Tks5 and SHIP2 Regulate Invadopodium Maturation, but Not Initiation, in Breast Carcinoma Cells

Abstract

Summary Background Tks5 regulates invadopodium formation, but the precise timing during invadopodium lifetime (initiation, stabilization, maturation) when Tks5 plays a role is not known. Results We report new findings based on high-resolution spatiotemporal live-cell imaging of invadopodium precursor assembly. Cortactin, N-WASP, cofilin, and actin arrive together to form the invadopodium precursor, followed by Tks5 recruitment. Tks5 is not required for precursor initiation but is needed for precursor stabilization, which requires the interaction of the phox homology (PX) domain of Tks5 with PI(3,4)P2. During precursor formation, PI(3,4)P2 is uniformly distributed but subsequently starts accumulating at the precursor core 3–4 min after core initiation, and conversely, PI(3,4,5)P3 gets enriched in a ring around the precursor core. SHIP2, a 5′-inositol phosphatase, localizes at the invadopodium core and regulates PI(3,4)P2 levels locally at the invadopodium. The timing of SHIP2 arrival at the invadopodium precursor coincides with the onset of PI(3,4)P2 accumulation. Consistent with its late arrival, we found that SHIP2 inhibition does not affect precursor formation but does cause decreases in mature invadopodia and matrix degradation, whereas SHIP2 overexpression increases matrix degradation. Conclusions Together, these findings lead us to propose a new sequential model that provides novel insights into molecular mechanisms underlying invadopodium precursor initiation, stabilization, and maturation into a functional invadopodium.

The interplay between the proteolytic, invasive, and adhesive domains of invadopodia and their roles in cancer invasion

“…It is likely that this apparent discrepancy is attributable to differences between the cancer cell lines, or to variations in the persistence of the adhesion rings during the lifespan of the invadopodia. In fact, it was reported that the adhesive domain of invadopodia is formed soon after the formation of the core F-actin bundle, showing an oscillatory behavior, and occasionally, disappears at later stages, after stabilization of the structure 52,72 (and our unpublished data). The mechanisms underlying the interplay between the core and the adhesion ring, and their significance for ECM remodeling, are still poorly understood.…”

“…A new approach of high-resolution spatiotemporal live cell imaging, published recently, shows that cortactin, N-WASP, cofilin, and actin arrive simultaneously to form the invadopodia precursor, while TKS5 is recruited later and is required for the structure stabilization. 72 Actin polymerization in the core of invadopodia is regulated by small Rho-family GTPases, mostly, CDC42, 1,28 which can directly activate the N-WASP-WIP complex, which, in turn, drives actin polymerization by the Arp2/3 complex. [73][74][75][76] Arp 2/3 actin nucleation is promoted by the severing activity of cofillin.…”

Sorting nexin 9 negatively regulates invadopodia formation and function in cancer cells

“…The molecular composition of invadopodia is highly similar to other actin-related structures. Therefore, to unambiguously identify these structures we used the specific invadopodial marker, TKS5 (tyrosine kinase substrate with five SH3 domains), an adaptor protein that is required for invadopodia function (Sharma et al, 2013). Using total internal reflection fluorescence (TIRF) microscopy, which selectively illuminates molecules localized within ∼100 nm of the ventral cell surface, we found that transiently expressed mCherry-SNX9 colocalized with TKS5-GFP (Fig.…”

“…As only mature invadopodia are able to degrade the matrix and TKS5 is a marker for mature invadopodia (Sharma et al, 2013), we also stained for endogenous TKS5 in control and SNX9-depleted MDA-MB-231 cells. In accordance with the previous results, the number of TKS5-and actin-positive structures, indicative of mature invadopodia (Fig.…”

SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration in 1321 N1 glioblastoma cells

“…SHIP2 also regulates important biological mechanisms such as cell adhesion (Prasad et al, 2002), migration (Yu et al, 2010) or endocytosis at the leading edge of the cells (Boucrot et al, 2015). It is accepted that PI(3,4,5)P3 is the main SHIP2 substrate (Giuriato et al, 2002;Nakatsu et al, 2010;Taylor et al, 2000) and its reaction product phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] is a lipid involved in endocytosis, lamellipodia formation (Krause and Gautreau, 2014), invadopodium maturation (Sharma et al, 2013) and ruffle formation (Hasegawa et al, 2011). In addition to PI(3,4,5)P3, PI(4,5)P2 is also a substrate of SHIP2 although evidence of this in intact cells has been more difficult to establish, although it has been observed in a very few models, e.g.…”