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The FAK-Arp2/3 interaction promotes leading edge advance and haptosensing by coupling nascent adhesions to lamellipodia actin.

Swaminathan V, Fischer RS, Waterman CM - Mol. Biol. Cell (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8019.

No MeSH data available.


Related in: MedlinePlus

FAK couples leading edge protrusion to NAs independent of Y397 phosphorylation. (A) Left, representative DIC micrographs of FAK−/− knockout (FAK-KO) MEFs expressing eGFP-tagged wild-type (FAK-KO + wtFAK; top) and FAK-KO cells expressing eGFP-tagged FAKY397F mutant (FAK-KO + FAKY397F; bottom). Cells were imaged 4–6 h after plating on 10 μg/ml 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 1 min (right). (B) Box plot of velocities (μm/min) of protrusion (Vp) and retraction (Vr) of FAK-KO cells expressing the noted FAK constructs. Color coding in B–J: FAK-KO + wtFAK (red), FAK-KO (blue), and FAK-KO + FAKY397F (yellow) cells. (C) Box plot of distances (μm) of protrusion (Dp) and retraction (Dr) of FAK-KO cells expressing the noted FAK constructs. (D) Box plot of protrusion efficiency (%) of FAK-KO + wtFAK, FAK-KO, and FAK-KO + FAKY397F cells. (E) Box plot of net edge advance (μm/min) of FAK-KO + wtFAK, FAK-KO, and FAK-KO + FAKY397F cells (10–12 cells/condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test. (F) Left, representative TIRF micrographs of FAK-KO cells expressing either EGFP-wtFAK (top) or EGFP-FAKY397F (bottom; scale bar, 10 μm). Contrast inverted. Blue box indicates area zoomed in for right images, a TIRF time-lapse image sequence of eGFP-FAK (top)– or eGFP-FAKY397F (bottom)–marked adhesions at the leading edge. Time in seconds. Scale bar, 5 μm. Far right, kymograph analysis of NA dynamics from preceding images. White open arrowheads, extremely short-lived NA; white closed arrowheads, longer-lived NA. Scale bars, distance 2 μm, time 2 min. (G) Distribution of NA lifetimes in FAK-KO cells expressing either eGFP-wtFAK (FAK-KO + wtFAK; top) or EGFP-FAKY397F (FAK-K + FAKY397F; bottom). Middle, distribution of EGFP-paxillin–marked NA in FAK-KO cells (50–70 NAs in five or six cells/condition). (H) Box plots of quantification of NA lifetimes (seconds) from distributions in G. (I) Box plot for maturation fraction (dimensionless, NAs formed/NAs that mature) among >100 NAs in protruding lamellipodia of FAK-KO + EGFP-FAK, FAK-KO, and FAK-KO + EGFPFAKY397F cells (10–12 protrusions, five cells/condition). (J) Box plot of NA formation density (number/μm2 lamellipodia protrusion area) in protruding lamellipodia marked by eGFP-FAK in FAK-KO + FAK cells, eGFP-paxillin in FAK-KO cells or EGFP-FAKY397F in FAK-KO + FAKY397F cells (17–20 protrusions, five or six cells per condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test. Note: FAK-KO data in all plots are the same data as presented in Figure 1.
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Figure 3: FAK couples leading edge protrusion to NAs independent of Y397 phosphorylation. (A) Left, representative DIC micrographs of FAK−/− knockout (FAK-KO) MEFs expressing eGFP-tagged wild-type (FAK-KO + wtFAK; top) and FAK-KO cells expressing eGFP-tagged FAKY397F mutant (FAK-KO + FAKY397F; bottom). Cells were imaged 4–6 h after plating on 10 μg/ml 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 1 min (right). (B) Box plot of velocities (μm/min) of protrusion (Vp) and retraction (Vr) of FAK-KO cells expressing the noted FAK constructs. Color coding in B–J: FAK-KO + wtFAK (red), FAK-KO (blue), and FAK-KO + FAKY397F (yellow) cells. (C) Box plot of distances (μm) of protrusion (Dp) and retraction (Dr) of FAK-KO cells expressing the noted FAK constructs. (D) Box plot of protrusion efficiency (%) of FAK-KO + wtFAK, FAK-KO, and FAK-KO + FAKY397F cells. (E) Box plot of net edge advance (μm/min) of FAK-KO + wtFAK, FAK-KO, and FAK-KO + FAKY397F cells (10–12 cells/condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test. (F) Left, representative TIRF micrographs of FAK-KO cells expressing either EGFP-wtFAK (top) or EGFP-FAKY397F (bottom; scale bar, 10 μm). Contrast inverted. Blue box indicates area zoomed in for right images, a TIRF time-lapse image sequence of eGFP-FAK (top)– or eGFP-FAKY397F (bottom)–marked adhesions at the leading edge. Time in seconds. Scale bar, 5 μm. Far right, kymograph analysis of NA dynamics from preceding images. White open arrowheads, extremely short-lived NA; white closed arrowheads, longer-lived NA. Scale bars, distance 2 μm, time 2 min. (G) Distribution of NA lifetimes in FAK-KO cells expressing either eGFP-wtFAK (FAK-KO + wtFAK; top) or EGFP-FAKY397F (FAK-K + FAKY397F; bottom). Middle, distribution of EGFP-paxillin–marked NA in FAK-KO cells (50–70 NAs in five or six cells/condition). (H) Box plots of quantification of NA lifetimes (seconds) from distributions in G. (I) Box plot for maturation fraction (dimensionless, NAs formed/NAs that mature) among >100 NAs in protruding lamellipodia of FAK-KO + EGFP-FAK, FAK-KO, and FAK-KO + EGFPFAKY397F cells (10–12 protrusions, five cells/condition). (J) Box plot of NA formation density (number/μm2 lamellipodia protrusion area) in protruding lamellipodia marked by eGFP-FAK in FAK-KO + FAK cells, eGFP-paxillin in FAK-KO cells or EGFP-FAKY397F in FAK-KO + FAKY397F cells (17–20 protrusions, five or six cells per condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test. Note: FAK-KO data in all plots are the same data as presented in Figure 1.

Mentions: To determine the mechanism by which FAK couples lamellipodial protrusion to NAs, we first examined the role of FAK phosphorylation. Autophosphorylation on tyrosine 397 (Y397) mediates relief of autoinhibition and activation of FAK kinase activity and induces its binding to SH2 domain–containing signaling proteins (Frame et al., 2010). We used time-lapse DIC imaging to compare leading edge dynamics of FAK-KO cells with FAK-KO cells expressing GFP fusions of either wtFAK or nonphosphorylatable FAK (FAKY397F) at near-endogenous levels (Supplemental Figure S1A and Supplemental Movie S4). Quantitative analysis of kymographs showed that expression of either wtFAK or FAKY397F in FAK-KO cells rescued the increased edge protrusion and retraction velocities and distances that were induced by loss of FAK (Figure 3, B and C). Furthermore, expression of either wtFAK or FAKY397F in FAK-KO cells was sufficient to increase both the protrusion efficiency and net edge advance compared with FAK-KO (Figure 3, D and E) and restore it to levels similar to those in control cells (Figure 1, D and E). These results show that FAK is required to promote protrusion efficiency and net edge advance independently of Y397 phosphorylation.


The FAK-Arp2/3 interaction promotes leading edge advance and haptosensing by coupling nascent adhesions to lamellipodia actin.

Swaminathan V, Fischer RS, Waterman CM - Mol. Biol. Cell (2016)

FAK couples leading edge protrusion to NAs independent of Y397 phosphorylation. (A) Left, representative DIC micrographs of FAK−/− knockout (FAK-KO) MEFs expressing eGFP-tagged wild-type (FAK-KO + wtFAK; top) and FAK-KO cells expressing eGFP-tagged FAKY397F mutant (FAK-KO + FAKY397F; bottom). Cells were imaged 4–6 h after plating on 10 μg/ml 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 1 min (right). (B) Box plot of velocities (μm/min) of protrusion (Vp) and retraction (Vr) of FAK-KO cells expressing the noted FAK constructs. Color coding in B–J: FAK-KO + wtFAK (red), FAK-KO (blue), and FAK-KO + FAKY397F (yellow) cells. (C) Box plot of distances (μm) of protrusion (Dp) and retraction (Dr) of FAK-KO cells expressing the noted FAK constructs. (D) Box plot of protrusion efficiency (%) of FAK-KO + wtFAK, FAK-KO, and FAK-KO + FAKY397F cells. (E) Box plot of net edge advance (μm/min) of FAK-KO + wtFAK, FAK-KO, and FAK-KO + FAKY397F cells (10–12 cells/condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test. (F) Left, representative TIRF micrographs of FAK-KO cells expressing either EGFP-wtFAK (top) or EGFP-FAKY397F (bottom; scale bar, 10 μm). Contrast inverted. Blue box indicates area zoomed in for right images, a TIRF time-lapse image sequence of eGFP-FAK (top)– or eGFP-FAKY397F (bottom)–marked adhesions at the leading edge. Time in seconds. Scale bar, 5 μm. Far right, kymograph analysis of NA dynamics from preceding images. White open arrowheads, extremely short-lived NA; white closed arrowheads, longer-lived NA. Scale bars, distance 2 μm, time 2 min. (G) Distribution of NA lifetimes in FAK-KO cells expressing either eGFP-wtFAK (FAK-KO + wtFAK; top) or EGFP-FAKY397F (FAK-K + FAKY397F; bottom). Middle, distribution of EGFP-paxillin–marked NA in FAK-KO cells (50–70 NAs in five or six cells/condition). (H) Box plots of quantification of NA lifetimes (seconds) from distributions in G. (I) Box plot for maturation fraction (dimensionless, NAs formed/NAs that mature) among >100 NAs in protruding lamellipodia of FAK-KO + EGFP-FAK, FAK-KO, and FAK-KO + EGFPFAKY397F cells (10–12 protrusions, five cells/condition). (J) Box plot of NA formation density (number/μm2 lamellipodia protrusion area) in protruding lamellipodia marked by eGFP-FAK in FAK-KO + FAK cells, eGFP-paxillin in FAK-KO cells or EGFP-FAKY397F in FAK-KO + FAKY397F cells (17–20 protrusions, five or six cells per condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test. Note: FAK-KO data in all plots are the same data as presented in Figure 1.
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Figure 3: FAK couples leading edge protrusion to NAs independent of Y397 phosphorylation. (A) Left, representative DIC micrographs of FAK−/− knockout (FAK-KO) MEFs expressing eGFP-tagged wild-type (FAK-KO + wtFAK; top) and FAK-KO cells expressing eGFP-tagged FAKY397F mutant (FAK-KO + FAKY397F; bottom). Cells were imaged 4–6 h after plating on 10 μg/ml 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 1 min (right). (B) Box plot of velocities (μm/min) of protrusion (Vp) and retraction (Vr) of FAK-KO cells expressing the noted FAK constructs. Color coding in B–J: FAK-KO + wtFAK (red), FAK-KO (blue), and FAK-KO + FAKY397F (yellow) cells. (C) Box plot of distances (μm) of protrusion (Dp) and retraction (Dr) of FAK-KO cells expressing the noted FAK constructs. (D) Box plot of protrusion efficiency (%) of FAK-KO + wtFAK, FAK-KO, and FAK-KO + FAKY397F cells. (E) Box plot of net edge advance (μm/min) of FAK-KO + wtFAK, FAK-KO, and FAK-KO + FAKY397F cells (10–12 cells/condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test. (F) Left, representative TIRF micrographs of FAK-KO cells expressing either EGFP-wtFAK (top) or EGFP-FAKY397F (bottom; scale bar, 10 μm). Contrast inverted. Blue box indicates area zoomed in for right images, a TIRF time-lapse image sequence of eGFP-FAK (top)– or eGFP-FAKY397F (bottom)–marked adhesions at the leading edge. Time in seconds. Scale bar, 5 μm. Far right, kymograph analysis of NA dynamics from preceding images. White open arrowheads, extremely short-lived NA; white closed arrowheads, longer-lived NA. Scale bars, distance 2 μm, time 2 min. (G) Distribution of NA lifetimes in FAK-KO cells expressing either eGFP-wtFAK (FAK-KO + wtFAK; top) or EGFP-FAKY397F (FAK-K + FAKY397F; bottom). Middle, distribution of EGFP-paxillin–marked NA in FAK-KO cells (50–70 NAs in five or six cells/condition). (H) Box plots of quantification of NA lifetimes (seconds) from distributions in G. (I) Box plot for maturation fraction (dimensionless, NAs formed/NAs that mature) among >100 NAs in protruding lamellipodia of FAK-KO + EGFP-FAK, FAK-KO, and FAK-KO + EGFPFAKY397F cells (10–12 protrusions, five cells/condition). (J) Box plot of NA formation density (number/μm2 lamellipodia protrusion area) in protruding lamellipodia marked by eGFP-FAK in FAK-KO + FAK cells, eGFP-paxillin in FAK-KO cells or EGFP-FAKY397F in FAK-KO + FAKY397F cells (17–20 protrusions, five or six cells per condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test. Note: FAK-KO data in all plots are the same data as presented in Figure 1.
Mentions: To determine the mechanism by which FAK couples lamellipodial protrusion to NAs, we first examined the role of FAK phosphorylation. Autophosphorylation on tyrosine 397 (Y397) mediates relief of autoinhibition and activation of FAK kinase activity and induces its binding to SH2 domain–containing signaling proteins (Frame et al., 2010). We used time-lapse DIC imaging to compare leading edge dynamics of FAK-KO cells with FAK-KO cells expressing GFP fusions of either wtFAK or nonphosphorylatable FAK (FAKY397F) at near-endogenous levels (Supplemental Figure S1A and Supplemental Movie S4). Quantitative analysis of kymographs showed that expression of either wtFAK or FAKY397F in FAK-KO cells rescued the increased edge protrusion and retraction velocities and distances that were induced by loss of FAK (Figure 3, B and C). Furthermore, expression of either wtFAK or FAKY397F in FAK-KO cells was sufficient to increase both the protrusion efficiency and net edge advance compared with FAK-KO (Figure 3, D and E) and restore it to levels similar to those in control cells (Figure 1, D and E). These results show that FAK is required to promote protrusion efficiency and net edge advance independently of Y397 phosphorylation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8019.

No MeSH data available.


Related in: MedlinePlus