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. 2006 Mar 1;90(5):1810-20.
doi: 10.1529/biophysj.105.064600. Epub 2005 Dec 2.

Weak force stalls protrusion at the leading edge of the lamellipodium

Sophie Bohnet  1 , Revathi Ananthakrishnan, Alex Mogilner, Jean-Jacques Meister, Alexander B Verkhovsky
Affiliations

Weak force stalls protrusion at the leading edge of the lamellipodium

Sophie Bohnet et al. Biophys J. .

Abstract

Protrusion, the first step of cell migration, is driven by actin polymerization coupled to adhesion at the cell's leading edge. Polymerization and adhesive forces have been estimated, but the net protrusion force has not been measured accurately. We arrest the leading edge of a moving fish keratocyte with a hydrodynamic load generated by a fluid flow from a micropipette. The flow arrests protrusion locally as the cell approaches the pipette, causing an arc-shaped indentation and upward folding of the leading edge. The effect of the flow is reversible upon pipette removal and dependent on the flow direction, suggesting that it is a direct effect of the external force rather than a regulated cellular response. Modeling of the fluid flow gives a surprisingly low value for the arresting force of just a few piconewtons per micrometer. Enhanced phase contrast, fluorescence, and interference reflection microscopy suggest that the flow does not abolish actin polymerization and does not disrupt the adhesions formed before the arrest but rather interferes with weak nascent adhesions at the very front of the cell. We conclude that a weak external force is sufficient to reorient the growing actin network at the leading edge and to stall the protrusion.

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Figures

FIGURE 1
FIGURE 1
Reversible arrest of the leading edge by a hydrodynamic load. (A) (0–30) As the cell approaches the pipette tip, the protrusion becomes locally arrested by the flow, resulting in the arc-shaped indentation of the leading edge. (37–104) The leading edge recovers its initial shape when the pipette is removed. (B) Traces of the position of the leading edge and the front boundary of the cell body. Whereas the leading edge of the cell is arrested by the flow and then recovers after the pipette removal, the cell body translocation is unaffected. (C) Flow parallel to the leading edge neither stops the protrusion nor affects cell motility in general. L stands for the lamellipodium and B for the cell body. Bar, 10 μm; time in seconds.
FIGURE 2
FIGURE 2
Effect of the flow on actin polymerization and substrate adhesion. (A) The flow does not affect actin polymerization: TRITC-phalloidin staining of the keratocyte fixed at the moment of the protrusion arrest shows F-actin accumulation at the site of the arrest and a fine crisscross pattern of the F-actin network with the highest density at the leading edge and a gradual decrease toward the nucleus. (B) Integrin β-1 immunostaining of the keratocyte fixed at the moment of the protrusion arrest shows that integrin β-1 is enriched in the lifted part of the edge as well as in a narrow rim along the intact leading edge. (C) F-actin network assembled before the arrest is not displaced with respect to the substrate: enhanced phase contrast microscopy of the lamellipodium reveals distinct features of the F-actin network (arrows) remaining nearly stationary with respect to the substrate. (D) IRM demonstrates that the adhesion pattern formed before the arrest (arrows on the left) is not affected by the flow; however, the flow interferes with the nascent adhesions at the very tip of the lamellipodium, which lifts up (narrow bright zone indicated by the arrow on the right). (E) Model: hydrodynamic load interferes with the weak nascent adhesions and reorients branching and elongation of the leading edge actin filaments away from the substrate. Bar, 5 μm; time in seconds.
FIGURE 3
FIGURE 3
Recovery of the substrate adhesion after the arrest of the leading edge. (A) IRM demonstrates that the adhesion of the leading edge is first reestablished at a small region at the tip of the edge (arrow) and then zips in from both sides, generating a continuous adhesion zone. Simultaneous phase contrast (B) and IRM (C) imaging confirms that adhesion is first reestablished at the very tip of the leading edge. Bar, 5 μm; time in seconds.
FIGURE 4
FIGURE 4
Recovery of the leading edge shape. (A) After the readhesion, protrusion of the formerly arrested part of the leading edge results in the recovery of the initial shape. (B) TRITC-phalloidin staining of the keratocyte fixed at the moment of recovery shows F-actin accumulation at the site of the indentation. (C) Normal extension from both sides of the indentation explains the recovery of the leading edge shape in agreement with the GRE model. Bar, 10 μm; time in seconds.
FIGURE 5
FIGURE 5
3D finite element simulation of the stationary flow from the pipette. (A) The parallelepiped within which the flow was computed is shown. The computed velocity field is illustrated with the arrows. The flow impinges on the lamellipodium step. The gray scale on the lamellipodium surface illustrates the computed shear stress. (B) The computed (color-coded) shear stress on the lamellipodium surface—view from above. A few stress level curves (on which the stress is constant) are shown. (C) The computed flow impinges on the cell body represented by the half-ellipsoid. (D) 2D simulation of the flow impinging on the lifted lamellipodium tip. The gray scale shows the velocity magnitude.
FIGURE 6
FIGURE 6
Estimation of the flow velocity and comparison with computed flow field. (A) The length of a bead trace divided by the exposure time gives the horizontal component of the flow speed. Bar, 10 μm. (B) Circles show the data for the horizontal component of the flow velocity measured at 2 μm from the substrate as a function of the horizontal distance from the pipette tip. The data represent the measurements corrected for systematic errors as described in Materials and Methods. The curve is the computed corresponding velocity distribution obtained from the numerical simulations of the Navier-Stokes equation. The discrepancy between theoretical and experimental data at small distances (0–10 μm) is due to the systematic errors described in Materials and Methods.
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