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Eps8 controls Src- and FAK-dependent phenotypes in squamous carcinoma cells.

Schoenherr C, Serrels B, Proby C, Cunningham DL, Findlay JE, Baillie GS, Heath JK, Frame MC - J. Cell. Sci. (2014)

Bottom Line: Eps8 is an actin regulatory scaffold protein whose expression is increased in squamous cell carcinoma (SCC) cells.Here, we describe two new roles for Eps8.Therefore, Eps8 is a crucial mediator of Src- and FAK-regulated processes; it participates in specific biochemical complexes and promotes actin re-arrangements that determine the spatial localization of Src, and modulates the functions of Src and FAK during invasive migration.

View Article: PubMed Central - PubMed

Affiliation: Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South, EH4 2XR Edinburgh, UK.

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Eps8 is required for active Src localization to autophagosomes. (A) FAK WT and FAK−/− cells were transiently transfected with Eps8 siRNA (siEps8 #1 and #2; siControl, control siRNA), fixed and stained with anti-Eps8 and anti-Src pY416 antibodies. The quantification is representative of three independent experiments. Solid arrows indicate focal adhesions. Dashed arrows indicate active Src in autophagosomes. Scale bars: 20 µm. Results are mean±s.d. *P<0.01 (Student's t-test). (B) FAK−/− SCC cells were infected with either Eps8 (shE) or non-targeting (shC) shRNA and LC3B was immunoprecipitated (IP) from cell lysates. Samples were subjected to western blot analysis using anti-Eps8 and anti-Src pY416 antibodies. (C) FAK−/− cells were transiently transfected with two independent Eps8 siRNAs, fixed and stained with anti-Src pY416 antibody and TRITC–phalloidin. Solid arrows indicate active Src localization at focal adhesions. Dashed arrows indicate active Src in intracellular puncta. Scale bars: 20 µm. Insets show a magnified view of the boxed area.
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f05: Eps8 is required for active Src localization to autophagosomes. (A) FAK WT and FAK−/− cells were transiently transfected with Eps8 siRNA (siEps8 #1 and #2; siControl, control siRNA), fixed and stained with anti-Eps8 and anti-Src pY416 antibodies. The quantification is representative of three independent experiments. Solid arrows indicate focal adhesions. Dashed arrows indicate active Src in autophagosomes. Scale bars: 20 µm. Results are mean±s.d. *P<0.01 (Student's t-test). (B) FAK−/− SCC cells were infected with either Eps8 (shE) or non-targeting (shC) shRNA and LC3B was immunoprecipitated (IP) from cell lysates. Samples were subjected to western blot analysis using anti-Eps8 and anti-Src pY416 antibodies. (C) FAK−/− cells were transiently transfected with two independent Eps8 siRNAs, fixed and stained with anti-Src pY416 antibody and TRITC–phalloidin. Solid arrows indicate active Src localization at focal adhesions. Dashed arrows indicate active Src in intracellular puncta. Scale bars: 20 µm. Insets show a magnified view of the boxed area.

Mentions: We next investigated whether Eps8 plays a key role in the trafficking of active Src to autophagosomes or whether it was co-trafficked as a ‘passenger’. We used two independent siRNAs to efficiently suppress expression of endogenous Eps8 as judged by immunofluorescence and immunoblotting (Fig. 1E; Fig. 2A; Fig. 5A). Knockdown of Eps8 in FAK WT SCC cells had no effect on the localization of active Src, which remained at focal adhesions (Fig. 5A, top panel, solid arrows). However, in FAK−/− SCC cells, Eps8 knockdown significantly reduced the number of Src-positive intracellular puncta (Fig. 5A, bottom panels, dashed arrows). Active Src was now predominantly re-localized to focal adhesions as indicated by co-staining with paxillin (Fig. 5A, bottom panel, solid arrows and supplementary material Fig. S3C). Similar results were obtained by shRNA-mediated stable knockdown of Eps8 (supplementary material Fig. S3D,E). Furthermore, we found that the biochemical complex between active Src and LC3B in SCC FAK−/− cells was reduced upon stable knockdown of Eps8, providing biochemical evidence that Eps8 function is required to induce the binding of Src to LC3B during autophagic targeting of Src. Eps8 did not affect general autophagic flux as judged by immunoblotting for LC3B (Fig. 5B), including after chloroquine treatment (supplementary material Fig. S4A,B). These results indicate that Eps8 is required for the selective trafficking of active Src to autophagosomes.


Eps8 controls Src- and FAK-dependent phenotypes in squamous carcinoma cells.

Schoenherr C, Serrels B, Proby C, Cunningham DL, Findlay JE, Baillie GS, Heath JK, Frame MC - J. Cell. Sci. (2014)

Eps8 is required for active Src localization to autophagosomes. (A) FAK WT and FAK−/− cells were transiently transfected with Eps8 siRNA (siEps8 #1 and #2; siControl, control siRNA), fixed and stained with anti-Eps8 and anti-Src pY416 antibodies. The quantification is representative of three independent experiments. Solid arrows indicate focal adhesions. Dashed arrows indicate active Src in autophagosomes. Scale bars: 20 µm. Results are mean±s.d. *P<0.01 (Student's t-test). (B) FAK−/− SCC cells were infected with either Eps8 (shE) or non-targeting (shC) shRNA and LC3B was immunoprecipitated (IP) from cell lysates. Samples were subjected to western blot analysis using anti-Eps8 and anti-Src pY416 antibodies. (C) FAK−/− cells were transiently transfected with two independent Eps8 siRNAs, fixed and stained with anti-Src pY416 antibody and TRITC–phalloidin. Solid arrows indicate active Src localization at focal adhesions. Dashed arrows indicate active Src in intracellular puncta. Scale bars: 20 µm. Insets show a magnified view of the boxed area.
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Related In: Results  -  Collection

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f05: Eps8 is required for active Src localization to autophagosomes. (A) FAK WT and FAK−/− cells were transiently transfected with Eps8 siRNA (siEps8 #1 and #2; siControl, control siRNA), fixed and stained with anti-Eps8 and anti-Src pY416 antibodies. The quantification is representative of three independent experiments. Solid arrows indicate focal adhesions. Dashed arrows indicate active Src in autophagosomes. Scale bars: 20 µm. Results are mean±s.d. *P<0.01 (Student's t-test). (B) FAK−/− SCC cells were infected with either Eps8 (shE) or non-targeting (shC) shRNA and LC3B was immunoprecipitated (IP) from cell lysates. Samples were subjected to western blot analysis using anti-Eps8 and anti-Src pY416 antibodies. (C) FAK−/− cells were transiently transfected with two independent Eps8 siRNAs, fixed and stained with anti-Src pY416 antibody and TRITC–phalloidin. Solid arrows indicate active Src localization at focal adhesions. Dashed arrows indicate active Src in intracellular puncta. Scale bars: 20 µm. Insets show a magnified view of the boxed area.
Mentions: We next investigated whether Eps8 plays a key role in the trafficking of active Src to autophagosomes or whether it was co-trafficked as a ‘passenger’. We used two independent siRNAs to efficiently suppress expression of endogenous Eps8 as judged by immunofluorescence and immunoblotting (Fig. 1E; Fig. 2A; Fig. 5A). Knockdown of Eps8 in FAK WT SCC cells had no effect on the localization of active Src, which remained at focal adhesions (Fig. 5A, top panel, solid arrows). However, in FAK−/− SCC cells, Eps8 knockdown significantly reduced the number of Src-positive intracellular puncta (Fig. 5A, bottom panels, dashed arrows). Active Src was now predominantly re-localized to focal adhesions as indicated by co-staining with paxillin (Fig. 5A, bottom panel, solid arrows and supplementary material Fig. S3C). Similar results were obtained by shRNA-mediated stable knockdown of Eps8 (supplementary material Fig. S3D,E). Furthermore, we found that the biochemical complex between active Src and LC3B in SCC FAK−/− cells was reduced upon stable knockdown of Eps8, providing biochemical evidence that Eps8 function is required to induce the binding of Src to LC3B during autophagic targeting of Src. Eps8 did not affect general autophagic flux as judged by immunoblotting for LC3B (Fig. 5B), including after chloroquine treatment (supplementary material Fig. S4A,B). These results indicate that Eps8 is required for the selective trafficking of active Src to autophagosomes.

Bottom Line: Eps8 is an actin regulatory scaffold protein whose expression is increased in squamous cell carcinoma (SCC) cells.Here, we describe two new roles for Eps8.Therefore, Eps8 is a crucial mediator of Src- and FAK-regulated processes; it participates in specific biochemical complexes and promotes actin re-arrangements that determine the spatial localization of Src, and modulates the functions of Src and FAK during invasive migration.

View Article: PubMed Central - PubMed

Affiliation: Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South, EH4 2XR Edinburgh, UK.

Show MeSH
Related in: MedlinePlus