[PDF] - Muscle Muscle Muscle Muscle Cytoskeleton Cytoskeleton PDF Document

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http://www.rsc.org/images/b901714c-400-FOR-TRIDION_tcm18-152053.jpg

Muscle Muscle Muscle Muscle Cytoskeleton Cytoskeleton Cytoskeleton Cytoskeleton II II II II

Muscle Biophysics Summer School Semmelweis University, Budapest, Hungary 8/28/2018 – 8/30/2018

Balazs Kiss

Postdoc at Granzier Lab Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ 85721 Email: kissb@email.arizona.edu

Lecture outline

2

4. Nebulin

M-band

3. Titin

Z-disk Z-disk

1. Thin filament
2. Thick filament

Muscle Cytoskeleton in details:

unique protein components: Z-line, M-line, costamere, intermediate filaments,
length regulation of thick and thin filaments: titin and nebulin as “rulers”,
specific biophysical approaches to study striated muscle structure and function:

X-ray diffraction, superresolution microscopy.

based on Labeit et al., 2010.

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Muscle cytoskeleton at the Z-disks

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Z-disks: borders of contractile units:

thin filament anchoring: α-actinin, filamin, myotilin,
force transmission: via titin and nebulin,
signaling node: muscle LIM protein (MLP), muscle ankyrin repeat proteins

(MARPs), myopodin, myopalladin.

Henderson and Gomez et al, 2017.

α-Actinin

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α-actinin 2: cardiac muscle
α-actinin 3: fast skeletal and cardiac muscle
head-to-tail antiparallel homodimers
contour length: 35 nm
N-term: actin binding (ABD)
C-term: 2 EF hand domains: titin binding
4 spectrin-repeats (SR)
mutations: ACTA2: DCM, HCM

ACTA3: nonsense - 16% of humans!

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α-Actinin at the Z-disks

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Z-disk I-band I-band

Grison et al, 2017.

α-Actinin-Titin interaction at the Z-disk

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Titin at the Z-disk:

Z1-Z2 (Ig-domains): binds telethonin
Z-repeats 1-7: could bind α-actinin

Joseph et al, 2001

M Z

Luther, 2009

Testing the mechanical stability:

using optical trap
dsDNA spacer towards the microbeads
short covalent linker between titin Z-

repeat and α-actinin EF domain

Grison et al, 2017.

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Analysis of kinetic parameters – Hidden Markov Model

Life of a Grad Student 3-state model

lab
coffee-shop
bar

Analysis of kinetic parameters – 3 state model

A: folded T: bound (FB) A: folded T: unbound (FU) A: unfolded T: unbound (FB) fixed trap distance (passive mode)

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α-Actinin-Titin interaction model

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different

Z-repeats have different affinities to α- actinin,

avidity:

accumulated strength

f

multiple, parallel bonds/interactions.

Grison et al, 2017. 10

Filamin-C and Myotilin at Z-disk

Rognoni et al, 2012.

Filamin-C:

connecting element to integrins,
affinity to its targets might be modulates

by mechanical forces,

nonsense mutation: myofibrillar myopathy.

Myotilin:

homodimers,
prevents actin depolymerization,
interacts with multiple Z-disk proteins
nonsense mutation: “myotilinopathies”.

Sanfilippo and Di Rosa, 2016.

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M-band: hub for mechanosensing

Gautel and Djinović-Carugo, 2016.

M-band

M-bridges: stabilize myosin in the

center of the sarcomere

Myomesin-1: all striated

muscles

Myomesin-2 (M-protein): fast

(type IIb/x) muscle fibers

Myomesin-3: intermediate

twitch fibers

antiparallel homodimers,

dimerization through the C-terminal Ig-domain

domains are able to stretch to 2.5x

their original length: “elastic band”

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M-band titin: titin kinase unfolding

AFM-based molecular combing

Mártonfalvi and Kellermayer, 2014.

titin kinase might be

unfolded with receding meniscus: mechanosensor

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Costamere: protects against mechanical stress

Bi-directionally links the cytoskeleton to the extracellular matrix: vinculin- talin- integrin system dystrophin glycoprotein complex

Henderson and Gomez et al, 2017. 14

Costamere pathology: myopathies

Henderson et al, 2017.

BMD: Becker muscular dystrophy
DMD: Duchenne muscular dystrophy
CMD1C-1D: congenital muscular

dystrophy type 1C-1D

FCMD, Fukuyama congenital muscular

dystrophy

LGMD2C-2F: limb-girdle muscular

dystrophy type 2C-2F

LAMA2: laminin alpha 2 chain or

merosin-deficient muscular dystrophy

MEB: muscle-eye-brain disease
WWS: Walker–Warburg syndrome

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Intermediate filaments: scaffold for cell organelles

diameter: 8-10 nm
coiled-coil dimer:
main components in muscle:
desmin (2% of total protein in

heart)

cytokeratins (K8K19)
desmin knockout mice: myofibril

misalignment, loss of nuclear shape, impaired force generation

mutations: “desminopathies”

head tail

16

Molecular elasticity of desmin

Kiss et al, 2011.

AFM-based shape fluctuation analysis

curve fit: persistence length (P): ~0.45 µm for lateral bending

Desmin assembly with Mg2+

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Nebulin in the sarcomere

Nebulin

M-band Tmod Z-disk Z-disk

~850 kDa, ~1µm
only in skeletal muscle
35 aa residue modules (M)
super repeat (SR) from 7M
mutations: nemaline myopathy

Kontrogianni-Konstantopoulos et al, 2009. 18

Putative role: thin filament stabilizer?

Trinick, 1992.

Nebulin (one strand, simulation) Actin subunit Tropomyosin Troponin

Squire et al, 2004.

(projected image) 38.5 nm 2.73 nm

Nebulin interacts stronglywith thin filament:

7 actin monomer binding motifs per SR,
1 troponin and tropomyosin binding

subunit per SR,

nebulin binds along the long pitch helix
f the thin filament,
2 nebulin molecules per thin filament?

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Problem #1: nebulin cannot be expressed due to its giant size. Problem #2: isolation of native nebulin is currently not possible. Solution: mechanical effect of nebulin on the thin filament could be assessed by directly measuring thin filament compliance in living muscle.

How to measure thin filament compliance?

Background: thin filaments of striated muscle are longitudinally extensible!

Myosin spacing change (%) Actin spacing change (%)

Huxley et al, 1994. Brunello et al, 2014. Huxley et al, 1994. Brunello et al, 2014.

X-ray diffraction in muscle research

Early studies on sarcomere structure and function: based on X-ray diffraction:

inter-thick filament spacing,
sliding filament theory of contraction; moving cross-bridges,
tropomyosin: steric blocking model,
longitudinal extensibility of thin and thick filaments.

“Then as now, x-ray diffraction remains the only technique to detect nanometer-scale structural changes in real physiological time in living muscle.”

Hitchcock-DeGregori and Irving, 2014. Hugh Esmor Huxley

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X-ray diffraction: (bio)physical basis

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Bragg’s Law: 2d sin θ = nλ

d: lattice spacing θ: angle of incidence λ: wavelength of the electromagnetic wave

when λ=constant: d ~

real space

reciprocal space

X-ray diffraction on fibers

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X-ray

Fibrous protein: naturally ordered structure real space reciprocal space

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X-ray diffraction on skeletal muscle

real space real space 1/P subunit spacing meridional pattern P lattice spacing equatorial pattern 1/P P skeletal muscle (fiber) X-ray reciprocal space reciprocal space

1 8.7 2 250

meridian equator

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Structural data from the diffraction image

27.3 Å 28.6 Å 51 Å 59 Å 1,1 1,0

meridian equator

d10, d11: lattice spacing I10, I11: cross-bridge

Milman, 1998.

27.3 Å = 2.73 nm: actin subunit repeat

Ebashi et al., 1969.

28.6 Å = 2.86 nm: myosin backbone spacing 143 Å 5th order: 28.6 Å

Needham, 1971.

1 8.7 2 250

Volkmann et al., 2001.

51 Å and 59 Å: thin filament helix meridional pattern equatorial pattern

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Higher order reflections: fine structure of proteins

25 Linari et al, 2000. Park-Holohan et al, 2012.

rest tetanus

Huxley and Brown, 1967.

~43-nm fundamental axial periodicity sensitive to the helix perturbations axial repeat of myosin HEAD (14.3 nm) periodic mass distribution in myosin BACKBONE (7.2 nm)

fundamental axial periodicity N.b. changes in spacing and intensity!

26

Experimental strategy

Ctrl Neb cKO

and

50-70 days old
Neb cKO: few percent

nebulin expression

m. soleus
force- and tension deficit in Neb cKO

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What is the mechanical effect of nebulin on the TF?

thin filaments of Neb cKO are ~3-times

more compliant than Ctrl

nebulin stiffens the thin filament via its

actin subunits

Nebulin (one strand, simulation) Actin subunit

Squire et al, 2004.

0.0027%/kPa 0.0075%/kPa 2.73nm = 27.3Å: actin subunit repeat

27Å

Kiss et al, in press 28

Nebulin increases single thin filament stiffness.

Single thin filament stiffness:

Ctrl: 30.3 pN/nm
Neb cKO: 10.0 pN/nm

EM X-ray Ctrl Neb cKO

30 60 60 80 100 Void Mito Myo ECM **** ****

significantly

reduced mitochondrial area in Neb cKO

significantly increased myofibril area in

Neb cKO

Ultrastructure of muscle cross-section (EM)

Kiss et al, in press

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What happens to the thin filament helix?

51 and 59Å: thin filament pitch

Volkmann et al., 2001.

59Å 51Å

51Å spacing at rest (nm)

1 2 3 4 5 6 7 8 5.09 5.11 5.13 5.15 Time (s)

Ctrl Neb cKO

rest tetanus Short pitch helix

59Å spacing (nm)

Ctrl Neb cKO 5.86 5.88 5.90 5.92 5.94

**** rest tetanus Long pitch helix

twisted helix in

Neb cKO at rest

twisted helix in

Neb cKO during tetanus

Kiss et al, in press

Rest Difference

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What happens to the thin filament regulators?

Nebulin Actin subunit Tropomyosin Troponin

Squire et al, 2004.

TN3 2ALL

2ALL intensity (a.u.)

Tropomyosin (2ALL) Troponin (TN3)

thin filament regulators have

dominant pattern during contraction

decreased 2ALL and TN3

intensity in Neb cKO

nebulin promotes tropomyosin

movement and troponin activity

Kiss et al, in press

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Does nebulin affect cross-bridges?

1,0 1,1 4MLL

Thick filament Thin filament

Resting Tetanized

Percentage of myosin heads

interactingwith actin: 46% in Ctrl 34% in Neb cKO

nebulin promotes cross-bridge

formation Resting Tetanized

Ctrl Neb cKO Ctrl Neb cKO 100 200 300 50 100 150 4MLL intensity at rest Tetanic 4MLL intensity

**

4MLL intensity ratio

Myosin heads around thick filament backbone (4MLL)

0.29 0.44 Ctrl Neb cKO Ctrl Neb cKO 2 4 6 8 2 4 6 8 I1,1 / I1,0 I1,1 / I1,0

**** ****

Resting Tetanized Mass at thin filaments (I11/I10)

Kiss et al, in press 32

Model: nebulin, a thin filament stabilizer

Ca2+ Ca2+ Ca2+

Neb cKO

Ca2+ Ca2+ Ca2+ Ca2+

Thick filament Thin filament Nebulin Tropomyosin

TnI TnC

Troponin

Ctrl fewer cross-bridges are formed nebulin deficiency decreased actin subunit stiffness impaired thin filament helix impaired troponin/tropomyosin movement

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Is nebulin a thin filament ruler?

positive correlation between thin

filament length (TFL) and molecule weight

f

nebulin across different species

Kruger, Wright and Wang 1991.

Z-line

Fully nebulin-coated segment

Ruler model

Is nebulin only a thin filament stabilizer?

Two-segment model

Gokhin and Fowler, 2013. Pappas CT, Krieg PA, Gregorio CC, 2010.

Neb-coated, stabilized no Neb, dynamic Z-line Neb-N / Phalloidin

Castillo et al, 2009.

muscles from rabbit:

Z

Mouse models to test nebulin’s role in thin filament length (TFL) regulation

Deletion involving just a part of a SR (Exon 55 in S9) is lethal (Ottenheijm et al and Granzier, 2013).

S9 S10 S11

1) SR 9-11 are deleted: NebΔSR9-11 2) SR 9-11 are duplicated: NebdupSR9-11

S9 S10 S11 S9 S10 S11

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Present: Two mouse models Past:

Kiss et al, unpublished

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NebΔSR9-11

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Nebulin expression in the mouse models

NebdupSR9-11

Nebulin / MHC

WT titin T2 MHC WT HOM WT HOM

85 kDa

+85 kDa

Kiss et al, unpublished

EDL: m. extensor digitorum longus

Nebulin / MHC 36

m. EDL

Measuring nebulin length in skeletal muscle

skinned m. EDL fiber bundle preparation

fixing (10% formalin, 3-5h)
cryosectioning (~4 μm thickness)
primary and secondary immunolabeling

Superresolution Microscopy ~30% stretch

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A-band (Ti102) Phalloidin Neb N-term (goat) Tmod1

Superresolution microscopy (SR-SIM)

2 μm

Phalloidin Ti102 Neb N-term Tmod1

modified from Gokhin et al., 2013. Kiss et al, unpublished 38

Nebulin rules thin filaments in EDL

3x SR = -115.5 nm

2200 2400 2600 2800 3000 3200 600 700 800 900 1000 1100 1200 1600 1800 2000 SL (nm) 2000 2200 2400 2600 2800 3000 3200 SL (nm)

Ctrl NebΔSR9-11

Distance from Z-disk (nm) A-band width (nm)

1628 nm 1093 nm 1074 nm 1026 nm 989 nm 966 nm 914 nm 1622 nm

Neb

109

Phall

108

Tmod

104

Δ (nm) Ti102 Tmod1 Phall Neb-N

Kiss et al, unpublished

shorter nebulin and thin filaments in NebΔSR9-11 EDL muscle

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+3x SR = +115.5 nm

2200 2400 2600 2800 3000 3200 600 700 800 900 1000 1100 1200 1300 1400 1600 1800 2000 SL (nm)

Ctrl NebdupSR9-11

Distance from Z-disk (nm) A-band width (nm)

Nebulin rules thin filaments in EDL

Ti102 Tmod1 Phall Neb-N Neb +123 Phall +104 Tmod +99 Δ (nm)

1589 nm 1093 nm 1074 nm 1026 nm 1192 nm 1178 nm 1149 nm 1622 nm

Kiss et al, unpublished

longer nebulin and thin filaments in NebdupSR9-11 EDL muscle

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A-band titin: interaction with MyBP-C

Spudich, 2015. Lin et al, 2013. Previs et al, 2015. Previs et al, 2013.

MyBP-C interacts with the thick, thin and

titin filament systems

43 nm

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Titin: thick filament ruler in striated muscle

Myhre et al, 2014. Kiss and Tonino et al, 2017.

C1 and C2 C-zone repeats

are deleted from A-band titin (TtnΔC1-2)

(both skeletal and cardiac

muscle)

A-band width and thick

filament length was measured with EM/SR- SIM

2x 43 nm

42

Titin: thick filament ruler in striated muscle

shorter A-band width by ~164 nm in TtnΔC1-2 cardiac and skeletal muscles
thick filament length regulation is not constrained to the tip of the thick filament

Kiss and Tonino et al, 2017.

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Functional consequences of shorter thick filaments

reduced thick filament length: lower

maximal calcium-induced force by 12%

steeper descending limb due to the

reduced length of the cross-bridge bearing region of the thick filaments

Kiss and Tonino et al, 2017. Gordon et al., 1966 44