Ena/VASP and fascin collaborate in the self- organization of actin filaments Jonathan Winkelman Mini...
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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
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
Slide 2
Actin monomers assemble into polar filaments Pointed end (P)
Barbed end (B) In vitro: TIRF microscopy
Slide 3
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 Svitkina et al JCB 2003
Steph Nowotarski, Peifer lab (UNC) Dorsal closure in Drosophila
GFP-actin Capping protein Ena Arp 2/3 complex
ActinMembraneFascin
Slide 6
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)
Slide 7
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
Slide 8
Ena is a protein with multiple conserved domains EVH2
Slide 9
Ena stimulates actin assembly In vitro: TIRF microscopy 125 nM
Ena(ProEVH2) Actin only
Slide 10
Ena stimulates actin assembly In vitro: TIRF microscopy 125 nM
Ena(ProEVH2) Actin only How is Ena stimulating actin assembly?
Slide 11
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
Slide 12
Ena remains processively associated with elongating barbed ends
In vitro: 2 Color TIRF microscopy Length of processive runs 50 pM
SNAP-549-Ena( L)
Slide 13
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
Slide 14
Processive Ena units are tetramers Bleaching StepsFrequency of
Steps 50 pM SNAP-549-Ena( L)
Slide 15
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
Slide 16
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
Slide 17
Ena gathers and elongates multiple barbed ends 0.05 nM
SNAP-549-Ena( L) 5 nM Ena( L) Svitkina et al., 2003
Slide 18
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
Slide 19
19 How does Ena behave on actin filaments bundled by Fascin?
Capping protein Ena Arp 2/3 complex ActinMembraneFascin
Slide 20
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
Slide 21
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
Slide 22
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!
Slide 23
Ena has a much higher affinity for bundled actin 23 50 nM
fascin + 5 nM Snap-549-Ena( L)
Slide 24
Ena has increased affinity for the sides of bundled actin 1
filament2-filament bundle4-filament bundle
Slide 25
Increased dwell time on filament sides may promote barbed-end
loading of Ena 25 Hansen et al., JCB
Slide 26
26 Interaction of FAB with neighboring filament could stabilize
trailing barbed end association FAB GAB Colied-Coil Proline-rich
EVH1
Slide 27
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
Slide 28
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
Slide 29
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)
Slide 30
Reconstitute the transition of arp2/3 complex- generated
branched networks into filopodial-like networks 30 Reymann et al.,
2010
Slide 31
31
Slide 32
Fascin enhances protection from capping protein in the presence
of Ena 32