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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
Contractile structure
  • Sarcomere structure
  • Contractile filament composition
    • Myosin (thick)
    • Actin (thin)
  • Anchors and attachments
    • Z-disk
    • Titin/nebulin
striated muscle
Striated muscle
  • “Light” and “dark” bands under visible light
  • Birefringence (uniaxial anisotropy)
birefringence
Birefringence
  • Light intensity due to refraction

Focused over

Focused under

Light and dark

bands swap

sources of striation
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
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
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)

2 d diffraction
2-D diffraction
  • Composition of 1-D
    • Radial symmetry
    • Miller notation

Unit cell

Major diffraction along (100)

Major diffraction along (110)

NaCl crystal

anisotropic crystals
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

slide10

Relaxed muscle

Rigor muscle

“End-on” diffraction pattern

Intensity shifts toward thin filaments

Fourier transformed data

Huxley, 1953

2 d diffraction1
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

high resolution tem
High resolution TEM

200 nm

Huxley, 1957

myosin molecule
Myosin molecule
  • Native hexamer
    • 2 heavy chains 180 kD
    • 4 light chains
  • Domains
    • Globular head
    • Helical tail
    • Tryptic fragments
s1 motor domain
S1 motor domain
  • ATPase
  • Actin binding
  • Sufficient for motility

Spudich lab movie

slide16

Actin filament points into page

(Lower 50 kD)

Holmes et al 2003

myosin filament

43 nm

14 nm

25 nm

Myosin filament
  • Triple helix
    • Diffraction symmetry
    • 14.5 nm repeat
  • Cryo-EM

Woodhead & al., 2005

slide18

MHC1

MHC2

ELC1

ELC2

RLC1

RLC2

m line
M-line
  • Thick filaments anchored at M-line
    • Myomesin
    • Obscurin

Thick Filament

Myomesin

Titin

actin
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
Actin filament polymerization
  • Asymmetric exchange of monomers

Myosin fragment

s1 decoration

Molecular

Model

S1 decoration

Actin filament

S-1 Fragments

actin filament regulation
Actin filament regulation
  • Troponin/tropomyosin
  • Nebulin
  • CapZ (barbed)

Weak myosin binding

Strong myosin binding

Actin

Tropomyosin

extra contractile support
Extra-contractile support
  • Extract contractile proteins
  • Intermediate filament ring around Z-disk
  • External scaffold (desmin)
  • Z-disk ghost

Wang & Ramirez-Mitchell, 1983

z disk
Z-disk
  • Thin filament anchor

Structural models

Transverse TEM

Long TEM

Rowe, 1971

Luther, 2009

z disk1
Z-disk
  • a-actinin
  • Titin
  • F-actin
titin
Titin
  • Molecular ruler
    • 3-4 MD
    • 30,000 AA
  • Modular spring
titin1
Titin
  • Modular spring
    • Fn repeats
    • Ig repeats
  • Kinase

Hoshijima 2006

Labeit & al 2003

summary
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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