Study of Cellular Adhesion by Means of Micropillar Surface Topologies

2011 ◽  
Vol 409 ◽  
pp. 105-110 ◽  
Author(s):  
Francesca Boccafoschi ◽  
Marco Rasponi ◽  
Cecilia Mosca ◽  
Erica Bocchi ◽  
Simone Vesentini
Keyword(s):  
Focal Adhesion ◽  
Vascular Tissue ◽  
Focal Adhesions ◽  
Traction Force ◽  
Cellular Adhesion ◽  
Research Field ◽  
Traction Forces ◽  
Adhesion Sites ◽  
Substrate Rigidity ◽  
Open Question

It is well-known that cellular behavior can be guided by chemical signals and physical interactions at the cell-substrate interface. The patterns that cells encounter in their natural environment include nanometer-to-micrometer-sized topographies comprising extracellular matrix, proteins, and adjacent cells. Whether cells transduce substrate rigidity at the microscopic scale (for example, sensing the rigidity between adhesion sites) or the nanoscopic scale remains an open question. Here we report that micromolded elastomeric micropost arrays can decouple substrate rigidity from adhesive and surface properties. Arrays of poly (dimethylsiloxane) (PDMS) microposts from microfabricated silicon masters have been fabricated. To control substrate rigidity they present the same post heights but different surface area and spacing between posts. The main advantage of micropost arrays over other surface modification solutions (i.e. hydrogels) is that measured subcellular traction forces could be attributed directly to focal adhesions. This would allow to map traction forces to individual focal adhesions and spatially quantify subcellular distributions of focal-adhesion area, traction force and focal-adhesion stress. Moreover, different adhesion intracellular pathways could be used by the cells to differentiate toward a proliferative or a contractile cellular phenotype, for instance. This particular application is advantageous for vascular tissue engineering applications, where mimicking as close as possible the vessels dynamics should be a step forward in this research field.

scholarly journals Correlation of cellular traction forces and dissociation kinetics of adhesive protein zyxin revealed by multi-parametric live cell microscopy

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0251411
Author(s):  
Lorena Sigaut ◽  
Micaela Bianchi ◽  
Catalina von Bilderling ◽  
Lía Isabel Pietrasanta
Keyword(s):  
Extracellular Matrix ◽  
Residence Time ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Traction Force ◽  
Correlation Spectroscopy ◽  
Substrate Stiffness ◽  
Dissociation Kinetics ◽  
Traction Forces ◽  
Cellular Division

Cells exert traction forces on the extracellular matrix to which they are adhered through the formation of focal adhesions. Spatial-temporal regulation of traction forces is crucial in cell adhesion, migration, cellular division, and remodeling of the extracellular matrix. By cultivating cells on polyacrylamide hydrogels of different stiffness we were able to investigate the effects of substrate stiffness on the generation of cellular traction forces by Traction Force Microscopy (TFM), and characterize the molecular dynamics of the focal adhesion protein zyxin by Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). As the rigidity of the substrate increases, we observed an increment of both, cellular traction generation and zyxin residence time at the focal adhesions, while its diffusion would not be altered. Moreover, we found a positive correlation between the traction forces exerted by cells and the residence time of zyxin at the substrate elasticities studied. We found that this correlation persists at the subcellular level, even if there is no variation in substrate stiffness, revealing that focal adhesions that exert greater traction present longer residence time for zyxin, i.e., zyxin protein has less probability to dissociate from the focal adhesion.

Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK

1993 ◽  
Vol 13 (2) ◽  
pp. 785-791
Author(s):  
M D Schaller ◽  
C A Borgman ◽  
J T Parsons
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Protein Tyrosine Kinase ◽  
Signal Transduction Pathway ◽  
Focal Adhesions ◽  
Cellular Adhesion ◽  
Protein Tyrosine ◽  
Intracellular Signals ◽  
Embryo Cells

Integrins play a central role in cellular adhesion and anchorage of the cytoskeleton and participate in the generation of intracellular signals, including tyrosine phosphorylation. We have recently isolated a cDNA encoding a unique, focal adhesion-associated protein tyrosine kinase (FAK) that is a component of an integrin-mediated signal transduction pathway. Here we report the isolation of cDNAs encoding the C-terminal, noncatalytic domain of the FAK kinase, termed FRNK (FAK-related nonkinase). Both the FAK- and FRNK-encoded polypeptides, pp125FAK and p41/p43FRNK, are expressed in normal chicken embryo cells. pp125FAK and p41/p43FRNK were localized to focal adhesions, suggesting that pp125FAK is directed to the focal adhesions by sequences within its C-terminal domain. We also show that the fibronectin-dependent increase in tyrosine phosphorylation of pp125FAK is accompanied by a concomitant posttranslational modification of p41FRNK.

scholarly journals Force-independent interactions of talin and vinculin govern integrin-mediated mechanotransduction

2019 ◽  
Author(s):  
Paul Atherton ◽  
Franziska Lausecker ◽  
Alexandre Carisey ◽  
Andrew Gilmore ◽  
David Critchley ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Critical Role ◽  
Traction Force ◽  
Full Length ◽  
Mitochondrial Targeting ◽  
Integrin Activation ◽  
Traction Forces ◽  
The Matrix ◽  
Core Components ◽  
Dynamic Link

Talin, vinculin and paxillin are core components of the dynamic link between integrins and actomyosin. Here we study the mechanisms that mediate their activation and association using a mitochondrial-targeting assay, structure-based mutants, and advanced microscopy. As expected, full-length vinculin and talin are auto-inhibited and do not interact with each other in this state. Contrary to previous models that propose a critical role for forces driving talin-vinculin association, our data show that force-independent relief of auto-inhibition is sufficient to mediate their tight interaction. Interestingly, paxillin can bind to both talin and vinculin when either is inactive. Further experiments demonstrate that adhesions containing paxillin and vinculin can form without talin following integrin activation. However, these are largely deficient in exerting traction forces to the matrix. Our observations lead to a model whereby paxillin contributes to talin and vinculin recruitment into nascent adhesions. Activation of the talin-vinculin axis subsequently leads to the engagement with the traction force-machinery and focal adhesion maturation.

Osteoblast Adhesion on Tissue Engineering Scaffolds Made by Bio-Manufacturing Techniques

2005 ◽  
Author(s):  
T. Dutta Roy ◽  
J. J. Stone ◽  
W. Sun ◽  
E. H. Cho ◽  
S. J. Lockett ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Focal Adhesion ◽  
Cellular Response ◽  
Focal Adhesions ◽  
Cellular Adhesion ◽  
Tissue Engineering Scaffolds ◽  
3D Scaffolds ◽  
Imaging Results ◽  
Proliferation And Differentiation ◽  
Manufacturing Techniques

Scientific exploration into understanding and developing relationships between three-dimensional (3D) scaffolds prepared by rapid prototyping (RP) and cellular response has focused primarily on end results targeting osteoblast proliferation and differentiation. Here at the National Institute of Standards and Technology (NIST), we take a systems approach to developing relationships between material properties and quantitative biological responses. This study in particular focuses on the screening of parameters controlled by RP techniques and their ability to trigger signalling events leading to cell adhesion. This pioneering research in our group also characterizes the in vitro cell-material interactions of 2D films and 3D scaffolds. From there, one can postulate on contributory factors leading to cell migration, proliferation, and differentiation. In summary, we believe that the quantitative information from this fundamental investigation will enhance our knowledge of the interactions between cells and 3D material interfaces with respect to formation of focal adhesions. This work consists of two sections — the application of imaging techniques for 3D characterization of properties and culturing of osteoblasts for size and shape determination. This includes quantifying the number of focal adhesion sites. We are using 3D RP polycaprolactone (PCL) scaffolds as this surrogate model in which to compare 2D to 3D material performance and cell interactions. Using RP bio-manufacturing techniques to fabricate tissue engineering scaffolds allows for control of pore size, strut size, and layer thickness, therefore providing adjustable parameters to study which can potentially influence, or even dynamically modulate, cellular adhesion. Imaging results after culturing for 24 h showed differences in cell morphology and spreading relative to the different structures. The focal adhesion response also varied, indicating an apparent loss of organization in 3D scaffolds compared to 2D surfaces. See Results and Discussion for details.

Traction Forces During Cell Migration Predicted by the Multiphysics Model

2011 ◽  
Author(s):  
Sangyoon J. Han ◽  
Nathan J. Sniadecki
Keyword(s):  
Computational Models ◽  
Temporal Dynamics ◽  
Focal Adhesions ◽  
Traction Force ◽  
Leading Edge ◽  
Traction Forces ◽  
Spatial And Temporal Dynamics ◽  
Trailing Edges ◽  
Cytoskeletal Tension ◽  
Biochemical Activation

Cells rely on traction forces in order to crawl across a substrate. These traction forces come from dynamic changes in focal adhesions, cytoskeletal structures, and chemical and mechanical signals from the extracellular matrix. Several computational models have been developed that help explain the trajectory or accumulation of cells during migration, but little attention has been placed on traction forces during this process. Here, we investigated the spatial and temporal dynamics of traction forces by using a multiphysics model that describes the cycle of steps for a migrating cell on an array of posts. The migration cycle includes extension of the leading edge, formation of new adhesions at the front, contraction of the cytoskeleton, and the release of adhesions at the rear. In the model, an activation signal triggers the assembly of actin and myosin into a stress fiber, which generates a cytoskeletal tension in a manner similar to Hill’s muscle model. In addition, the role that adhesion dynamics has in regulating cytoskeletal tension has been added to the model. The multiphysics model was simulated in Matlab for 1-D simulations, and in Comsol for 2-D simulations. The model was able to predict the spatial distribution of traction forces observed with previous experiments in which large forces were seen at the leading and trailing edges. The large traction force at the trailing edge during the extension phase likely contributes to detachment of the focal adhesion by overcoming its adhesion strength with the post. Moreover, the model found that the mechanical work of a migrating cell underwent a cyclic relationship that rose with the formation of a new adhesion and fell with the release of an adhesion at its rear. We applied a third activation signal at the time of release and found it helped to maintain a more consistent level of work during migration. Therefore, the results from both our 1-D and 2-D migration simulations strongly suggest that cells use biochemical activation to supplement the loss in cytoskeletal tension upon adhesion release.

scholarly journals Substrate Resistance to Traction Forces Controls Fibroblast Polarization

2020 ◽  
Author(s):  
D. Missirlis ◽  
T. Haraszti ◽  
L. Heckmann ◽  
J. P. Spatz
Keyword(s):  
Mechanical Properties ◽  
Surface Layer ◽  
Focal Adhesion ◽  
Human Fibroblasts ◽  
Traction Force ◽  
Cell Polarization ◽  
Cell Physiology ◽  
Traction Forces ◽  
Cellular Processes ◽  
Cell Mechanosensing

AbstractThe mechanics of fibronectin-rich extracellular matrix regulate cell physiology in a number of diseases, prompting efforts to elucidate cell mechanosensing mechanisms at the molecular and cellular scale. Here, the use of fibronectin-functionalized silicone elastomers that exhibit considerable frequency-dependence in viscoelastic properties unveiled the presence of two cellular processes that respond discreetly to substrate mechanical properties. Soft elastomers supported efficient focal adhesion maturation and fibroblast spreading due to an apparent stiff surface layer. However, soft elastomers did not enable cytoskeletal and fibroblast polarization; elastomers with high cross-linking and low deformability were required for polarization. The underlying reason for this behavior was the inability of soft elastomeric substrates to resist traction forces, rather than a lack of sufficient traction force generation; accordingly, mild inhibition of actomyosin contractility rescued fibroblast polarization even on the softer elastomers. Our findings help reconcile previously proposed local and global models of cell mechanosensing by demonstrating the differential dependence of substrate mechanics on distinct cellular processes.Statement of SignificanceThe mechanisms cells employ to sense and respond to the mechanical properties of their surroundings remain incompletely understood. In this study we used a commercial silicone elastomer formulation to prepare compliant, fibronectin-coated substrates and investigate the adhesion and polarization of human fibroblasts. Our results suggest the existence of at least two discrete mechanosensing processes regulated at different time and length (force) scales. Focal adhesion assembly and cell spreading were promoted by a stiff surface layer independent from bulk viscoelasticity, whereas effective cell polarization required elevated elastomer stiffness, sufficient to resist applied cell traction. The results presented here have implications on the use of elastomeric substrates as biomaterials for mechanosensing studies or clinical applications.

scholarly journals Quantifying force transmission through fibroblasts: changes of traction forces under external shearing

2021 ◽  
Author(s):  
Steven Huth ◽  
Johannes W. Blumberg ◽  
Dimitri Probst ◽  
Jan Lammerding ◽  
Ulrich S. Schwarz ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Shear Force ◽  
Apical Cell ◽  
Traction Force ◽  
Traction Force Microscopy ◽  
Force Transmission ◽  
Traction Forces ◽  
Force Microscopy ◽  
Adhesion Sites

AbstractMammalian cells have evolved complex mechanical connections to their microenvironment, including focal adhesion clusters that physically connect the cytoskeleton and the extracellular matrix. This mechanical link is also part of the cellular machinery to transduce, sense and respond to external forces. Although methods to measure cell attachment and cellular traction forces are well established, these are not capable of quantifying force transmission through the cell body to adhesion sites. We here present a novel approach to quantify intracellular force transmission by combining microneedle shearing at the apical cell surface with traction force microscopy at the basal cell surface. The change of traction forces exerted by fibroblasts to underlying polyacrylamide substrates as a response to a known shear force exerted with a calibrated microneedle reveals that cells redistribute forces dynamically under external shearing and during sequential rupture of their adhesion sites. Our quantitative results demonstrate a transition from dipolar to monopolar traction patterns, an inhomogeneous distribution of the external shear force to the adhesion sites as well as dynamical changes in force loading prior to and after the rupture of single adhesion sites. Our strategy of combining traction force microscopy with external force application opens new perspectives for future studies of force transmission and mechanotransduction in cells.

scholarly journals The biochemical composition of the actomyosin network sets the magnitude of cellular traction forces

2021 ◽  
Vol 32 (18) ◽  
pp. 1737-1748
Author(s):  
Somanna Kollimada ◽  
Fabrice Senger ◽  
Timothée Vignaud ◽  
Manuel Théry ◽  
Laurent Blanchoin ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Biochemical Composition ◽  
Force Production ◽  
Traction Force ◽  
Traction Force Microscopy ◽  
Traction Forces ◽  
Force Microscopy ◽  
Cell Force

The endogenous content of proteins associated with force production and the resultant traction forces were quantified in the same cells using a new traction force-microscopy assay. Focal adhesion size correlated with force in stationary cells. Relative numbers of motors and cross-linkers per actin required an optimum to maximize cell force production.

scholarly journals Does cellular adaptation to force loading rate determine the biphasic vs monotonic response of actin retrograde flow with substrate rigidity?

2021 ◽  
Author(s):  
Partho Sakha De ◽  
Rumi De
Keyword(s):  
Cancer Progression ◽  
Loading Rate ◽  
Focal Adhesions ◽  
Traction Force ◽  
Leading Edge ◽  
Substrate Stiffness ◽  
Retrograde Flow ◽  
Force Loading ◽  
Substrate Rigidity ◽  
Cell Traction

AbstractThe transmission of cytoskeletal forces to the extracellular matrix through focal adhesion complexes is essential for a multitude of biological processes such as cell migration, differentiation, tissue development, cancer progression, among others. During migration, focal adhesions arrest the actin retrograde flow towards the cell interior, allowing the cell front to move forward. Here, we address a puzzling observation of the existence of two distinct phenomena: a biphasic relationship of the retrograde flow and cell traction force with increasing substrate rigidity, with maximum traction force and minimum retrograde flow velocity being present at an optimal substrate stiffness; in contrast, a monotonic relationship between them where the retrograde flow decreases and traction force increases with substrate stiffness. We propose a theoretical model for cell-matrix adhesions at the leading edge of a migrating cell, incorporating a novel approach in force loading rate sensitive binding and reinforcement of focal adhesions assembly and the subsequent force-induced slowing down of actin flow. Our model unravels both biphasic and monotonic responses of the retrograde flow and cell traction force with increasing substrate rigidity, owing to the cell’s ability to sense and adapt to the fast-growing forces. Moreover, we also elucidate how the viscoelastic properties of the substrate regulate these nonlinear responses and alter cellular behaviours.

scholarly journals A Focal Adhesion Filament Cross-correlation Kit for fast, automated segmentation and correlation of focal adhesions and actin stress fibers in cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0250749
Author(s):  
Lara Hauke ◽  
Shwetha Narasimhan ◽  
Andreas Primeßnig ◽  
Irina Kaverina ◽  
Florian Rehfeldt
Keyword(s):  
Focal Adhesion ◽  
Cross Correlation ◽  
Focal Adhesions ◽  
Force Production ◽  
Traction Force ◽  
Stress Fibers ◽  
Coupled Analysis ◽  
Matrix Remodeling ◽  
Fiber System ◽  
Actin Stress Fibers

Focal adhesions (FAs) and associated actin stress fibers (SFs) form a complex mechanical system that mediates bidirectional interactions between cells and their environment. This linked network is essential for mechanosensing, force production and force transduction, thus directly governing cellular processes like polarization, migration and extracellular matrix remodeling. We introduce a tool for fast and robust coupled analysis of both FAs and SFs named the Focal Adhesion Filament Cross-correlation Kit (FAFCK). Our software can detect and record location, axes lengths, area, orientation, and aspect ratio of focal adhesion structures as well as the location, length, width and orientation of actin stress fibers. This enables users to automate analysis of the correlation of FAs and SFs and study the stress fiber system in a higher degree, pivotal to accurately evaluate transmission of mechanocellular forces between a cell and its surroundings. The FAFCK is particularly suited for unbiased and systematic quantitative analysis of FAs and SFs necessary for novel approaches of traction force microscopy that uses the additional data from the cellular side to calculate the stress distribution in the substrate. For validation and comparison with other tools, we provide datasets of cells of varying quality that are labelled by a human expert. Datasets and FAFCK are freely available as open source under the GNU General Public License.

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