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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 the rate and directionality of chemotactic cells.A, Hypothetical chemotactic paths of three cells (square, diamond or triangle) based on five time measurements (numbered). The forward migration index (FMI) is calculated as the distance “h”, if a cell theoretically travelled directly towards the chemoattractant source over time-points 1 to 5, divided by “b”, the direct vector from the cell’s start (time-point 1) to end (time-point 5). A cell moving in a straight, direct line towards a chemoattractant would have an FMI = 1. KO MEF have increased FMI (B) and velocity (C) compared to WT MEF. *, p<0.05, **, p<0.01 for at least 20 cells/time-point/condition.
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pone-0111534-g002: SSeCKS inhibits the rate and directionality of chemotactic cells.A, Hypothetical chemotactic paths of three cells (square, diamond or triangle) based on five time measurements (numbered). The forward migration index (FMI) is calculated as the distance “h”, if a cell theoretically travelled directly towards the chemoattractant source over time-points 1 to 5, divided by “b”, the direct vector from the cell’s start (time-point 1) to end (time-point 5). A cell moving in a straight, direct line towards a chemoattractant would have an FMI = 1. KO MEF have increased FMI (B) and velocity (C) compared to WT MEF. *, p<0.05, **, p<0.01 for at least 20 cells/time-point/condition.

Mentions: Chemotaxis efficiency is affected by both the directionality and velocity of cell migration, parameters associated with the number, orientation and type of protrusions at the leading edge, as well as their interaction with dynamic changes in the remodeling of the actin cytoskeleton [14]. We addressed whether SSeCKS could regulate chemotaxis through control of migration directionality or velocity, given our results that it affects the dynamics of F-actin stress fiber and leading-edge protrusion assembly (Fig. 1). Cell movement of WT or KO MEF towards chemoattractant-laden agarose spots was measured over time, where directionality was calculated by a forward motion index (FMI) based on the distance traveled by a cell (origin to endpoint) when vectored directly toward the agarose spot (h), divided by the vector distance from the actual origin to endpoint (b) (Fig. 2A). Our data indicate that KO MEF have roughly 1.5-fold greater directionality than WT controls, and a small, but significant increase in velocity (Fig. 2B). These data suggest that SSeCKS controls chemotaxis by attenuating chemoattractant sensing and/or cytoskeletal reorganization mechanisms.


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 the rate and directionality of chemotactic cells.A, Hypothetical chemotactic paths of three cells (square, diamond or triangle) based on five time measurements (numbered). The forward migration index (FMI) is calculated as the distance “h”, if a cell theoretically travelled directly towards the chemoattractant source over time-points 1 to 5, divided by “b”, the direct vector from the cell’s start (time-point 1) to end (time-point 5). A cell moving in a straight, direct line towards a chemoattractant would have an FMI = 1. KO MEF have increased FMI (B) and velocity (C) compared to WT MEF. *, p<0.05, **, p<0.01 for at least 20 cells/time-point/condition.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214753&req=5

pone-0111534-g002: SSeCKS inhibits the rate and directionality of chemotactic cells.A, Hypothetical chemotactic paths of three cells (square, diamond or triangle) based on five time measurements (numbered). The forward migration index (FMI) is calculated as the distance “h”, if a cell theoretically travelled directly towards the chemoattractant source over time-points 1 to 5, divided by “b”, the direct vector from the cell’s start (time-point 1) to end (time-point 5). A cell moving in a straight, direct line towards a chemoattractant would have an FMI = 1. KO MEF have increased FMI (B) and velocity (C) compared to WT MEF. *, p<0.05, **, p<0.01 for at least 20 cells/time-point/condition.
Mentions: Chemotaxis efficiency is affected by both the directionality and velocity of cell migration, parameters associated with the number, orientation and type of protrusions at the leading edge, as well as their interaction with dynamic changes in the remodeling of the actin cytoskeleton [14]. We addressed whether SSeCKS could regulate chemotaxis through control of migration directionality or velocity, given our results that it affects the dynamics of F-actin stress fiber and leading-edge protrusion assembly (Fig. 1). Cell movement of WT or KO MEF towards chemoattractant-laden agarose spots was measured over time, where directionality was calculated by a forward motion index (FMI) based on the distance traveled by a cell (origin to endpoint) when vectored directly toward the agarose spot (h), divided by the vector distance from the actual origin to endpoint (b) (Fig. 2A). Our data indicate that KO MEF have roughly 1.5-fold greater directionality than WT controls, and a small, but significant increase in velocity (Fig. 2B). These data suggest that SSeCKS controls chemotaxis by attenuating chemoattractant sensing and/or cytoskeletal reorganization mechanisms.

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