Force generated by actomyosin contraction builds bridges between adhesive contacts

145

163

Podosomes are unique cellular entities specifically found in macrophages and involved in cell-matrix interactions, matrix degradation, and 3D migration. They correspond to a core of F-actin surrounded at its base by matrix receptors. To investigate the structure/function relationships of podosomes, soft lithography, atomic force microscopy (AFM), and correlative fluorescence microscopy were used to characterize podosome physical properties in macrophages differentiated from human blood monocytes. Podosome formation was restricted to delineated areas with micropatterned fibrinogen to facilitate AFM analyses. Podosome height and stiffness were measured with great accuracy in living macrophages (578 ± 209 nm and 43.8 ± 9.3 kPa) and these physical properties were independent of the nature of the underlying matrix. In addition, time-lapse AFM revealed that podosomes harbor two types of overlapping periodic stiffness variations throughout their lifespan, which depend on F-actin and myosin II activity. This report shows that podosome biophysical properties are amenable to AFM, allowing the study of podosomes in living macrophages at nanoscale resolution and the analysis of their intimate dynamics. Such an approach opens up perspectives to better understand the mechanical functionality of podosomes under physiological and pathological contexts.cytoskeleton | Young's modulus | microcontact printing

Section: Discussionmentioning

confidence: 67%

Dynamics of podosome stiffness revealed by atomic force microscopy

Labernadie

Thibault

Vieu

et al. 2010

145

163

“…Cell spreading is known to be mediated by actin treadmilling (36,37), which controls the extension of the lamellipodium (38). An effect of treadmilling is the net flow of filamentous actin from the lamellipodial region to the proximal part of the actomyosin cytoskeleton between adhesions (37, 39), which persists even if the cell and its adhesions are immobile (18). Thus, the length of the cell is regulated by a combination of the myosin-driven contraction rate − _ « of the cytoskeleton (the retrograde flow described above) and the speed v t at which newly polymerized actin is incorporated into the cortex.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Etienne

Fouchard

Mitrossilis

et al. 2015

116

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...

“…The actin filaments in stress fibers constantly sustain tensile force at focal adhesions and may undergo polymerization and depolymerization (32)(33)(34)(35). Although rupture of actin filaments under tension (28) and the growth of an actin network under compression (36) have been studied, the effects of tensile force on actin depolymerization have not been investigated.…”

Section: Discussionmentioning

confidence: 99%

Actin depolymerization under force is governed by lysine 113:glutamic acid 195-mediated catch-slip bonds

Lee

Lou

Wen

et al. 2013

As a key element in the cytoskeleton, actin filaments are highly dynamic structures that constantly sustain forces. However, the fundamental question of how force regulates actin dynamics is unclear. Using atomic force microscopy force-clamp experiments, we show that tensile force regulates G-actin/G-actin and G-actin/F-actin dissociation kinetics by prolonging bond lifetimes (catch bonds) at a low force range and by shortening bond lifetimes (slip bonds) beyond a threshold. Steered molecular dynamics simulations reveal force-induced formation of new interactions that include a lysine 113(K113):glutamic acid 195 (E195) salt bridge between actin subunits, thus suggesting a molecular basis for actin catch-slip bonds. This structural mechanism is supported by the suppression of the catch bonds by the single-residue replacements K113 to serine (K113S) and E195 to serine (E195S) on yeast actin. These results demonstrate and provide a structural explanation for actin catchslip bonds, which may provide a mechanoregulatory mechanism to control cell functions by regulating the depolymerization kinetics of force-bearing actin filaments throughout the cytoskeleton.single-molecule force spectroscopy | mechanotransduction | mechanosensing | nemaline myopathy T he actin cytoskeleton, primarily a force-bearing structure, controls the morphology, motility, and adhesion of the cell (1-4). Its core filamentous component, assembled from actin monomers via noncovalent interactions (5), undergoes rapid and controlled polymerization and depolymerization, allowing the dynamic reorganization of the actin cytoskeleton (1, 2).In cells, this dynamic process can be modulated by forces, and this is crucial to mechanosensitivity, mechanotransduction, and cellular adaptations to mechanical stresses (3, 6-8). For example, the assembly, stabilization, and reorganization of the actin stress fiber and the focal adhesion, where actin filaments constantly sustain tension, are induced by externally applied forces (9-12) dependent on myosin-generated contractility (4,8,13,14) and sensitive to substrate rigidity (3, 15, 16). These observations led us to investigate the molecular mechanism by which actin dynamics are regulated by force.The force-regulated kinetics of several molecular interactions important to adhesion and force-bearing functions of cells are governed by catch-slip bonds, in which the interaction is stabilized by tensile force in a low range and destabilized when force exceeds a threshold (17)(18)(19)(20)(21)(22). Various mechanisms, such as the allosteric model based on intramolecular conformational change under forces (23,24) and the sliding-rebinding model based on force-induced formation of new interactions due to intermolecular interface sliding (18,25), have been proposed to provide structural explanations for catch-slip bonds in different molecular interactions.Here we use atomic force microscopy (AFM) force-clamp experiments to determine how force regulates the off-rate of actin depolymerization and to elucidate the structural ...