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Contractile structure

Contractile structure. Sarcomere structure Contractile filament composition Myosin (thick) Actin (thin) Anchors and attachments Z-disk Titin/nebulin. Basic Structure. M. Z. 1 um. Striated muscle. “Light” and “dark” bands under visible light Birefringence (uniaxial anisotropy).

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Contractile structure

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  1. Contractile structure • Sarcomere structure • Contractile filament composition • Myosin (thick) • Actin (thin) • Anchors and attachments • Z-disk • Titin/nebulin

  2. Basic Structure M Z 1 um

  3. Striated muscle • “Light” and “dark” bands under visible light • Birefringence (uniaxial anisotropy)

  4. Birefringence • Light intensity due to refraction Focused over Focused under Light and dark bands swap

  5. Sources of striation • Darker and lighter material bands • Wavy or crimped filaments • Bands of different refractive index • Disappears during contraction • Disappears by soaking in salt Phase-contrast image of myofibril Directly broken-up muscle After extraction (Hanson & Huxley, 1953) 5 um

  6. Extractions • High salt extracts of muscle coagulate over time • Myosin+actin+ATPActomyosin + ADP • Seems to be the active stuff of muscle • Flow-induced birefringence • Time-varying viscosity • Contraction results from • Polymerization (fibrin) • Rod shaped particles

  7. X-ray Diffraction • 1-D diffraction • Constructive/destructive interference • ml = d sin q • Bragg diffraction • Reflection • nl = 2 d sin q • X-ray l~1-10Å (Soren Pedersen) d q d sin(q)

  8. 2-D diffraction • Composition of 1-D • Radial symmetry • Miller notation Unit cell Major diffraction along (100) Major diffraction along (110) NaCl crystal

  9. Anisotropic crystals • 2-D image (still some symmetry) • Depends on illumination window 1,1 1,0 Myofilament image Living 45 nm 3 axes of symmetry in point-illuminated image H.E. Huxley’s image, near the axis of living muscle fiber shows strong reflection @ 45nm & weak reflection 22.5 nm. In rigor, these intensities reverse. Rigor 22 nm Huxley, 1953

  10. Relaxed muscle Rigor muscle “End-on” diffraction pattern Intensity shifts toward thin filaments Fourier transformed data Huxley, 1953

  11. 2-D diffraction • Point-source images are more complicated • 14.3 nm, 43.0 nm triple-symmetry • Mostly due to myosin Magid & Reedy, 1980 Huxley 1953

  12. High resolution TEM 200 nm Huxley, 1957

  13. Transverse TEM

  14. Myosin molecule • Native hexamer • 2 heavy chains 180 kD • 4 light chains • Domains • Globular head • Helical tail • Tryptic fragments

  15. S1 motor domain • ATPase • Actin binding • Sufficient for motility Spudich lab movie

  16. Actin filament points into page (Lower 50 kD) Holmes et al 2003

  17. 43 nm 14 nm 25 nm Myosin filament • Triple helix • Diffraction symmetry • 14.5 nm repeat • Cryo-EM Woodhead & al., 2005

  18. MHC1 MHC2 ELC1 ELC2 RLC1 RLC2

  19. M-line • Thick filaments anchored at M-line • Myomesin • Obscurin Thick Filament Myomesin Titin

  20. Actin • Disk shaped • Adenine nucleotide binding • ATPase activity • Nucleotide exchange • Promoted by Profilin • Inhibited by Cofilin • Filament formation • Barbed/Pointed end • Myosin S-1 “decoration” • ADP maturation

  21. Actin filament polymerization • Asymmetric exchange of monomers Myosin fragment

  22. Molecular Model S1 decoration Actin filament S-1 Fragments

  23. Actin filament regulation • Troponin/tropomyosin • Nebulin • CapZ (barbed) Weak myosin binding Strong myosin binding Actin Tropomyosin

  24. Extra-contractile support • Extract contractile proteins • Intermediate filament ring around Z-disk • External scaffold (desmin) • Z-disk ghost Wang & Ramirez-Mitchell, 1983

  25. Z-disk • Thin filament anchor Structural models Transverse TEM Long TEM Rowe, 1971 Luther, 2009

  26. Z-disk • a-actinin • Titin • F-actin

  27. Titin • Molecular ruler • 3-4 MD • 30,000 AA • Modular spring

  28. Titin • Modular spring • Fn repeats • Ig repeats • Kinase Hoshijima 2006 Labeit & al 2003

  29. Summary • Sarcomere • Z-I-A-I-Z • Interdigitating arrays of thick & thin filaments • Myosin motors • Actin rails • Z-disk anchors • Titin skeleton

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