Contact guidance requires spatial control of leading-edge protrusion

Abstract

In vivo, geometric cues from the extracellular matrix (ECM) are critical for the regulation of cell shape, adhesion, and migration. During contact guidance, the fibrillar architecture of the ECM promotes an elongated cell shape and migration along the fibrils. The subcellular mechanisms by which cells sense ECM geometry and translate it into changes in shape and migration direction are not understood. Here we pattern linear fibronectin features to mimic fibrillar ECM and elucidate the mechanisms of contact guidance. By systematically varying patterned line spacing, we show that a 2-μm spacing is sufficient to promote cell shape elongation and migration parallel to the ECM, or contact guidance. As line spacing is increased, contact guidance increases without affecting migration speed. To elucidate the subcellular mechanisms of contact guidance, we analyze quantitatively protrusion dynamics and find that the structured ECM orients cellular protrusions parallel to the ECM. This spatial organization of protrusion relies on myosin II contractility, and feedback between adhesion and Rac-mediated protrusive activity, such that we find Arp2/3 inhibition can promote contact guidance. Together our data support a model for contact guidance in which the ECM enforces spatial constraints on the lamellipodia that result in cell shape elongation and enforce migration direction.

FIGURE 1:

(A, B) Fluorescence images of paxillin, fibronectin, and F-actin of NIH 3T3 fibroblasts plated on uniform (0 μm) and linear ECM geometries. ECM patterns consist of 2-μm-wide stripes spaced at 3, 5, and 10 μm, respectively. Scale bar, 30 μm. (C) Migration trajectories of cells on uniform (0 μm) and linear ECM spaced at 3, 5, and 10 μm, respectively. (D–G) Cell elongation, orientation, instantaneous speed, and guidance as a function of ECM line spacing. Elongation is measured by taking the ratio of long over short cellular axes. Orientation is determined by measuring the angle, θ, between the long axis of the cell and a line parallel to the ECM and calculating the orientation parameter, P = cos²θ. Guidance for τ = 300 min is plotted. Mean and SD for >100 cells are shown for each condition. Insets, results of pairwise statistical testing: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (see Supplemental Table S3 for exact p values).

FIGURE 2:

(A) GFP-stargazin images of cells on linear ECM patterns with uniform (0 µm) and 2-, 3-, 5-, and 10-μm line spacings. Contour plots show cell outlines obtained every 1 min over 30 min. (B) Schematic representation of analysis of leading-edge protrusions. From cell contours at time t and t + t₀ (left), areas of leading-edge growth are identified as protrusions (middle). Protrusion orientation is found by measuring the angle between a line connecting the protrusion center with the cell center and a line parallel to the ECM. Final orientation values are obtained by calculating the orientation parameter, P = cos²θ. (C, D) Area and orientation of cell protrusion as a function of ECM line spacing. Mean and SEM for ≥12 cells. Insets, results of pairwise statistical testing: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (see Supplemental Table S3 for exact p values). (E) Phase-contrast images of NIH 3T3 fibroblasts spreading on uniform (0 μm) and ECM striped patterns spaced at 5 and 10 μm, respectively. Images correspond to 1, 5, 10, 15, and 30 min; contour plots show the entire time lapse. Left, control cells treated with dimethyl sulfoxide (DMSO); right, cells treated with 20 µM Y-27632. Scale bar, 20 μm. (F, G) Cell elongation and orientation during cell spreading for uniform (blue), 5-μm (black), and 10-μm (red) patterns. DMSO- and Y-27632–treated cells are shown by closed and open symbols, respectively. Data are presented as the mean and SD for ≥15 cells.

FIGURE 3:

(A, D) Top, F-actin cells on patterns treated with 20 µM Y-27632 (A), transfected with RacQ61L (B), an shRNA to β-pix (C), or treated with 100 µM CK-666 (D). Bottom, migration trajectories for cells in these conditions over 10 h. (E–G) Cell elongation, orientation, and guidance as a function of line spacing for cells treated with 20 µM Y-27632 (solid line). Light gray symbols and dotted line show data for cells treated with DMSO. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, t tests (see Supplemental Table S3 for exact p values). (H–J) Fold change in elongation, orientation, and guidance of cells with pharmacological or genetic perturbations: RacQ61L, shRNA β-pix, 100 µM CK-666, or 20 µM Y-27632. All data are plotted as a ratio with respect to their controls: WT, shNT, CK-689, or DMSO, respectively.

FIGURE 4:

(A–D) Phase contrast images of cells with specified perturbations on uniform and 5- and 10-μm patterns. Contour plots show cell outlines for a 30-min time interval during migration. Cells shown were treated with 20 µM Y-27632 (A), transfected with RacQ61L (B), shRNA to β-pix (C), or treated with 100 µM CK-666 (D). Scale bar, 30 µm. (E, F) Area and orientation of protrusions as a function of ECM line spacing for cells treated with 20 µM Y-27632 (solid line). Gray data points and dashed line are for control cells treated with DMSO. Mean and SEM for ≥8 cells. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, t tests (see Supplemental Table S3 for exact p values). (G, H) Fold change in area and orientation of individual protrusions as a function of ECM line spacing for cells transfected with RacQ61L, shRNA to β-pix, treated with 100 µM CK-666, or 20 µM Y-27632 relative to their respective controls: WT, shNT, CK-689, or DMSO.

FIGURE 5:

(A) Schematic representation of feedbacks that regulate protrusion area and orientation during contact guidance. At line spacings ≤5 µm, myosin II contractility inhibits protrusion from straight edges, and activation of Rac at adhesions promotes protrusion initiation parallel to the ECM. At spacings ≥5 µm, constraints in lamellipodia size limit stabilization of protrusions perpendicular to the ECM, promoting further contact guidance. (B) Schematic representation of the model for contact guidance. The guidance response depends on two protrusion physical parameters: area and orientation.