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

Striated Muscle: Structural Studie ies

Massimo Reconditi PhysioLab University of Florence, Italy

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

Just look at the thing!

The diffraction limit

𝑒 = λ 𝑜 ∙ sin(θ) = λ NA 𝑜

λ=wavelength n=refractive index NA=Numerical Aperture A point source is imaged as a disk with diameter d

LENS

d

2d d 0.8 d 0.4 d

The diffraction limit

Airy disk Rayleigh resolution limit Sparrow resolution limit

How a lens produces an image

OBJECT INCIDENT LIGHT DIFFRACTED LIGHT Described by the Fourier Transform of the object IMAGE LENS Performs the Fourier synthesis

The electron microscope

Image from http://www.tutorsglobe.com

λ = ℎ 𝑛 ∙ 𝑤 de Broglie hypothesis: Example: since 𝑤 = 2𝑓𝑊/𝑛 , if V = 10kV, λ ≈ 0.01 nm

λ=wavelength h=Planck constant m=electron mass e=electron charge

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 OBJECT DIFFRACTED LIGHT Described by the Fourier Transform of the object

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: d∙sin(θ) = n λ Known also as the grating equation Rearranged: λ/d = sin(θ) < 1

The diffraction grating

….sort of analogous to the diffraction limit…

θ d

To the detector Undiffracted beam Plane wave, wavelength λ

    / ) sin( ) sin( ) sin( ) (

2

=              = R d R d R N R I

The diffraction grating θ d

To the detector Undiffracted beam Plane wave, wavelength λ

The sarcomeres form a diffraction grating

Single fibre from skeletal muscle Cardiac myocytes

The striated muscle seen with the optical microscope

20 μm

2 μm

The structural unit of the striated muscle: the sarcomere

100 nm actin myosin Z-line M-line

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

15 nm 5 nm

Subfragment S1 or Myosin ‘head’

Rayment et al. 1993, Science 261:50-58

The crystallographic model of the myosin motor

The myosin head is the molecular motor that pulls the overlapping actin filament toward the centre of the sarcomere and hydrolyses ATP Motor Domain Essential Light Chain Regulatory Light Chain

17

The decorated actin and the docking of the myosin motor

Holmes et al. 2003, Nature 425:423-427 Fit of crystallographic molecular models of F-actin and myosin subfragment 1 into the reconstructed density. The molecular models (ribbon representation) of myosin and F-actin are shown docked into the experimental density Electron cryo-microscopy and image processing of the complex of F-actin and myosin subfragment 1 (decorated actin)

The crystallographic model of the working stroke

Geeves and Holmes 1999, Annu Rev Biochem 68:687–728 Z-line

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

Bare zone The thick filament is bipolar, with two arrays of heads separated by a “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

actin monomer tropomyosin troponin complex

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.

The myosin-binding protein C (MyBP-C)

Luther et al. 2011, PNAS 108:11423-11428 (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 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. 200 nm

The myosin-binding protein C (MyBP-C)

Luther et al. 2011, PNAS 108:11423-11428 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.

The complexity of the half-sarcomere

stimulating electrode muscle fibre

Motor Force transducer Striation follower hs length changes (<1nm, 2s) Laser Changes in fibre length (100m, 50s) Force (10µN - 10mN, 5s)

Half-sarcomere mechanics in single fibres

14.5 nm

stimulating electrode muscle fibre

Motor Force transducer Striation follower hs length changes (<1nm, 2s) Laser Changes in fibre length (100m, 50s)

mica window X-rays

Small angle X-ray diffraction (0.1nm, 100s)

M3

Force (10µN - 10mN, 5s)

14.5 nm

Combining half-sarcomere mechanics and X-ray diffraction in single fibres

Small angle X-ray diffraction from intact muscle fibre

1,0 1,1 M3 M6 ML1 AL6

REST ISOMETRIC CONTRACTION 1s exposure time for both patterns. Data collected at ID2, ESRF, on a CCD detector M3

Small angle X-ray diffraction from an intact muscle fibre

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Reciprocal space (nm-1)

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

Intensity (a.u.)

200 400 600 800 1000 1200 1400 1600

Reciprocal space (nm-1)

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

Intensity (a.u.)

200 400 600 800 1000

Intensity distribution along the meridional axis

REST ACTIVE M1/C1 T1 M2 M3 M4 M5 M6 M1/C1 T1 M3 M6

Reciprocal space (nm-1)

0.02 0.03 0.04 0.05 0.06

Intensity (a.u.)

100 200 300 400

Reciprocal space (nm-1)

0.02 0.03 0.04 0.05 0.06

Intensity (a.u.)

100 200 300 400

Intensity distribution along the equatorial axis

REST ACTIVE 1,0 1,1 1,0 1,1

37

X-rays measurements of filament compliance

Huxley et al. 1994 Biophys J 67:2411-2421 Wakabayashi et al. 1994 Biophys J 67:2422-2435 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).

  / ) sin( | | ) (

2

= = R F R I

()

Origin of the M3 reflection and modulation of its intensity

    / ) sin( ) sin( ) sin( | | ) (

2 2

=               = R d R d R N F R I

() d

Origin of the M3 reflection and modulation of its intensity

    / ) sin( ) sin( ) sin( | | ) (

2 2

=               = R d R d R N F R I

() d

Origin of the M3 reflection and modulation of its intensity

    / ) sin( ) sin( ) sin( | | ) (

2 2

=               = R d R d R N F R I

() d

Origin of the M3 reflection and modulation of its intensity

    / ) sin( ) sin( ) sin( | | ) (

2 2

=               = R d R d R N F R I

() d

Origin of the M3 reflection and modulation of its intensity

    / ) sin( ) sin( ) sin( | | ) (

2 2

=               = R d R d R N F R I

() d

Origin of the M3 reflection and modulation of its intensity

    / ) sin( ) sin( ) sin( | | ) (

2 2

=               = R d R d R N F R I

() d

Origin of the M3 reflection and modulation of its intensity

45

    / ) sin( ) sin( ) sin( | | ) (

2 2

=               = R d R d R N F R I

() d

Origin of the M3 reflection and modulation of its intensity

The intensity of a reflection depends on both the conformation and the number of the molecules that contribute to it.

Force generation is synchronous with conformational changes in the myosin motor

Huxley et al. 1981 PNAS 78:2297-2301 Irving et al. 1992 Nature 357:156-158

REST ISOMETRIC CONTRACTION

Small angle X-ray diffraction from intact muscle fibres of frog at high spatial resolution

M3 M6

stimulus force 200 kPa

Origin of the meridional reflection fine structure: recovering the phase information

Origin of the meridional reflection fine structure: recovering the phase information

Determining the amplitude of the working stroke in situ

Myosin motors move towards the centre of the sarcomere until they detach from actin at the end of the working stroke Reconditi et al 2004 Nature 428:578-581 LA HA RM3=IHA/ILA

52

Determining the amplitude of the working stroke in situ

The motor stroke is slower and smaller at higher load Reconditi et al 2004 Nature 428:578-581

Huxley et al 2006 J Mol Biol 363:743-761 Direct plot of total intensity of M3 reflection against ratio of the heights of the two interference peaks, for five experiments and several different computed models Tension record (red) during a series of quick releases, each followed 3–4 ms later by a re-stretch to the original length. Length steps (blue) were applied at intervals of 65 ms. Green trace shows the ion current in an in-line X-ray detector, with a fast shutter opening only for 1–2ms immediately after each release, i.e. before the re-stretch.

Intensity and fine structure of M3 indicate that not all heads move

The intensity of M3 scales with sarcomere length

These data indicate that in the non-overlap region

• f the thick filament the detached myosin

molecules do not contribute to the M3 reflection in the active muscle: thus the non-moving heads in the overlap region must be the detached partner head of an attached motor.

SL (μm)

Muscle during the plateau of an isometric tetanus Reconditi et al 2014 J Physiol 592:1119-1137 Heads at rest (OFF) Attached dimers: one motor works while the other heads is detached (low dispersion) Detached dimers: both heads have high conformational dispersion Z line M line

3D reconstruction of tarantula thick filament

Woodhead et al. 2005, Nature 436:1195-1199 Toward the centre of the thick filament J motif or Interacting Heads Motif (IHM) S2 S1 S1

The textbook model of regulation

[Ca2+]

Actin filament Myosin filament troponin

The textbook model of regulation

?

[Ca2+]

Actin filament Myosin filament troponin

Myosin filament mechanosensing

Linari et al. 2015 Nature 528:276-279

Myosin filament mechanosensing

Linari et al. 2015 Nature 528:276-279

Myosin filament mechanosensing

Linari et al. 2015 Nature 528:276-279

Myosin filament mechanosensing

Linari et al. 2015 Nature 528:276-279

Myosin filament mechanosensing

Linari et al. 2015 Nature 528:276-279

Two mechanisms for regulation of skeletal muscle

[Ca2+] Force

Actin filament Myosin filament troponin

How are the X-rays produced?

Schematic of a synchrotron

Third generation synchrotrons

ESRF (European Synchrotron Radiation Facility) at Grenoble, France APS (Advanced Photon Source) at Argonne, IL, U.S.A.

Two SAXS beamlines

ID02 at ESRF (before upgrade) BioCAT at APS