title: 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.
authors.affiliation: 1Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium. jan.gettemans@ugent.be.
authors.forename: Isabel
authors.initials: I
authors.lastname: Van Audenhove
authors.forename: Ciska
authors.initials: C
authors.lastname: Boucherie
authors.forename: Leen
authors.initials: L
authors.lastname: Pieters
authors.forename: Olivier
authors.initials: O
authors.lastname: Zwaenepoel
authors.forename: Berlinda
authors.initials: B
authors.lastname: Vanloo
authors.forename: Evelien
authors.initials: E
authors.lastname: Martens
authors.forename: Charlotte
authors.initials: C
authors.lastname: Verbrugge
authors.forename: Gholamreza
authors.initials: G
authors.lastname: Hassanzadeh-Ghassabeh
authors.forename: Joël
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: Maria
authors.initials: M
authors.lastname: Cornelissen
authors.forename: Ariane
authors.initials: A
authors.lastname: De Ganck
authors.forename: Jan
authors.initials: J
authors.lastname: Gettemans
mesh_terms: D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002465:Cell Movement; D022081:Cell Surface Extensions / Q000378:metabolism* / Q000648:ultrastructure; D051356:Cortactin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D000939:Epitopes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D009369:Neoplasms / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding; D011554:Pseudopodia / Q000378:metabolism / Q000648:ultrastructure; D061905:Single-Domain Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013816:Thermodynamics; D018909:src Homology Domains
chemical_list: D000199:Actins; D002352:Carrier Proteins; D051356:Cortactin; D003598:Cytoskeletal Proteins; D000939:Epitopes; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D061905:Single-Domain Antibodies; C109903:WIPF1 protein, human; C020324:fascin; D049452:Green Fluorescent Proteins; D020780:Matrix Metalloproteinase 9
keywords: cytoskeletal protein modulation; heavy-chain only antibodies; invasive protrusions; oncotargets