Contractile structure

• Sarcomere structure• Contractile filament composition

– Myosin (thick)– Actin (thin)

• Anchors and attachments– Z-disk– Titin/nebulin

Basic StructureZM

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 myofibrilDirectly broken-up muscle

After extraction (Hanson & Huxley, 1953)5 um

Extractions

• High salt extracts of muscle coagulate over time– Myosin+actin+ATPActomyosin + 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 notationNaCl crystal

Unit cellMajor 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 reflection @ 45nm & weak reflection 22.5 nm. In rigor, these intensities reverse.

Huxley, 1953

3 axes of symmetry in point-illuminated image

1,0

1,1

“End-on” diffraction pattern

Fourier transformed data

Relaxed muscle Rigor 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 FilamentMyomesinTitin

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

MolecularModel

Actin filament

S-1 Fragments

Actin filament regulation

• Troponin/tropomyosin• Nebulin• CapZ (barbed)

Weak myosin bindingStrong myosin bindingActinTropomyosin

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 TEM Long 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