1.Structural Basis of Actin Nucleations :Lessons from bacterial actin nucleator

2. Actin 101Actin is one of the most abundant, highly conserved and well characterized ofall eukaryotic proteinsMonomeric 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 theregulation of various cellular processCytokinesisDevelopmentsCell Motility TransportPhagocytosis Vesicle 5. Spatial and temporal regulation of Actin filament remodelingWhen? How long?Where? Type of actin filaments 6. Remodeling of actin filaments New Filament GenerationsDepolymerizations/SeveringCapping/DecappingVarious Actin Binding Proteins (ABP) mediate actin filament remodeling 7. Nucleation Seed Formation is the rate limiting step for actin polymerizationRapidPolymerization Rate LimitingSlowSlow FastStabilization of actin nucleation seed is critical for theRapid actin polymerizationsActin nucleators : Family of proteins stabilize actin nucleations 8. Each actin nucleator determine specific type of:59 Structure actinAnnual Reviews AR131-19REGULATION OF ACTIN FILAMENT NETWORK651 Arp2/3 complexFormins SpireBranched actin filamentsure 1 Electron micrographs of branched actin laments. (a) Electron micrograph of Straight actin bundlesleading edge of a migrating Xenopus keratocyte prepared by detergent extraction andin Lamellipodiary shadowing. (b) Detail of boxed region from (a), with a branched lament network In Stress fibers and filopodiaed in blue. (c) Gold-labeled antibody to ARPC2 localized to a branch point in thement network at the leading edge. (d ) Branch formed in vitro by pure actin laments Acanthamoeba Arp2/3 complex. (ac) modied from 14. (d ) Modied from 13.p2/3 complex (hereafter referred to as nucleation promoting factors), are being 9. Arp2/3 complex : 6 Protein complex makes branched networksBoczkowska et al., Structure 2008Requires activator proteins called nucleation promotingfactor (NPF) 10. ForminOtomo et al., Nature 2005Responsible for the straight bundles of filaments (Stress fiber, Fillopodia)Caps fast growing ends (barbed end) of actin filament and enhance elongation rates 11. New Class of Actin Nucleators:Tandem W domain containing actin nucleatorsOocyte maturation, Endocytosis (Drosophila, mouse, human)Brain specific actin nucleator (mouse, human)Vibrio parahemolyticus / Vibrio choleraRicketssia sp. 12. WASP-homology domain 2 (WH2):Common Actin binding motifs in actin nucleators and NPF~25aa long conserved sequencesPresent in many actin Nucleation Promoting Factor(WASP, N-WASP, WAVE) and actin nucleatorsBinds hydrophobic cleft of actinPresent in various Nucleation Promoting FactorsAnd tandem WH2 domain containing nucleatorsActin -crosslinked first WH2 domain structureRebowski and Namgoong, J. Mol. Biol. 2010 13. Mechanism of tandem W domain containing nucleators Initial model of actin nucleation of spire (2005, Quinlan et al.,)SpireKINDWWW W S FYVE zinc-finger Actin Template Modelubiquitin-L Pro-richCC-?W WWCobl152229 325425560 600 11811337VopLW W WVCD 99 484Sca2FH2-like ? P W W WCD34671 700872 1020 1086 1544Similar Organization: More or less similar mechanism? 14. W domain containing nucleators is not created as equal..Nucleation mechanism of each protein will be different.. 15. QuestionsMechanism of tandom W domain nucleators- Huge differences in nucleation activities suggest theDifferences innucleation mechanismIs Tandom WH2 domain sufficient for nucleationactivity?- Involvement of other domains in nucleation? 16. Vibrio parahemolyticus VopL as model system for actinNucleation mechanism- Encoded by bacteria Vibrio parahemolyticus(main agents for food poisoning)- It injects ~20 effector proteins into host cellusing Type III secretion machinary (VopL is one of them)- Robust actin nucleation activity in vitro & in vivo- Actin Cytoskeleton is usual target for various effectorproteins 17. Bacterial Type III Secretion System and EffectorsAnd host cell actin cytoskeleton 18. Listeria monocytogenesActin-based motility 19. Mapping of minimal actin nucleaton requirement in VopL 3W domains and C-terminal domain are both essential for actin nucleation 20. Actin polymerization assay using pyrene labeled actins(Industry Standard actin polymerization assay)Higher F-actinActivity Fluorescence LowerActivity Actin onlyG-actin Pyrene labeled actin at Cys-374 Time 21. Limitation of pyrene labeled actin assayGeneration of more nucleation seeds Enhancement of elongation speeds(Example : Arp2/3 Complex) (Example : Formins)Traditional fluorescence-based bulk assay cannot distinguish thedifference between two 22. Single-Molecule Actin Polymerization Assay usingTotal Internal Reflection Fluorescence Microscopy (TIRFM) Oregon-Green Labeled ActinMyosin S1 headLaser excitationHigh resolution imaging sufficient for visualizing single actin filaments is possible.. 23. Single molecule TIRFM assay confirms that VopL is genuine actin nucleators (more filament seeds, no change in elongation speed)Actin only1.0P-3W-VCD 31.0W W WVCD3W-VCD 27.0W W W VCD VCD1.0VCDNamgoong et al., Nature Struct. Mol. Biol. 2011 24. Generation of more nucleation seeds Enhancement of elongation speeds 25. C-terminal domain of VopL (VCD) is essential for the actinnucleation. (By its own it has negligible nucleationactivity) What is the functional roles of VCD in actin nucleation ofVopL ? 26. Leucine-Zipper like coiled coil is predicted in C-terminalDoes VopL exist as a dimer? 27. VopL is dimerize by VCD and dimerization is essential for robust nucleation activityMulti angle light scattering-SizePyrene-actin Polymerization AssayExclusion chromatogrphy (SEC-MALS)But dimerization difficient mutants (3W-VCD*) still retains trace ofnucleation activityVCD is doing more than dimerization? Namgoong et al., Nature Struct. Mol. Biol. 2011 28. VCD can bind F-actin and 1W-VCD+Actin forms complex of 2:2 29. Two different binding mode of Actin nucleatorsArp2/3 complex stay on the pointed end (slow growing end) of actin filamentsFormins remains processively bound on barbed end (fast growing end) 30. QD FH2P PYeast Formin (Bni1) labeled with Quantum dotFormin moves along with fast-growing end of actin filamentsPaul and Pollard, J.Biol. Chem. 2009 31. Bound and Stay Rapid Dissociationat the pointed endNucleation Detachment Q Q D D W W W W W W W W W W W P PP WPActin Nucleations by VopL is initiated from the pointed end of actinFormed filaments is rapidly dissociated from VopLNamgoong et al., Nature Struct. Mol. Biol. 2011 32. VCD is globular domainPoly-ProW-1 W-2 PP W-3 DD No homologs in PDBPredicted AsCoiled- Mostly alpha-helicalCoil C-terminal is predicted as coiled-coil 33. Crystallization of VCD VopL VopFCrystal is readily obtained, but poor diffractions..Crystal is very sensitive for the cryoprotection (weak diffraction 6-7)- 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 34. Tails Base ArmsArms Namgoong et al., Nature Struct. Mol. Biol. 2011 35. Namgoong et al., Nature Struct. Mol. Biol. 2011 36. Namgoong et al., Nature Struct. Mol. Biol. 2011 37. SAXS reconstitution of Dimerization Domains and actin+VCD complex Namgoong et al., Nature Struct. Mol. Biol. 2011 38. Model of actin binding with VopL VCDNamgoong et al., Nature Struct. Mol. Biol. 2011 39. Namgoong et al., Nature Struct. Mol. Biol. 2011 40. Dimerization of other W domain contains nucleators enhance nucleations Namgoong et al., Nature Struct. Mol. Biol. 2011 41. Dimerization of W containing nucleators are common strategies in different nucleators 42. ConclusionVopL utilizes unique VCD domain as a platform foractin nucleationUnique catalytic nature of actin nucleatorDimerization of actin nucleators are universal activationMechanism : actin filament has two strands, so dimericNucleator is needed. - Spire - Formin - arp2/3 complex 43. AcknowledgementsDominguez Lab in University of PennsylvaniaRoberto DominguezGrzegorz RebowskiBoczkowska MalgorzataUniversity of ChicagoMicheal GlistaDavid KovarAdvanced Photon Source (IMCA-CAT, 17-BM / Bio-CAT)CHESS A1 44. 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 Samecrystal with different orientations (360 frameseachs) merged and scaled using first 240 frames oftwo datasets 45. Locate 6 Se atoms using SHELXD Solvent flattenned experimental map Identification of C-terminal coiled-coil Build a initial model 46. Identification ofNoncrystallographicsymmetry from initialmodel and operators andmask generationsNCS averaged mapRefine with PHENIX usingsecondary structure/ NCSrestrainsFinal Refined 2Fo-Fc map