Also in the Article

This protocol is extracted from research article:
Regulation and dynamics of force transmission at individual cell-matrix adhesion bonds

Procedure

In the reversible cross-linker model, F-actin was treated as a network of filaments connected by noncompliant dynamic cross-linkers (Fig. 4). These cross-linkers bind and unbind at rates kx,on and kx,off. Each clutch is bound to an individual filament, and the number of motors per filament is dictated by simulation parameters (table S6). Force traces were averaged over 1-s time steps to reflect the time scale of the processed force traces from the single-molecule measurement. Individual clutches experience load routed from different combinations of motors at any given instant, where individual loads are dictated by both the force-velocity relationships of individual motors and the loading history of the clutches within the cluster. The forces on individual clutches build until the F-actin retrograde flow rate is close to 0 and the motor stall force and clutch force are nearly equal. When a cross-linker binds or unbinds, the forces on the associated clutches are no longer balanced, and the F-actin velocities adjust to reestablish mechanical equilibrium.

In the resulting simulations, dynamic clusters of clutches continuously stretch and relax, oscillating around force plateaus for periods of 10 to 60 s. Ramp and step transitions are observed throughout these simulations, in a manner consistent with our experimental observation: A step transition occurs when a binding clutch quickly builds force or when an unbinding clutch instantaneously returns to 0 force. More gradual ramp transitions occur in neighboring clutches as the associated loads readjust to achieve a force balance within the cluster.

Although a variety of models were explored, the dynamic F-actin network best captured the behavior observed experimentally. In contrast to the other models, the force plateaus persisted the longest with minimal fluctuations, and the single-clutch dynamics were consistent across simulation parameters (i.e., relatively insensitive to small parameter changes). Although further testing of the dynamic F-actin network model is still needed and alternate models have not been definitively ruled out, the dynamic F-actin model best captures the experimentally observed behavior of the individual sensor force distribution and force dynamics.

Energy dissipation was calculated as the sum of stored energy in anchors upon unbinding. When an unbinding event occurred, the stored energy in the anchor was calculated using the spring constant of the anchor and its displacement$E=12kcx2$

The running sum of this value was recorded for the duration of the experiment. This does not account for energy dissipation within the motor-actin system.

Note: The content above has been extracted from a research article, so it may not display correctly.

Q&A