An invasion-related complex of cortactin, paxillin and PKCμ associates with invadopodia at sites of extracellular matrix degradation

Abstract

Invasive breast cancer cells have the ability to extend membrane protrusions, invadopodia, into the extracellular matrix (ECM). These structures are associated with sites of active matrix degradation. The amount of matrix degradation associated with the activity of these membrane protrusions has been shown to directly correlate with invasive potential. We demonstrate here that microinjection of polyclonal anti-cortactin antibodies blocks matrix degradation at invadopodia supporting the hypothesis that cortactin has a direct role in invasive behavior. MDA-MB-231, invasive breast cancer cells were sheared from the surface of a gelatin matrix to isolate invadopodia. Cortactin, paxillin and protein kinase C (PKC) m, a serine kinase, were co-immunoprecipitated as a complex from invadopodia-enriched membranes. We con®rmed the subcellular distribution of these proteins by immunolocalization and Western blotting. We also determined that, in contrast to its presence in invasive cells, this complex of proteins was not detected in lysates from non-invasive cells that do not form invadopodia. Taken together, these data suggest that the formation of this cortactin-containing complex correlates with cellular invasiveness. We hypothesize that this complex of molecules has a role in the formation and function of invadopodia during cellular invasion.

Human cortactin as putative cancer antigen

“…Additionally, recent studies have shown the overexpression of cortactin in NIH3T3 cells to result in increased cell invasion/migration in vitro (Patel et al, 1998;Huang et al, 1998). Moreover, cortactin takes part in formation and function of invadopodias in (Bowden et al, 1999) supporting the hypothesis of direct involvement of cortactin in metastasis.…”

Autonomous regulation of osteosarcoma cell invasiveness by Wnt5a/Ror2 signaling

“…Invasive tumor cells form actin-rich protrusions, called invadopodia, that degrade and extend into ECMs (Bowden et al, 1999;Onodera et al, 2005;Weaver, 2006;Cortesio et al, 2008). To evaluate the ability of invadopodia formation during in vitro invasion of SaOS-2 cells, Alexa-labeled gelatin (red) was utilized as ECM (see Materials and Methods).…”

“…Fluorescent images were obtained using a laser scanning confocal imaging system (LSM510, Carl Zeiss, Go¨ttingen, Germany). For ECM degradation assay, glass-bottom dishes were coated with gelatin (Type A with 300 Bloom; Sigma) conjugated with Alexa 594 (Invitrogen) (Alexa-gelatin) and then treated with 0.5% glutaraldehyde, as described earlier (Bowden et al, 1999;Onodera et al, 2005). Cells were cultured on these glassbottom dishes in DMEM, fixed and stained with anti-cortactin antibody or Alexa 488-phalloidin.…”

A role for the podosome/invadopodia scaffold protein Tks5 in tumor growth in vivo

“…Fluorescently-labeled gelatin-coated coverslips were prepared as described by Berdeaux et al (2004), as originally described by Bowden et al (1999). In brief, coverslips were coated with Oregon green 488-conjugated gelatin (Chemicon, Temecula, CA), cross-linked 15 min in 0.5% glutaraldehyde in phosphate-buffered saline (PBS), and incubated for 3 min with 5 mg/ml NaBH 4 in PBS.…”

“…They are regulated by various proteins, including the Arp2/3 complex, N-WASp, WAVE1, Cdc42, ASAP1, cortactin, gelsolin, and cofilin (Bowden et al, 1999(Bowden et al, , 2006Baldassarre et al, 2003;Yamaguchi et al, 2005;Artym et al, 2006;Chellaiah, 2006;Bharti et al, 2007;Clark et al, 2007). Podosomes/invadopodia are found in various mammalian cells such as macrophages, osteoclasts and vascular smooth muscle cells (Linder and Aepfelbacher, 2003).…”