Limits...
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.

Show MeSH

Related in: MedlinePlus

Enrichment of Frabin at the leading edge of KO MEF directs increased chemotaxis.A, IFA for Frabin or F-actin in resting vs. chemotactic (arrows) WT and KO MEF. Scale bar, 10 µm. B, IFA of GFP or Frabin in KO MEF re-expressing FL-, ΔPBD-, or ΔSrc-SSeCKS-GFP. Scale bar, 10 µm. Arrow, chemotaxis direction. C, IB analysis of SSeCKS, Frabin or Gapd levels in WT vs. KO MEF, relative to markers (left). D, IB analysis of Frabin or Gapdh protein levels in KO MEF cell lysates transfected with scrambled (scr) or Frabin-specific siRNA. E, IFA analysis of F-actin in KO MEF transfected with scr- or Frabin-siRNA. Scale bar, 5 µm. Arrow, chemotaxis direction. F, Quantification of KO MEF with leading edge lamellipodia vs. filopodia after transfection with scr- or Frabin-siRNA. Error bars, S.E. of 5 visual microscope fields with at least 10 cells/field in two independent experiments. **, p<0.005. G, Effect of scr- or Frabin-siRNA on WT or KO MEF chemotaxis. Error bars, S.E. of triplicates from two independent experiments. **, p<0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4214753&req=5

pone-0111534-g007: Enrichment of Frabin at the leading edge of KO MEF directs increased chemotaxis.A, IFA for Frabin or F-actin in resting vs. chemotactic (arrows) WT and KO MEF. Scale bar, 10 µm. B, IFA of GFP or Frabin in KO MEF re-expressing FL-, ΔPBD-, or ΔSrc-SSeCKS-GFP. Scale bar, 10 µm. Arrow, chemotaxis direction. C, IB analysis of SSeCKS, Frabin or Gapd levels in WT vs. KO MEF, relative to markers (left). D, IB analysis of Frabin or Gapdh protein levels in KO MEF cell lysates transfected with scrambled (scr) or Frabin-specific siRNA. E, IFA analysis of F-actin in KO MEF transfected with scr- or Frabin-siRNA. Scale bar, 5 µm. Arrow, chemotaxis direction. F, Quantification of KO MEF with leading edge lamellipodia vs. filopodia after transfection with scr- or Frabin-siRNA. Error bars, S.E. of 5 visual microscope fields with at least 10 cells/field in two independent experiments. **, p<0.005. G, Effect of scr- or Frabin-siRNA on WT or KO MEF chemotaxis. Error bars, S.E. of triplicates from two independent experiments. **, p<0.01.

Mentions: We previously reported that although SSeCKS could attenuate v-Src-induced RhoA and Cdc42 activation, thereby inhibiting podosome/invadopodia formation, there was no interaction between these GTPases and SSeCKS based on co-immunoprecipitation [35]. The activation of Rho family GTPases is mediated by the translocation of GEFs to the membrane through their binding to PIPs [6]. We hypothesized that in the absence of SSeCKS, the localization of a Cdc42-specific GEF in filopodia structures at the leading edge, facilitated by the enrichment of PIP2 and PIP3, might drive the selective activation of Cdc42. Frabin (FGD1-related F-actin binding protein), a known Cdc42-specific GEF [57] that binds PIPs via two PH and one FYVE domain [58], can induce Cdc42 activation in the vicinity of actin structures leading to filopodia formation [59]. In the absence of a chemoattractant gradient, Frabin was distributed in a reticulate, cytoplasmic pattern with little association with plasma membrane structures in both WT and KO MEF (Fig. 7A; left panel). WT MEF exhibited increased levels of perinuclear Frabin compared to KO MEF. In the context of a chemoattractant gradient, however, Frabin enriched to the tips of filopodia in the leading edges of KO cells, whereas in WT MEF, it was relatively absent from the lamellipodia, and instead, concentrated in perinuclear regions (Fig. 7A, right panel). The ectopic expression of FL-SSeCKS in KO MEF restored leading edge lamellipodia formation, yet these structures were relatively devoid of Frabin staining (Fig. 7B). Similar results were obtained using an SSeCKS mutant, ΔSrc, deleted of its Src-binding domain (a.a. 153–166; ref. [27]). In contrast, ΔPBD-SSeCKS expression failed to induce lamellipodia, resulting in filopodia protrusions at the leading edge containing terminal enrichments of Frabin. Although SSeCKS affects the localization of Frabin, the level of total cellular Frabin (MWt. = 105 kDa) was not changed by SSeCKS (Fig. 7C). Importantly, we could not show Frabin-SSeCKS interactions in WT MEF lysates based on co-IP.


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)

Enrichment of Frabin at the leading edge of KO MEF directs increased chemotaxis.A, IFA for Frabin or F-actin in resting vs. chemotactic (arrows) WT and KO MEF. Scale bar, 10 µm. B, IFA of GFP or Frabin in KO MEF re-expressing FL-, ΔPBD-, or ΔSrc-SSeCKS-GFP. Scale bar, 10 µm. Arrow, chemotaxis direction. C, IB analysis of SSeCKS, Frabin or Gapd levels in WT vs. KO MEF, relative to markers (left). D, IB analysis of Frabin or Gapdh protein levels in KO MEF cell lysates transfected with scrambled (scr) or Frabin-specific siRNA. E, IFA analysis of F-actin in KO MEF transfected with scr- or Frabin-siRNA. Scale bar, 5 µm. Arrow, chemotaxis direction. F, Quantification of KO MEF with leading edge lamellipodia vs. filopodia after transfection with scr- or Frabin-siRNA. Error bars, S.E. of 5 visual microscope fields with at least 10 cells/field in two independent experiments. **, p<0.005. G, Effect of scr- or Frabin-siRNA on WT or KO MEF chemotaxis. Error bars, S.E. of triplicates from two independent experiments. **, p<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4214753&req=5

pone-0111534-g007: Enrichment of Frabin at the leading edge of KO MEF directs increased chemotaxis.A, IFA for Frabin or F-actin in resting vs. chemotactic (arrows) WT and KO MEF. Scale bar, 10 µm. B, IFA of GFP or Frabin in KO MEF re-expressing FL-, ΔPBD-, or ΔSrc-SSeCKS-GFP. Scale bar, 10 µm. Arrow, chemotaxis direction. C, IB analysis of SSeCKS, Frabin or Gapd levels in WT vs. KO MEF, relative to markers (left). D, IB analysis of Frabin or Gapdh protein levels in KO MEF cell lysates transfected with scrambled (scr) or Frabin-specific siRNA. E, IFA analysis of F-actin in KO MEF transfected with scr- or Frabin-siRNA. Scale bar, 5 µm. Arrow, chemotaxis direction. F, Quantification of KO MEF with leading edge lamellipodia vs. filopodia after transfection with scr- or Frabin-siRNA. Error bars, S.E. of 5 visual microscope fields with at least 10 cells/field in two independent experiments. **, p<0.005. G, Effect of scr- or Frabin-siRNA on WT or KO MEF chemotaxis. Error bars, S.E. of triplicates from two independent experiments. **, p<0.01.
Mentions: We previously reported that although SSeCKS could attenuate v-Src-induced RhoA and Cdc42 activation, thereby inhibiting podosome/invadopodia formation, there was no interaction between these GTPases and SSeCKS based on co-immunoprecipitation [35]. The activation of Rho family GTPases is mediated by the translocation of GEFs to the membrane through their binding to PIPs [6]. We hypothesized that in the absence of SSeCKS, the localization of a Cdc42-specific GEF in filopodia structures at the leading edge, facilitated by the enrichment of PIP2 and PIP3, might drive the selective activation of Cdc42. Frabin (FGD1-related F-actin binding protein), a known Cdc42-specific GEF [57] that binds PIPs via two PH and one FYVE domain [58], can induce Cdc42 activation in the vicinity of actin structures leading to filopodia formation [59]. In the absence of a chemoattractant gradient, Frabin was distributed in a reticulate, cytoplasmic pattern with little association with plasma membrane structures in both WT and KO MEF (Fig. 7A; left panel). WT MEF exhibited increased levels of perinuclear Frabin compared to KO MEF. In the context of a chemoattractant gradient, however, Frabin enriched to the tips of filopodia in the leading edges of KO cells, whereas in WT MEF, it was relatively absent from the lamellipodia, and instead, concentrated in perinuclear regions (Fig. 7A, right panel). The ectopic expression of FL-SSeCKS in KO MEF restored leading edge lamellipodia formation, yet these structures were relatively devoid of Frabin staining (Fig. 7B). Similar results were obtained using an SSeCKS mutant, ΔSrc, deleted of its Src-binding domain (a.a. 153–166; ref. [27]). In contrast, ΔPBD-SSeCKS expression failed to induce lamellipodia, resulting in filopodia protrusions at the leading edge containing terminal enrichments of Frabin. Although SSeCKS affects the localization of Frabin, the level of total cellular Frabin (MWt. = 105 kDa) was not changed by SSeCKS (Fig. 7C). Importantly, we could not show Frabin-SSeCKS interactions in WT MEF lysates based on co-IP.

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