1. Stratifying fascin and cortactin function in invadopodium formation using inhibitory nanobodies and targeted subcellular delocalization
- Author
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Maria Cornelissen, Ariane De Ganck, Jan Gettemans, Evelien Martens, Joël Vandekerckhove, Charlotte Verbrugge, Gholamreza Hassanzadeh-Ghassabeh, Ciska Boucherie, Isabel Van Audenhove, Olivier Zwaenepoel, Leen Pieters, Berlinda Vanloo, and Cellular and Molecular Immunology
- Subjects
Invadopodium ,Blotting, Western ,Green Fluorescent Proteins ,Protein domain ,Arp2/3 complex ,macromolecular substances ,Biochemistry ,heavy-chain only antibodies ,src Homology Domains ,Epitopes ,Microscopy, Electron, Transmission ,Cell Movement ,Cell Line, Tumor ,Neoplasms ,Genetics ,Humans ,Pseudopodia ,Molecular Biology ,Actin ,Fascin ,biology ,Cell Membrane ,Microfilament Proteins ,Intracellular Signaling Peptides and Proteins ,Single-Domain Antibodies ,invasive protrusions ,Actins ,Cell biology ,Cytoskeletal Proteins ,cytosketal protein modulation ,HEK293 Cells ,Matrix Metalloproteinase 9 ,Microscopy, Fluorescence ,Invadopodia ,biology.protein ,Thermodynamics ,oncotargets ,Cell Surface Extensions ,Carrier Proteins ,Cortactin ,Extracellular Matrix Degradation ,Protein Binding ,Biotechnology - 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.
- Published
- 2014