Invadopodia are hair-like membrane protrusions projecting from the ventral side of the plasma membrane of tumor cells invading into an extracellular matrix (ECM). Formation of invadopodia and phagocytosis of partially degraded ECM is correlated with invasiveness of cancer cells. Many proteins associated with actin-rich punctae associated with invadopodia have been identified. However, the dynamic temporal and spatial relationship of invadopodia-related proteins and the mechanisms required for invadopodia formation remain largely unknown. Total Internal Reflection Fluorescence (TIRF) microscopy provides a powerful tool to directly visualize the dynamic membrane transportation of invadopodia-related, GFP-tagged proteins and to simultaneously monitor invadopodia formation by observation of localized degradation and phagocytosis of fluorescently labeled gelatin. Cell-substratum contacts can be visualized using a related technique, Interference Reflection Microscopy (IRM). In this chapter, we provide detailed methodologies to monitor the dynamic localizations of c-Src-eGFP using two-color TIRF microscopy along with IRM to simultaneously visualize translocation of c-Src-eGFP and invadopodia formation by degradation of AlexaFluor� 568-labeled gelatin.
