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

Basic Structure ZM 1 um

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

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

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

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

X-ray Diffraction 1-D diffraction – Constructive/destructive interference – m = d sin  Bragg diffraction – Reflection – n = 2 d sin  – X-ray ~1-10Å (Soren Pedersen) d  d sin(  )

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

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

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

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

High resolution TEM 200 nm Huxley, 1957

Transverse TEM

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

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

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

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

MHC1 MHC2 ELC1 ELC2 RLC1 RLC2

M-line Thick filaments anchored at M-line – Myomesin – Obscurin Thick Filament Myomesin Titin

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

Actin filament polymerization Asymmetric exchange of monomers Myosin fragment

S1 decoration Molecular Model Actin filament S-1 Fragments

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

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

Z-disk Thin filament anchor Transverse TEMLong TEM Structural models Luther, 2009Rowe, 1971

Z-disk a-actinin Titin F-actin

Titin Molecular ruler – 3-4 MD – 30,000 AA Modular spring

Titin Modular spring – Fn repeats – Ig repeats Kinase Labeit & al 2003 Hoshijima 2006

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