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

• Published in: The FASEB journal Vol. 28; no. 4; p. 1805
• Main Authors: Van Audenhove, Isabel, Boucherie, Ciska, Pieters, Leen, Zwaenepoel, Olivier, Vanloo, Berlinda, Martens, Evelien, Verbrugge, Charlotte, Hassanzadeh-Ghassabeh, Gholamreza, Vandekerckhove, Joël, Cornelissen, Maria, De Ganck, Ariane, Gettemans, Jan
• Format: Journal Article
• Language: English
• Published: United States 01.04.2014
• Subjects: Actins - metabolism; Blotting, Western; Carrier Proteins - genetics; Carrier Proteins - immunology; Carrier Proteins - metabolism; Cell Line, Tumor; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cell Movement; Cell Surface Extensions - metabolism; Cell Surface Extensions - ultrastructure; Cortactin - genetics; Cortactin - immunology; Cortactin - metabolism; Cytoskeletal Proteins - metabolism; Epitopes - genetics; Epitopes - immunology; Epitopes - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins - metabolism; Matrix Metalloproteinase 9 - metabolism; Matrix Metalloproteinase 9 - secretion; Microfilament Proteins - genetics; Microfilament Proteins - immunology; Microfilament Proteins - metabolism; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Neoplasms - metabolism; Neoplasms - pathology; Protein Binding; Pseudopodia - metabolism; Pseudopodia - ultrastructure; Single-Domain Antibodies - genetics; Single-Domain Antibodies - immunology; Single-Domain Antibodies - metabolism; src Homology Domains; Thermodynamics; cytoskeletal protein modulation; invasive protrusions; heavy-chain only antibodies; oncotargets
• ISSN: 1530-6860
• DOI: 10.1096/fj.13-242537

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.