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A direct interaction between fascin and microtubules contributes to adhesion dynamics and cell migration.

Villari G, Jayo A, Zanet J, Fitch B, Serrels B, Frame M, Stramer BM, Goult BT, Parsons M - J. Cell. Sci. (2015)

Bottom Line: However, potential non-actin bundling roles for fascin remain unknown.Here, we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling.These findings shed light on new non actin-dependent roles for fascin and might have implications for the design of therapies to target fascin in metastatic disease.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, Guys Campus, London SE1 1UL, UK.

No MeSH data available.


Related in: MedlinePlus

Fascin regulates activation of FAK. (A) Lysates from control (ctrl) and fascinKD MDA MB 231 cells that were either untreated (UT), treated with NOC for 20 min (NOC) or at 30 or 60 min post-NOC washout were subjected to western blot analysis for pY397-FAK (pYFAK) or total FAK. The graph beneath the blots shows densitometry quantification of pY397-FAK/total FAK levels from three independent experiments. Mean±s.e.m. values are shown. (B) Representative confocal images of regions of control MDA MB 231 cells that were untreated (UT) or following NOC washout (60′W) fixed and stained for endogenous fascin (green), FAK (red) and tubulin (blue). Merged images are shown. Scale bar: 2 μm. A profile of staining along the indicated line is shown underneath the images. (C) Example images of FAK staining (red) at focal adhesions at the periphery of fascinKD MDA MB 231 cells expressing the specified fascin–GFP constructs (green) co-stained for tubulin (blue). Merged images are shown in top panels, and fascin and tubulin channels are shown as black-and-white images below. Scale bar: 15 μm. (D) Western blot of lysates from fascinKD or the specified GFP–fascin rescued MDA MB 231 cells at 60 min post-NOC washout, probed for pYFAK, FAK or tubulin. Numbers beneath blots are a mean±s.e.m. densitometry quantification of pY397-FAK/total FAK levels from three independent experiments. (E) Western blots of lysates from control cells treated with NOC or Taxol and immunoprecipitated (IP) with control (IgG) or anti-FAK antibodies. Blots were probed for specified proteins. Relative fascin and pY397-FAK levels in each immunoprecipitation lane were quantified over four independent experiments (mean±s.e.m. densitometry values are denoted below each respective lane). *P<0.01 compared to control (A,D); *P<0.001 compared to untreated controls (E) (A,D, one-way ANOVA; E, Student's t-test).
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JCS175760F5: Fascin regulates activation of FAK. (A) Lysates from control (ctrl) and fascinKD MDA MB 231 cells that were either untreated (UT), treated with NOC for 20 min (NOC) or at 30 or 60 min post-NOC washout were subjected to western blot analysis for pY397-FAK (pYFAK) or total FAK. The graph beneath the blots shows densitometry quantification of pY397-FAK/total FAK levels from three independent experiments. Mean±s.e.m. values are shown. (B) Representative confocal images of regions of control MDA MB 231 cells that were untreated (UT) or following NOC washout (60′W) fixed and stained for endogenous fascin (green), FAK (red) and tubulin (blue). Merged images are shown. Scale bar: 2 μm. A profile of staining along the indicated line is shown underneath the images. (C) Example images of FAK staining (red) at focal adhesions at the periphery of fascinKD MDA MB 231 cells expressing the specified fascin–GFP constructs (green) co-stained for tubulin (blue). Merged images are shown in top panels, and fascin and tubulin channels are shown as black-and-white images below. Scale bar: 15 μm. (D) Western blot of lysates from fascinKD or the specified GFP–fascin rescued MDA MB 231 cells at 60 min post-NOC washout, probed for pYFAK, FAK or tubulin. Numbers beneath blots are a mean±s.e.m. densitometry quantification of pY397-FAK/total FAK levels from three independent experiments. (E) Western blots of lysates from control cells treated with NOC or Taxol and immunoprecipitated (IP) with control (IgG) or anti-FAK antibodies. Blots were probed for specified proteins. Relative fascin and pY397-FAK levels in each immunoprecipitation lane were quantified over four independent experiments (mean±s.e.m. densitometry values are denoted below each respective lane). *P<0.01 compared to control (A,D); *P<0.001 compared to untreated controls (E) (A,D, one-way ANOVA; E, Student's t-test).

Mentions: We next asked how fascin–MT binding might mechanistically impact on focal adhesion signalling. FAK is a key component of focal adhesions and is known to recruit the non-receptor tyrosine kinase Src following auto-phosphorylation at Tyr397. The activation of FAK and induction of the FAK-Src complex has previously been shown to play a key role in adhesion dynamics through clathrin-dependent endocytosis of integrins and other adhesion components (Ezratty et al., 2009, 2005; Wang et al., 2011). Furthermore, MT-induced disassembly of adhesions requires activation of the FAK-Src pathway, and disruption of the FAK phosphorylation cycle leads to enhanced focal adhesion growth and stability (Hamadi et al., 2005; Wang et al., 2011). Thus, we postulated that fascin might operate upstream of the FAK–Src pathway to regulate adhesion dynamics. In order to determine whether fascin might regulate focal adhesion dynamics through this pathway, we quantified active FAK levels in lysates from control or fascinKD cells throughout the NOC washout assay. Data demonstrated that fascin-depleted cells exhibited significantly lower active FAK under basal conditions and also failed to activate FAK (through phosphorylation of Y397) at 60 min after drug washout and focal adhesion disassembly (Fig. 5A). Confocal imaging and linescan analysis of WT-fascin-expressing cells further revealed that a population of endogenous fascin colocalised with FAK and MTs at peripheral focal adhesion sites following NOC washout (Fig. 5B). Investigation of FAK and tubulin in cells expressing fascin mutants further revealed larger MT bundles overlapping with FAK-positive adhesions in S274D-fascin-expressing cells, and partial colocalisation between S39A-fascin and FAK, but not the other fascin mutants (Fig. 5C). Moreover, biochemical analysis of FAK phosphorylated at tyrosine (pY-FAK) levels following NOC washout in these cells demonstrated that re-expression of S274D-fascin did not support activation of FAK upon MT re-growth to the extent seen in cells expressing all other fascin forms (Fig. 5D). These data together suggest that fascin is required for activation of FAK during MT-induced adhesion disassembly, and that co-ordination between these functions likely requires a dynamic cycle between fascin–actin and fascin–MT binding.Fig. 5.


A direct interaction between fascin and microtubules contributes to adhesion dynamics and cell migration.

Villari G, Jayo A, Zanet J, Fitch B, Serrels B, Frame M, Stramer BM, Goult BT, Parsons M - J. Cell. Sci. (2015)

Fascin regulates activation of FAK. (A) Lysates from control (ctrl) and fascinKD MDA MB 231 cells that were either untreated (UT), treated with NOC for 20 min (NOC) or at 30 or 60 min post-NOC washout were subjected to western blot analysis for pY397-FAK (pYFAK) or total FAK. The graph beneath the blots shows densitometry quantification of pY397-FAK/total FAK levels from three independent experiments. Mean±s.e.m. values are shown. (B) Representative confocal images of regions of control MDA MB 231 cells that were untreated (UT) or following NOC washout (60′W) fixed and stained for endogenous fascin (green), FAK (red) and tubulin (blue). Merged images are shown. Scale bar: 2 μm. A profile of staining along the indicated line is shown underneath the images. (C) Example images of FAK staining (red) at focal adhesions at the periphery of fascinKD MDA MB 231 cells expressing the specified fascin–GFP constructs (green) co-stained for tubulin (blue). Merged images are shown in top panels, and fascin and tubulin channels are shown as black-and-white images below. Scale bar: 15 μm. (D) Western blot of lysates from fascinKD or the specified GFP–fascin rescued MDA MB 231 cells at 60 min post-NOC washout, probed for pYFAK, FAK or tubulin. Numbers beneath blots are a mean±s.e.m. densitometry quantification of pY397-FAK/total FAK levels from three independent experiments. (E) Western blots of lysates from control cells treated with NOC or Taxol and immunoprecipitated (IP) with control (IgG) or anti-FAK antibodies. Blots were probed for specified proteins. Relative fascin and pY397-FAK levels in each immunoprecipitation lane were quantified over four independent experiments (mean±s.e.m. densitometry values are denoted below each respective lane). *P<0.01 compared to control (A,D); *P<0.001 compared to untreated controls (E) (A,D, one-way ANOVA; E, Student's t-test).
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JCS175760F5: Fascin regulates activation of FAK. (A) Lysates from control (ctrl) and fascinKD MDA MB 231 cells that were either untreated (UT), treated with NOC for 20 min (NOC) or at 30 or 60 min post-NOC washout were subjected to western blot analysis for pY397-FAK (pYFAK) or total FAK. The graph beneath the blots shows densitometry quantification of pY397-FAK/total FAK levels from three independent experiments. Mean±s.e.m. values are shown. (B) Representative confocal images of regions of control MDA MB 231 cells that were untreated (UT) or following NOC washout (60′W) fixed and stained for endogenous fascin (green), FAK (red) and tubulin (blue). Merged images are shown. Scale bar: 2 μm. A profile of staining along the indicated line is shown underneath the images. (C) Example images of FAK staining (red) at focal adhesions at the periphery of fascinKD MDA MB 231 cells expressing the specified fascin–GFP constructs (green) co-stained for tubulin (blue). Merged images are shown in top panels, and fascin and tubulin channels are shown as black-and-white images below. Scale bar: 15 μm. (D) Western blot of lysates from fascinKD or the specified GFP–fascin rescued MDA MB 231 cells at 60 min post-NOC washout, probed for pYFAK, FAK or tubulin. Numbers beneath blots are a mean±s.e.m. densitometry quantification of pY397-FAK/total FAK levels from three independent experiments. (E) Western blots of lysates from control cells treated with NOC or Taxol and immunoprecipitated (IP) with control (IgG) or anti-FAK antibodies. Blots were probed for specified proteins. Relative fascin and pY397-FAK levels in each immunoprecipitation lane were quantified over four independent experiments (mean±s.e.m. densitometry values are denoted below each respective lane). *P<0.01 compared to control (A,D); *P<0.001 compared to untreated controls (E) (A,D, one-way ANOVA; E, Student's t-test).
Mentions: We next asked how fascin–MT binding might mechanistically impact on focal adhesion signalling. FAK is a key component of focal adhesions and is known to recruit the non-receptor tyrosine kinase Src following auto-phosphorylation at Tyr397. The activation of FAK and induction of the FAK-Src complex has previously been shown to play a key role in adhesion dynamics through clathrin-dependent endocytosis of integrins and other adhesion components (Ezratty et al., 2009, 2005; Wang et al., 2011). Furthermore, MT-induced disassembly of adhesions requires activation of the FAK-Src pathway, and disruption of the FAK phosphorylation cycle leads to enhanced focal adhesion growth and stability (Hamadi et al., 2005; Wang et al., 2011). Thus, we postulated that fascin might operate upstream of the FAK–Src pathway to regulate adhesion dynamics. In order to determine whether fascin might regulate focal adhesion dynamics through this pathway, we quantified active FAK levels in lysates from control or fascinKD cells throughout the NOC washout assay. Data demonstrated that fascin-depleted cells exhibited significantly lower active FAK under basal conditions and also failed to activate FAK (through phosphorylation of Y397) at 60 min after drug washout and focal adhesion disassembly (Fig. 5A). Confocal imaging and linescan analysis of WT-fascin-expressing cells further revealed that a population of endogenous fascin colocalised with FAK and MTs at peripheral focal adhesion sites following NOC washout (Fig. 5B). Investigation of FAK and tubulin in cells expressing fascin mutants further revealed larger MT bundles overlapping with FAK-positive adhesions in S274D-fascin-expressing cells, and partial colocalisation between S39A-fascin and FAK, but not the other fascin mutants (Fig. 5C). Moreover, biochemical analysis of FAK phosphorylated at tyrosine (pY-FAK) levels following NOC washout in these cells demonstrated that re-expression of S274D-fascin did not support activation of FAK upon MT re-growth to the extent seen in cells expressing all other fascin forms (Fig. 5D). These data together suggest that fascin is required for activation of FAK during MT-induced adhesion disassembly, and that co-ordination between these functions likely requires a dynamic cycle between fascin–actin and fascin–MT binding.Fig. 5.

Bottom Line: However, potential non-actin bundling roles for fascin remain unknown.Here, we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling.These findings shed light on new non actin-dependent roles for fascin and might have implications for the design of therapies to target fascin in metastatic disease.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, Guys Campus, London SE1 1UL, UK.

No MeSH data available.


Related in: MedlinePlus