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Suppression of chemotaxis by SSeCKS via scaffolding of phosphoinositol phosphates and the recruitment of the Cdc42 GEF, Frabin, to the leading edge.

Ko HK, Guo LW, Su B, Gao L, Gelman IH - PLoS ONE (2014)

Bottom Line: Frabin knockdown in SSeCKS- MEF restores leading edge lamellipodia and chemotaxis inhibition.However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin.Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York, United States of America.

ABSTRACT
Chemotaxis is controlled by interactions between receptors, Rho-family GTPases, phosphatidylinositol 3-kinases, and cytoskeleton remodeling proteins. We investigated how the metastasis suppressor, SSeCKS, attenuates chemotaxis. Chemotaxis activity inversely correlated with SSeCKS levels in mouse embryo fibroblasts (MEF), DU145 and MDA-MB-231 cancer cells. SSeCKS loss induced chemotactic velocity and linear directionality, correlating with replacement of leading edge lamellipodia with fascin-enriched filopodia-like extensions, the formation of thickened longitudinal F-actin stress fibers reaching to filopodial tips, relative enrichments at the leading edge of phosphatidylinositol (3,4,5)P3 (PIP3), Akt, PKC-ζ, Cdc42-GTP and active Src (SrcpoY416), and a loss of Rac1. Leading edge lamellipodia and chemotaxis inhibition in SSeCKS- MEF could be restored by full-length SSeCKS or SSeCKS deleted of its Src-binding domain (ΔSrc), but not by SSeCKS deleted of its three MARCKS (myristylated alanine-rich C kinase substrate) polybasic domains (ΔPBD), which bind PIP2 and PIP3. The enrichment of activated Cdc42 in SSeCKS- leading edge filopodia correlated with recruitment of the Cdc42-specific guanine nucleotide exchange factor, Frabin, likely recruited via multiple PIP2/3-binding domains. Frabin knockdown in SSeCKS- MEF restores leading edge lamellipodia and chemotaxis inhibition. However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin. Consistent with the notion that chemotaxis is controlled by SSeCKS-PIP (vs. -Src) scaffolding activity, constitutively-active phosphatidylinositol 3-kinase could override the ability of the Src inhibitor, SKI-606, to suppress chemotaxis and filopodial enrichment of Frabin in SSeCKS- MEF. Our data suggest a role for SSeCKS in controlling Rac1 vs. Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement.

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SSeCKS inhibits chemotaxis and affects leading edge protrusions.A, Relative chemotaxis of MEF (WT or KO), DU145 (transfected with control [con] or human SSeCKS-siRNA [si]) and MDA-MB-231 cells (transfected with empty vector [V] or an SSeCKS-GFP expression plasmid [SS]), as measured in Boyden chamber assays using serum as the chemoattractant. Error bars, S.E. of triplicate assays. *, p<0.02, **, p<0.005. B, IB analysis of SSeCKS and GAPDH levels in the cells described in Panel A. C, Relative chemotaxis of WT or KO MEF using PDGF as the chemoattractant in Boyden chamber assays. Error bars, S.E. of three wounding fields in two independent experiments. **, p<0.02. D, Relative ability of WT or KO MEF to close wound scratches, based on measuring three wound field gaps at a given time in triplicate experiments. E, Agarose chemotaxis spot assay. Top: left panel- cartoon of motile cells (black) moving towards (red arrows) an agarose spot containing chemoattractant; right panels- example of assay without (“culture media” plus PBS; left) or with chemoattractant gradient (“serum-free media” plus EGF/PDGF in spot; right). Bottom: Leading edges of chemotactic WT cells predominantly display lamellipodia whereas those of KO cells predominantly display filopodia-like extensions. Left panels- phase contrast microscopy of chemotactic cells. Open-head black arrows, filopodia; closed-head black arrows, lamellipodia; white arrows, chemotaxis direction. Middle and right panels- IFA staining of SSeCKS or F-actin in WT or KO MEF. Arrows, chemotaxis direction. Scale bar, 10 µm. F, Fraction of chemotactic WT or KO cells with leading edge lamellipodia or filopodia. Error bars, S.E. of 5 visual fields with at least 10 cells/field in three independent experiments. *, p<0.02, **, p<0.005. G, Percentage of chemotactic WT or KO cells in Panel F with <1, 1–3 or >3 filopodia/leading edge. **, p<0.005. H, Induction of lamellipodia formation in MDA-MB-231 cells transfected with SSeCKS-GFP (vs. GFP vector alone), as shown by IFA for GFP (left panels) or F-actin (center), or following quantification (graph, right). Arrows, chemotaxis direction. Scale bar, 10 µm. Error bars, S.E. of 5 visual fields with at least 10 cells/field in three independent experiments. I, IFA for fascin and F-actin in WT and KO MEF. Short arrows, fascin-staining filopodia. Long arrows, chemotaxis direction.
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pone-0111534-g001: SSeCKS inhibits chemotaxis and affects leading edge protrusions.A, Relative chemotaxis of MEF (WT or KO), DU145 (transfected with control [con] or human SSeCKS-siRNA [si]) and MDA-MB-231 cells (transfected with empty vector [V] or an SSeCKS-GFP expression plasmid [SS]), as measured in Boyden chamber assays using serum as the chemoattractant. Error bars, S.E. of triplicate assays. *, p<0.02, **, p<0.005. B, IB analysis of SSeCKS and GAPDH levels in the cells described in Panel A. C, Relative chemotaxis of WT or KO MEF using PDGF as the chemoattractant in Boyden chamber assays. Error bars, S.E. of three wounding fields in two independent experiments. **, p<0.02. D, Relative ability of WT or KO MEF to close wound scratches, based on measuring three wound field gaps at a given time in triplicate experiments. E, Agarose chemotaxis spot assay. Top: left panel- cartoon of motile cells (black) moving towards (red arrows) an agarose spot containing chemoattractant; right panels- example of assay without (“culture media” plus PBS; left) or with chemoattractant gradient (“serum-free media” plus EGF/PDGF in spot; right). Bottom: Leading edges of chemotactic WT cells predominantly display lamellipodia whereas those of KO cells predominantly display filopodia-like extensions. Left panels- phase contrast microscopy of chemotactic cells. Open-head black arrows, filopodia; closed-head black arrows, lamellipodia; white arrows, chemotaxis direction. Middle and right panels- IFA staining of SSeCKS or F-actin in WT or KO MEF. Arrows, chemotaxis direction. Scale bar, 10 µm. F, Fraction of chemotactic WT or KO cells with leading edge lamellipodia or filopodia. Error bars, S.E. of 5 visual fields with at least 10 cells/field in three independent experiments. *, p<0.02, **, p<0.005. G, Percentage of chemotactic WT or KO cells in Panel F with <1, 1–3 or >3 filopodia/leading edge. **, p<0.005. H, Induction of lamellipodia formation in MDA-MB-231 cells transfected with SSeCKS-GFP (vs. GFP vector alone), as shown by IFA for GFP (left panels) or F-actin (center), or following quantification (graph, right). Arrows, chemotaxis direction. Scale bar, 10 µm. Error bars, S.E. of 5 visual fields with at least 10 cells/field in three independent experiments. I, IFA for fascin and F-actin in WT and KO MEF. Short arrows, fascin-staining filopodia. Long arrows, chemotaxis direction.

Mentions: Our group reported previously that SSeCKS suppressed chemotaxis in rat MATLyLu prostate cancer cells without affecting cell motility in monolayer wounding assays [26]. Because chemotaxis assays typically measure parameters of individual cell migration whereas monolayer wounding reflects collective movements of cell sheets, this suggests that SSeCKS controls specific aspects of individual cell movement. To further address the role of SSeCKS in controlling chemotaxis, we compared motility of cells in Boyden chambers assays consisting of chemoattractants in the bottom chamber. SSeCKS- (KO)-MEF displayed increased chemotaxis relative to matched WT MEF against serum (Fig. 1A) or PDGF-BB (20 ng/ml) (Fig. 1C). SSeCKS levels in DU145 and MDA-MB-231 are considered downregulated compared to untransformed epithelial cells [36], although to a greater extent in MDA-MB-231 cells, and thus, we sought to knockdown SSeCKS/AKAP12 levels in DU145 and overexpress SSeCKS in MDA-MB-231 cells. The siRNA-mediated knockdown of human SSeCKS/AKAP12 in DU145 prostate cancer cells resulted in increased chemotaxis to serum, whereas its overexpression in MDA-MB-231 breast cancer cells inhibited chemotaxis (Fig. 1A). The knockdown of both endogenous α and β SSeCKS isoforms (upper and lower bands, respectively; ref. [37]) or overexpression of ectopic αSSeCKS isoform in MDA-MB-231 cells was verified by immunoblotting (Fig. 1B). Significantly, the loss of SSeCKS had no effect on cell migration involving the closure of monolayer wounds (Fig. 1D). Taken together, these data suggest that SSeCKS regulates specialized motility such as chemotaxis in both untransformed and cancer cells.


Suppression of chemotaxis by SSeCKS via scaffolding of phosphoinositol phosphates and the recruitment of the Cdc42 GEF, Frabin, to the leading edge.

Ko HK, Guo LW, Su B, Gao L, Gelman IH - PLoS ONE (2014)

SSeCKS inhibits chemotaxis and affects leading edge protrusions.A, Relative chemotaxis of MEF (WT or KO), DU145 (transfected with control [con] or human SSeCKS-siRNA [si]) and MDA-MB-231 cells (transfected with empty vector [V] or an SSeCKS-GFP expression plasmid [SS]), as measured in Boyden chamber assays using serum as the chemoattractant. Error bars, S.E. of triplicate assays. *, p<0.02, **, p<0.005. B, IB analysis of SSeCKS and GAPDH levels in the cells described in Panel A. C, Relative chemotaxis of WT or KO MEF using PDGF as the chemoattractant in Boyden chamber assays. Error bars, S.E. of three wounding fields in two independent experiments. **, p<0.02. D, Relative ability of WT or KO MEF to close wound scratches, based on measuring three wound field gaps at a given time in triplicate experiments. E, Agarose chemotaxis spot assay. Top: left panel- cartoon of motile cells (black) moving towards (red arrows) an agarose spot containing chemoattractant; right panels- example of assay without (“culture media” plus PBS; left) or with chemoattractant gradient (“serum-free media” plus EGF/PDGF in spot; right). Bottom: Leading edges of chemotactic WT cells predominantly display lamellipodia whereas those of KO cells predominantly display filopodia-like extensions. Left panels- phase contrast microscopy of chemotactic cells. Open-head black arrows, filopodia; closed-head black arrows, lamellipodia; white arrows, chemotaxis direction. Middle and right panels- IFA staining of SSeCKS or F-actin in WT or KO MEF. Arrows, chemotaxis direction. Scale bar, 10 µm. F, Fraction of chemotactic WT or KO cells with leading edge lamellipodia or filopodia. Error bars, S.E. of 5 visual fields with at least 10 cells/field in three independent experiments. *, p<0.02, **, p<0.005. G, Percentage of chemotactic WT or KO cells in Panel F with <1, 1–3 or >3 filopodia/leading edge. **, p<0.005. H, Induction of lamellipodia formation in MDA-MB-231 cells transfected with SSeCKS-GFP (vs. GFP vector alone), as shown by IFA for GFP (left panels) or F-actin (center), or following quantification (graph, right). Arrows, chemotaxis direction. Scale bar, 10 µm. Error bars, S.E. of 5 visual fields with at least 10 cells/field in three independent experiments. I, IFA for fascin and F-actin in WT and KO MEF. Short arrows, fascin-staining filopodia. Long arrows, chemotaxis direction.
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Related In: Results  -  Collection

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pone-0111534-g001: SSeCKS inhibits chemotaxis and affects leading edge protrusions.A, Relative chemotaxis of MEF (WT or KO), DU145 (transfected with control [con] or human SSeCKS-siRNA [si]) and MDA-MB-231 cells (transfected with empty vector [V] or an SSeCKS-GFP expression plasmid [SS]), as measured in Boyden chamber assays using serum as the chemoattractant. Error bars, S.E. of triplicate assays. *, p<0.02, **, p<0.005. B, IB analysis of SSeCKS and GAPDH levels in the cells described in Panel A. C, Relative chemotaxis of WT or KO MEF using PDGF as the chemoattractant in Boyden chamber assays. Error bars, S.E. of three wounding fields in two independent experiments. **, p<0.02. D, Relative ability of WT or KO MEF to close wound scratches, based on measuring three wound field gaps at a given time in triplicate experiments. E, Agarose chemotaxis spot assay. Top: left panel- cartoon of motile cells (black) moving towards (red arrows) an agarose spot containing chemoattractant; right panels- example of assay without (“culture media” plus PBS; left) or with chemoattractant gradient (“serum-free media” plus EGF/PDGF in spot; right). Bottom: Leading edges of chemotactic WT cells predominantly display lamellipodia whereas those of KO cells predominantly display filopodia-like extensions. Left panels- phase contrast microscopy of chemotactic cells. Open-head black arrows, filopodia; closed-head black arrows, lamellipodia; white arrows, chemotaxis direction. Middle and right panels- IFA staining of SSeCKS or F-actin in WT or KO MEF. Arrows, chemotaxis direction. Scale bar, 10 µm. F, Fraction of chemotactic WT or KO cells with leading edge lamellipodia or filopodia. Error bars, S.E. of 5 visual fields with at least 10 cells/field in three independent experiments. *, p<0.02, **, p<0.005. G, Percentage of chemotactic WT or KO cells in Panel F with <1, 1–3 or >3 filopodia/leading edge. **, p<0.005. H, Induction of lamellipodia formation in MDA-MB-231 cells transfected with SSeCKS-GFP (vs. GFP vector alone), as shown by IFA for GFP (left panels) or F-actin (center), or following quantification (graph, right). Arrows, chemotaxis direction. Scale bar, 10 µm. Error bars, S.E. of 5 visual fields with at least 10 cells/field in three independent experiments. I, IFA for fascin and F-actin in WT and KO MEF. Short arrows, fascin-staining filopodia. Long arrows, chemotaxis direction.
Mentions: Our group reported previously that SSeCKS suppressed chemotaxis in rat MATLyLu prostate cancer cells without affecting cell motility in monolayer wounding assays [26]. Because chemotaxis assays typically measure parameters of individual cell migration whereas monolayer wounding reflects collective movements of cell sheets, this suggests that SSeCKS controls specific aspects of individual cell movement. To further address the role of SSeCKS in controlling chemotaxis, we compared motility of cells in Boyden chambers assays consisting of chemoattractants in the bottom chamber. SSeCKS- (KO)-MEF displayed increased chemotaxis relative to matched WT MEF against serum (Fig. 1A) or PDGF-BB (20 ng/ml) (Fig. 1C). SSeCKS levels in DU145 and MDA-MB-231 are considered downregulated compared to untransformed epithelial cells [36], although to a greater extent in MDA-MB-231 cells, and thus, we sought to knockdown SSeCKS/AKAP12 levels in DU145 and overexpress SSeCKS in MDA-MB-231 cells. The siRNA-mediated knockdown of human SSeCKS/AKAP12 in DU145 prostate cancer cells resulted in increased chemotaxis to serum, whereas its overexpression in MDA-MB-231 breast cancer cells inhibited chemotaxis (Fig. 1A). The knockdown of both endogenous α and β SSeCKS isoforms (upper and lower bands, respectively; ref. [37]) or overexpression of ectopic αSSeCKS isoform in MDA-MB-231 cells was verified by immunoblotting (Fig. 1B). Significantly, the loss of SSeCKS had no effect on cell migration involving the closure of monolayer wounds (Fig. 1D). Taken together, these data suggest that SSeCKS regulates specialized motility such as chemotaxis in both untransformed and cancer cells.

Bottom Line: Frabin knockdown in SSeCKS- MEF restores leading edge lamellipodia and chemotaxis inhibition.However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin.Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York, United States of America.

ABSTRACT
Chemotaxis is controlled by interactions between receptors, Rho-family GTPases, phosphatidylinositol 3-kinases, and cytoskeleton remodeling proteins. We investigated how the metastasis suppressor, SSeCKS, attenuates chemotaxis. Chemotaxis activity inversely correlated with SSeCKS levels in mouse embryo fibroblasts (MEF), DU145 and MDA-MB-231 cancer cells. SSeCKS loss induced chemotactic velocity and linear directionality, correlating with replacement of leading edge lamellipodia with fascin-enriched filopodia-like extensions, the formation of thickened longitudinal F-actin stress fibers reaching to filopodial tips, relative enrichments at the leading edge of phosphatidylinositol (3,4,5)P3 (PIP3), Akt, PKC-ζ, Cdc42-GTP and active Src (SrcpoY416), and a loss of Rac1. Leading edge lamellipodia and chemotaxis inhibition in SSeCKS- MEF could be restored by full-length SSeCKS or SSeCKS deleted of its Src-binding domain (ΔSrc), but not by SSeCKS deleted of its three MARCKS (myristylated alanine-rich C kinase substrate) polybasic domains (ΔPBD), which bind PIP2 and PIP3. The enrichment of activated Cdc42 in SSeCKS- leading edge filopodia correlated with recruitment of the Cdc42-specific guanine nucleotide exchange factor, Frabin, likely recruited via multiple PIP2/3-binding domains. Frabin knockdown in SSeCKS- MEF restores leading edge lamellipodia and chemotaxis inhibition. However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin. Consistent with the notion that chemotaxis is controlled by SSeCKS-PIP (vs. -Src) scaffolding activity, constitutively-active phosphatidylinositol 3-kinase could override the ability of the Src inhibitor, SKI-606, to suppress chemotaxis and filopodial enrichment of Frabin in SSeCKS- MEF. Our data suggest a role for SSeCKS in controlling Rac1 vs. Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement.

Show MeSH
Related in: MedlinePlus