Microplates-based rheometer for a single living cell

Desprat, Nicolas; Guiroy, Axel; Asnacios, Atef

doi:10.1063/1.2202921

Cited by 82 publications

(94 citation statements)

References 29 publications

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“…After a few seconds, the two microplates were simultaneously and smoothly lifted to 60 μm from the chamber's bottom to get the desired configuration of one cell adherent between two parallel plates. One of the plates was rigid, and the other could be used as a nanonewton force sensor (44). By using flexible microplates of different stiffness values, we were able to characterize the effect of rigidity on force generation up to a stiffness of about 200 nN/μm.…”

Section: Methodsmentioning

confidence: 89%

“…By using flexible microplates of different stiffness values, we were able to characterize the effect of rigidity on force generation up to a stiffness of about 200 nN/μm. To measure forces at an even higher stiffness, we used a flexible microplate of stiffness ' 10 nN/μm but controlled the plate-to-plate distance using a feedback loop, maintaining it constant regardless of cell force and plate deflection (14,44). Concurrently, we visualized cell spreading under bright-light illumination at an angle perpendicular to the plane defined by the main axis of the two microplates and analyzed the dynamics.…”

Section: Methodsmentioning

confidence: 99%

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Cells as liquid motors: Mechanosensitivity emerges from collective dynamics of actomyosin cortex

Etienne

Fouchard

Mitrossilis

et al. 2015

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

116

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Living cells adapt and respond actively to the mechanical properties of their environment. In addition to biochemical mechanotransduction, evidence exists for a myosin-dependent purely mechanical sensitivity to the stiffness of the surroundings at the scale of the whole cell. Using a minimal model of the dynamics of actomyosin cortex, we show that the interplay of myosin power strokes with the rapidly remodeling actin network results in a regulation of force and cell shape that adapts to the stiffness of the environment. Instantaneous changes of the environment stiffness are found to trigger an intrinsic mechanical response of the actomyosin cortex. Cortical retrograde flow resulting from actin polymerization at the edges is shown to be modulated by the stress resulting from myosin contractility, which in turn, regulates the cell length in a forcedependent manner. The model describes the maximum force that cells can exert and the maximum speed at which they can contract, which are measured experimentally. These limiting cases are found to be associated with energy dissipation phenomena, which are of the same nature as those taking place during the contraction of a whole muscle. This similarity explains the fact that single nonmuscle cell and wholemuscle contraction both follow a Hill-like force-velocity relationship. -5). This behavior is strongly dependent on the contractile activity of the actomyosin network (6-10). One of the cues driving the cell response to its environment is rigidity (11). Cells are able to sense not only the local rigidity of the material with which they are in contact (12) but also, the one associated with distant cell substrate contacts. This ability has been demonstrated by tracking the amount of extra force needed to achieve a given displacement of microplates between which the cell is placed (13, 14) (Fig. 1B), of an atomic force microscope (AFM) cantilever (15, 16) or elastic micropillars (17). This cell-scale rigidity sensing is totally dependent on myosin-II activity (13). A working model of the molecular mechanisms at play in the actomyosin cortex is available (18), where myosin contraction, actin treadmilling, and actin cross-linker turnover are the main ingredients. Phenomenological models (19, 20) of mechanosensing have been proposed but could not bridge the gap between the molecular microstructure and this cell-scale phenomenology. Here, we show that the collective dynamics of actin, actin cross-linkers, and myosin molecular motors are sufficient to explain cellscale rigidity sensing: depending on the tension that can be borne by the environment, there is a change of the fraction of myosin molecules which perform mechanical work that is effectively transmitted rather than dissipated. The model derivation is analogous to the one of rubber elasticity of transiently cross-linked networks (21), with the addition of active crosslinkers accounting for the myosin. It involves four parameters only: myosin contractile stress, speed of actin treadmilling, elastic modulus, and viscoela...

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Section: Methodsmentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

Cells as liquid motors: Mechanosensitivity emerges from collective dynamics of actomyosin cortex

Etienne

Fouchard

Mitrossilis

et al. 2015

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

116

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“…We let single, isolated C2.7 myoblast cells spread between two parallel glass plates of a custom-made microrheometer (23). One plate was rigid, the other was flexible and could be used as a nano-Newton force sensor.…”

Section: Resultsmentioning

confidence: 99%

“…To measure forces at higher stiffness values, we could not simply use flexible plates of higher stiffness because the plate deflections would have been too small for accurate force measurement. To overcome this difficulty, we controlled the position of a ≈10 nN/μm flexible plate such that the plate-to-plate distance was constant regardless of cell force and plate deflection (23), which was equivalent to an infinite plate stiffness. Under these conditions, we observed shape evolutions and force curves very similar to those obtained at stiffness of 60 and 176 nN/μm, confirming that 300 nN was the maximum force generated by these cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Single-cell response to stiffness exhibits muscle-like behavior

Mitrossilis

Fouchard

Guiroy

et al. 2009

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

210

254

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Living cells sense the rigidity of their environment and adapt their activity to it. In particular, cells cultured on elastic substrates align their shape and their traction forces along the direction of highest stiffness and preferably migrate towards stiffer regions. Although numerous studies investigated the role of adhesion complexes in rigidity sensing, less is known about the specific contribution of acto-myosin based contractility. Here we used a custom-made single-cell technique to measure the traction force as well as the speed of shortening of isolated myoblasts deflecting microplates of variable stiffness. The rate of force generation increased with increasing stiffness and followed a Hill force-velocity relationship. Hence, cell response to stiffness was similar to muscle adaptation to load, reflecting the force-dependent kinetics of myosin binding to actin. These results reveal an unexpected mechanism of rigidity sensing, whereby the contractile acto-myosin units themselves can act as sensors. This mechanism may translate anisotropy in substrate rigidity into anisotropy in cytoskeletal tension, and could thus coordinate local activity of adhesion complexes and guide cell migration along rigidity gradients.mechanosensing | adaptation to load | cell migration | cell spreading | cell mechanics A s part of their normal physiological functions, most cells in the organism need to respond to mechanical stimuli such as deformations, forces, and the geometry and stiffness of the extracellular matrix (1, 2). Aberrant mechanical responsiveness is often associated with severe diseases, including cardiovascular disorders, asthma, fibrotic diseases, or cancer metastasis.Since the early 1980s, several techniques have been developed to characterize the forces generated by living cells (3-5) and to investigate the effect of the mechanical properties of twodimensional (2D) substrates (6-8). It was shown that cells are able to sense and respond to the rigidity of their surroundings. For instance, cells cultured on elastic substrates with a rigidity gradient preferably locomote towards stiffer regions and align their shape, their cytoskeletal structures, and their traction forces along the direction of highest stiffness (9-11). Moreover, it has been demonstrated that matrix rigidity could direct stem cells' lineage specification (12).It is generally assumed that rigidity sensing is based on mechanochemical signal-transduction pathways. The search for the mechanosensing element has generated numerous plausible candidates (reviewed in ref.2). The most prominent of them is the focal adhesion complex (13,14). These molecular assemblies consist of numerous proteins that are associated with integrin adhesion receptors (15) and provide the pathway of force transmission from the cytoskeleton to the extracellular matrix (16). Adhesion of integrins to extracellular matrix proteins triggers the formation of focal adhesions, their connection to actin, and the contraction of the cytoskeleton by myosin II (17-19). On a soft substrat...

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“…The creep function of a single living cell is measured by means of a home-made stretching rheometer, which has been described in detail elsewhere [11,28]. This setup takes advantage of a simple uniaxial geometry, since the cell is stretched between two glass microplates, a rigid one and a flexible one [9].…”

Section: -Measurements Of the Creep Function J(t)mentioning

confidence: 99%

Power laws in microrheology experiments on living cells: Comparative analysis and modeling

et al. 2006

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We compare and synthesize the results of two microrheological experiments on the cytoskeleton of single cells. In the first one, the creep function J(t) of a cell stretched between two glass plates is measured after applying a constant force step. In the second one, a microbead specifically bound to transmembrane receptors is driven by an oscillating optical trap, and the viscoelastic coefficient G e (ω) is retrieved. Both J(t) and G e (ω) exhibit power law

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Microplates-based rheometer for a single living cell

Cited by 82 publications

References 29 publications

Cells as liquid motors: Mechanosensitivity emerges from collective dynamics of actomyosin cortex

Cells as liquid motors: Mechanosensitivity emerges from collective dynamics of actomyosin cortex

Single-cell response to stiffness exhibits muscle-like behavior

Power laws in microrheology experiments on living cells: Comparative analysis and modeling

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