PURICH_EMA_6580 - The Actoclampins: Affinity-Modulated...

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Unformatted text preview: The Actoclampins: Affinity-Modulated Motors that Power Actin-Based Motility Motors Dan Purich Department of Biochemistry & Molecular Biology, College of Medicine University of Florida, Gainesville 32610 dlpurich@ufl.edu April 20, 2009 Guest Lecture in EMA 6580 SCIENCE OF BIOMATERIALS Phagocyte chasing a S. aureus (Wright,1955) Phagocyte aureus (Wright,1955) Cellular Tensegrity Model – Donald Ingber Entire cell is a prestressed tensegrity structure e.g., clathrin-coated vesicles resemble geodesic domes clathrin Cell’s mechanical stiffness is based on their material properties, with forces exerted by cytoskeleton Forces are balanced by interconnected structures resisting compression, most notably internal microtubule struts and extracellular matrix adhesions. Individual filaments experience tension or compression: e.g. Contractile actin microfilaments Tension Tension Actomyosin Actomyosin e.g. Expansile actin microfilaments Compression Compression Actoclampin Actoclampin Additional passive contributions to prestress from: Cell distension (adhesions to ECM & other cells) Osmotic forces (acting on cell membrane & vesicles) Interconnections (cytoskeleton & attached organelles) 1 Actin Filament Elongation Powers the “Expansile Apparatus” the Expansile Apparatus 4 µm = 4 sec sec filopod lamellipod Leading edge protrudes Leading at rate of 1 µm / sec sec > 400 subunits / sec 400 sec add to each filament add ? The Now-Obvious Solution The to a Long-Standing Puzzle – to Dickinson & Purich (2002) Biophys. J. 82, 615-627. Dickinson Biophys. Cell motility is powered by Clamped-Filament Motors that Clamped-Filament define the “Expansile Apparatus” Expansile Apparatus Hydrolysis of filament-bound Actin·ATP can facilitate clamp ATP release during elongation. release 2 Energy Available for Actin Motility if motility is driven by ATP hydrolysis if ΔGhydrolysis/molecule = Workreversible/molecule /molecule Work ~ 38 kJoules ÷ Avogadro’s Number ~ 38,000 Newton·meter / 6×1023 molecules 38,000 Newton molecules ~ 60 pN·nm 60 ~ 11 pN exerted 11 over 5.4 nm over 5.4 nm nm … if work is completely reversible reversible Energy Available for Actin Motility T F·ADP ·P i ADP F·ADP ATP G·ATP T Penultimate ATP T D D D Hydrolysis T DDD G·ADP TDDD TDDDD Translocation 3 End-Tracking Motor Proteins — the key component VASODILATORSTIMULATED PHOSPHOPROTEIN N-terminal surface-tethering domain binds to receptor on motile object Proline-rich cargo to be propelled) (specifies the profilin-localizing motif binds up to 24 mol Profilin or Carboxyl-terminal clamp Profilin·Actin·ATP per mol ctin binds to elongating a VASP Tetramerization domain rare RH coiled-coiltsegion filament at/near i r (+)-end makes for a mechanically strong tetramer Kuhnel et al. (2004) PNAS 101, 17027. Kuhnel et PNAS GFP-VASP dynamics at the leading edge– VASP exhibits the ideal spatiotemporal properties for an actoclampin end-tracking motor. (Time C ompression = 80 ×) ompression 80 Rottner, Behrendt, Small & Wehland (1999) Nature Cell Biol. 1, 321. Small Nature 4 Clamped-Filament Elongation Motor Cargo-Docking Domain Domain Dickinson & Purich (2002) Biophys. J. 82, 615. Dickinson Biophys. Motile Object Listeria Lamellipodia/Filipodia Endosomes/Lysosomes Phagosomes VASP F(E/D)FPPPPX(E/D)LE where X = P or T Listeria ActA Protein Zyxin Host-Cell Proteins Vinculin Purich & Southwick Migfilin (1997) BBRC 231, 686. (1997) BBRC Fedorov, Fedorov, Gertler & Almo (1999) (1999) Nature Structural Biology 6, 661. Vogtherr, Grimme, Pescatore & Schwalbe (2003) Grimme (2003) Journal of Biomolecular NMR 27, 189. Journal NMR Ena/VASP Homology Domain-1 Clamped-Filament Elongation Motor PROFILIN Motile Object Profilin-Docking Domain Actin·ATP VASP has twenty-four XPPPPP Sequences (where X = G, A, L, P or S) Purich & Southwick (1997) BBRC 231, 686. Mahoney, Rozwarski, Fedorov, Fedoro &, Almo (1999) Nat Struct. Biol. 6, 666. Struct 5 Clamped-Filament Elongation Motor Actin·ATP Clamping Domain Actin·ADP·Pi (or Actin·ADP) Motile Object Key assumptions: Growing end of EVERY filament is tightly Growing EVERY bound to a clamping protein. Hydrolysis of filament-bound ATP rapidly attenuates clamp binding affinity. Dickinson & Purich (2002) Biophys. J. 82, 615. Dickinson Biophys. “Lock, Load & Fire” Mechanism: New Actin·ATP ATP monomers Load Clamp in energy well at terminal subunits of actin filament 5.4-nm shift Clamp Occupies New Terminal Energy Well Remnant Energy Well New Energy Well (unoccupied) at new End of Filament Clamp in Penultimate Energy Well ATP hydrolysis weakens clamp binding affinity clamp 2 Clamp Shifts & Re-locks onto Actin·ATP at ATP new terminus new Clamp is “Lifted” into Now Shallow Energy Well Dickinson & Purich (2002) Biophys. J. 82, 615. Dickinson Biophys. 6 Propulsion by an Ensemble Propulsion of Clamped Filaments… compressed filaments Why didn’t the object move? motile surface exert expansile force force lagging taut filament exerts tensile force that restrains object Dickinson & Purich (2002) Biophys. J. 82, 615. Dickinson Biophys. A simplified biophysical perspective z0 z1 Compressive Force: Compressive F = – κ (z - z0) Uncompressed clamp F 5.4 nm z1 Potential Energy Potential Energy Compressed clamp z1 5.4 nm Dickinson & Purich (2002) Biophys. J. 82, 615. Dickinson Biophys. 7 A more complete treatment Df ≡ Filament Diffusivity N ≡ Number of Filaments t ≡ time dW ≡ Wiener Process Increment zs ≡ Position of Surface κ ≡ Compressive Stiffness κΤ ≡ Tensile Stiffness z0,i ≡ Equilibrium End Position Force from Filament i : Force −κ T (z0,i − zs ) Fi = −κ ( z0,i − z s ) zs ≥ z0,i z ≥ z 0,i Motion of Motile Surface: dzs = Df N ∑ F dt + 2 Df / N dW NkT i =1 i Mean Translocation Time τ : Mean z +d κ ( x −z 0 ) 2 x −κ ( y− z 0 ) 2 11 2kT τ= dxe ∫ dye 2kT Df ∫1 z z1 Actoclampin model also model explains the shape of explains the shape motile vesicles – motile Taunton (2000) J. Cell Biol. 148, 519. Dickinson & Purich (2006) Biophys. J, Dickinson Biophys. 91, 1548. 91, (a) Distorted vesicle shape arises from push/pull forces on motile vesicle surface. forces (b) Reduced availability of actin monomer to interior motors generates the shape. (c) Model predicts 9-10 pN/filament stall force – close to 8-9 pN/filament experimental value. 8 Actoclampin model also model explains the shape of explains the shape motile vesicles – motile phase-contrast microscopy fluorescent-actin microscopy His-Tagged ActA Protein Immobilized on Vesicles His-Tagged (90% phosphatidylcholine + 10% Nickel-chelating Lipid) 10% Upadhyaya, Chabot, Andreeva,Samadani & van Oudenaarden (2003) PNAS 100, 4521. PNAS Diffusion-limited clamped-filament Diffusion-limited elongation deforms vesicle’s shape. elongation Actin Monomer Concentration Gradient 100% 90% bulk solution 80% rocket tail region 70% motile vesicle vesicle Dickinson & Purich (2006) Biophys. J. 91, 1548. Dickinson 91, 9 Diffusion-Limitations Explain Actin-Based Propulsion of Hard & Deformable Particles Dickinson & Purich (2006) Biophys. J. 91, 1548. Dickinson 91, ActA Clamp “senses” γ-phosphoryl of Actin·ATP Clamp Dickinson & Purich (2002) Biophys. J. 82, 615. Dickinson Biophys. Graceffa & Dominguez (2003) JBC 278, 34172. Graceffa JBC 278 10 Energy Available for Actin Motility if motility is drive by ATP hydrolysis if ΔGhydrolysis/molecule = Workreversible/molecule /molecule Work ~ 38 kJoules ÷ Avogadro’s Number ~ 38,000 Newton·meter / 6×1023 molecules 38,000 Newton molecules ~ 60 pN·nm 60 ~ 11 pN exerted 11 over 5.4 nm over 5.4 nm nm Energetic Tuning Each motor’s clamping affinity governs magnitude of force generated as well of as the elongation rate. as Fstall = ΔGeff 5.4 nm [Etot]kcate-Load/Fstall v= Km 1+ [Profilin·Actin·ATP] [Profilin·Actin·ATP] Very High Affinity ~8 ΔGeff kcal Clamp affinity Moderate Affinity 4 - 5 kcal Low Affinity 2 - 3 kcal Free < 1kcal 11 Actoclampin comes in different “flavors” comes in different Actoclampin EMV Actin Filament Filament Ena Ena Drosophila Enabled gene product Mena Mammalian Ena Ena or or + Actoclampin F Actin + Filament or VASP Formin or M-Dia Actoclampin W WASP Actin + or or Filament N-WASP N-WASP Vasodilator-Stimulated Phosphoprotein Yeast Bud-Forming Protein Mammalian Diaphanous Protein Wiscott-Aldrich Syndrome Protein yndrome Neuronal WASP euronal WASP Cell Crawling Exploits Coordinated Control of Actomyosin & Actoclampin Motors Push/Pull Force-Balance Exerted on the Cell’s Leading Edge Actoclampin Provides Cells with a Push/Pull Force-Balance Actoclampin Motor Membrane-tethering domain Membrane-tethering Actomyosin Motor substratum (a) Actomyosin is the working unit of the “contractile apparatus”. (b) Actoclampin is the working unit of the “expansile apparatus”. 12 ...
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