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Ena/VASP and fascin collaborate in the self-organization of actin filaments

Ena/VASP and fascin collaborate in the self-organization of actin filaments. Jonathan Winkelman Mini Retreat, April 10 th , 2014 Department of Molecular Genetics and Cell Biology. Actin monomers assemble into polar filaments. Barbed end (B). Pointed end (P). In vitro : TIRF microscopy.

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Ena/VASP and fascin collaborate in the self-organization of actin filaments

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  1. Ena/VASP and fascin collaborate in the self-organization of actin filaments Jonathan Winkelman Mini Retreat, April 10th, 2014 Department of Molecular Genetics and Cell Biology

  2. Actin monomers assemble into polar filaments Barbed end (B) Pointed end (P) In vitro: TIRF microscopy

  3. Actin assembly drives diverse cellular processes Complex actin structures self-organize due to the coordinated interaction of actin and actin-binding proteins

  4. Actin assembly drives directed cell motility Filopodia Lamellipodium

  5. Filopodia are exploratory organelles Dorsal closure in Drosophila GFP-actin StephNowotarski, Peifer lab (UNC) Capping protein Arp 2/3 complex Svitkina et al JCB 2003 Ena Actin Membrane Fascin

  6. Ena/VASP proteins facilitate filopodia formation Drosophila cell culture (D16), expressing GFP-actin Ena Knockdown Ena-mCherry Overexpression WT Colleen Bilanciaand Mark Peifer (UNC)

  7. Question: How do Ena’s biochemical properties facilitate the assembly of filopodia? Capping protein Arp 2/3 complex Ena Actin Membrane Fascin Strategy: Purify Ena and characterize its actin assembly properties in vitro

  8. Ena is a protein with multiple conserved domains EVH2

  9. Ena stimulates actin assembly In vitro: TIRF microscopy Actin only 125 nM Ena(ProEVH2)

  10. Ena stimulates actin assembly In vitro: TIRF microscopy How is Ena stimulating actin assembly? Actin only 125 nM Ena(ProEVH2)

  11. Ena increases filament elongation rate 3-fold during processive runs 0.25 nM Ena(ProEVH2) 1.25 nM Ena(ProEVH2) 5.0 nM Ena(ProEVH2) In vitro: TIRF Microscopy Actin only (9.3 sub/s) (13.8 sub/s) (15.6 sub/s) (27.1 sub/s) 25 nM Ena(ProEVH2) Length of fast (processive) runs 0.25 nM Ena(ProEVH2)

  12. Ena remains processively associated with elongating barbed ends In vitro: 2 Color TIRF microscopy 50 pM SNAP-549-Ena(DL) Length of processive runs

  13. Ena’s barbed-end processive run length increases when adsorbed to a surface 50 pM SNAP-549-Ena(DL) Length of processive runs Fraction bound Time, sec Immobilized Ena

  14. Processive Ena units are tetramers 50 pM SNAP-549-Ena(DL) Bleaching Steps Frequency of Steps

  15. How do Ena’s biochemical properties facilitate the assembly of filopodia? • Ena binds filament barbed ends with high affinity • Processive tetramer that increases the elongation rate ~3-fold • Immobilization increases processivity Capping protein Arp 2/3 complex Ena Actin Membrane

  16. Ena protects barbed ends from capping protein Seeds only +4 nM Capping Protein +1 nM SNAP-549-Ena(DL) +Ena & CP Seeded assembly length TMR-actin seeds Ena Actin

  17. Ena gathers and elongates multiple barbed ends 5 nMEna(DL) Svitkina et al., 2003 0.05 nM SNAP-549-Ena(DL)

  18. Ena’s biochemical properties • Ena binds filament barbed ends with high affinity • Processive tetramer that increases the elongation rate ~3-fold • Immobilization increases processivity • Gathers barbed ends • Protects growing barbed ends from capping protein Capping protein Arp 2/3 complex Ena Actin Membrane

  19. How does Ena behave on actin filaments bundled by Fascin? Capping protein Arp 2/3 complex Ena Actin Membrane Fascin

  20. Ena and Fascin cooperate to stimulate the assembly of filopodia-like bundled actin filaments 400 nM fascin + 50 pM Snap-549-Ena(DL) In vitro: TIRF Microscopy

  21. Ena drives barbed-end alignment in fascin bundles Barbed ends: 50 nM fascin + 50 pM Snap-549-Ena(DL) Trailing Leading Pointed ends Time In vitro: TIRF microscopy length Aligned barbed ends 1 2 Leading barbed end, low processivity 3 Trailing barbed end, high processivity

  22. Ena drives barbed-end alignment in fascin bundles Barbed ends 50 nM fascin + 50 pM Snap-549-Ena(DL) Trailing Leading Pointed ends Time In vitro: TIRF microscopy length Time to next processive run Length of processive runs Leading BE, 14 +/- 0.4 s Kon= 120 uM-1s-1 Trailing BE, 50 +/- 0.8 s Ena has a 10-fold higher affinity for trailing(0.1 nM) ends vs. leading (1.0 nM) ends! Kon= 230 uM-1s-1

  23. Ena has a much higher affinity for bundled actin 50 nM fascin + 5 nM Snap-549-Ena(DL)

  24. Ena has increased affinity for the sides of bundled actin 1 filament 2-filament bundle 4-filament bundle

  25. Increased dwell time on filament sides may promote barbed-end loading of Ena Hansen et al., JCB

  26. Interaction of FAB with neighboring filament could stabilize trailing barbed end association EVH1 Proline-rich Colied-Coil FAB GAB

  27. Ena’s increased affinity for trailing ends results in self-alignment of barbed ends Trailing ends are preferentially elongated Barbed end alignment results. Ena can elongate two multiple barbed ends simultaneously Leading barbed end elongation is unstable

  28. Ena and Fascin cooperate to stimulate the assembly of filopodia-like bundled actin filaments • Ena binds filament barbed ends with high affinity • Processive tetramer that increases the elongation rate ~3-fold • Protects growing barbed ends from capping protein • Gathers barbed ends • Fascin enhances Ena’s actin assembly properties (longer and more frequent processive runs) • Fascin and Ena cooperate in a positive-feedback loop to drive assembly of polarized bundled filaments Capping protein Arp 2/3 complex Ena Actin Membrane Fascin

  29. KovarLab: Jenna Christensen Tom Burke YujieLi Jen Sees Cristian Suarez Dennis Zimmermann Thanks to my committee- Ed Munro, Michael Glotzer, Rick Fehonand MagaretGardel. Thanks to Ed, Margaret, andRoberto Dominguez, for helpful conversations about paper Thanks to Jon Staley and Kristine Gaston for organizing the mini retreat Colleen Bilancia and Mark Peifer (UNC – Chapel Hill)

  30. Reconstitute the transition of arp2/3 complex- generated branched networks into filopodial-like networks Reymann et al., 2010

  31. Fascin enhances protection from capping protein in the presence of Ena

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