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

FASEB J. 2014 Apr;28(4):1805-18. doi: 10.1096/fj.13-242537. Epub 2014 Jan 10.

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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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
  • 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*
  • Thermodynamics
  • src Homology Domains

Substances

  • Actins
  • Carrier Proteins
  • Cortactin
  • Cytoskeletal Proteins
  • Epitopes
  • Intracellular Signaling Peptides and Proteins
  • Microfilament Proteins
  • Single-Domain Antibodies
  • WIPF1 protein, human
  • fascin
  • Green Fluorescent Proteins
  • Matrix Metalloproteinase 9