetic ECM substrates of precisely defined elasticity. Perhaps the most widely used of these platforms is based on synthesis and biofunctionalization of polyacrylamide ͑PA͒ hydrogels. 7,8 In this system, the elasticity is determined by the ratio of the monomer ͑acrylamide͒ to the crosslinker ͑bisacrylamide͒, and the cell adhesive function is derived by grafting full-length ECM proteins onto the surface at defined coverage. Meaningful interpretation of results from this system relies on accurate knowledge of the effective elasticity experienced by the cell at the hydrogel surface. Traditionally, this value has been inferred from bulk mechanical properties of the hydrogel, for example, as measured by macroscale extensional 8 or oscillatory 6 rheometry. While relatively high-throughput in practice, these measurements depend strongly on sample geometry, strain rate, and other experimental details. Moreover, cell-ECM interactions occur at interfaces and involve cellular mechanosensors and ECM proteins that are micro-and nanoscale in size, raising questions about the appropriateness of bulk approaches. Thus, atomic force micrsoscopy ͑AFM͒ has gained favor for measuring ECM elasticity; here the AFM tip is used to indent the ECM surface, and the force versus indentation depth profile is fit to an indentational model to extract the Young's modulus. However, AFM is skill-intensive, low-throughput, and requires expensive instrumentation. These limitations are particularly p...

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Bulk and Micropatterned Conjugation of Extracellular Matrix Proteins to Characterized Polyacrylamide Substrates for Cell Mechanotransduction Assays

Cited by 132 publications

References 13 publications

Regulation of cell motile behavior by crosstalk between cadherin- and integrin-mediated adhesions

Regulation of cell motile behavior by crosstalk between cadherin- and integrin-mediated adhesions

Insights into the Role of Focal Adhesion Modulation in Myogenic Differentiation of Human Mesenchymal Stem Cells

High-throughput indentational elasticity measurements of hydrogel extracellular matrix substrates

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