1. Structural Basis of Actin Nucleations :
Lessons from bacterial actin nucleator
2. Actin 101
Actin is one of the most abundant, highly conserved and well characterized of
all eukaryotic proteins
Monomeric Actin (42kDa) Filamentous Actin
(G-Actin) (F-Actin)
3. Actin Filament is the main „building block‟ of cytoskeleton
Borish & Svitskina
4. Dynamic actin filament remodeling is the key for the
regulation of various cellular process
Cytokinesis
Developments
Cell Motility Transport
Phagocytosis
Vesicle
5. Spatial and temporal regulation of Actin filament remodeling
When?
How long?
Where?
Type of actin filaments
6. Remodeling of actin filaments
New Filament Generations
Depolymerizations/Severing
Capping/Decapping
Various Actin Binding Proteins (ABP) mediate actin filament remodeling
7. Nucleation Seed Formation is the rate limiting step
for actin polymerization
Rapid
Polymerization
Rate Limiting
Slow Slow Fast
Stabilization of actin nucleation seed is critical for the
Rapid actin polymerizations
Actin nucleators : Family of proteins stabilize actin nucleations
8. Each actin nucleator determine specific type of
:59
Structure actin
Annual Reviews AR131-19
REGULATION OF ACTIN FILAMENT NETWORK 651
Arp2/3 complex Formins Spire
Branched actin filaments
ure 1 Electron micrographs of branched actin filaments. (a) Electron micrograph of
Straight actin bundles
leading edge of a migrating Xenopus keratocyte prepared by detergent extraction and
in Lamellipodia
ry shadowing. (b) Detail of boxed region from (a), with a branched filament network
In Stress fibers and filopodia
ed in blue. (c) Gold-labeled antibody to ARPC2 localized to a branch point in the
ment network at the leading edge. (d ) Branch formed in vitro by pure actin filaments
Acanthamoeba Arp2/3 complex. (a–c) modified from 14. (d ) Modified from 13.
p2/3 complex (hereafter referred to as nucleation promoting factors), are being
9. Arp2/3 complex : 6 Protein complex makes branched networks
Boczkowska et al., Structure 2008
Requires activator proteins called nucleation promoting
factor (NPF)
10.
11.
12. Formin
Otomo et al., Nature 2005
Responsible for the straight bundles of filaments (Stress fiber, Fillopodia)
Caps fast growing ends (barbed end) of actin filament and enhance elongation rates
13. New Class of Actin Nucleators:
Tandem W domain containing actin nucleators
Oocyte maturation, Endocytosis (Drosophila, mouse, human)
Brain specific actin nucleator (mouse, human)
Vibrio parahemolyticus / Vibrio cholera
Ricketssia sp.
14. WASP-homology domain 2 (WH2):
Common Actin binding motifs in actin nucleators and NPF
~25aa long conserved sequences
Present in many actin Nucleation Promoting Factor
(WASP, N-WASP, WAVE) and actin nucleators
Binds hydrophobic cleft of actin
Present in various Nucleation Promoting Factors
And tandem WH2 domain containing nucleators
Actin -crosslinked first WH2 domain structure
Rebowski and Namgoong, J. Mol. Biol. 2010
15. Mechanism of tandem W domain containing nucleators
Initial model of actin nucleation of spire (2005, Quinlan et al.,)
Spire KIND WWW W S FYVE zinc-finger
‘Actin Template Model’
ubiquitin-L Pro-rich CC-? W W W
Cobl 152 229 325 425 560 600 1181 1337
VopL W W W VCD
99 484
Sca2 FH2-like ? P W W W CD
34 671 700 872 1020 1086 1544
Similar Organization: More or less similar mechanism?
16. W domain containing nucleators is not created as equal..
Nucleation mechanism of each protein will be different..
17. Questions
Mechanism of tandom W domain nucleators
- Huge differences in nucleation activities suggest the
Differences innucleation mechanism
Is Tandom WH2 domain sufficient for nucleation
activity?
- Involvement of other domains in nucleation?
18. Vibrio parahemolyticus VopL as model system for actin
Nucleation mechanism
- Encoded by bacteria Vibrio parahemolyticus
(main agents for food poisoning)
- It injects ~20 effector proteins into host cell
using Type III secretion machinary (VopL is one of them)
- Robust actin nucleation activity in vitro & in vivo
- Actin Cytoskeleton is usual target for various effector
proteins
19. Bacterial Type III Secretion System and Effectors
And host cell actin cytoskeleton
21. Mapping of minimal actin nucleaton requirement in VopL
3W domains and C-terminal
domain are both essential
for actin nucleation
22. Actin polymerization assay using pyrene labeled actins
(“Industry Standard” actin polymerization assay)
Higher
F-actin Activity
Fluorescence Lower
Activity
Actin only
G-actin
Pyrene labeled actin at Cys-374
Time
23. Limitation of pyrene labeled actin assay
Generation of more nucleation seeds Enhancement of elongation speeds
(Example : Arp2/3 Complex) (Example : Formins)
Traditional fluorescence-based bulk assay cannot distinguish the
difference between two
24. Single-Molecule Actin Polymerization Assay using
Total Internal Reflection Fluorescence Microscopy (TIRFM)
Oregon-Green
Labeled Actin
Myosin S1 head
Laser excitation
High resolution imaging sufficient for visualizing single actin filaments is possible..
25. Single molecule TIRFM assay confirms that VopL is genuine actin
nucleators (more filament seeds, no change in elongation speed)
Actin only 1.0
P-3W-VCD 31.0
W W W VCD
3W-VCD 27.0
W W W VCD
VCD 1.0
VCD
Namgoong et al., Nature Struct. Mol. Biol. 2011
27. • C-terminal domain of VopL (VCD) is essential for the actin
nucleation. (By its own it has negligible nucleation
activity)
• What is the functional roles of VCD in actin nucleation of
VopL ?
29. VopL is dimerize by VCD
and dimerization is essential for robust nucleation activity
Multi angle light scattering-Size
Pyrene-actin Polymerization Assay
Exclusion chromatogrphy (SEC-MALS)
But dimerization difficient mutants (3W-VCD*) still retains trace of
nucleation activity
VCD is doing more than dimerization?
Namgoong et al., Nature Struct. Mol. Biol. 2011
30. VCD can bind F-actin and 1W-VCD+Actin forms complex of 2:2
31. Two different binding mode of Actin nucleators
Arp2/3 complex stay on the „pointed end‟ (slow growing end) of actin filaments
Formins remains processively bound on „barbed end‟ (fast growing end)
32. QD
FH2
P
P
Yeast Formin (Bni1) labeled with Quantum dot
Formin moves along with fast-growing end of actin filaments
Paul and Pollard, J.Biol. Chem. 2009
33. Bound and Stay
Rapid Dissociation at the pointed end
Nucleation Detachment
Q Q
D D
W W
W W
W W
W W
W
W W
P P
P W P
Actin Nucleations by VopL is initiated from the pointed end of actin
Formed filaments is rapidly dissociated from VopL
Namgoong et al., Nature Struct. Mol. Biol. 2011
34. VCD is globular domain
Poly-Pro W-1 W-2 PP W-3
DD
• No homologs in PDB Predicte
d As
Coiled-
• Mostly alpha-helical Coil
• C-terminal is predicted as coiled-coil
35. Crystallization of VCD
VopL VopF
Crystal is readily obtained, but poor diffractions..
Crystal is very sensitive for the cryoprotection (weak diffraction 6-7Å)
- Without any cryoprotections, diffraction is even poorer..
- Most cryoprotectants (Glycerol, PEG400, Oil, Ethylene glycol, Sucrose etc) are not effective
- Stepwise increase of cryoprotectant
- Dihydrations somewhat improve diffraction in Native, but not in SeMet Crystal..
- Room temp diffraction did not works..
‘Flash dipping’ of crystal (less than 1 sec) in mother liquid + 15% PEG400
36.
37. Tails
Base
Arms
Arms
Namgoong et al., Nature Struct. Mol. Biol. 2011
43. Dimerization of other W domain contains nucleators enhance nucleations
Namgoong et al., Nature Struct. Mol. Biol. 2011
44. Dimerization of W containing nucleators are common strategies in different nucleators
45.
46. Conclusion
VopL utilizes unique VCD domain as a ‘platform’ for
actin nucleation
Unique ‘catalytic’ nature of actin nucleator
Dimerization of actin nucleators are universal activation
Mechanism : actin filament has two strands, so dimeric
Nucleator is needed.
- Spire
- Formin
- arp2/3 complex
47. Acknowledgements
Dominguez Lab in University of Pennsylvania
Roberto Dominguez
Grzegorz Rebowski
Boczkowska Malgorzata
University of Chicago
Micheal Glista
David Kovar
Advanced Photon Source (IMCA-CAT, 17-BM / Bio-CAT)
CHESS A1
48. • SAD data collections at CHESS A1
- Fixed wavelength at 0.9772
- Relatively week diffractions (3.1-3.2A)
- Radiation damages
• Two datasets are collected from the Same
crystal with different orientations (360 frames
eachs)
• merged and scaled using first 240 frames of
two datasets
49. • Locate 6 Se atoms using SHELXD
• Solvent flattenned experimental map
• Identification of C-terminal coiled-coil
• Build a initial model
