The FAK-Arp2/3 interaction promotes leading edge advance and haptosensing by coupling nascent adhesions to lamellipodia actin.
Although FAK is known to be required for cell migration through effects on focal adhesions, its role in NA formation and lamellipodial dynamics is unclear.Haptosensing of extracellular matrix (ECM) concentration during migration requires the interaction between FAK and Arp2/3, whereas FAK phosphorylation modulates mechanosensing of ECM stiffness during spreading.Taken together, our results show that mechanistically separable functions of FAK in NA are required for cells to distinguish distinct properties of their environment during migration.
Affiliation: Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8019.
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Figure 4: FAK–Arp2/3 interaction promotes recruitment of Arp2/3 to NA to mediate efficient protrusion and leading edge advance. (A) Left, representative confocal micrographs of FAK−/− knockout (FAK-KO) MEF cells transfected with mCherry-tagged wild-type FAK (FAK-KO + wtFAK; top), eGFP-tagged FAKY397F mutant (FAK-KO + FAKY397F; middle) or mCherry tagged FAKK38AR86A mutant (FAK-KO + FAKK38AR86A; bottom), showing respective FAK channel in red and immunostaining for ArpC2 subunit of the Arp2/3 complex (green), with color overlay. Zoom of the red box is shown in the inset of each channel. Blue box indicates the position of the line scan used for quantification of fluorescence intensities in right panel and B, top. White box indicates the position of the line scan used for quantification of colocalization coefficients in B, bottom left and bottom right. Scale bar, 5 μm. Right, representative line scans of fluorescence intensities of mCherry-FAK or mCherry FAKK38AR86A (top and bottom; red) or EGFP- FAKY397F (middle; red) and intensity of immunostained ArpC2 subunit of Arp2/3 complex (green). (B) Top, box plot of distance between the peak of fluorescence intensity of line scans of the ArpC2 subunit of Arp2/3 complex immunostaining to the peak of fluorescence intensity of line scans of the indicated expressed FAK mutant, FAK-KO + wtFAK (red), FAK-KO + FAKY397F (yellow), and FAK-KO + FAKK38AR86A (light green; 38 protrusions/condition, five to seven cells/condition). Bottom left, bar plot of the Manders coefficient of colocalization for indicated expressed FAK constructs with immunostained ArpC2 subunit of the Arp2/3 complex, FAK-KO + wtFAK (red), FAK-KO + FAKY397F (yellow), and FAK-KO + FAKK38AR86A (light green). Bottom right, bar plot of the Manders coefficient of colocalization for immunostained ArpC2 subunit for the Arp2/3 complex with indicated expressed FAK construct. Error bars indicate SD (five cells/condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test. (C) Representative DIC micrograph of FAK-KO MEFs expressing mCherry-tagged FAKK38AR86A imaged 4 h after plating on fibronectin-coated coverslips. Black line indicates the line along which the kymograph on the right was obtained. Scale bars, 10 μm (left), distance 2 μm, time 2 min (right). (D) Box plot of velocities (μm/min) of protrusion (Vp) and retraction (Vr) of FAK-KO cells expressing the noted FAK construct (10–12 cells/condition). Color coding in D–G, I, J: FAK-KO + wtFAK cells (red), FAK-KO + FAKK38AR86A cells (light green), and FAK-KO cells (blue). (E) Box plot of distances (μm) of protrusion (Dp) and retraction (Dr) of FAK-KO cells expressing the noted FAK construct (10–12 cells/condition). (F) Box plot of protrusion efficiency (%) for FAK-KO + FAK, FAK-KO, and FAK-KO + FAKK38AR86A cells (10–12 cells/condition). (G) Box plot of net edge advance (μm/min) of FAK-KO + FAK, FAK-KO, and FAK-KO + FAKK38AR86A cells (10–12 cells/condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test. Note: FAK-KO data are the same as in Figure 1. (H) Left, representative inverted-contrast confocal micrographs of eGFP-actin in FAK+/+ MEFs expressing eGFP-actin (control, top) and in FAK-KO (second row), FAK-KO + FAK (third row), or FAK-KO + FAKK38AR86A cells (bottom). The red bar indicates the line along which the kymograph (right) of eGFP-actin was done, in which the red line shows an example of a slope of retrograde actin flow used to measure actin flow velocity. (I) Box plot of speed of retrograde actin flow (μm/min) from protrusions in FAK+/+ (control, dark green in I and J), FAK-KO, FAK-KO + wtFAK, and FAK-KO + FAKK38AR86A cells (15–23 cells/condition). (J) Box plot of actin polymerization rate (μm/min) calculated as the sum of protrusion rate and retrograde actin flow rate in control, FAK-KO, FAK-KO + wtFAK, and FAK-KO + FAKK38AR86A cells (15–23 cells/ condition). ). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test.
We first examined the effect of FAK phosphorylation and the effect of the FERM-domain mutant on the localizations of FAK and Arp2/3 in lamellipodia. Immunofluorescence localization of endogenous ArpC2 subunit of the Arp2/3 complex in FAK-KO cells reconstituted with fluorescence-tagged FAK mutants expressed at near-endogenous levels (Supplemental Figure S1A) showed that Arp2/3, wtFAK, FAKY397F, and FAKK38AR86A all localized in punctate distributions in thin bands along the leading edge of lamellipodia. Examination of color overlay images of Arp2/3 together with either wtFAK or FAKY397F in FAK-KO cells showed that a subset of Arp2/3 puncta partially colocalized with FAK-containing NAs in spite of the reduced NA density in cells containing FAKY397F (Figure 4, A and B). In addition, in cells reconstituted with wtFAK, line scan analysis showed that FAK and Arp2/3 exhibited indistinguishable peaks of localization at the leading edge, whereas in cells reconstituted with FAKY397F, Arp2/3 exhibited two peaks near the leading edge, with FAKY397F colocalizing with the proximal peak. In addition, Manders analysis of lamellipodia showed that both wtFAK and FAKY397F exhibited a high degree of colocalization (Figure 4B). In contrast, in FAK-KO cells reconstituted with FAKK38AR86A, although Arp2/3 remained localized along the leading edge and FAKK38AR86A localized to NAs, color overlay images showed a reduction in colocalization of Arp2/3 and FAKK38AR86A, line scans showed that the peak of FAKK38AR86A was displaced just proximal to the peak of Arp2/3 at the leading edge, and a Manders colocalization analysis revealed a significant reduction in FAKK38AR86A–Arp2/3 colocation (Figure 4B). These results suggest that the FAK–Arp2/3 interaction promotes localization of Arp2/3 to NAs, and yet Arp2/3 targets to lamellipodia and FAK targets to NAs independent of their interaction with each other.