ERK reinforces actin polymerization to power persistent edge protrusion during motility

Abstract

Cells move through perpetual protrusion and retraction cycles at the leading edge. These cycles are coordinated with substrate adhesion and retraction of the cell rear. We tracked spatial and temporal fluctuations in the molecular activities of individual moving cells to elucidate how extracellular signal-regulated kinase (ERK) signaling controlled the dynamics of protrusion and retraction cycles. ERK is activated by many cell surface receptors, and we found that ERK signaling specifically reinforced cellular protrusions so that they translated into rapid, sustained forward motion of the leading edge. Using quantitative fluorescent speckle microscopy and cross-correlation analysis, we showed that ERK controlled the rate and timing of actin polymerization by promoting the recruitment of the actin nucleator Arp2/3 to the leading edge. These findings support a model in which surges in ERK activity induced by extracellular cues enhance Arp2/3-mediated actin polymerization to generate protrusion power phases with enough force to counteract increasing membrane tension and to promote sustained motility.

Fig. 1. ERK signaling extends membrane protrusion velocity and persistence time

(A) Displacement distributions of cancer cells migrating after treatment with DMSO or AZD6244 (MEK inhibitor). n = number of cells tracked in 3 independent experiments. (B) Representative images of the migration of a DMSO- or AZD6244-treated PtK1 monolayer after scratch wounding. (C) Representative overlay of single cell edge dynamics tracked for 10–20 min post-treatment. Blue: early time points; red: late time points. (D) Representative profile (1 of 5 cells in each treatment group) of the fraction of the cell edge in protrusion, retraction, quiescent states. (E) Velocity and (F) persistence time distributions of m significant protrusion events in n = 5 DMSO- and AZD6244-treated cells tracked in 3–4 independent experiments. The “> 75^th Percentile” graphs plot the distribution of the top 25% of events in the corresponding “All” graphs. For A, E and F, gray area indicates total smoothed distribution and the boxes’ upper and lower edges represent the 75^th and 25^th distribution percentiles, respectively. Central horizontal line indicates the median. Error bars about the median indicate 95% confidence interval. (G) Maximum velocity compared to persistence time of all protrusion events tracked in 3–4 independent experiments. Encircled areas indicate events within the 1–75^th percentile of the joint velocity and persistence distribution. Shaded areas indicate protrusions within the 1–99^th percentile.

Fig. 2. ERK promotes actin flow and assembly in protrusions

(A) Raw image of fluorescent actin speckles. The red outline defines the computationally segmented cell mask. (B) Instantaneous flow vectors of speckles identified in A, calculated with qFSM software (38). Longer and red vectors indicate faster flow rate. Scale bar, 5 μm. (C to F) Normalized instantaneous rates of actin retrograde flow (C), polymerization (D, E), and depolymerization (F) calculated as average rate over all identified speckles as a function of distance from the cell edge. Data from a single representative control cell and a MEK-inhibitor-treated cell before and after treatment. Shaded areas are 95% confidence interval. On average, within a micron from the cell edge, DMSO-treatment reduced flow 18% +/− 1.1%, actin assembly 2% +/− 3.3%, and actin disassembly 9% +/− 7.3% (n = 6 cells tracked in 4 independent experiments). AZD6244 treatment reduced flow rates 28% +/− 0.8%, actin assembly 31% +/− 5.7%, and actin disassembly 38% +/− 3.1% (n = 4 cells tracked in 4independent experiments).

Fig. 3. ERK regulates actin dynamics during protrusion power phase

(A) Temporal cross-correlation of actin flow with edge velocity along entire edge, including retractions and protrusions. Marks 1 and 2 indicate negative and positive extrema, respectively. (B and C) Temporal cross-correlation of actin flow specifically in retractions (B) and protrusions (C). Marks 1′ and 2′ indicate that the negative and positive extrema of the combined cross-correlation in (A) are associated with retraction and protrusion events, respectively. (D and E) Temporal cross-correlation of actin assembly with edge velocity in retractions (D) and protrusions (E). Retraction velocity was computed with negative values. Positive time-lags in x-axis indicate that actin dynamics were delayed relative to edge motion. The corresponding model diagrams interpret the cross-correlation scores as readouts of temporal coordination between actin dynamics and edge velocity. E.V., edge velocity. R.F., retrograde flow. A.A., actin assembly. Orange arrows indicate how the indicated parameter of actin dynamics is altered relative to the edge velocity upon MEK inhibition. In control DMSO-treated cells, actin assembly correlates with protrusive edge motion with a cross-correlation score of 0.4 and a delay of 20 sec. In cells treated with AZD6244 and lacking ERK activity, actin assembly dynamics are less correlated with edge motion (cross-correlation score of 0.2) and this correlation occurs with a delay of only 10 sec. (F) Revised model of the sequence of mechanical processes during protrusion and retraction events. Actin retrograde flow correlates with the cell edge twice during a protrusion-retraction cycle: once when peak retraction velocities are attained and once 20 seconds after protrusion initiation, which coincides with peak actin assembly.

Fig. 4. ERK controls actin assembly and protrusion power phase by enhancing Arp2/3 recruitment

(A to E) GFP-Arp3 localization at cell edge. Green arrows indicate protrusion events (A). Normalized mean Arp3 intensity (m = 2779 protrusion events from n = 5 cells tracked in 3 independent experiments) in cells treated with DMSO (blue) or MEK inhibitor (red) at protrusion initiation (B), point of maximum velocity (C), power phase (~20 s after maximal protrusion velocity) (D), and protrusion end (E). GFP intensity at the cell edge (within 0–0.5 microns) was normalized to intensity in the lamella (3–3.5 microns). Shaded areas indicate 95% confidence intervals. (F) Cross-correlation of Arp2/3 intensity and cell edge velocity shows reduced correlation and delayed recruitment with MEK inhibitor. (G) Model of the role of ERK in controlling edge protrusion. Following protrusion initiation, ERK controls an intensity rheostat of WAVE regulatory complex (WRC) activation and Arp2/3 recruitment to induce actin polymerization power for sustained events.