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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

Phosphorylation of fascin regulates MT and adhesion dynamics. (A) Example images of Coomassie and silver-stained gels from co-sedimentation analysis of purified WT versus mutant fascin alone (top gel) or with tubulin (bottom gel) in the supernatant (S) or pellet (P) fractions. Values beneath show the mean±s.e.m. percentage of fascin in the pellet from three independent experiments. (B) Example fluorescence lifetime maps of fascinKD cells expressing S39D- or S274D-fascin–GFP and tubulin–mCherry (shown in inset panels). A pseudocolour lifetime scale is shown next to each image. Graph shows quantification of FRET efficiency for WT, S39D- and S274D-fascin-expressing cells from 15 cells per condition over two independent experiments. Mean±s.e.m. values are shown. (C) Quantification of MT re-growth from images of fixed MDA MB 231 FascinKD cells expressing WT or mutant GFP–fascin at 60 min post-NOC washout. n=30 cells were quantified across three independent experiments. (D) Analysis of MT growth rate (left graph) and catastrophe events/min (right graph) in fascinKD HeLa cells re-expressing WT or mutant GFP–fascin variants as specified. Values are calculated from 25 MT per cell in five cells per experiment over three independent experiments. Example movies are shown in Movie 4. *P<0.05; **P<0.01; ***P<0.001 compared to controls (one-way ANOVA).
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JCS175760F3: Phosphorylation of fascin regulates MT and adhesion dynamics. (A) Example images of Coomassie and silver-stained gels from co-sedimentation analysis of purified WT versus mutant fascin alone (top gel) or with tubulin (bottom gel) in the supernatant (S) or pellet (P) fractions. Values beneath show the mean±s.e.m. percentage of fascin in the pellet from three independent experiments. (B) Example fluorescence lifetime maps of fascinKD cells expressing S39D- or S274D-fascin–GFP and tubulin–mCherry (shown in inset panels). A pseudocolour lifetime scale is shown next to each image. Graph shows quantification of FRET efficiency for WT, S39D- and S274D-fascin-expressing cells from 15 cells per condition over two independent experiments. Mean±s.e.m. values are shown. (C) Quantification of MT re-growth from images of fixed MDA MB 231 FascinKD cells expressing WT or mutant GFP–fascin at 60 min post-NOC washout. n=30 cells were quantified across three independent experiments. (D) Analysis of MT growth rate (left graph) and catastrophe events/min (right graph) in fascinKD HeLa cells re-expressing WT or mutant GFP–fascin variants as specified. Values are calculated from 25 MT per cell in five cells per experiment over three independent experiments. Example movies are shown in Movie 4. *P<0.05; **P<0.01; ***P<0.001 compared to controls (one-way ANOVA).

Mentions: Our data have demonstrated that fascin can associate with MTs directly, and that binding to MTs and F-actin might occur in a mutually exclusive manner. We therefore next asked whether the known sites in fascin that control actin binding might also contribute to the formation of the fascin–MT complex. To determine this, we repeated the MT co-sedimentation assays with purified WT fascin or the previously characterised mutants of fascin to mimic phosphorylated (S>D) or non-phosphorylated (S>A) states at S39 or S274 (Zanet et al., 2012). S39D, S274A and S274D are all unable to bundle F-actin as efficiently as WT fascin, whereas S39A fascin forms highly stable F-actin bundles in vitro and in cells (Ono et al., 1997; Vignjevic et al., 2006; Zanet et al., 2012). All fascin mutant proteins showed a degree of co-sedimentation with MTs (Fig. 3A); however, S274D-fascin showed significantly higher binding compared to WT protein and other mutants (Fig. 3A), as was also observed for the MT1-fascin mutant (Fig. 2C). To further confirm that S274D-fascin was more strongly associated with MT in cells, we performed FRET-FLIM analysis between fascin and tubulin in cells as described in Fig. 2D. FRET efficiency data from multiple cells demonstrated a direct interaction between S274D-fascin–GFP and tubulin–mCherry that was significantly higher than both WT fascin and S39D-fascin (Fig. 3B). This data again supports the notion that F-actin-binding mutants of fascin do not also lead to impaired fascin–MT association, suggesting the two cytoskeletal binding events can be independently controlled.Fig. 3.


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)

Phosphorylation of fascin regulates MT and adhesion dynamics. (A) Example images of Coomassie and silver-stained gels from co-sedimentation analysis of purified WT versus mutant fascin alone (top gel) or with tubulin (bottom gel) in the supernatant (S) or pellet (P) fractions. Values beneath show the mean±s.e.m. percentage of fascin in the pellet from three independent experiments. (B) Example fluorescence lifetime maps of fascinKD cells expressing S39D- or S274D-fascin–GFP and tubulin–mCherry (shown in inset panels). A pseudocolour lifetime scale is shown next to each image. Graph shows quantification of FRET efficiency for WT, S39D- and S274D-fascin-expressing cells from 15 cells per condition over two independent experiments. Mean±s.e.m. values are shown. (C) Quantification of MT re-growth from images of fixed MDA MB 231 FascinKD cells expressing WT or mutant GFP–fascin at 60 min post-NOC washout. n=30 cells were quantified across three independent experiments. (D) Analysis of MT growth rate (left graph) and catastrophe events/min (right graph) in fascinKD HeLa cells re-expressing WT or mutant GFP–fascin variants as specified. Values are calculated from 25 MT per cell in five cells per experiment over three independent experiments. Example movies are shown in Movie 4. *P<0.05; **P<0.01; ***P<0.001 compared to controls (one-way ANOVA).
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Related In: Results  -  Collection

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JCS175760F3: Phosphorylation of fascin regulates MT and adhesion dynamics. (A) Example images of Coomassie and silver-stained gels from co-sedimentation analysis of purified WT versus mutant fascin alone (top gel) or with tubulin (bottom gel) in the supernatant (S) or pellet (P) fractions. Values beneath show the mean±s.e.m. percentage of fascin in the pellet from three independent experiments. (B) Example fluorescence lifetime maps of fascinKD cells expressing S39D- or S274D-fascin–GFP and tubulin–mCherry (shown in inset panels). A pseudocolour lifetime scale is shown next to each image. Graph shows quantification of FRET efficiency for WT, S39D- and S274D-fascin-expressing cells from 15 cells per condition over two independent experiments. Mean±s.e.m. values are shown. (C) Quantification of MT re-growth from images of fixed MDA MB 231 FascinKD cells expressing WT or mutant GFP–fascin at 60 min post-NOC washout. n=30 cells were quantified across three independent experiments. (D) Analysis of MT growth rate (left graph) and catastrophe events/min (right graph) in fascinKD HeLa cells re-expressing WT or mutant GFP–fascin variants as specified. Values are calculated from 25 MT per cell in five cells per experiment over three independent experiments. Example movies are shown in Movie 4. *P<0.05; **P<0.01; ***P<0.001 compared to controls (one-way ANOVA).
Mentions: Our data have demonstrated that fascin can associate with MTs directly, and that binding to MTs and F-actin might occur in a mutually exclusive manner. We therefore next asked whether the known sites in fascin that control actin binding might also contribute to the formation of the fascin–MT complex. To determine this, we repeated the MT co-sedimentation assays with purified WT fascin or the previously characterised mutants of fascin to mimic phosphorylated (S>D) or non-phosphorylated (S>A) states at S39 or S274 (Zanet et al., 2012). S39D, S274A and S274D are all unable to bundle F-actin as efficiently as WT fascin, whereas S39A fascin forms highly stable F-actin bundles in vitro and in cells (Ono et al., 1997; Vignjevic et al., 2006; Zanet et al., 2012). All fascin mutant proteins showed a degree of co-sedimentation with MTs (Fig. 3A); however, S274D-fascin showed significantly higher binding compared to WT protein and other mutants (Fig. 3A), as was also observed for the MT1-fascin mutant (Fig. 2C). To further confirm that S274D-fascin was more strongly associated with MT in cells, we performed FRET-FLIM analysis between fascin and tubulin in cells as described in Fig. 2D. FRET efficiency data from multiple cells demonstrated a direct interaction between S274D-fascin–GFP and tubulin–mCherry that was significantly higher than both WT fascin and S39D-fascin (Fig. 3B). This data again supports the notion that F-actin-binding mutants of fascin do not also lead to impaired fascin–MT association, suggesting the two cytoskeletal binding events can be independently controlled.Fig. 3.

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