Development of a Novel Magnetic-Driven Micropillar Substrate for Mechanical Stimulation at Individual Focal Adhesions of Cells

2012 ◽  
Author(s):  
Kazuaki Nagayama ◽  
Yasuhiro Hamada ◽  
Takuya Inoue ◽  
Takeo Matsumoto
Keyword(s):  
Mechanical Stimulation ◽  
Essential Role ◽  
Flexible Substrate ◽  
Focal Adhesions ◽  
Traction Force ◽  
Cellular Functions ◽  
Traction Forces ◽  
Local Displacement ◽  
Cellular Mechanotransduction ◽  
Microfabricated Arrays

Traction force generated at focal adhesions (FAs) of cells plays an essential role in regulating various cellular functions. The force can be measured by plating cells on a flexible substrate to observe local displacement of the substrate caused by the forces (1–100 nN) [1]. Approaches employing this method include using microfabricated arrays of poly(dimethylsiloxane) (PDMS) micropillars that bend by cellular traction forces [2]. If you could apply forces to individual FAs independently by actively moving micropillars, it should become a powerful tool to delineate the cellular mechanotransduction mechanisms.

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 Black Dots: Microcontact-Printed, Reference-Free Traction Force Microscopy

2021 ◽  
Author(s):  
Kevin M Beussman ◽  
Molly Y Mollica ◽  
Andrea Leonard ◽  
Jeffrey Miles ◽  
John Hocter ◽  
...  
Keyword(s):  
Flexible Substrate ◽  
Traction Force ◽  
Human Platelets ◽  
High Yield ◽  
Traction Forces ◽  
Cooperative Effects ◽  
Force Microscopy ◽  
Interaction Terms ◽  
Spread Area ◽  
Insight Into

Measuring the traction forces produced by cells provides insight into their behavior and physiological function. Here, we developed a technique (dubbed 'black dots') that microcontact prints a fluorescent micropattern onto a flexible substrate to measure cellular traction forces without constraining cell shape or needing to detach the cells. To demonstrate our technique, we assessed human platelets, which can generate a large range of forces within a population. We find platelets that exert more force have more spread area, are more circular, and have more uniformly distributed F-actin filaments. As a result of the high yield of data obtainable by this technique, we were able to evaluate multivariate mixed effects models with interaction terms and conduct a clustering analysis to identify clusters within our data. These statistical techniques demonstrated a complex relationship between spread area, circularity, F-actin dispersion, and platelet force, including cooperative effects that significantly associate with platelet traction forces.

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 Protein kinase A activity is regulated by actomyosin contractility during cell migration and is required for durotaxis

2020 ◽  
Vol 31 (1) ◽  
pp. 45-58 ◽  
Author(s):  
Andrew J. McKenzie ◽  
Kathryn V. Svec ◽  
Tamara F. Williams ◽  
Alan K. Howe
Keyword(s):  
Cell Migration ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Mechanical Stimulation ◽  
Traction Forces ◽  
Cell Matrix ◽  
Actomyosin Contractility ◽  
Cellular Mechanotransduction ◽  
Kinase A ◽  
Migrating Cells

Here, we show that localized PKA activity in migrating cells is regulated by cell–matrix tension, correlates with cellular traction forces, is enhanced by acute mechanical stimulation, and is required for durotaxis. This establishes PKA as an effector of cellular mechanotransduction and as a regulator of mechanically guided cell migration.

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.

scholarly journals A Dynamic Stochastic Model of Frequency-Dependent Stress Fiber Alignment Induced by Cyclic Stretch

2009 ◽  
Author(s):  
Hui-Ju Hsu ◽  
Chin-Fu Lee ◽  
Roland Kaunas
Keyword(s):  
Cell Shape ◽  
Principal Direction ◽  
Focal Adhesions ◽  
Cyclic Stretch ◽  
Cell Alignment ◽  
Cellular Functions ◽  
Fiber Alignment ◽  
Dynamic Stochastic ◽  
Dynamic Structures ◽  
Point Level

Actin stress fibers (SFs) are bundles of actin filaments anchored at each end via focal adhesions. Myosin-generated contraction leads to the development of tension, which extends SFs beyond their unloaded lengths. In human aortic ECs, the level of SF extension is maintained at a set-point level of ∼1.10 (1). SFs are also dynamic structures and their continuous assembly and disassembly is critical to cellular functions involving changes in cell shape. Further, deformation of the extracellular matrix perturbs SF extension, leading to compensatory responses such as the gradual alignment of SFs perpendicular to the principal direction of cyclic stretch. The extent of cell alignment has been shown to depend on the pattern of matrix stretch; however, it is unclear how cells distinguish between different patterns of stretch to determine their unique responses.

scholarly journals Dissipation of contractile forces: the missing piece in cell mechanics

2017 ◽  
Vol 28 (14) ◽  
pp. 1825-1832 ◽  
Author(s):  
Laetitia Kurzawa ◽  
Benoit Vianay ◽  
Fabrice Senger ◽  
Timothée Vignaud ◽  
Laurent Blanchoin ◽  
...  
Keyword(s):  
Cell Mechanics ◽  
Force Production ◽  
Traction Force ◽  
Structural Rearrangements ◽  
Force Transmission ◽  
Traction Forces ◽  
Cell Traction Forces ◽  
Cell Traction ◽  
Contractile Forces ◽  
Fiber Contraction

Mechanical forces are key regulators of cell and tissue physiology. The basic molecular mechanism of fiber contraction by the sliding of actin filament upon myosin leading to conformational change has been known for decades. The regulation of force generation at the level of the cell, however, is still far from elucidated. Indeed, the magnitude of cell traction forces on the underlying extracellular matrix in culture is almost impossible to predict or experimentally control. The considerable variability in measurements of cell-traction forces indicates that they may not be the optimal readout to properly characterize cell contractile state and that a significant part of the contractile energy is not transferred to cell anchorage but instead is involved in actin network dynamics. Here we discuss the experimental, numerical, and biological parameters that may be responsible for the variability in traction force production. We argue that limiting these sources of variability and investigating the dissipation of mechanical work that occurs with structural rearrangements and the disengagement of force transmission is key for further understanding of cell mechanics.

scholarly journals Mechanical stimulation promotes enthesis injury repair by mobilizing Prx1+ cells via ciliary TGF-β signaling

2021 ◽  
Author(s):  
Han Xiao ◽  
Tao Zhang ◽  
Chang Jun Li ◽  
Yong Cao ◽  
Lin Feng Wang ◽  
...  
Keyword(s):  
Cell Migration ◽  
Rotator Cuff ◽  
Single Cell ◽  
Mechanical Stimulation ◽  
Essential Role ◽  
Primary Cilia ◽  
Underlying Mechanism ◽  
Rna Sequence ◽  
Injury Repair ◽  
Tgf Β1

Proper mechanical stimulation can improve rotator cuff enthsis injury repair. However, the underlying mechanism of mechanical stimulation promoting injury repair is still unknown. In this study, we found that Prx1+ cell was essential for murine rotator cuff enthesis development identified by single-cell RNA sequence and involved in the injury repair. Proper mechanical stimulation could promote the migration of Prx1+ cells to enhance enthesis injury repair. Meantime, TGF-β signaling and primary cilia played an essential role in mediating mechanical stimulation signaling transmission. Proper mechanical stimulation enhanced the release of active TGF-β1 to promote migration of Prx1+ cells. Inhibition of TGF-β signaling eliminated the stimulatory effect of mechanical stimulation on Prx1+ cell migration and enthesis injury repair. In addition, knockdown of Pallidin to inhibit TGF-βR2 translocation to the primary cilia or deletion of IFT88 in Prx1+ cells also restrained the mechanics-induced Prx1+ cells migration. These findings suggested that mechanical stimulation could increase the release of active TGF-β1 and enhance the mobilization of Prx1+ cells to promote enthesis injury repair via ciliary TGF-β signaling.

scholarly journals RAB6 and microtubules restrict protein secretion to focal adhesions

2019 ◽  
Vol 218 (7) ◽  
pp. 2215-2231 ◽  
Author(s):  
Lou Fourriere ◽  
Amal Kasri ◽  
Nelly Gareil ◽  
Sabine Bardin ◽  
Hugo Bousquet ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Protein Secretion ◽  
Golgi Complex ◽  
Essential Role ◽  
Focal Adhesions ◽  
Secreted Proteins ◽  
Cell Compartments ◽  
Secretion Assay

To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.

scholarly journals Cell prestress. II. Contribution of microtubules

2002 ◽  
Vol 282 (3) ◽  
pp. C617-C624 ◽  
Author(s):  
Dimitrije Stamenović ◽  
Srboljub M. Mijailovich ◽  
Iva Marija Tolić-Nørrelykke ◽  
Jianxin Chen ◽  
Ning Wang
Keyword(s):  
Flexible Substrate ◽  
Traction Force ◽  
Airway Smooth Muscle Cells ◽  
Force Microscopy ◽  
Intracellular Ca ◽  
Post Buckling ◽  
Contractile Stress ◽  
Energetic Analysis ◽  
Work Done ◽  
Tensegrity Model

The tensegrity model hypothesizes that cytoskeleton-based microtubules (MTs) carry compression as they balance a portion of cell contractile stress. To test this hypothesis, we used traction force microscopy to measure traction at the interface of adhering human airway smooth muscle cells and a flexible polyacrylamide gel substrate. The prediction is that if MTs balance a portion of contractile stress, then, upon their disruption, the portion of stress balanced by MTs would shift to the substrate, thereby causing an increase in traction. Measurements were done first in maximally activated cells (10 μM histamine) and then again after MTs had been disrupted (1 μM colchicine). We found that after disruption of MTs, traction increased on average by ∼13%. Because in activated cells colchicine induced neither an increase in intracellular Ca2+ nor an increase in myosin light chain phosphorylation as shown previously, we concluded that the observed increase in traction was a result of load shift from MTs to the substrate. In addition, energy stored in the flexible substrate was calculated as work done by traction on the deformation of the substrate. This result was then utilized in an energetic analysis. We assumed that cytoskeleton-based MTs are slender elastic rods supported laterally by intermediate filaments and that MTs buckle as the cell contracts. Using the post-buckling equilibrium theory of Euler struts, we found that energy stored during buckling of MTs was quantitatively consistent with the measured increase in substrate energy after disruption of MTs. This is further evidence supporting the idea that MTs are intracellular compression-bearing elements.

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