Substrate deformations induce directed keratinocyte migration

Abstract

Cell migration is an essential part of many (patho)physiological processes, including keratinocyte re-epithelialization of healing wounds. Physical forces and mechanical cues from the wound bed (in addition to biochemical signals) may also play an important role in the healing process. Previously, we explored this possibility and found that polyacrylamide (PA) gel stiffness affected human keratinocyte behaviour and that mechanical deformations in soft (approx. 1.2 kPa) PA gels produced by neighbouring cells appeared to influence the process of de novo epithelial sheet formation. To clearly demonstrate that keratinocytes do respond to such deformations, we conducted a series of experiments where we observed the response of single keratinocytes to a prescribed local substrate deformation that mimicked a neighbouring cell or evolving multicellular aggregate via a servo-controlled microneedle. We also examined the effect of adding either Y27632 or blebbistatin on cell response. Our results indicate that keratinocytes do sense and respond to mechanical signals comparable to those that originate from substrate deformations imposed by neighbouring cells, a finding that could have important implications for the process of keratinocyte re-epithelialization that takes place during wound healing. Furthermore, the Rho/ROCK pathway and the engagement of NM II are both essential to substrate deformation-directed keratinocyte migration.

Keywords: actomyosin; mechanobiology; mechanosensing; non-muscle myosin II; polyacrylamide gel.

Figure 1.

Experimental set-up to apply controlled mechanical deformations to PA gels. (a) The PA gel is placed within a microscope-mounted micro-chamber contained with an environmentally controlled enclosure. A servo-controlled liner actuator drives needle movement via a high-precision micromanipulator. (b) A single keratinocyte is identified and the needle is inserted a prescribed distance away from the centroid of the cell, in this instance approximately 100 µm. (c) Substrate deformation in response to needle movement are calculated by tracking the movement of embedded fluorescent microspheres at each node (red points) of a user-defined grid superimposed on the image. (d) A displacement field can be calculated from the nodal displacements. Here, the series of images depicts the evolution of substrate displacement vectors, where the arrows indicate direction and the displacement magnitudes are colour-coded. Large red arrows denote the position of the migrating cell. Note that although the needle moves at the prescribed rate of 1 µm min⁻¹, the rate of gel displacement decreases with distance from the needle. (e) Displacement fields for a prototypical evolving multicellular aggregate (*) at equivalent time points. The three single cells denoted with large red arrows eventually join the aggregate (adapted from [42]). (f) Histograms showing the corresponding substrate displacements displayed in (d,e). (g) Histograms showing the corresponding substrate displacement rates corresponding to (d,e). (h) Comparison of the substrate displacement rates in the PA gel initially located approximately 100 µm from the microneedle tip. (Online version in colour.)

Figure 2.

Direction of keratinocyte migration in response to needle-induced substrate deformations at t = 0, t = 45, and t = 90 min. Red arrows indicate the current direction of cell movement. (a) For Soft-100, the cell, which is initially migrating away from the needle gradually reorients and moves in the direction in which the needle is pulling the substrate. (b) For Soft-Y27632 and (c) Soft-Bleb, the cells move in a manner that is unresponsive to the substrate deformations produced by needle movement. Also, note that exposure to both drugs perceptibly altered cell morphology. (Online version in colour.)

Figure 3.

Adjusted cell motility on PA gels. Each colour line shows the trace of , the position vector of the cell due to cell migration effects alone (see equation (2.2)). Each cell path is colour-coded to show the relative time along the path from its beginning at the origin (dark blue) to the final tracked position (yellow). The majority of cells moved toward the displacing needle for needles nominally positioned either 100 or 200 µm from the cell centroid at the start of the experiment (i.e. Soft-100 and Soft-200, respectively). Exposure to the Rho kinase inhibitor Y27632 or the actin–myosin inhibitor blebbistatin interrupted this directed migration. (Online version in colour.)

Figure 4.

Polar histograms showing the combined angle distributions in degrees of adjusted cell displacement (red) and substrate displacement (blue) for all 10 replicates of the (a) Soft-100, (b) Soft-200, (c) Soft-Y27632 and (d) Soft-Bleb experiments. Each distribution is normalized by the total number of observations with the black radial text associated with and the blue radial text associated with . The dashed line shows the mean and standard deviation of the angle of needle movement. For all four conditions, the displacements of the substrate aligned with the direction of needle movement. In addition, for Soft-100 and Soft-200, V-tests indicate that the direction of cell movement is coincident with that of the needle-induced substrate deformations, but not for soft-Y27632 or Soft-Bleb (p < 0.001). (Online version in colour.)