Ena/VASP processive elongation is modulated by avidity on actin filaments bundled by the filopodia cross-linker fascin

Mol Biol Cell. 2019 Mar 21;30(7):851-862. doi: 10.1091/mbc.E18-08-0500. Epub 2019 Jan 2.


Ena/VASP tetramers are processive actin elongation factors that localize to diverse F-actin networks composed of filaments bundled by different cross-linking proteins, such as filopodia (fascin), lamellipodia (fimbrin), and stress fibers (α-actinin). Previously, we found that Ena takes approximately threefold longer processive runs on trailing barbed ends of fascin-bundled F-actin. Here, we used single-molecule TIRFM (total internal reflection fluorescence microscopy) and developed a kinetic model to further dissect Ena/VASP's processive mechanism on bundled filaments. We discovered that Ena's enhanced processivity on trailing barbed ends is specific to fascin bundles, with no enhancement on fimbrin or α-actinin bundles. Notably, Ena/VASP's processive run length increases with the number of both fascin-bundled filaments and Ena "arms," revealing avidity facilitates enhanced processivity. Consistently, Ena tetramers form more filopodia than mutant dimer and trimers in Drosophila culture cells. Moreover, enhanced processivity on trailing barbed ends of fascin-bundled filaments is an evolutionarily conserved property of Ena/VASP homologues, including human VASP and Caenorhabditis elegans UNC-34. These results demonstrate that Ena tetramers are tailored for enhanced processivity on fascin bundles and that avidity of multiple arms associating with multiple filaments is critical for this process. Furthermore, we discovered a novel regulatory process whereby bundle size and bundling protein specificity control activities of a processive assembly factor.

MeSH terms

  • Actin Cytoskeleton / metabolism
  • Actinin / metabolism
  • Actins / metabolism*
  • Actins / physiology
  • Animals
  • Carrier Proteins / metabolism
  • Carrier Proteins / physiology*
  • Cell Line
  • Cytoskeletal Proteins / metabolism*
  • Cytoskeleton / metabolism
  • Kinetics
  • Mice
  • Microfilament Proteins / metabolism
  • Microfilament Proteins / physiology*
  • Microscopy, Fluorescence / methods
  • Peptide Chain Elongation, Translational / physiology
  • Protein Binding
  • Pseudopodia / physiology


  • Actins
  • Carrier Proteins
  • Cytoskeletal Proteins
  • Enah protein, mouse
  • Microfilament Proteins
  • Actinin
  • fascin