Transcript Sarcomere 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)
Birefringence
• Light intensity due to refraction
Focused over
Focused under
Light and dark
bands swap
Sources of striation
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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
5 um
After extraction
(Hanson & Huxley, 1953)
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
– ml = d sin q
• Bragg diffraction
(Soren Pedersen)
– Reflection
– nl = 2 d sin q
– X-ray l~1-10Å
d
q
d sin(q)
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
1,1
Living
45 nm
1,0
Rigor
22 nm
Huxley, 1953
3 axes of symmetry in pointilluminated 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.
Relaxed muscle
Rigor muscle
“End-on” diffraction
pattern
Fourier transformed
data
Intensity shifts
toward thin
filaments
Huxley, 1953
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
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
Actin filament points into page
(Lower 50 kD)
Holmes et al 2003
Myosin filament
• Triple helix
– Diffraction
symmetry
– 14.5 nm repeat
• Cryo-EM
43 nm
14 nm
25 nm
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
Actin filament
Molecular
Model
S-1 Fragments
Actin filament regulation
• Troponin/tropomyosin
• Nebulin
• CapZ (barbed)
Weak myosin binding
Strong myosin binding
Actin
Tropomyosin
Extra-contractile support
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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
Rowe, 1971
Long TEM
Structural models
Luther, 2009
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
Hoshijima 2006
Labeit & al 2003
Summary
• Sarcomere
– Z-I-A-I-Z
– Interdigitating arrays of thick & thin filaments
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Myosin motors
Actin rails
Z-disk anchors
Titin skeleton