As stiffness of the extracellular matrix increases, the mechanical properties of the actin cytoskeleton shifts from fluid to solid.
These findings were published in Nature Communications in 2015.
Gupta M, Sarangi BR, Deschamps J, Nematbakhsh Y, Callan-Jones A, Margadant F, Mège RM, Lim CT, Voituriez R, Ladoux B. (2015) Adaptive rheology and ordering of cell cytoskeleton govern matrix rigidity sensing. Nature Communications, 6:7525. doi: 10.1038/ncomms8525
More information on the Ladoux Lab.
Figure: As substrate stiffness increases, the arrangement of actin filaments progressively changes, from an orthoradial pattern around the nucleus to a parallel arrangement of stress fibers.
This work shows the change in rheological properties of actin from liquid to solid with increasing substrate stiffness. The researchers attribute the viscoelastic transition to the dynamics of Focal Adhesions (FAs) on soft and stiff substrates. On soft substrates, cells form smaller FAs that are short lived and therefore cells experience low substrate friction. In contrast to this, on harder substrates, FAs form stable links with the actin generating higher tension and traction forces.
Understanding the basics
The actin cytoskeleton is physically connected with the cell exterior, e.g., ECM or other cells, through a multiprotein complex known as the focal adhesion complex. Interactions between cell surface molecules, e.g., integrins, and the actin cytoskeleton are bidirectional, with the focal adhesion complex forming the link between them. Actin filaments and their associated focal adhesion complexes act as information handling machines or mechanosensors: they convert both the strength of the adhesion and the tensile forces along the linked network of actin filaments (and associated proteins) into biochemical signals that control actin extension and cell migration. Focal adhesions are subject to continuous pulling forces and the force differences due to external stress or the chemical nature, rigidity, and topography of the exterior components will influence the assembly and organization of the actin cytoskeleton. The orientation of individual actin filaments in the cytoskeleton is a force-driven evolutionary process that contributes to the elastic behavior of the network and influences whether a filament will deform by compression, bending or extension. Cross-linked actin networks initially become more elastic under low force as a result of filament resistance to the direction of compression. As the force increases, individual filaments inherently resist being compressed and/or cross-linking proteins become more extended, which causes the cytoskeleton network to become more rigid.
Cells exert traction forces on the ECM and generate tension at focal adhesions through actin stress fibers, which are higher-order structures in the cytoplasm that consist of parallel contractile bundles of actin and myosin filaments. Stress fibers are linked at their ends to the ECM through focal adhesion complexes. Cell tension is generated along the actin filaments by the movement of myosin II motor proteins along the filaments. Forces produced by the contraction of stress fibers not only helps the cell body to translocate during migration, but they also serve as a vital “inside-out” feedback system to regulate actin filament initiation, cell growth and motility, and formation/maturation of focal adhesion complexes. Forces produced by stress fibers also stabilize the cell structure and contribute to establishing the cell polarity and they help determine the cell fate.
To study the influence of substrate rigidity on cell function, cells can be grown and observed on microfabricated pillars of varying stiffness. Rigidity of the pillars is controlled by varying their dimensions. Shorter pillars are stiffer and taller pillars are softer. The tips of the micropillars are often functionalized with ECM proteins to promote cell adhesion. Forces exerted by the cell can also be determined by using an array of elastomeric micropillars. In these experiments the cells adhere onto the ECM-coated tips and deflect the pillars as they move. This deflection can be measured as local force (F), determined by the equation shown in the figure where r, L, E and delta x are the radius, the length, the Young’s modulus, and the deflection of the pillar, respectively.
The Study in Detail
- On soft micropillar substrates, cells displayed a non-polarized circular shape with actin filaments arranged in an orthoradial pattern around the nucleus. Focal adhesions (FAs) were smaller compared with those on stiffer substrates. Consequently cells experience low substrate friction on soft substrates.
- As substrate stiffness increased, cells became more elongated and developed stress fibers. Cells on the stiffest substrate had well-developed stress fibers aligned in the same direction. On moderately stiff substrates microdomains of ordered actin filaments were observed.
- On soft substrates actin moved radially from the cell periphery to the nucleus and displayed a fluid like behavior as compared to stiff substrates where the stress fibers showed no movement and displayed solid-like behavior.
- The change in organization of actin from disorganized filaments to organized stress fibers is characterized by a transition from isotropic to nematic order, akin to liquid crystals, leading to higher order and higher tension on stiffer substrates.
- Microfabricated pillar substrates were used to provide substrates of varying stiffness by changing the height of the micropillars. The smaller the pillars, the stiffer the substrate becomes. The micropillars also allow for measurement of cellular traction forces.
- Atomic Force Microscopy (AFM) was employed to perform creep tests on cells adhered to the soft and stiff substrates in order to measure fluidity of cell cytoskeleton on these substrates.
- The findings on rigidity sensing may be extended to other mechanosensing processes. For instance, the radial flow of actin favouring circular cellular shapes and low traction forces on soft substrates have been observed in neuronal growth cones in vitro. Neurons in fact evolve in soft environments.
- This finding on the role of substrate stiffness in remodeling cytoskeletal properties sheds light on how cells make the decision to migrate during development and metastasis.
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