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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

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

slide3

Actin assembly drives diverse cellular processes

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

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 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)

slide12

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

slide19

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

slide26

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

slide28

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

slide29

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)

slide30
Reconstitute the transition of arp2/3 complex- generated branched networks into filopodial-like networks

Reymann et al., 2010

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