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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

Microtubule-dependent adhesion dynamics are regulated through a fascin–FAK–Src signalling pathway. (A) Western blots of lysates from fascinKD cells expressing WT, S39A-, S274D- or MT1-fascin–GFP immunoprecipitated (IP) with control (IgG) or anti-FAK antibodies. Blots of immunoprecipitations (left panel) were re-probed for specified proteins. Values are denoted below each respective lane and represent relative intensity of each band as a mean±s.e.m. of the four experiments. (B) Western blots of lysates from fascinKD cells expressing GFP-tagged WT, S39A-, S274D- or MT1-fascin probed with the specified antibodies. (C) Representative images of vinculin staining in fascinKD HeLa cells expressing GFP-tagged WT Src (WTSrc) or constitutively active S527F Src (527FSrc) with WT, S39A or 274D-fascin–mFP at 60 min post-NOC washout. Scale bars: 20 μm. (D) The percentage focal adhesion coverage per cell was quantified from cells treated with NOC for 20 min (NOC) or at 60 min following NOC washout (60′W). n=45 cells were quantified across three independent experiments. Mean values±s.e.m. are shown. *P<0.05, **P<0.01, compared to untreated controls (A); *P<0.01 compared to WT fascin (D) (one-way ANOVA).
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JCS175760F6: Microtubule-dependent adhesion dynamics are regulated through a fascin–FAK–Src signalling pathway. (A) Western blots of lysates from fascinKD cells expressing WT, S39A-, S274D- or MT1-fascin–GFP immunoprecipitated (IP) with control (IgG) or anti-FAK antibodies. Blots of immunoprecipitations (left panel) were re-probed for specified proteins. Values are denoted below each respective lane and represent relative intensity of each band as a mean±s.e.m. of the four experiments. (B) Western blots of lysates from fascinKD cells expressing GFP-tagged WT, S39A-, S274D- or MT1-fascin probed with the specified antibodies. (C) Representative images of vinculin staining in fascinKD HeLa cells expressing GFP-tagged WT Src (WTSrc) or constitutively active S527F Src (527FSrc) with WT, S39A or 274D-fascin–mFP at 60 min post-NOC washout. Scale bars: 20 μm. (D) The percentage focal adhesion coverage per cell was quantified from cells treated with NOC for 20 min (NOC) or at 60 min following NOC washout (60′W). n=45 cells were quantified across three independent experiments. Mean values±s.e.m. are shown. *P<0.05, **P<0.01, compared to untreated controls (A); *P<0.01 compared to WT fascin (D) (one-way ANOVA).

Mentions: To determine whether FAK–fascin complex formation was regulated by fascin–MT binding, we performed FAK immunoprecipitations from fascinKD cells expressing WT, S39A, S274D or MT1 mutant forms of GFP–fascin. Data showed similar levels of WT and S39A-fascin in complex with FAK; however, the S274D and MT1-fascin mutants, that both exhibit increased MT binding, both showed significantly lower association in the complex (Fig. 6A). Moreover, re-probes of these immunoprecipitation complexes revealed the presence of Src, which also showed lower co-association with FAK in cells expressing S274D- or MT1-fascin (Fig. 6A). Taken together, these data suggest that fascin, FAK and Src are able to form a complex and that this is dependent upon both fascin–MT binding and the presence of a dynamic MT cytoskeleton.Fig. 6.


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)

Microtubule-dependent adhesion dynamics are regulated through a fascin–FAK–Src signalling pathway. (A) Western blots of lysates from fascinKD cells expressing WT, S39A-, S274D- or MT1-fascin–GFP immunoprecipitated (IP) with control (IgG) or anti-FAK antibodies. Blots of immunoprecipitations (left panel) were re-probed for specified proteins. Values are denoted below each respective lane and represent relative intensity of each band as a mean±s.e.m. of the four experiments. (B) Western blots of lysates from fascinKD cells expressing GFP-tagged WT, S39A-, S274D- or MT1-fascin probed with the specified antibodies. (C) Representative images of vinculin staining in fascinKD HeLa cells expressing GFP-tagged WT Src (WTSrc) or constitutively active S527F Src (527FSrc) with WT, S39A or 274D-fascin–mFP at 60 min post-NOC washout. Scale bars: 20 μm. (D) The percentage focal adhesion coverage per cell was quantified from cells treated with NOC for 20 min (NOC) or at 60 min following NOC washout (60′W). n=45 cells were quantified across three independent experiments. Mean values±s.e.m. are shown. *P<0.05, **P<0.01, compared to untreated controls (A); *P<0.01 compared to WT fascin (D) (one-way ANOVA).
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JCS175760F6: Microtubule-dependent adhesion dynamics are regulated through a fascin–FAK–Src signalling pathway. (A) Western blots of lysates from fascinKD cells expressing WT, S39A-, S274D- or MT1-fascin–GFP immunoprecipitated (IP) with control (IgG) or anti-FAK antibodies. Blots of immunoprecipitations (left panel) were re-probed for specified proteins. Values are denoted below each respective lane and represent relative intensity of each band as a mean±s.e.m. of the four experiments. (B) Western blots of lysates from fascinKD cells expressing GFP-tagged WT, S39A-, S274D- or MT1-fascin probed with the specified antibodies. (C) Representative images of vinculin staining in fascinKD HeLa cells expressing GFP-tagged WT Src (WTSrc) or constitutively active S527F Src (527FSrc) with WT, S39A or 274D-fascin–mFP at 60 min post-NOC washout. Scale bars: 20 μm. (D) The percentage focal adhesion coverage per cell was quantified from cells treated with NOC for 20 min (NOC) or at 60 min following NOC washout (60′W). n=45 cells were quantified across three independent experiments. Mean values±s.e.m. are shown. *P<0.05, **P<0.01, compared to untreated controls (A); *P<0.01 compared to WT fascin (D) (one-way ANOVA).
Mentions: To determine whether FAK–fascin complex formation was regulated by fascin–MT binding, we performed FAK immunoprecipitations from fascinKD cells expressing WT, S39A, S274D or MT1 mutant forms of GFP–fascin. Data showed similar levels of WT and S39A-fascin in complex with FAK; however, the S274D and MT1-fascin mutants, that both exhibit increased MT binding, both showed significantly lower association in the complex (Fig. 6A). Moreover, re-probes of these immunoprecipitation complexes revealed the presence of Src, which also showed lower co-association with FAK in cells expressing S274D- or MT1-fascin (Fig. 6A). Taken together, these data suggest that fascin, FAK and Src are able to form a complex and that this is dependent upon both fascin–MT binding and the presence of a dynamic MT cytoskeleton.Fig. 6.

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