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Stratifying fascin and cortactin function in invadopodium formation using inhibitory nanobodies and targeted subcellular delocalization

Invadopodia are actin‐rich protrusions arising through the orchestrated regulation of precursor assembly, stabilization, and maturation, endowing cancer cells with invasive properties. Using nanobodies (antigen‐binding domains of Camelid heavy‐chain antibodies) as perturbators of intracellular functions and/or protein domains at the level of the endogenous protein, we examined the specific contribution of fascin and cortactin during invadopodium formation in MDA‐MB‐231 breast and PC‐3 prostate cancer cells. A nanobody (Kd~35 nM, 1:1 stoichiometry) that disrupts fascin F‐actin bundling emphasizes the importance of stable actin bundles in invadopodium array organization and turnover, matrix degradation, and cancer cell invasion. Cortactin‐SH3 dependent WIP recruitment toward the plasma membrane was specifically inhibited by a cortactin nanobody (Kd~75 nM, 1:1 stoichiometry). This functional domain is shown to be important for formation of properly organized invadopodia, MMP‐9 secretion, matrix degradation, and cancer cell invasion. Notably, using a subcellular delocalization strategy to trigger protein loss of function, we uncovered a fascin‐bundling‐independent role in MMP‐9 secretion. Hence, we demonstrate that nanobodies enable high resolution protein function mapping in cells.—Van Audenhove, I., Boucherie, C., Pieters, L., Zwaenepoel, O., Vanloo, B., Martens, E., Verbrugge, C., Hassanzadeh‐Ghassabeh, G., Vandekerckhove, J., Cornelissen, M., De Ganck, A., Gettemans, J. Stratifying fascin and cortactin function in invadopodium formation using inhibitory nanobodies and targeted subcellular delocalization. FASEB J. 28, 1805–1818 (2014). www.fasebj.org