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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 attenuates PIP3 enrichment at the leading edge.A, Chemotactic WT or KO MEF (long arrows: chemotaxis direction) transiently expressing the PIP3 reporter, GFP-PH-AKT, and stained for F-actin, showing enrichment of PIP3 in the leading edge filopodia of KO cells. Short arrows, leading edge lamellipodia (WT cells) or filopodia (KO cells). B, Re-expression of FL-, but not ΔPBD-, SSeCKS-GFP rescues leading edge lamellipodia formation in KO MEF and suppresses leading edge enrichment of the GFP-PH-AKT reporter. Scale bar, 10 µm. C, Quantification of leading edge GFP-PH-AKT levels (normalized to total cell GFP) determined for 3 fields containing at least 10 cells/field in 2 independent experiments. Error bars, S.E. *, p<0.01, **, p<0.005. D, PIP2 or PIP3 in chemotactic leading edges of WT or KO MEF by IFA or quantified as in E. **, p<0.01. Scale bar, 10 µm.
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pone-0111534-g004: SSeCKS attenuates PIP3 enrichment at the leading edge.A, Chemotactic WT or KO MEF (long arrows: chemotaxis direction) transiently expressing the PIP3 reporter, GFP-PH-AKT, and stained for F-actin, showing enrichment of PIP3 in the leading edge filopodia of KO cells. Short arrows, leading edge lamellipodia (WT cells) or filopodia (KO cells). B, Re-expression of FL-, but not ΔPBD-, SSeCKS-GFP rescues leading edge lamellipodia formation in KO MEF and suppresses leading edge enrichment of the GFP-PH-AKT reporter. Scale bar, 10 µm. C, Quantification of leading edge GFP-PH-AKT levels (normalized to total cell GFP) determined for 3 fields containing at least 10 cells/field in 2 independent experiments. Error bars, S.E. *, p<0.01, **, p<0.005. D, PIP2 or PIP3 in chemotactic leading edges of WT or KO MEF by IFA or quantified as in E. **, p<0.01. Scale bar, 10 µm.

Mentions: Given that SSeCKS functions as a scaffolding protein and that PBD domains are critical for the ability of SSeCKS to inhibit chemotaxis, it is conceivable that SSeCKS suppresses chemotaxis by controlling the enrichment of phosphoinositol phosphates at the leading edge through its scaffolding function. To address this, WT and KO MEF were transfected with a GFP-PH-AKT probe, which preferentially binds PI(3,4,5)P3 [49] and which shows enriched staining in the leading edge lamellipodia of fibroblasts chemotaxing to PDGF [50]. Consistent with their differences in leading edge protrusions, WT MEF showed concentrated GFP-PH-AKT staining at the leading edges of lamellipodia whereas KO MEF showed enrichments in filopodia-like extensions (Fig. 4A). Re-expression of full-length (FL)-SSeCKS in KO MEF restored lamellipodia formation, similar to the effects found in MDA-MB-231 cells (Fig. 1H), as well as the GFP-PH-AKT edge staining pattern found in WT MEF (Fig. 4B). In contrast, ΔPBD-SSeCKS failed to alter the filopodial staining pattern in KO MEF. Interestingly, the pattern of F-actin stress fibers protruding to the leading edge filopodia in KO MEF was altered by FL-SSeCKS overexpression, such that stress fibers ended prior to the leading edge lamellipodia (Fig. 4A). However, ΔPBD-SSeCKS overexpression failed to alter the typical stress fiber formation in KO MEF. The ability of SSeCKS to alter PIP3 levels at the leading edge was assessed by measuring relative GFP-PH-AKT staining intensities 0.5 µm from the leading cell edge as a fraction of total cell GFP-PH-AKT staining, normalized for cell volume. KO MEF exhibited 2.5-fold more PIP3 enrichment at their leading edges compared to levels in WT MEF (Fig. 4C). The finding that FL-SSeCKS, but not ΔPBD-SSeCKS, decreases PIP3 leading edge staining levels to those in WT cells strongly suggests that SSeCKS attenuates chemotaxis by scaffolding PIP3 away from leading edge structures. We also compared the localization of endogenous PIP2/PIP3 at the leading edge of WT and KO MEF by immunofluorescence staining using PIP2- or PIP3-specific antibodies (Fig. 4D). Although WT and KO MEF had similar PIP2 levels in their leading edge protrusions, KO MEF had 2-fold higher relative levels of PIP3 in their leading edge filopodia compared to levels in the WT leading edge lamellipodia (Figs. 4D & E).


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 attenuates PIP3 enrichment at the leading edge.A, Chemotactic WT or KO MEF (long arrows: chemotaxis direction) transiently expressing the PIP3 reporter, GFP-PH-AKT, and stained for F-actin, showing enrichment of PIP3 in the leading edge filopodia of KO cells. Short arrows, leading edge lamellipodia (WT cells) or filopodia (KO cells). B, Re-expression of FL-, but not ΔPBD-, SSeCKS-GFP rescues leading edge lamellipodia formation in KO MEF and suppresses leading edge enrichment of the GFP-PH-AKT reporter. Scale bar, 10 µm. C, Quantification of leading edge GFP-PH-AKT levels (normalized to total cell GFP) determined for 3 fields containing at least 10 cells/field in 2 independent experiments. Error bars, S.E. *, p<0.01, **, p<0.005. D, PIP2 or PIP3 in chemotactic leading edges of WT or KO MEF by IFA or quantified as in E. **, p<0.01. Scale bar, 10 µm.
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pone-0111534-g004: SSeCKS attenuates PIP3 enrichment at the leading edge.A, Chemotactic WT or KO MEF (long arrows: chemotaxis direction) transiently expressing the PIP3 reporter, GFP-PH-AKT, and stained for F-actin, showing enrichment of PIP3 in the leading edge filopodia of KO cells. Short arrows, leading edge lamellipodia (WT cells) or filopodia (KO cells). B, Re-expression of FL-, but not ΔPBD-, SSeCKS-GFP rescues leading edge lamellipodia formation in KO MEF and suppresses leading edge enrichment of the GFP-PH-AKT reporter. Scale bar, 10 µm. C, Quantification of leading edge GFP-PH-AKT levels (normalized to total cell GFP) determined for 3 fields containing at least 10 cells/field in 2 independent experiments. Error bars, S.E. *, p<0.01, **, p<0.005. D, PIP2 or PIP3 in chemotactic leading edges of WT or KO MEF by IFA or quantified as in E. **, p<0.01. Scale bar, 10 µm.
Mentions: Given that SSeCKS functions as a scaffolding protein and that PBD domains are critical for the ability of SSeCKS to inhibit chemotaxis, it is conceivable that SSeCKS suppresses chemotaxis by controlling the enrichment of phosphoinositol phosphates at the leading edge through its scaffolding function. To address this, WT and KO MEF were transfected with a GFP-PH-AKT probe, which preferentially binds PI(3,4,5)P3 [49] and which shows enriched staining in the leading edge lamellipodia of fibroblasts chemotaxing to PDGF [50]. Consistent with their differences in leading edge protrusions, WT MEF showed concentrated GFP-PH-AKT staining at the leading edges of lamellipodia whereas KO MEF showed enrichments in filopodia-like extensions (Fig. 4A). Re-expression of full-length (FL)-SSeCKS in KO MEF restored lamellipodia formation, similar to the effects found in MDA-MB-231 cells (Fig. 1H), as well as the GFP-PH-AKT edge staining pattern found in WT MEF (Fig. 4B). In contrast, ΔPBD-SSeCKS failed to alter the filopodial staining pattern in KO MEF. Interestingly, the pattern of F-actin stress fibers protruding to the leading edge filopodia in KO MEF was altered by FL-SSeCKS overexpression, such that stress fibers ended prior to the leading edge lamellipodia (Fig. 4A). However, ΔPBD-SSeCKS overexpression failed to alter the typical stress fiber formation in KO MEF. The ability of SSeCKS to alter PIP3 levels at the leading edge was assessed by measuring relative GFP-PH-AKT staining intensities 0.5 µm from the leading cell edge as a fraction of total cell GFP-PH-AKT staining, normalized for cell volume. KO MEF exhibited 2.5-fold more PIP3 enrichment at their leading edges compared to levels in WT MEF (Fig. 4C). The finding that FL-SSeCKS, but not ΔPBD-SSeCKS, decreases PIP3 leading edge staining levels to those in WT cells strongly suggests that SSeCKS attenuates chemotaxis by scaffolding PIP3 away from leading edge structures. We also compared the localization of endogenous PIP2/PIP3 at the leading edge of WT and KO MEF by immunofluorescence staining using PIP2- or PIP3-specific antibodies (Fig. 4D). Although WT and KO MEF had similar PIP2 levels in their leading edge protrusions, KO MEF had 2-fold higher relative levels of PIP3 in their leading edge filopodia compared to levels in the WT leading edge lamellipodia (Figs. 4D & E).

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