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

Abstract

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 (K(d)~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 (K(d)~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.

Fascin Rigidity and L-plastin Flexibility Cooperate in Cancer Cell Invadopodia and Filopodia

“…Nanobody Generation, cDNA Cloning, and Recombinant Protein Productions-Nanobody generation by the VIB Nanobody service facility, further cloning and recombinant production of nanobodies and antigens was described (14,30).…”

“…4 M nanobody was preincubated with the bundlers for 30 min. Samples were evaluated with a transmission electron microscope (Jeol 1200 EXII; Jeol Ltd., Akishima, Japan) at an accelerating voltage of 100 kV and ϫ100,000 view and analyzed as described (14).…”

“…Transient expression was achieved with Jetprime (Polyplus Transfection Inc., New York, NY). Stable expression was achieved by transduction, making use of the Lenti-X Tet-On advanced system (Clontech, Mountain View, CA) (14). Expression was induced by addition of 500 ng/ml of doxycycline.…”

“…Recently, it was also described as an important stabilizer of invadopodia (13,14). Fascin contains several actin binding sites (15), of which the N-terminal one can be inhibited by phosphorylation of a conserved serine (Ser-39) by means of PKC (16).…”

“…The fascin nanobody FASNb5 3 (K d ϳ35 nM) disrupts fascinmediated actin bundling resulting in impeded filopodium formation, unstable invadopodia, and reduced degradation and invasiveness (14). The L-plastin nanobody LPLNb5 reduces L-plastin bundling by binding the hinge region between the actin binding repeats (K d ϳ40 nM), resulting in disrupted filopodia formation.…”