The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration

Abstract

The Arp2/3 complex nucleates the formation of the dendritic actin network at the leading edge of motile cells, but it is still unclear if the Arp2/3 complex plays a critical role in lamellipodia protrusion and cell motility. Here, we differentiated motile fibroblast cells from isogenic mouse embryonic stem cells with or without disruption of the ARPC3 gene, which encodes the p21 subunit of the Arp2/3 complex. ARPC3(-/-) fibroblasts were unable to extend lamellipodia but generated dynamic leading edges composed primarily of filopodia-like protrusions, with formin proteins (mDia1 and mDia2) concentrated near their tips. The speed of cell migration, as well as the rates of leading edge protrusion and retraction, were comparable between genotypes; however, ARPC3(-/-) cells exhibited a strong defect in persistent directional migration. This deficiency correlated with a lack of coordination of the protrusive activities at the leading edge of ARPC3(-/-) fibroblasts. These results provide insights into the Arp2/3 complex's critical role in lamellipodia extension and directional fibroblast migration.

Figure 1.

ARPC3^−/− fibroblast cells are deficient in lamellipodia formation. (A and B) Representative phase-contrast images showing the morphology of ARPC3^+/+ (A) and ARPC3^−/− (B) fibroblast cells after 7 d of differentiation. Arrows indicate lamellipodia (wild type) and FLPs (mutant). Bar, 50 µm. (C and D) Time-lapse montage showing spreading morphology of ARPC3^+/+ (C) and ARPC3^−/− cells (D) on 5 mg/ml fibronectin-coated glass surface. Frame interval is 15 min. Bars, 15 µm. (E) Quantification of cell area from experiments as described in C and D. Plots show mean and standard error of the mean (SEM) from 13 (APRC3^+/+) and 11 (APRC3^−/−) spreading cell movies made in three different experiments.

Figure 2.

Localization of Arp2 and mDia1 formin in ARPC3^+/+ and ARPC3^−/− fibroblasts. Spreading (A) or polarized (B) ARPC3^+/+ and ARPC3^−/− cells were fixed and stained with AF546 phalloidin (red), the indicated antibodies (green), and DAPI (blue). Bars, 25 µm.

Figure 3.

ARPC3^−/− fibroblast cells in wound healing and their leading edge dynamics. (A and B) Wound-healing montage of ARPC3^+/+ and ARPC3^−/− fibroblast cells. Bars, 50 µm. (C) Quantification of wound area as a function of time. Plots show mean and standard deviation from six different regions along the wound per experiment and three experiments per genotype. (D) Cell boundary segmentation and illustration of leading edge kymograph line selection. Bars, 25 µm. (E) Each kymograph (left) was digitized (center) and then smoothed for assignment of protrusion and retraction segments. (F) Angular correlation functions were computed as described in Materials and methods. (G) The smaller width at half maximum of the angular correlation function for mutant indicates less coordinated motion of the leading edge than for wild type. Small box shows the mean, line shows median, large box the SEM, and whiskers show SD.

Figure 4.

Migration behavior of individual ARPC3^+/+ and ARPC3^−/− fibroblast cells during wound closure. (A and B) Aggregated trajectories of ARPC3^+/+ (A) and ARPC3^−/− (B) fibroblast cells migrating for 10 h toward the wound center. (C) Visual demonstration of path length vs. displacement for speed and velocity calculations. (D) Cell speed, (E) velocity, and (F) straightness of wild-type and mutant cells. Notation is as in Fig. 3 G. (G) A plot of MSD vs. time shift and (H) α values from MSD curve fits indicating more directed motion for ARPC3^+/+ cells (solid line) than for ARPC3^−/− cells (dashed line). Dotted line represents the case for α = 1 indicating pure random motion.

Figure 5.

Chemotaxis of ARPC3^+/+ and ARPC3^−/− fibroblast cells in response to an EGF gradient. The ARPC3^+/+ and ARPC3^−/− cells were plated on fibronectin-coated μ-Slide and analyzed for 12 h in the presence of EGF gradient (500 ng/ml at source). (A and B) Aggregated trajectories of individual ARPC3^+/+ (A) and ARPC3^−/− (B) cells in the presence of the EGF gradient. (C) Cell speed, (D) velocity, (E) straightness, and (F) MSD α values for wild-type and mutant cells presented as in Fig. 4. The data came from tracking of 43 (two experiments) ARPC3^+/+ and 115 (five experiments) ARPC3^−/− cells.