Studie ies Massimo Reconditi PhysioLab University of Florence, - PowerPoint PPT Presentation

Striated Muscle: Structural Studie ies Massimo Reconditi PhysioLab University of Florence, Italy

Just look at the thing! It is very easy to answer many [ … ] fundamental biological questions; you just look at the thing! [ … ] Unfortunately, the present microscope sees at a scale which is just a bit too crude. Make the microscope one hundred times more powerful, and many problems of biology would be made very much easier. I exaggerate, of course [ … ] Plenty of Room at the Bottom Richard P. Feynman December 1959 http://calteches.library.caltech.edu/1976/1/1960Bottom.pdf

The diffraction limit A point source is imaged as a disk with diameter d 𝑜 d LENS λ =wavelength 𝑜 ∙ sin(θ) = λ λ 𝑒 = n =refractive index NA NA=Numerical Aperture

The diffraction limit Rayleigh resolution limit 2d d Airy disk 0.8 d 0.4 d Sparrow resolution limit

How a lens produces an image LENS Performs the Fourier synthesis INCIDENT LIGHT IMAGE DIFFRACTED LIGHT OBJECT Described by the Fourier Transform of the object

The electron microscope de Broglie hypothesis: ℎ λ = 𝑛 ∙ 𝑤 Example: since 𝑤 = 2𝑓𝑊/𝑛 , if V = 10kV, λ ≈ 0.01 nm λ =wavelength h =Planck constant m =electron mass e =electron charge Image from http://www.tutorsglobe.com

The electron microscope Electron Microscope: Electrons are easily absorbed by matter. Thin samples in vacuum. X-rays: No proper lens exists. Image from http://www.tutorsglobe.com

X-ray diffraction INCIDENT LIGHT DIFFRACTED LIGHT OBJECT Described by the Fourier Transform of the object

The diffraction grating Undiffracted beam To the detector The different path length along the direction at the angle θ with the undiffracted beam of the beam diffracted by two next diffractors separated by a distance d is d ∙sin ( θ ). When the path difference equals an integer multiple of the wavelength, then the diffracted waves interfere constructivelly: θ d ∙sin ( θ ) = n λ Known also as the grating equation Rearranged: λ / d = sin( θ ) < 1 …. sort of analogous to the diffraction limit … d Plane wave, wavelength λ

The diffraction grating 2       sin( ) N R d =   Undiffracted beam ( ) I R      To the detector   sin( ) R d =   sin( ) / R θ d Plane wave, wavelength λ

The sarcomeres form a diffraction grating

The striated muscle seen with the optical microscope Single fibre from skeletal muscle Cardiac myocytes 20 μ m

The structural unit of the striated muscle: the sarcomere 2 μ m M-line Z-line actin myosin 100 nm

The cross-bridges as seen with EM HE Huxley, 2004, Eur. J. Biochem. 271 :1403 – 1415

The myosin II molecule Enzymatically defined structural components of the myosin dimer

The crystallographic model of the myosin motor Subfragment S1 or Myosin ‘head’ 5 nm Motor Domain 15 nm Essential Light Chain Regulatory Light Chain Rayment et al. 1993, Science 261 :50-58 The myosin head is the molecular motor that pulls the overlapping actin filament toward the centre of the sarcomere and hydrolyses ATP

The decorated actin and the docking of the myosin motor Fit of crystallographic molecular models of F-actin and Electron cryo-microscopy and image myosin subfragment 1 into the reconstructed density. processing of the complex of F-actin and The molecular models (ribbon representation) of myosin subfragment 1 (decorated actin) myosin and F-actin are shown docked into the experimental density 17 Holmes et al. 200 3, Nature 425 :423-427

The crystallographic model of the working stroke Z-line Geeves and Holmes 1999, Annu Rev Biochem 68 :687 – 728

The crystallographic model of the working stroke Z-line Geeves and Holmes 1999, Annu Rev Biochem 68 :687 – 728

The crystallographic model of the working stroke Z-line Geeves and Holmes 1999, Annu Rev Biochem 68 :687 – 728

The crystallographic model of the working stroke Z-line Geeves and Holmes 1999, Annu Rev Biochem 68 :687 – 728

The crystallographic model of the working stroke 11 nm Z-line Geeves and Holmes 1999, Annu Rev Biochem 68 :687 – 728

The crystallographic model of the working stroke 11 nm Z-line The quasi-crystalline structure of the sarcomere allows quantitative interpretation of cell-level measurements at filament/motor level in situ Geeves and Holmes 1999, Annu Rev Biochem 68 :687 – 728

The helical arrangement of the myosin motors in the thick filament The thick filament is bipolar, with two arrays of heads separated by a “bare zone”. Bare zone

The helical arrangement of the myosin motors in the thick filament Myosin motors emerge from the thick filament backbone at an axial distance of 14.5 nm in crowns of three pairs of motors at angles of 120°. Each successive crown is twisted by 40°, to form a three-stranded helix with 43 nm helical periodicity. On each half thick filament there are 49 crowns, or 49x3=147 myosin molecules, or 147x2=294 myosin ‘heads’. In the figure, only 10 out of 49 crowns per half filament are shown for convenience.

The thick filament is surrounded by six thin filaments In the overlap region, the thick and thin filament are arranged in a double hexagonal lattice. Each thick filament is surrounded by 6 thin filaments. Each thin filament is surrounded by 3 thick filament. The ratio thin/thick filament is 2.

The structure of the actin-containing thin filament The actin monomers are arranged in the thin filament to give the overall appearance of a two stranded helix with 37.5 nm repeat. Along the actin helix runs the tropomyosin, that in the muscle at rest covers the sites of actin for the interaction with myosin. At its end tropomyonin is attached with the troponin complex. actin monomer tropomyosin troponin complex

The myosin-binding protein C (MyBP-C) (A) Electron micrograph of frog sartorius muscle. Transverse stripes of 43 nm periodicity (numbered 1 – 11) are due to MyBP-C and other nonmyosin proteins, and fine lines of 14.3 nm repeat are due to 200 nm myosin heads. Layer lines in the Fourier transform (inset; third and sixth marked) indicate good preservation of myosin head helical order. (B) Mean profile plot of several boxed regions similar to that in (A). M, M-band; stripe 1 to 5, P-zone; stripe 5 to 11, C-zone; and stripe 11 to edge of A-band, D-zone. Luther et al . 2011, PNAS 108 :11423-11428

The myosin-binding protein C (MyBP-C) Tomographic reconstruction of thick filament. (A, B) Interleaved stereo images of averaged, surface-rendered frog muscle thick filament tomogram;(A) face view, (B) tilted 20°. MyBP-C is present at stripes S5 – S11, corresponding to the stripe numbers in the previous figure). Between these stripe levels are two layers of density due to crowns of myosin heads, with a periodicity of about 14.3 nm (labeled c2 andc3). (Inset) Density representation (in stereo, tilted forward 20°) of averaged tomogram of stripes 7 – 9, showing that MyBP-C density is weak compared to the myosin head crowns. Luther et al . 2011, PNAS 108 :11423-11428

The complexity of the half-sarcomere

Half-sarcomere mechanics in single fibres Force (10µN - 10mN, 5  s) Laser Force 14.5 nm transducer stimulating electrode muscle fibre Motor Changes in fibre length (100  m, 50  s) Striation follower hs length changes (<1nm, 2  s)

Combining half-sarcomere mechanics and X-ray diffraction in single fibres Force (10µN - 10mN, 5  s) Laser mica Force 14.5 nm window transducer stimulating electrode M3 X-rays muscle fibre Small angle X-ray diffraction (0.1nm, 100  s) Motor Changes in fibre length (100  m, 50  s) Striation follower hs length changes (<1nm, 2  s)

Small angle X-ray diffraction from intact muscle fibre AL6 M6 M3 ML1 1,1 1,0

Small angle X-ray diffraction from an intact muscle fibre REST ISOMETRIC CONTRACTION M3 1s exposure time for both patterns. Data collected at ID2, ESRF, on a CCD detector

Intensity distribution along the meridional axis 1600 REST 1400 1200 M3 Intensity (a.u.) 1000 800 600 M1/C1 400 M6 M2 M5 T1 M4 200 0 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12 0.13 0.14 Reciprocal space (nm -1 ) 1000 ACTIVE 800 Intensity (a.u.) 600 M3 400 M1/C1 200 M6 T1 0 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12 0.13 0.14 35 Reciprocal space (nm -1 )

Intensity distribution along the equatorial axis REST 1,0 400 Intensity (a.u.) 300 200 1,1 100 0 0.02 0.03 0.04 0.05 0.06 Reciprocal space (nm -1 ) ACTIVE 400 Intensity (a.u.) 300 200 1,0 1,1 100 0 0.02 0.03 0.04 0.05 0.06 Reciprocal space (nm -1 )

X-rays measurements of filament compliance The spacing changes of the meridional reflections measure the length change of the myofilaments. If a filament increases its length, on the detector the reflection moves closer to the centre of the pattern (since the reflection is in the reciprocal space). 37 Huxley et al . 1994 Biophys J 67 :2411-2421 Wakabayashi et al . 1994 Biophys J 67 :2422-2435

Origin of the M3 reflection and modulation of its intensity = 2 ( ) | | I R F (  ) =  /  sin( ) R