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.
Keywords:
cytoskeletal protein modulation; heavy-chain only antibodies; invasive protrusions; oncotargets.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Actins / metabolism
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Blotting, Western
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Carrier Proteins / genetics
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Carrier Proteins / immunology
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Carrier Proteins / metabolism*
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Cell Line, Tumor
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Cell Membrane / metabolism
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Cell Membrane / ultrastructure
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Cell Movement
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Cell Surface Extensions / metabolism*
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Cell Surface Extensions / ultrastructure
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Cortactin / genetics
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Cortactin / immunology
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Cortactin / metabolism*
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Cytoskeletal Proteins / metabolism
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Epitopes / genetics
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Epitopes / immunology
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Epitopes / metabolism
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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HEK293 Cells
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Humans
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Intracellular Signaling Peptides and Proteins / metabolism
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Matrix Metalloproteinase 9 / metabolism
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Microfilament Proteins / genetics
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Microfilament Proteins / immunology
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Microfilament Proteins / metabolism*
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Microscopy, Electron, Transmission
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Microscopy, Fluorescence
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Neoplasms / metabolism
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Neoplasms / pathology
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Protein Binding
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Pseudopodia / metabolism
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Pseudopodia / ultrastructure
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Single-Domain Antibodies / genetics
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Single-Domain Antibodies / immunology
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Single-Domain Antibodies / metabolism*
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Thermodynamics
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src Homology Domains
Substances
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Actins
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Carrier Proteins
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Cortactin
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Cytoskeletal Proteins
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Epitopes
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Intracellular Signaling Peptides and Proteins
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Microfilament Proteins
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Single-Domain Antibodies
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WIPF1 protein, human
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fascin
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Green Fluorescent Proteins
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Matrix Metalloproteinase 9