contractile structure sarcomere structure contractile filament composition – myosin (thick) –...
TRANSCRIPT
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