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 funct...

Full description

Saved in:

Bibliographic Details
Published in:	The FASEB journal Vol. 28; no. 4; pp. 1805 - 1818
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 The Federation of American Societies for Experimental Biology 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 protein modulation Cytoskeletal Proteins - metabolism Epitopes - genetics Epitopes - immunology Epitopes - metabolism Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism heavy‐chain only antibodies HEK293 Cells Humans Intracellular Signaling Peptides and Proteins - metabolism invasive protrusions 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 oncotargets 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
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	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
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0892-6638 1530-6860
DOI:	10.1096/fj.13-242537