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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Relationship between predicted adhesion forces and F-actin-vinculin interactions during protrusion and retraction. (a)Top-left panel: adhesion force magnitude (color-coded) and boundary force (cyan vectors); Top-right panel: dual-color FSM image of F-actin (red) and vinculin (green) (see Video 2). Bottom-left panel: Zoom of the middle sector M showing F-actin flow (yellow vectors), adhesion forces (red vectors) overlaid to adhesion force magnitude (color-coded). Bottom-right panel: eGFP-vinculin signal. (b)Top row: Adhesion force magnitude (color-coded) and F-actin/vinculin signal in two time-points between which the cell edge in the bottom sector (first) and top sector (second) retracts and the adhesions slide (asterisk). Bottom-left column: Kymograph display of vinculin signal along three profiles indicated in the top panels. Bottom-right column: Time courses of the average adhesion (ADHF; red) and boundary forces (BNDF; cyan) in the Top, Bottom and Middle sectors. Also shown are time courses of the direction-coupling score (DCS, green) and the velocity-magnitude-coupling score (VMCS, blue) of F-actin and vinculin speckle motion4. Arrows: onset of decreasing adhesion and boundary forces, which correlates in time with increased motion coupling of F-actin and vinculin speckles. Asterisks: time-points of minimal adhesion forces (correspond to asterisks in top row). (c) Time montages of the adhesion force (ADHF) and the velocity magnitude coupling score (VMCS) in the middle and bottom sectors. (d) Models of a vinculin-mediated clutch. Scale bars: 5 μm.
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Figure 3: Relationship between predicted adhesion forces and F-actin-vinculin interactions during protrusion and retraction. (a)Top-left panel: adhesion force magnitude (color-coded) and boundary force (cyan vectors); Top-right panel: dual-color FSM image of F-actin (red) and vinculin (green) (see Video 2). Bottom-left panel: Zoom of the middle sector M showing F-actin flow (yellow vectors), adhesion forces (red vectors) overlaid to adhesion force magnitude (color-coded). Bottom-right panel: eGFP-vinculin signal. (b)Top row: Adhesion force magnitude (color-coded) and F-actin/vinculin signal in two time-points between which the cell edge in the bottom sector (first) and top sector (second) retracts and the adhesions slide (asterisk). Bottom-left column: Kymograph display of vinculin signal along three profiles indicated in the top panels. Bottom-right column: Time courses of the average adhesion (ADHF; red) and boundary forces (BNDF; cyan) in the Top, Bottom and Middle sectors. Also shown are time courses of the direction-coupling score (DCS, green) and the velocity-magnitude-coupling score (VMCS, blue) of F-actin and vinculin speckle motion4. Arrows: onset of decreasing adhesion and boundary forces, which correlates in time with increased motion coupling of F-actin and vinculin speckles. Asterisks: time-points of minimal adhesion forces (correspond to asterisks in top row). (c) Time montages of the adhesion force (ADHF) and the velocity magnitude coupling score (VMCS) in the middle and bottom sectors. (d) Models of a vinculin-mediated clutch. Scale bars: 5 μm.

Mentions: The force reconstruction algorithm distinguishes between boundary forces FI and domain forces FII+III. To infer the contribution of adhesion and contraction to the domain force transients, we defined the cone rule (Figure 2c, d; Supplementary Note 6) assuming that in adhesion-dominated regions forces are anti-parallel to network flow while force and flow vectors are approximately parallel in contraction-dominated regions. Accordingly, in Figure 2e the lamellipodium at the cell edge is an adhesion-dominant region whereas contraction-dominant regions distribute throughout the lamella. Figure 2e-I highlights a mixed-force region where adhesion forces are gradually overcome by contraction forces as the flow field is deflected into centers of high contractile activity. Further evaluation of the proposed model and numerical approach to force reconstruction was performed on simulated force fields with simulated measurement noise (Supplementary Note 7). We also tested the colocalization of contraction forces with myosin-II motors (Supplementary Note 8, Fig. S4 and Video 1) and of adhesion forces with eGFP-vinculin (Fig 3a), following previous reports of correlations between vinculin density with traction force5, 24. Overall, these analyses established the possibility to predict from F-actin flow adhesion, contraction, and boundary force transients that mediate morphological changes during protrusion and retraction.


Fluctuations of intracellular forces during cell protrusion.

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

Relationship between predicted adhesion forces and F-actin-vinculin interactions during protrusion and retraction. (a)Top-left panel: adhesion force magnitude (color-coded) and boundary force (cyan vectors); Top-right panel: dual-color FSM image of F-actin (red) and vinculin (green) (see Video 2). Bottom-left panel: Zoom of the middle sector M showing F-actin flow (yellow vectors), adhesion forces (red vectors) overlaid to adhesion force magnitude (color-coded). Bottom-right panel: eGFP-vinculin signal. (b)Top row: Adhesion force magnitude (color-coded) and F-actin/vinculin signal in two time-points between which the cell edge in the bottom sector (first) and top sector (second) retracts and the adhesions slide (asterisk). Bottom-left column: Kymograph display of vinculin signal along three profiles indicated in the top panels. Bottom-right column: Time courses of the average adhesion (ADHF; red) and boundary forces (BNDF; cyan) in the Top, Bottom and Middle sectors. Also shown are time courses of the direction-coupling score (DCS, green) and the velocity-magnitude-coupling score (VMCS, blue) of F-actin and vinculin speckle motion4. Arrows: onset of decreasing adhesion and boundary forces, which correlates in time with increased motion coupling of F-actin and vinculin speckles. Asterisks: time-points of minimal adhesion forces (correspond to asterisks in top row). (c) Time montages of the adhesion force (ADHF) and the velocity magnitude coupling score (VMCS) in the middle and bottom sectors. (d) Models of a vinculin-mediated clutch. Scale bars: 5 μm.
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Figure 3: Relationship between predicted adhesion forces and F-actin-vinculin interactions during protrusion and retraction. (a)Top-left panel: adhesion force magnitude (color-coded) and boundary force (cyan vectors); Top-right panel: dual-color FSM image of F-actin (red) and vinculin (green) (see Video 2). Bottom-left panel: Zoom of the middle sector M showing F-actin flow (yellow vectors), adhesion forces (red vectors) overlaid to adhesion force magnitude (color-coded). Bottom-right panel: eGFP-vinculin signal. (b)Top row: Adhesion force magnitude (color-coded) and F-actin/vinculin signal in two time-points between which the cell edge in the bottom sector (first) and top sector (second) retracts and the adhesions slide (asterisk). Bottom-left column: Kymograph display of vinculin signal along three profiles indicated in the top panels. Bottom-right column: Time courses of the average adhesion (ADHF; red) and boundary forces (BNDF; cyan) in the Top, Bottom and Middle sectors. Also shown are time courses of the direction-coupling score (DCS, green) and the velocity-magnitude-coupling score (VMCS, blue) of F-actin and vinculin speckle motion4. Arrows: onset of decreasing adhesion and boundary forces, which correlates in time with increased motion coupling of F-actin and vinculin speckles. Asterisks: time-points of minimal adhesion forces (correspond to asterisks in top row). (c) Time montages of the adhesion force (ADHF) and the velocity magnitude coupling score (VMCS) in the middle and bottom sectors. (d) Models of a vinculin-mediated clutch. Scale bars: 5 μm.
Mentions: The force reconstruction algorithm distinguishes between boundary forces FI and domain forces FII+III. To infer the contribution of adhesion and contraction to the domain force transients, we defined the cone rule (Figure 2c, d; Supplementary Note 6) assuming that in adhesion-dominated regions forces are anti-parallel to network flow while force and flow vectors are approximately parallel in contraction-dominated regions. Accordingly, in Figure 2e the lamellipodium at the cell edge is an adhesion-dominant region whereas contraction-dominant regions distribute throughout the lamella. Figure 2e-I highlights a mixed-force region where adhesion forces are gradually overcome by contraction forces as the flow field is deflected into centers of high contractile activity. Further evaluation of the proposed model and numerical approach to force reconstruction was performed on simulated force fields with simulated measurement noise (Supplementary Note 7). We also tested the colocalization of contraction forces with myosin-II motors (Supplementary Note 8, Fig. S4 and Video 1) and of adhesion forces with eGFP-vinculin (Fig 3a), following previous reports of correlations between vinculin density with traction force5, 24. Overall, these analyses established the possibility to predict from F-actin flow adhesion, contraction, and boundary force transients that mediate morphological changes during protrusion and retraction.

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