Figure 7.
A TKS5/FGD1/CDC42 signaling module directs the assembly of collagenolytic invadopodia in breast cancer cells.(A) Upper panel: Cells were plated on a layer of fibrillary collagen (gray) and stained for SHIP2 (red) and cortactin (green), and nuclei were stained with DAPI (blue). Scale bar, 10 µm; zoom-in of boxed region, 5 µm. Middle panel: Fluorescence (Fluo) intensity profiles for SHIP2 and cortactin were recorded along an invadopodium (boxed region in upper panel) and normalized to the maximum fluorescence intensity set to 100. Lower panel: Correlation of SHIP2 (x axis) and cortactin (y axis) fluorescence intensity along the invadopodial structures. inv, number of invadopodia analyzed; pix, total number of pixels analyzed for both markers. (B) Correlation of p130CAS (red) and cortactin (green) as in A. (C) Correlation of TKS5 (blue) and overexpressed FLAG-tagged inositol polyphosphate 4-phosphatase type II (green) as in A. (D) Model of feed-forward loop self-assembly mechanisms for invadopodia formation and maturation. Surface-exposed MT1-MMP in association with the collagen fibers within the matrix may contribute to the early production of PI(3,4)P2, the phosphoinositide ligand of the TKS5 PX domain, by interacting with a p130CAS/SHIP2 complex. Accumulation of PI(3,4)P2 at collagen–cell contact sites leads to the recruitment of TKS5 to the forming invadopodial plasma membrane. The carboxy-terminal SH3 domains of TKS5 interact with and recruit the highly specific CDC42-GEF, FGD1. GTP-bound active CDC42 interacts with several downstream effectors, which are known to contribute to invadopodia assembly and function including TKS5 itself, N-WASP, the polarity protein, IQGAP1, and FBP17. Arp2/3 complex activation by N-WASP leads to the assembly of the invadopodia branched actin core structure. FBP17 also positively influences branched actin assembly and may lead to PI(3,4)P2 production by interacting with SHIP2. Recruitment of IQGAP1 at invadopodia contributes to the polarized recycling of MT1-MMP from endolysosomal compartments (Sakurai-Yageta et al., 2008). In addition, IQGAP1 scaffolds a network of phosphoinositide kinases, potentially leading to further phosphatidylinositol (3,4,5)-trisphosphate and PI(3,4)P2 accumulation (Choi et al., 2016). This network of interactions further promotes invadopodia assembly and function in a feed-forward loop with contributions of actin assembly and membrane trafficking. Cat, catalytic domain; Hpx, hemopexin domain; PIPK, phosphoinositide kinase; PM, plasma membrane. The PX and SH3 domains #1 to #5 of TKS5 are depicted.

A TKS5/FGD1/CDC42 signaling module directs the assembly of collagenolytic invadopodia in breast cancer cells. (A) Upper panel: Cells were plated on a layer of fibrillary collagen (gray) and stained for SHIP2 (red) and cortactin (green), and nuclei were stained with DAPI (blue). Scale bar, 10 µm; zoom-in of boxed region, 5 µm. Middle panel: Fluorescence (Fluo) intensity profiles for SHIP2 and cortactin were recorded along an invadopodium (boxed region in upper panel) and normalized to the maximum fluorescence intensity set to 100. Lower panel: Correlation of SHIP2 (x axis) and cortactin (y axis) fluorescence intensity along the invadopodial structures. inv, number of invadopodia analyzed; pix, total number of pixels analyzed for both markers. (B) Correlation of p130CAS (red) and cortactin (green) as in A. (C) Correlation of TKS5 (blue) and overexpressed FLAG-tagged inositol polyphosphate 4-phosphatase type II (green) as in A. (D) Model of feed-forward loop self-assembly mechanisms for invadopodia formation and maturation. Surface-exposed MT1-MMP in association with the collagen fibers within the matrix may contribute to the early production of PI(3,4)P2, the phosphoinositide ligand of the TKS5 PX domain, by interacting with a p130CAS/SHIP2 complex. Accumulation of PI(3,4)P2 at collagen–cell contact sites leads to the recruitment of TKS5 to the forming invadopodial plasma membrane. The carboxy-terminal SH3 domains of TKS5 interact with and recruit the highly specific CDC42-GEF, FGD1. GTP-bound active CDC42 interacts with several downstream effectors, which are known to contribute to invadopodia assembly and function including TKS5 itself, N-WASP, the polarity protein, IQGAP1, and FBP17. Arp2/3 complex activation by N-WASP leads to the assembly of the invadopodia branched actin core structure. FBP17 also positively influences branched actin assembly and may lead to PI(3,4)P2 production by interacting with SHIP2. Recruitment of IQGAP1 at invadopodia contributes to the polarized recycling of MT1-MMP from endolysosomal compartments (Sakurai-Yageta et al., 2008). In addition, IQGAP1 scaffolds a network of phosphoinositide kinases, potentially leading to further phosphatidylinositol (3,4,5)-trisphosphate and PI(3,4)P2 accumulation (Choi et al., 2016). This network of interactions further promotes invadopodia assembly and function in a feed-forward loop with contributions of actin assembly and membrane trafficking. Cat, catalytic domain; Hpx, hemopexin domain; PIPK, phosphoinositide kinase; PM, plasma membrane. The PX and SH3 domains #1 to #5 of TKS5 are depicted.

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