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Actin-based motility of Listeria monocytogenes

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  1. Actin-based motility ofListeria monocytogenes Scot C. Kuo Department of Biomedical Engineering Johns Hopkins University Baltimore, MD 21205

  2. Listeria monocytogenes strains Dictyostelium discoideum strains Dan Portnoy, U. California, Berkeley Justin Skoble, U. California, Berkeley Peter Devreotes, Johns Hopkins John Hammer, NIH Polymer Physics Tom Mason, CalTech Denis Wirtz, Johns Hopkins Jim Harden, Johns Hopkins Reconstitution & Extracts Dyche Mullins, U. California, SF Tim Mitchison, Harvard Narat John Eungdamrong, Harvard Frank Gertler, MIT People(Animations: //www.bme.jhu.edu/~skuo/) Scot C. Kuo, Johns Hopkins James L. McGrath (now U. Rochester)Fay Peng (actin gels; Listeria) Charles Fisher (Listeria) Soichiro Yamada (COS7 cells)Rooshin Dalal (Dictyostelium phago)Karthik Ganesan (LTM device) Support: NSF (MCB), NIH (GM), Whitaker Foundation

  3. Outline • Biology: actin-based cell motility • Technology: laser-tracking and microrheology • Nanometer-scale stepping • Complexity of Listeria motility • Force-velocity relationship

  4. Svitkina et al. 1997 plus-end G-actin(monomer)~5.4 nm F-actin(all cells; muscle)200-4,000 G-actin subunitsnot covalently associated Cell structure determined by cytoskeleton: cytoskeleton = network of cross-linked filaments

  5. = plus-end Dynamics of actin (GÛF)

  6. spread listeriolysin host actin division propulsion Life Cycle of Listeria monocytogenes--penetration into adjacent cell (MDCK cells) Cellboundary Target cell MDCK columnar cells Robbins et al. 1999 J Cell Biol 146, 1333-49

  7. Listeria (ActA) Cells (WASp) Similar Biochemistry

  8. Outline • Biology: actin-based cell motility • Technology: laser-tracking and microrheology • Nanometer-scale stepping • Complexity of Listeria motility • Force-velocity relationship

  9. Wiggles reveal mechanical (viscoelasticity) environment around a particle For tracer particles(low vol fxn, larger than pores): big wiggles = soft/thin small wiggles = hard/thick Time-scale important: liquids different from solids *Theory: Mason & Weitz, 1995; Diffusing-wave spectroscopy (DWS)

  10. 2kBT Gd(w) » 3pa áDR2(t)ñ t=1/w ) ( d ln Gd(u) d ln Gd(u) p 1 d(w) » p 2 d ln u d ln u u=w 2kBT Gd(w) » G( ) d ln áDR2(t)ñ 3pa áDR2(t)ñ 1 + d ln t t=1/w ¥ ò u+w d(w) » d ln u ln u-w -¥ For General Viscoelastic Materials G* = Gd(w) exp[id(w)] Rough Approximation: Better Approximation: Wiggles2D=áDR2(t)ña=particle radius

  11. Laser-Tracking Instrument (a+b)-(c+d) (b+c)-(a+d) Dx = Dy = a+b+c+d a+b+c+d Laser-Tracking Microrheology (LTM) Mason et al. 1997 Phys Rev Lett 79, 3282-85 No optical forces (<0.1mW) --not an optical tweezers High resolution (latex beads, lipid droplets) ~0.2 nm spatial (ms) ~20 msec temporal

  12. Spatial Resolution Resolution (latex beads, lipid droplets) ~0.3 nm spatial (ms) ~20 msec temporal

  13. Proof-of-Principle: LTM of 3% PEO • Very accurate over 3.5 decades of moduli and 4.5 decades of frequency (<15% error)Mechanical rheometry (strain-controlled cone & plate) Diffusing wave spectroscopy (multiply-scattered light) • Phosphor latency of Newvicon video introduces major phase shift error Mason et al. 1997, Phys Rev Lett 79, 3282-5

  14. Non-invasive measurements:LTM in COS7 cells (not motile) oP • Natural “granules” (lipid droplets) ~300 nm -- spherical, rigid, and very refractile • Laser-tracking sensitivity gives non-invasive,in situ estimate of particle size (Mie-like) • Fast (3-30s, including calibration by PZT) ER L Lamellae (F-actin; 820, 28°)Endoplasmic Reticulum (vimentin; 330, 45°)Other perinuclear (~50, ~90°)F-actin, entangled (80 mM; 11, 23°) Moduli Values: Gd & d at 10 rad/s; Units: dyne/cm2 Yamada et al. 2000, Biophys J. 78, 1736-47

  15. Kuo Lab: Current Model Systems • How do proteins generate force? • Listeria monocytogenes – food borne pathogen that spreads by “hijacking” host cell’s actin-based motility– motility can be reconstituted using only purified proteins • How do cells respond to and generate forces? • Dictyostelium discoideum • Cell division (collab: D. Robinson) • Phagocytosis • Chemotaxis (collab: P. Devreotes) • Animal cells: adhesion, spreading, particle uptake(collab: L. Romer, C. Chen, K. Leong) • How do cells maintain tissue integrity? • Mouse keratinocytes (collab: P. Coulombe) • Validity of microrheology assumptions • Continuum? Ergodic?

  16. Listeria in COS7 cells Yamada et al. 2000, Biophys J. 78, 1736-47

  17. “Classic” Brownian ratchet (Peskin et al, 1993 Biophys J 65:316-324) Because of staggered filaments inF-actin, the intercalation distance isd=2.7nm (G-actin is 5.4nm). PredictionAt t>(d/velocity),Brownian fluctuations ³ d

  18. “Elastic” Brownian ratchet (Mogilner and Oster, 1996 Biophys J71:3030-45) Prediction: If flexing filament is bound, binding must be flexible enough to allow intercalation (>2.7nm).

  19. 2.7 Within living host cell -2.7 Perp (nm) -8.1 Parallel (nm) 2.2 7.6 13 18.4 23.8 29.2 34.6 40 45.4 50.8 56.2 61.6 67 72.4 77.8 2 mm Wiggles too small; Steps during motility INSIDE CELLS Kuo & McGrath (2000) Nature 407, 1026-9 RECONSTITUTED EXTRACTS (Methylcellulose)

  20. Speed controlled by duration of pauses • Despite presence of half-steps, the average distance between pauses is constant with speed(5.2±1.1 nm, n>650) • Duration of pauses increases as bacteria slow (power law= -1) Reconstituted extracts + methylcellulose

  21. Steps should not be observable! One filament is too soft, particularly if the end is fluctuatingmonomer dimensions to allow monomer intercalation. Hundreds of filaments should not be molecularly coordinated nor molecularly aligned!

  22. Models that generate “steps” #Tethering Filaments: One(Kuo & McGrath; Dickinson & Purich) Few (Mogilner & Oster) All (Mahadevan)

  23. Models that generate “steps” #Tethering Filaments: One(Kuo & McGrath; Dickinson & Purich) Few (Mogilner & Oster) All (Mahadevan)

  24. Models that generate “steps” #Tethering Filaments: One(Kuo & McGrath; Dickinson & Purich) Few (Mogilner & Oster) All (Mahadevan) Spatial periodicity of system

  25. VASP (Vasodilator-Activated Serine Phosphoprotein) • Delivers profilin-actin (ATP) • Protect barbed (+) ends? • Straighten actin filaments (debranch?) • Filopodia-like ARP2/3 (Actin-Related Protein) • Nucleates F-actin • Dendritic networks • Lamellapodia-like Biochemical Complexity:Two systems activated/recruited by ActA ARP2/3 VASP

  26. Removing VASP • Mutant ActA (GGG) that cannot bind VASP • Extract lacking VASP (MVD7 cell line) • Effects of removing VASP: • Slower speeds • Less directional persistence • Consistent with multiple (few) tethers without VASP

  27. Reduced System(no recycling): ActA (on beads) ARP2/3 G-actin Capping Protein Biochemical Complexity:Two systems activated/recruited by ActA ARP2/3 VASP

  28. Motility with Subset of Proteins:ARP2/3, actin, capping protein • Stepsizes not regular, but some stretches appear very regular • ~3nm steps appear often • Pure proteins very different from extract --Concentration of proteins?

  29. Models that generate “steps” #Tethering Filaments: One(Kuo & McGrath; Dickinson & Purich) Stretches ofregularity? Few (Mogilner & Oster) Jerkymotion? All (Mahadevan) Spatial periodicity of system

  30. 2kBT Gd(w) » 3pa áDR2(t)ñ t=1/w ) ( d ln Gd(u) d ln Gd(u) p 1 d(w) » p 2 d ln u d ln u u=w 2kBT Gd(w) » G( ) d ln áDR2(t)ñ 3pa áDR2(t)ñ 1 + d ln t t=1/w ¥ ò u+w d(w) » d ln u ln u-w -¥ For General Viscoelastic Materials G*(w) = Gd(w) exp[id(w)] ; |G*| = Gd Rough Approximation: Better Approximation: Wiggles2D=áDR2(t)ña=particle radius

  31. Listeria tail bead (0.5 mm) Strategy to measure forces-- use methylcellulose • Challenges: • Interfere with biochemistry? • Quantify moduli (hence Fdrag)? • Heterogeneity too thick to pipet: dissolve in situ15 min - NOT - ==> Local Measurement (tracer particles) (Laser-Tracking Microrheology)

  32. 8 pN45 nm/s 80 pN7.3 nm/s Fdrag=12(1.4)pa2|G*(w)|, w=v/2a Quantifying methylcellulose ‘load’on motility G a c3.3 Use Laser-Tracking Microrheology(LTM) to acquire complete viscoelastic spectra, despite heterogeneity (not at equilibrium).

  33. RNA polymerase (Wang et al. 1998) Skeletal Muscle, frog (Hill 1937) Kinesin (Visscher et al. 1999) c=4.5 Listeria velocity with methylcellulose ‘load’ Skeletal: c=1.3-4 Cardiac: c=3-6.1

  34. Why biphasic relationship? • Kinetics of “working” vs. “attached” filaments(Mogilner & Oster, 2003) • Biochemistry (VASP?)

  35. More actin in tail with loading Mogilner & Oster, 2003 McGrath et al, 2003

  36. Pure Proteins Stronger than Bovine Brain Extract Pure Proteins (no recycling): ARP2/3 Actin Capping Protein Problem: Agarose does not obey Cox-Merz rule.

  37. Microneedle MeasurementsMarcy et al. 2004 Biochemical Differences • Using purified proteins • WASP stimulation (not ActA) • Comparison: • Force-velocity relationship very gentle (not biphasic) • Tail wall “thickens” with load (similar to fluor.)

  38. Summary • Biology: actin-based cell motility • Technology: laser-tracking and microrheology • Nanometer-scale stepping • Complexity of Listeria motility • Force-velocity relationship

  39. ? spread listeriolysin host actin division propulsion Actin-based motility of pathogens-- Listeria monocytogenes & Shigella flexneri Listeria monocytogenes: food-borne infections Shigella flexneri: bacillary dysentery Rickettsiae conorii & R. rickettsiae: Rocky Mountain spotted fevers Vaccinia virus: related to smallpox virus

  40. Listeria Outbreak, 2002 • 40 cases in Northeast US, including 7 deaths • 10/9/02: Recall of 0.3 million pounds of cooked poultry deli meats (Pilgrim’s Pride/Wampler) • 10/14/02: Recall of additional 27.4 million pounds (Pilgrim’s Pride/Wampler); 6% of total turkey production== Largest recall in USDA history ==

  41. Dendritic Nucleation (Arp2/3)

  42. Reduced System(no recycling): ActA (on beads) ARP2/3 G-actin Capping Protein • VASP (Vasodilator-Activated Serine Phosphoprotein) • Delivers profilin-actin (ATP) • Protect barbed (+) ends? • Straighten actin filaments (debranch?) • Filopodia-like ARP2/3 (Actin-Related Protein) • Nucleates F-actin • Dendritic networks • Lamellapodia-like Biochemical Complexity:Two systems activated/recruited by ActA ARP2/3 VASP

  43. “Classic” Brownian ratchet (Peskin et al, 1993 Biophys J 65:316-324) Paradox: How can polymerization push?

  44. Listeria tail bead (0.5 mm) Strategy to measure forces -- use methylcellulose • Challenges: • Interfere with biochemistry? • Quantify moduli (hence Fdrag)?

  45. Methylcellulose does not affect biochemistry VCA (C-term of WASp) activates ARP2/3 1.5% MCL is highest methyl cellulose concentration Methyl cellulose has no effect on actin alone (not shown) and ARP2/3-induced actin polymerization kinetics.

  46. More rigorous model (Mogilner & Oster) • Two classes of actin filaments: • Attached (strain-dependent rate of dissociation) • Working (elastic Brownian ratchet; not attached)

  47. Microneedle MeasurementsMarcy et al. 2004

  48. Solvent Effects on Methylcellulose