Localization of I-BAR domain proteins in invasive cancer cells

An important step in cancer metastasis is the ability of the cancer cells to degrade the surrounding extracellular matrix (ECM) and migrate through it. To accomplish this, invasive cancer cells generate protrusive structures called invadopodia, which are dynamic cell protrusions that secrete matrix metalloproteinases (MMPs) responsible for the degradation of the ECM. Invadopodia formation employs the actin polymerization machinery and several cell adhesion, signal transduction and matrix degradation proteins. In addition, mechanisms involving membrane deformation may play a major role in their formation. Recently, an evolutionary conserved group of proteins involved in the formation of cell protrusions was characterized. These proteins were found to contain an inverse Bin-Amphiphysin-Rvs167 (I-BAR) domain. The I-BAR domain is capable of bending the plasma membrane outwards and via accessory domains, the I-BAR proteins link this membrane bending activity to actin cytoskeleton remodeling to generate cell protrusions. In the present study, the aim was to examine the localization of I-BAR domain proteins in invasive cancer cell lines. First, I studied the expression of the members of the I-BAR gene family (ABBA, IRTKS, IRSp53) in MDA-MB-231 breast cancer cells and SNB-19 glioma cells by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. The results indicated that I-BAR domain proteins were expressed at the messenger RNA (mRNA) and protein level in both cell lines. Then, to investigate the sub-cellular localization of these proteins to invasive sites, an ECM degradation assay was set up to distinguish invadopodias from other actin-rich sub-cellular structures. Using this assay, the localization of both endogenous and green fluorescent protein (GFP)-tagged I-BAR domain proteins to invadopodia was studied by light microscopy. Endogenous I-BAR domain proteins were examined both in two dimensional (2D) and three dimensional (3D) environments, but the antibodies used in this study appeared not to be suitable for their detection. However, the intensity profiles of invadopodia showed that in cells expressing I-BAR domain proteins fused to a GFP, these proteins accumulated either on the edge or at the centre of an invadopodium. The results suggest that the I-BAR domain proteins are likely to be recruited to distinct sites of invadopodia where they can induce or stabilize plasma membrane curvature and link it to actin cytoskeleton remodeling during the formation of invasive cell protrusions.