Limits...
Fluctuations of intracellular forces during cell protrusion.

Ji L, Lim J, Danuser G - Nat. Cell Biol. (2008)

Bottom Line: Surprisingly, the maxima in adhesion and boundary forces lag behind maximal edge advancement by about 40 s.Maximal F-actin assembly was observed about 20 s after maximal edge advancement.On the basis of these findings, we propose that protrusion events are limited by membrane tension and that the characteristic duration of a protrusion cycle is determined by the efficiency in reinforcing F-actin assembly and adhesion formation as tension increases.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

ABSTRACT
We present a model to estimate intracellular force variations from live-cell images of actin filament (F-actin) flow during protrusion-retraction cycles of epithelial cells in a wound healing response. To establish a mechanistic relationship between force development and cytoskelal dynamics, force fluctuations were correlated with fluctuations in F-actin turnover, flow and F-actin-vinculin coupling. Our analyses suggest that force transmission at focal adhesions requires binding of vinculin to F-actin and integrin (indirectly), which is modulated at the vinculin-integrin but not the vinculin-F-actin interface. Force transmission at focal adhesions is colocalized in space and synchronized in time with transient increases in the boundary force at the cell edge. Surprisingly, the maxima in adhesion and boundary forces lag behind maximal edge advancement by about 40 s. Maximal F-actin assembly was observed about 20 s after maximal edge advancement. On the basis of these findings, we propose that protrusion events are limited by membrane tension and that the characteristic duration of a protrusion cycle is determined by the efficiency in reinforcing F-actin assembly and adhesion formation as tension increases.

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Reconstruction of intracellular force transients from F-actin network flow. (a) Flow vectors measured by quantitative Fluorescent Speckle Microscopy (qFSM; Scale bar: 10 μm.). (b) Network flow is driven by forces at the cell boundary (∂ΩLE) and by domain forces within lamellipodium and lamella (Ω). These forces generate transient deformations of the network, observed as spatial gradients in the displacement of fluorescent speckles over the time interval δt between two consecutive frames (illustrated by the transformation of a rectangle, dashed, into a polygon, solid gray lines. Network flows without spatial gradients indicate force free areas. (c) Force prediction from transient deformations of elastic spring. (d) Model of the F-actin network as a transiently elastic material. Over time scales 1s≤δt≤10 s, cross-linked F-actin networks deform predominantly elastically19. Thus, the material behaves like an ensemble of springs. At longer time scales t2-t1’ δt, structures (cyan) break while others (light green) form (plastic behavior). As a result pre-stress in the network is relaxed, which preserves the elastic constant and resting length of the spring ensemble (L1 » L2); yet, the relationship between the deformation u and the actual force change from t1 to t2 is lost. When the extension is fast compared to the time scale of remodeling, as from t1 to t1+δt or from t2 to t2+δt force changes can be inferred as F1=k×u1/L1 and F2=k×u2/L2.
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Figure 1: Reconstruction of intracellular force transients from F-actin network flow. (a) Flow vectors measured by quantitative Fluorescent Speckle Microscopy (qFSM; Scale bar: 10 μm.). (b) Network flow is driven by forces at the cell boundary (∂ΩLE) and by domain forces within lamellipodium and lamella (Ω). These forces generate transient deformations of the network, observed as spatial gradients in the displacement of fluorescent speckles over the time interval δt between two consecutive frames (illustrated by the transformation of a rectangle, dashed, into a polygon, solid gray lines. Network flows without spatial gradients indicate force free areas. (c) Force prediction from transient deformations of elastic spring. (d) Model of the F-actin network as a transiently elastic material. Over time scales 1s≤δt≤10 s, cross-linked F-actin networks deform predominantly elastically19. Thus, the material behaves like an ensemble of springs. At longer time scales t2-t1’ δt, structures (cyan) break while others (light green) form (plastic behavior). As a result pre-stress in the network is relaxed, which preserves the elastic constant and resting length of the spring ensemble (L1 » L2); yet, the relationship between the deformation u and the actual force change from t1 to t2 is lost. When the extension is fast compared to the time scale of remodeling, as from t1 to t1+δt or from t2 to t2+δt force changes can be inferred as F1=k×u1/L1 and F2=k×u2/L2.

Mentions: Forces in lamellipodium and lamella of epithelial cells were inferred from the F-actin network flow field u(x, t) measured by quantitative FSM17, 18 (Fig. 1a). Spatiotemporal gradients in direction and/or magnitude of the flow vectors indicate local deformations of the F-actin network (Fig. 1b) associated with intracellular forces (Supplementary Note 1). Our goal was to predict the distribution of boundary forces FI at the leading edge, ∂ΩLE, and contraction and adhesion forces FII+III inside the cellular region, Ω, that would optimally explain the measured network deformation. The concept of force reconstruction can be understood by analogy of a Hookean spring under tension (Fig. 1c). The force F required to extend a spring is proportional to the extension u relative to the relaxation length L, i.e. the external force is balanced by spring-internal stresses. The ratio ε=u/L is referred to as the strain. Knowing the spring constant k and the strain, the force is F=k·ε.


Fluctuations of intracellular forces during cell protrusion.

Ji L, Lim J, Danuser G - Nat. Cell Biol. (2008)

Reconstruction of intracellular force transients from F-actin network flow. (a) Flow vectors measured by quantitative Fluorescent Speckle Microscopy (qFSM; Scale bar: 10 μm.). (b) Network flow is driven by forces at the cell boundary (∂ΩLE) and by domain forces within lamellipodium and lamella (Ω). These forces generate transient deformations of the network, observed as spatial gradients in the displacement of fluorescent speckles over the time interval δt between two consecutive frames (illustrated by the transformation of a rectangle, dashed, into a polygon, solid gray lines. Network flows without spatial gradients indicate force free areas. (c) Force prediction from transient deformations of elastic spring. (d) Model of the F-actin network as a transiently elastic material. Over time scales 1s≤δt≤10 s, cross-linked F-actin networks deform predominantly elastically19. Thus, the material behaves like an ensemble of springs. At longer time scales t2-t1’ δt, structures (cyan) break while others (light green) form (plastic behavior). As a result pre-stress in the network is relaxed, which preserves the elastic constant and resting length of the spring ensemble (L1 » L2); yet, the relationship between the deformation u and the actual force change from t1 to t2 is lost. When the extension is fast compared to the time scale of remodeling, as from t1 to t1+δt or from t2 to t2+δt force changes can be inferred as F1=k×u1/L1 and F2=k×u2/L2.
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Related In: Results  -  Collection

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Figure 1: Reconstruction of intracellular force transients from F-actin network flow. (a) Flow vectors measured by quantitative Fluorescent Speckle Microscopy (qFSM; Scale bar: 10 μm.). (b) Network flow is driven by forces at the cell boundary (∂ΩLE) and by domain forces within lamellipodium and lamella (Ω). These forces generate transient deformations of the network, observed as spatial gradients in the displacement of fluorescent speckles over the time interval δt between two consecutive frames (illustrated by the transformation of a rectangle, dashed, into a polygon, solid gray lines. Network flows without spatial gradients indicate force free areas. (c) Force prediction from transient deformations of elastic spring. (d) Model of the F-actin network as a transiently elastic material. Over time scales 1s≤δt≤10 s, cross-linked F-actin networks deform predominantly elastically19. Thus, the material behaves like an ensemble of springs. At longer time scales t2-t1’ δt, structures (cyan) break while others (light green) form (plastic behavior). As a result pre-stress in the network is relaxed, which preserves the elastic constant and resting length of the spring ensemble (L1 » L2); yet, the relationship between the deformation u and the actual force change from t1 to t2 is lost. When the extension is fast compared to the time scale of remodeling, as from t1 to t1+δt or from t2 to t2+δt force changes can be inferred as F1=k×u1/L1 and F2=k×u2/L2.
Mentions: Forces in lamellipodium and lamella of epithelial cells were inferred from the F-actin network flow field u(x, t) measured by quantitative FSM17, 18 (Fig. 1a). Spatiotemporal gradients in direction and/or magnitude of the flow vectors indicate local deformations of the F-actin network (Fig. 1b) associated with intracellular forces (Supplementary Note 1). Our goal was to predict the distribution of boundary forces FI at the leading edge, ∂ΩLE, and contraction and adhesion forces FII+III inside the cellular region, Ω, that would optimally explain the measured network deformation. The concept of force reconstruction can be understood by analogy of a Hookean spring under tension (Fig. 1c). The force F required to extend a spring is proportional to the extension u relative to the relaxation length L, i.e. the external force is balanced by spring-internal stresses. The ratio ε=u/L is referred to as the strain. Knowing the spring constant k and the strain, the force is F=k·ε.

Bottom Line: Surprisingly, the maxima in adhesion and boundary forces lag behind maximal edge advancement by about 40 s.Maximal F-actin assembly was observed about 20 s after maximal edge advancement.On the basis of these findings, we propose that protrusion events are limited by membrane tension and that the characteristic duration of a protrusion cycle is determined by the efficiency in reinforcing F-actin assembly and adhesion formation as tension increases.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

ABSTRACT
We present a model to estimate intracellular force variations from live-cell images of actin filament (F-actin) flow during protrusion-retraction cycles of epithelial cells in a wound healing response. To establish a mechanistic relationship between force development and cytoskelal dynamics, force fluctuations were correlated with fluctuations in F-actin turnover, flow and F-actin-vinculin coupling. Our analyses suggest that force transmission at focal adhesions requires binding of vinculin to F-actin and integrin (indirectly), which is modulated at the vinculin-integrin but not the vinculin-F-actin interface. Force transmission at focal adhesions is colocalized in space and synchronized in time with transient increases in the boundary force at the cell edge. Surprisingly, the maxima in adhesion and boundary forces lag behind maximal edge advancement by about 40 s. Maximal F-actin assembly was observed about 20 s after maximal edge advancement. On the basis of these findings, we propose that protrusion events are limited by membrane tension and that the characteristic duration of a protrusion cycle is determined by the efficiency in reinforcing F-actin assembly and adhesion formation as tension increases.

Show MeSH
Related in: MedlinePlus