Ena vasp and fascin collaborate in the self organization of actin filaments
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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. 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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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 10th, 2014

Department of Molecular Genetics and Cell Biology


Actin monomers assemble into polar filaments

Actin monomers assemble into polar filaments

Barbed end (B)

Pointed end (P)

In vitro: TIRF microscopy


Ena vasp and fascin collaborate in the self organization of actin filaments

Actin assembly drives diverse cellular processes

Complex actin structures self-organize due to the coordinated interaction of actin and actin-binding proteins


Ena vasp and fascin collaborate in the self organization of actin filaments

Actin assembly drives directed cell motility

Filopodia

Lamellipodium


Filopodia are exploratory organelles

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


Ena vasp proteins facilitate filopodia formation

Ena/VASP proteins facilitate filopodia formation

Drosophila cell culture (D16), expressing GFP-actin

Ena

Knockdown

Ena-mCherry

Overexpression

WT

Colleen Bilanciaand Mark Peifer (UNC)


Question how do ena s biochemical properties facilitate the assembly of filopodia

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


Ena is a protein with multiple conserved domains

Ena is a protein with multiple conserved domains

EVH2


Ena stimulates actin assembly

Ena stimulates actin assembly

In vitro: TIRF microscopy

Actin only

125 nM

Ena(ProEVH2)


Ena stimulates actin assembly1

Ena stimulates actin assembly

In vitro: TIRF microscopy

How is Ena stimulating actin assembly?

Actin only

125 nM

Ena(ProEVH2)


Ena increases filament elongation rate 3 fold during processive runs

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)


Ena vasp and fascin collaborate in the self organization of actin filaments

Ena remains processively associated with elongating barbed ends

In vitro: 2 Color TIRF microscopy

50 pM

SNAP-549-Ena(DL)

Length of processive runs


Ena s barbed end processive run length increases when adsorbed to a surface

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


Processive ena units are tetramers

Processive Ena units are tetramers

50 pM

SNAP-549-Ena(DL)

Bleaching Steps

Frequency of Steps


How do ena s biochemical properties facilitate the assembly of filopodia

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


Ena protects barbed ends from capping protein

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


Ena gathers and elongates multiple barbed ends

Ena gathers and elongates multiple barbed ends

5 nMEna(DL)

Svitkina et al., 2003

0.05 nM SNAP-549-Ena(DL)


Ena s biochemical properties

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


Ena vasp and fascin collaborate in the self organization of actin filaments

How does Ena behave on actin filaments bundled by Fascin?

Capping protein

Arp 2/3 complex

Ena

Actin

Membrane

Fascin


Ena and fascin cooperate to stimulate the assembly of filopodia like bundled actin filaments

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


Ena drives barbed end alignment in fascin bundles

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


Ena drives barbed end alignment in fascin bundles1

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


Ena has a much higher affinity for bundled actin

Ena has a much higher affinity for bundled actin

50 nM fascin + 5 nM Snap-549-Ena(DL)


Ena has increased affinity for the sides of bundled actin

Ena has increased affinity for the sides of bundled actin

1 filament

2-filament bundle

4-filament bundle


Increased dwell time on filament sides may promote barbed end loading of ena

Increased dwell time on filament sides may promote barbed-end loading of Ena

Hansen et al., JCB


Ena vasp and fascin collaborate in the self organization of actin filaments

Interaction of FAB with neighboring filament could stabilize trailing barbed end association

EVH1

Proline-rich

Colied-Coil

FAB

GAB


Ena s i ncreased affinity for trailing ends results in self alignment of barbed ends

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


Ena vasp and fascin collaborate in the self organization of actin filaments

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


Ena vasp and fascin collaborate in the self organization of actin filaments

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)


Ena vasp and fascin collaborate in the self organization of actin filaments

Reconstitute the transition of arp2/3 complex- generated branched networks into filopodial-like networks

Reymann et al., 2010


Fascin enhances protection from capping protein in the presence of ena

Fascin enhances protection from capping protein in the presence of Ena


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