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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

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

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

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Actin assembly drives directed cell motility Filopodia Lamellipodium

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Filopodia are exploratory organelles Svitkina et al JCB 2003 Steph Nowotarski, Peifer lab (UNC) Dorsal closure in Drosophila GFP-actin Capping protein Ena Arp 2/3 complex ActinMembraneFascin

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

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Question: How do Enas biochemical properties facilitate the assembly of filopodia? Strategy: Purify Ena and characterize its actin assembly properties in vitro Capping protein Ena Arp 2/3 complex ActinMembraneFascin

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Ena is a protein with multiple conserved domains EVH2

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

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

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

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

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

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Processive Ena units are tetramers Bleaching StepsFrequency of Steps 50 pM SNAP-549-Ena( L)

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

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

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

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Enas 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 Ena Arp 2/3 complex ActinMembrane

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

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

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

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length Time Pointed ends Trailing Leading Barbed ends Ena drives barbed-end alignment in fascin bundles In vitro: TIRF microscopy 50 nM fascin + 50 pM Snap-549-Ena( L) Length of processive runsTime to next processive run K on = 120 uM -1 s -1 K on = 230 uM -1 s -1 Leading BE, 14 +/- 0.4 s 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!

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

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Ena has increased affinity for the sides of bundled actin 1 filament2-filament bundle4-filament bundle

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

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

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

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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 Enas 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 Ena and Fascin cooperate to stimulate the assembly of filopodia-like bundled actin filaments Capping protein Ena Arp 2/3 complex ActinMembraneFascin

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Kovar Lab: Jenna Christensen Tom Burke Yujie Li Jen Sees Cristian Suarez Dennis Zimmermann Thanks to my committee - Ed Munro, Michael Glotzer, Rick Fehon and Magaret Gardel. Thanks to Ed, Margaret, and Roberto 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)

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

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Fascin enhances protection from capping protein in the presence of Ena 32