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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 formation and turnover of NAs in the lamellipodium. (A) Left, representative TIRF micrographs of FAK+/+ (control, top) and FAK−/− knockout (FAK-KO, bottom) MEF cells expressing eGFP-paxillin (contrast inverted). Scale bar, 10 μm. Blue box indicates area zoomed in for images to the right, a TIRF time-lapse image sequence of eGFP-paxillin–marked adhesions at the leading edge. Time in seconds. Scale bar, 2 μm. Black line indicates the line along which the kymograph on the far right was obtained. White open arrows show extremely short-lived NAs, and white closed arrows indicate medium and longer-lived NAs. Scale bars, distance 5 μm, time 1 min. (B) Left, representative confocal micrographs of control (top) and FAK-KO (bottom) cells showing Alexa 655–phalloidin staining of actin (green) and immunofluorescence of paxillin (red), along with the color merge. Scale bar, 10 μm. Right, quantification of focal adhesion size (area, μm2) from analysis of fluorescence images of paxillin in the noted cells (10 cells/condition). Color coding in B (right) and C– F: control (green), FAK-KO (blue), and FAK-KO expressing mCherry tagged wild-type FAK (FAK-KO + wtFAK; red). (C) Distribution of the lifetimes of eGFP-paxillin–marked NA in control (top), FAK-KO (middle), and FAK-KO + wtFAK (bottom) cells. Bin size, 20 s; 50–70 NAs in five or six cells/condition. (D) Box plots of quantification of NA lifetimes from distributions in C. (E) Box plot of NA formation density (number/μm2 lamellipodia protrusion area) marked by eGFP-paxillin in protruding lamellipodia of control, FAK-KO, and FAK-KO + wtFAK cells (17–20 protrusions, five or six cells/condition). (F) Box plot of maturation fraction (dimensionless, NAs formed − NAs disassembled/NAs that mature) among >100 NAs in protruding lamellipodia of control, FAK-KO, and FAK-KO + wtFAK cells (10–12 protrusions, five cells/condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test.
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Figure 2: FAK couples leading edge protrusion to formation and turnover of NAs in the lamellipodium. (A) Left, representative TIRF micrographs of FAK+/+ (control, top) and FAK−/− knockout (FAK-KO, bottom) MEF cells expressing eGFP-paxillin (contrast inverted). Scale bar, 10 μm. Blue box indicates area zoomed in for images to the right, a TIRF time-lapse image sequence of eGFP-paxillin–marked adhesions at the leading edge. Time in seconds. Scale bar, 2 μm. Black line indicates the line along which the kymograph on the far right was obtained. White open arrows show extremely short-lived NAs, and white closed arrows indicate medium and longer-lived NAs. Scale bars, distance 5 μm, time 1 min. (B) Left, representative confocal micrographs of control (top) and FAK-KO (bottom) cells showing Alexa 655–phalloidin staining of actin (green) and immunofluorescence of paxillin (red), along with the color merge. Scale bar, 10 μm. Right, quantification of focal adhesion size (area, μm2) from analysis of fluorescence images of paxillin in the noted cells (10 cells/condition). Color coding in B (right) and C– F: control (green), FAK-KO (blue), and FAK-KO expressing mCherry tagged wild-type FAK (FAK-KO + wtFAK; red). (C) Distribution of the lifetimes of eGFP-paxillin–marked NA in control (top), FAK-KO (middle), and FAK-KO + wtFAK (bottom) cells. Bin size, 20 s; 50–70 NAs in five or six cells/condition. (D) Box plots of quantification of NA lifetimes from distributions in C. (E) Box plot of NA formation density (number/μm2 lamellipodia protrusion area) marked by eGFP-paxillin in protruding lamellipodia of control, FAK-KO, and FAK-KO + wtFAK cells (17–20 protrusions, five or six cells/condition). (F) Box plot of maturation fraction (dimensionless, NAs formed − NAs disassembled/NAs that mature) among >100 NAs in protruding lamellipodia of control, FAK-KO, and FAK-KO + wtFAK cells (10–12 protrusions, five cells/condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test.

Mentions: The finding that FAK is required to reduce the distance of the retraction phase suggests a role for FAK in coupling leading edge protrusion to substrate adhesion, possibly through formation of NAs. We expressed enhanced green fluorescent protein (eGFP)–paxillin as a marker of NAs (Choi et al., 2008; Thievessen et al., 2013) in control and FAK-KO cells and imaged NA dynamics at 3-s intervals by total internal reflection fluorescence (TIRF) microscopy (Figure 2A). Examination of time-lapse movies showed that in control cells, each round of edge protrusion was accompanied by the formation of a row of tightly spaced NAs all along the cell edge, and the bulk of these NAs disassembled as the next cycle of protrusion ensued and the next row of NAs was formed. In contrast, in FAK-KO cells, protrusions formed with apparently fewer NAs, they were not organized in a row at the edge but instead sparsely distributed throughout the protrusion, and instead of disassembling, many of them grew into mature FAs or slid centripetally and disassembled (Supplemental Movie S2).


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 formation and turnover of NAs in the lamellipodium. (A) Left, representative TIRF micrographs of FAK+/+ (control, top) and FAK−/− knockout (FAK-KO, bottom) MEF cells expressing eGFP-paxillin (contrast inverted). Scale bar, 10 μm. Blue box indicates area zoomed in for images to the right, a TIRF time-lapse image sequence of eGFP-paxillin–marked adhesions at the leading edge. Time in seconds. Scale bar, 2 μm. Black line indicates the line along which the kymograph on the far right was obtained. White open arrows show extremely short-lived NAs, and white closed arrows indicate medium and longer-lived NAs. Scale bars, distance 5 μm, time 1 min. (B) Left, representative confocal micrographs of control (top) and FAK-KO (bottom) cells showing Alexa 655–phalloidin staining of actin (green) and immunofluorescence of paxillin (red), along with the color merge. Scale bar, 10 μm. Right, quantification of focal adhesion size (area, μm2) from analysis of fluorescence images of paxillin in the noted cells (10 cells/condition). Color coding in B (right) and C– F: control (green), FAK-KO (blue), and FAK-KO expressing mCherry tagged wild-type FAK (FAK-KO + wtFAK; red). (C) Distribution of the lifetimes of eGFP-paxillin–marked NA in control (top), FAK-KO (middle), and FAK-KO + wtFAK (bottom) cells. Bin size, 20 s; 50–70 NAs in five or six cells/condition. (D) Box plots of quantification of NA lifetimes from distributions in C. (E) Box plot of NA formation density (number/μm2 lamellipodia protrusion area) marked by eGFP-paxillin in protruding lamellipodia of control, FAK-KO, and FAK-KO + wtFAK cells (17–20 protrusions, five or six cells/condition). (F) Box plot of maturation fraction (dimensionless, NAs formed − NAs disassembled/NAs that mature) among >100 NAs in protruding lamellipodia of control, FAK-KO, and FAK-KO + wtFAK cells (10–12 protrusions, five cells/condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test.
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Figure 2: FAK couples leading edge protrusion to formation and turnover of NAs in the lamellipodium. (A) Left, representative TIRF micrographs of FAK+/+ (control, top) and FAK−/− knockout (FAK-KO, bottom) MEF cells expressing eGFP-paxillin (contrast inverted). Scale bar, 10 μm. Blue box indicates area zoomed in for images to the right, a TIRF time-lapse image sequence of eGFP-paxillin–marked adhesions at the leading edge. Time in seconds. Scale bar, 2 μm. Black line indicates the line along which the kymograph on the far right was obtained. White open arrows show extremely short-lived NAs, and white closed arrows indicate medium and longer-lived NAs. Scale bars, distance 5 μm, time 1 min. (B) Left, representative confocal micrographs of control (top) and FAK-KO (bottom) cells showing Alexa 655–phalloidin staining of actin (green) and immunofluorescence of paxillin (red), along with the color merge. Scale bar, 10 μm. Right, quantification of focal adhesion size (area, μm2) from analysis of fluorescence images of paxillin in the noted cells (10 cells/condition). Color coding in B (right) and C– F: control (green), FAK-KO (blue), and FAK-KO expressing mCherry tagged wild-type FAK (FAK-KO + wtFAK; red). (C) Distribution of the lifetimes of eGFP-paxillin–marked NA in control (top), FAK-KO (middle), and FAK-KO + wtFAK (bottom) cells. Bin size, 20 s; 50–70 NAs in five or six cells/condition. (D) Box plots of quantification of NA lifetimes from distributions in C. (E) Box plot of NA formation density (number/μm2 lamellipodia protrusion area) marked by eGFP-paxillin in protruding lamellipodia of control, FAK-KO, and FAK-KO + wtFAK cells (17–20 protrusions, five or six cells/condition). (F) Box plot of maturation fraction (dimensionless, NAs formed − NAs disassembled/NAs that mature) among >100 NAs in protruding lamellipodia of control, FAK-KO, and FAK-KO + wtFAK cells (10–12 protrusions, five cells/condition). **p < 0.0001, *p < 0.005; NS, not significant; Mann–Whitney U test.
Mentions: The finding that FAK is required to reduce the distance of the retraction phase suggests a role for FAK in coupling leading edge protrusion to substrate adhesion, possibly through formation of NAs. We expressed enhanced green fluorescent protein (eGFP)–paxillin as a marker of NAs (Choi et al., 2008; Thievessen et al., 2013) in control and FAK-KO cells and imaged NA dynamics at 3-s intervals by total internal reflection fluorescence (TIRF) microscopy (Figure 2A). Examination of time-lapse movies showed that in control cells, each round of edge protrusion was accompanied by the formation of a row of tightly spaced NAs all along the cell edge, and the bulk of these NAs disassembled as the next cycle of protrusion ensued and the next row of NAs was formed. In contrast, in FAK-KO cells, protrusions formed with apparently fewer NAs, they were not organized in a row at the edge but instead sparsely distributed throughout the protrusion, and instead of disassembling, many of them grew into mature FAs or slid centripetally and disassembled (Supplemental Movie S2).

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