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 6: Arp2/3 sequesters inactive FAK to couple lamellipodial protrusion to transient NA stabilization and turnover for efficient leading edge protrusion. (A) Representative confocal micrographs of FAK−/− knockout (FAK-KO) cells expressing either mCherry-tagged wild-type FAK (FAK-KO + wtFAK; top) or mCherry-tagged FAKK38AR86A (FAK-KO + FAKK38AR86A) mutant immunostained for Y397 phosphorylation site–specific FAK antibody (P-FAK397; left) and paxillin (middle). Right, fluorescence intensity ratio image of immunostained P-FAK397 to immunostained paxillin channels, color coded according to heat scale bar. Images were background subtracted and thresholded before ratio analysis. Magnification scale bar, 10 μm. (B) Box plot of fluorescence intensity ratio of immunostained P-FAK397 to immunostained paxillin in FAK-KO + wtFAK cells (red) or FAK-KO + FAKK38AR86A cells (light green; 290–379 adhesions, five or six cells/condition). (C) FAK-KO + wtFAK cells (left) or FAK-KO + FAKK38AR86A cells harvested at the time noted after plating on fibronectin and (top) Western blotted with antibodies to P-FAK397, total FAK, and tubulin. Bottom, bar plot showing quantification of ratio of P-FAK397 to total FAK from three Western blots. (D) Control cells were plated on fibronectin for 4 h and treated with either DMSO (left lane, cyan bar) or 100 μM CK-666 (right lane, red bar) and (top) Western blotted with antibodies to P-FAK397 and total FAK. Bottom, quantification of ratio of P-FAK397 to total FAK from three Western blots. (E) Representative DIC kymographs of protrusion dynamics of FAK-KO + wtFAK (top) or FAK-KO + FAKK38AR86A (bottom) MEFs after 1-h treatment with 1 μM PF-228 (FAK inhibitor). Scale bar, distance 5 μm, time 2 min. (F) Box plot of distances (μm) of protrusion (Dp) and retraction (Dr) of FAK-KO cells expressing indicated cDNA with and without FAK inhibitor (FAKinh). Color coding in F–I and K–N: FAK-KO + wtFAK (no inhibitor, red; FAK inhibitor, orange), FAK-KO + FAKK38AR86A (no inhibitor, light green; FAK inhibitor, yellow green; 10–12 cells/condition). (G) Box plot of velocities (μm/min) of protrusion (Vp) and retraction (Vr) of FAK-KO cells expressing indicated cDNA with and without FAK inhibitor (FAKinh; 10–12 cells/condition). (H) Box plot of protrusion efficiency (%) of FAK-KO + wtFAK and FAK-KO + FAKK38AR86A cells with and without FAK inhibitor (10–12 cells/condition). (I) Box plot of net edge advance (μm/min) of FAK-KO + FAK and FAK-KO + FAKK38AR86A cells with and without FAK inhibitor (10–12 cells/condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test. (J) Representative TIRF kymograph of eGFP-paxillin–marked adhesions in FAK-KO + wtFAK cells (top) or FAK-KO + FAKK38AR86A cells (bottom) after treatment with FAK inhibitor. Scale bar, distance 5 μm, time 5 min. (K) Distribution of lifetimes of eGFP-paxillin marked NAs in FAK-KO + wtFAK cells (top) or FAK-KO + FAKK38AR86A cells (bottom) after treatment with FAK inhibitor. (L) Box plots of quantification of NA lifetimes (seconds) from distributions in I. (M) Box plot of NA formation density (number/μm2 lamellipodia protrusion area) marked by eGFP-paxillin in protruding lamellipodia of FAK-KO + wtFAK and FAK-KO + FAKK38AR86A cells with and without FAK inhibitor (17–20 protrusions, five or six cells/condition). (N) Box plot for maturation fraction (dimensionless, NAs formed/NAs that mature) among >100 NAs in protruding lamellipodia of FAK-KO + wtFAK and FAK-KO + FAKK38AR86A cells with and without FAK inhibitor (20 protrusions, five or six cells/condition). Note: FAK-KO + FAK and FAK-KO + FAKK38AR86A data are the same as in corresponding plots of Figure 5. **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test.
Previous work showed that activation of FAK by Y397 phosphorylation disrupts its interaction with the Arp3 subunit of Arp2/3 (Serrels et al., 2007), suggesting the hypothesis that Arp2/3 sequestration of inactive FAK might be critical to coupling lamellipodial protrusion to NA formation. To test this, we first sought to determine the role of the FAK–Arp2/3 interaction and Arp2/3 activity in regulation of FAK activation in cells. We used immunofluorescence and Western blots with antibodies that specifically recognize FAK phosphorylated on Y397 (pY397 FAK) in FAK-KO cells expressing either wtFAK or FAKK38AR86A to disrupt its interaction with Arp2/3. Immunostaining and ratio imaging of pY397 FAK to total paxillin (Zaidel-Bar et al., 2007) showed an overall increase in FAK phosphorylation at Y397 in FAK-KO cells reconstituted with FAKK38AR86A compared with FAK-KO reconstituted with wtFAK (Figure 6A). Image segmentation and quantitative analysis confirmed this and showed that disruption of the FAK–Arp2/3 interaction with the FERM- domain mutant significantly increased levels of FAK Y397 phosphorylation in adhesions (Figure 6B). Western blot analysis of cell lysates showed that compared with FAK-KO cells reconstituted with wtFAK, FAK Y397 phosphorylation was significantly higher in FAK-KO cells reconstituted with FAKK38AR86A, independently of whether cells were in suspension or plated for 60 min on FN (Figure 6C). Thus the FAK–Arp2/3 interaction decreases FAK Y397 phosphorylation in NA.