Muscle Cytoskeleton I. Mikls Kellermayer Semmelweis University - - PowerPoint PPT Presentation
Muscle Biophysics Summer School - Budapest, 29 August, 2018 Muscle Cytoskeleton I. Mikls Kellermayer Semmelweis University Department of Biophysics and Radiation Biology Types of biological motion Collective motion Body motion (Leap of
Miklós Kellermayer
Semmelweis University Department of Biophysics and Radiation Biology Muscle Biophysics Summer School - Budapest, 29 August, 2018
Collective motion Body motion (“Leap of the century”) Organ motion
Autonomous cardiomyocyte Wound healing model - collective fibroblast movement
Dividing cell Moving spermatocytes Axonal (neurite) growth Chemotaxis Crawling keratinocyte Intracellular movement of pathogenic Listeria bacteria
Microtubules DNA Actin
interconnected filaments and tubules
independently, by Nikolai K. Koltsov (1903) and Paul Wintrebert (1931).
discovery of the composition, structure and function of the cytoskeleton.
scaffold for intracellular binding and transport processes.
complex functionality.
cytoskeleton is exclusive to the eukaryotic cell.
(MreB) and tubulin (FtsZ).
its cytoskeleton.
Brunó F. Straub
(Discoverer of actin)
Mary Osborn and Klaus Weber
(Labeling methods for intermediate filaments, microtubules)
which diversify their functions.
Microfilaments (Actin) (rhodamine-phalloidin) Microtubules (GFP-tubulin) Intermediate filaments (Vimentin, anti-vimentin)
microspikes, focal contacts, invagination)
cortex Stress fibers filopodium
Filamentous system of eukaryotic cells composed of tubulin and its associated proteins
filaments, found in most animal cell types.
Vimentin, Vic Small
Anti-keratin, PtK2 cell GFP-vimentin, 3T3 cell, R.D. Goldman
Epidermolysis bullosa Qin et al. J. Biomech. 43, 15, 2010
100 nm Neurofilament tail domain extending from surface (Ueli Aebi, Basel)
homology
membrane
segregation
MreB: actin homolog FtsZ: tubulin homolog
skeletal muscle fiber cardiac myocyte smooth muscle cell myoepithelial cell
Nucleus Myofibrils Myofibrils: The organelle-level structural and functional units of muscle.
striated muscle.
A I
Phenomenological mechanism: Sliding filament theory
Andrew F. Huxley, Jean Hanson, Hugh E. Huxley
Szarkomerhossz (µm)
1 2 3 4 5
The elastic component limits A-band asymmetry
Without elastic filament (two-filament sarcomere) With elastic filament (three-filament sarcomere)
A I I
Z Z M thick thin
The two-filament model on which the sliding-filament theory is founded (A. F. Huxley and Niedergerke 1954;
The S-filament, extending in between the tips of the thin (actin) filament (Hanson and Huxley 1956) The gap filament extending between the tips of the thin and thick filaments (Sjöstrand 1962) The C-filament model of Garamvölgyi (Garamvölgyi 1965) The T-filament stretching in between the Z-lines
The gap filament of Locker and Leet (Locker and Leet 1975), which stretches along the core of the thick filament and connects it to one of the Z-lines. Ferenc Guba (Fibrillin) (1919-2000) Miklós Garamvölgyi (C-filaments) (1932-1980) Károly Trombitás (C-filaments, titin)
desmin myosin actin Z-line M-line
serially-linked sarcomeres: basic unit
(and microtubular) system - links sarcomeres to the membrane and
Special cytoskeletal structures:
to the extracellular matrix.
together (important in cardiac myocytes).
skeletal muscle to tendon.
200 nm
Cardiac myocyte Immunoglobulin (Ig) domain PEVK domain Fibronectin (FN) domain Kinase domain Z-line Z-line M-line Thin filament (actin)
Muscle sacromere
Thick filament (myosin)
7 anti-parallel ß-strands
Myomesin Myomesin Kinase M1 M3 A170 M4 M 10 20 30 40 50 nm 60 M-protein M-protein Titin Titin
M-line complex
AFM structure of a single straightened titin molecule
Function:
Regulation:
Pathology:
Where else is titin?
sarcomere)
Hidalgo and Granzier,
da Silva Lopes et al, JCB 193, 785, 2011.
Diffusible titin pool: Titin-eGFP knockin (MEx6) Embryonic cardiomyocyte T1/2 ~ 3 h
Phosphorylation sites in titin:
TTN: 2q31
base-pair region: 179,390,715 - 179,672,149
364 exons - alternative splicing: cardiac- (N2B, N2BA) and skeletal-muscle isoforms
Titinopathies:
muscles affected.
Leinwald et al, Circ. Res. 111, 158, 2012.
Linke Annu Rev Physiol 2018
TTNTV - truncating variants DCM - dilated caradiomyopathy ExAC - Exome Aggregation Consortium PSI - percent spliced in
The titin “interactome”
Linke, Cardiovasc. Res. 2008. Henderson et al, Compr Physiol. 2017.
Force (F) Force (F) Extension (z) Extension (z) Rigid body: Hooke’s law Polymer chain: fluctuations, configurational entropy
Contour length (Lc)
Force (F) Extension (z)
R s Wormlike chain
LP = persistence length EI = bending rigidity kBT = thermal energy
Evan A. Evans and David A. Calderwood Science 316, 1148 (2007)
Under thermal activation: Under mechanical load:
ω = characteristic time Ea = activation energy Δx = distance between bound and transition states Conformational space Unfolded state Native state
Cantilever methods Field methods
Refractile bead Light beam P1
P2 ΔP F=ΔP/Δt F F
Objective lens Laser Refractile bead Scatter force (light pressure) Gradient force EQUILIBRIUM
bead in optical trap
Momentum-exchange between photons and refractile particle
Arthur Ashkin Developer of optical tweezers (1970) Steven Chu Atom cooling with
Nobel prize (1997) Tractor beam, Star Trek Escherichia coli bacterium grabbed with optical tweezers
Outgoing beam displacement Outgoing beam displacement Micropipette movement → → molecule extension → → bead displacement Micropipette Molecule Latex bead Latex bead Objective underfilled Objective underfilled Incoming laser beam Incoming laser beam
interaction between the atoms of the sample and the tip
cantilever by help of a laser beam reflected off of its back surface
cantilever) is moved in XYZ directions
repulsion attraction
contact mode non-contact (oscillating) mode
AFM tip diameter ~ 10 nm
500 nm
Height contrast Amplitude contrast Phase contrast
Phase difference between the sinusoidal driving signal and the detected cantilever
Cantilever resonence detuned upon the action of external forces
500 nm
400 nm
G G G G G
50 nm
200 nm
100 nm
50 nm
Movable micropipette Latex bead titin Optical tweezers: “virtual spring”
Non-linear force response
Force (pN) Extension (µm)
~28 nm
Domain-unfolding:
events
system
depends on mechanical stability Native state Unfolded state F=0 Large F Reaction coordinate G
Δx Transition state
Two-state system: Rate of force-driven domain unfolding:
Domain unfolding
Stretching single titin with constant rate
Katalin Naftz Pasquale Bianco Zsolt Mártonfalvi
N2-B I1 I15 I84 I105 N2-A I27 I79 PEVK I84 I105 I1 I15
differentially expressed tandem Ig region constitutively expressed tandem Ig region constitutively expressed tandem Ig region
I27 NH2 COOH
Cardiac N2-B
Unique sequences Ig domain PEVK domain
COOH NH2
Skeletal (soleus)
I55-62 I II III Cloned and mechanically manipulated titin fragments
Alternative splicing:
20 40 60 80 100 120 50 100 150 200 250 Force Force (pN (pN) Ex Exten ension ion (n (nm) m)
PE PEVKI VKI 0.5 1 1.5 2 2.5 50 100 150 200 250 300 350 Lp PEVKI Lp PEVKII Lp PEVKIII Ef Effec ectiv ive e persistence persistence len lengt gth (n (nm) m) Ionic Ionic stre strength ngth (mM) (mM)
Persistence length (Lp)
Entropic (wormlike) chain
Contraction driven by entropy maximization
Ionic strength (mM) Effective persistence length (nm)
Flexibility increases:
+ + + + + + + + + + + + + + + + + + + + + + +
+ + + + + +
Electrostatic stiffening contraction
Force (pN) Extension (nm)
Force spectroscopy
Attila Nagy Nagy, A. et al, Biophys. J. 89,329, 2005.
50 nm 200 pN 20 15 10 5
Frequency
400 300 200 100
Unfolding force (pN)
ΔL = 29.8 ± 3.5 nm Unfolding events
Force spectroscopy
László Grama Grama, L. et al. Croat. Chim. Acta 78, 405, 2005.
Trapped bead Moved bead
Force (pN) Extension (µm) Time (s)
120 pN
~28 nm
movable mikropipette
titin
Reference input (setpoint) Control (+/-) (piezo movement) PID Feedback Actuating (+/-) signal Σ Latex bead
Stretching single titin with constant force
Step size (nm) Frequency
Extension (nm) Force (pN) Stepwise extension
Time (s) Extension (µm) Time (s) Extension (µm)
Force (pN) lnkunfold
Force (pN) lnτF Folded-state lifetime (ms) Frequency
Monexponential fit
11 26 22 32 36 64 62
Bianco, P. et al, Biophys. J.
500 nm
1000 nm
AFM of titin molecules
receding meniscus
400 nm
G G G G G
50 nmTitin kinase
~30 nm
N-terminal ß-sheet ATP-binding pocket
Gap width (nm) Frequency
Surface- equilibrated titin molecules Overstretched titin molecule Normalized distance from M-line Frequency Autocorrelation
M Z 200 nm 300 nm 400 nm 500 nm
Zsolt Mártonfalvi Dorina Kőszegi
Mártonfalvi, Z. and Kellermayer, M. PLoS ONE 9(1):e85847, 2014; Bianco, P. et al, Biophys. J.
M
N C
ßC1 ßC2 ßC3
Titin kinase N-terminal ß-sheet Titin kinase (systematically unfolded)
MD simulation: Gräter & Grubmüller MPI
N-terminal ß-sheet ATP-binding pocket
Titin kinase Titin M-line domains
Apparently linear force response during stretch
Extension (µm) Force (pN)
0.06 µm/s 0.08 µm/s 0.20 µm/s 0.38 µm/s 3.12 µm/s
Force (pN) Extension (µm)
1.6 1.2 0.8 0.4 Extension (µm) 200 150 100 50 Time (s)
160 140 120 100 80 60 40 20 Time (s) 2.5 2.0 1.5 1.0 0.5 Extension (µm) 120 60 F (pN)
435 nm 443 nm 1s 1s 635 nm 5s 700 nm 10s ΔLc
120 60 F (pN) 400 300 200 100 Time (s) 2.5 2.0 1.5 1.0 0.5 Extension (µm)
860 nm 895 nm 15 s 30 s 1070 nm 45 s 1090 nm 60 s 90 s 1269 nm ΔLc
Refolded length (nm) Time (s)
Force (pN) Extension (µm) Time (s)
4 pN ~700 nm
Fluctuations Domain unfolding
Large fluctuations
Time (s) Force (pN) Extension (µm)
Rapid conformational transitions?
Entropic collapse 2.5 2.0 1.5 1.0 0.5 Extension (µm) 60 50 40 30 20 10 Time (s)
10 pN 5 pN 3pN 1 pN
Refolding against constant force
Constant pipette position
Titin contracts and generates force by refolding In 4M urea, contraction is abolished, but fluctuations persist
Monte-Carlo simulation
Extra contractility!
Energetic topology of the three-state folding model
Folded state
Unfolded state
Reaction coordinate (length)
Molten- globule state
N-term → C-term N-term → C-term
most of the H- bonds in place, except the ones in the core
Residue contact map
unfolded folded molten globule
Mártonfalvi et al. Prot. Sci. 2017. DOI 10.1002/pro.3117
György Ferenczy
500 nm 400 nm 500 nm 500 nm 500 nm 100 nm
Zsombor Papp Brennan Decker Eszter Lakatos
Tonino et al. Nat Commun 2018
Titin oligomers Titin monomers Stretched titin oligomer Stretched titin monomers Myosin monomers Myosin filaments
Kellermayer et al. J.Struct.Biol 2018
500 nm 500 nm 400 nm 400 nm 400 nm
300 mM KCl 200 mM KCl 150 mM KCl 150 mM KCl 150 mM KCl To To To To Tm Tm Tm Tm TF TF TF TF TF TF TF TF M M
400 nm 400 nm 400 nm 400 nm 300 nm 400 nm
To Tm TF M To Tm M TF TF To M M
Titin-myosin mixture Titin-myosin mixture stretched with meniscus force
1 µm 4 µm 1 µm 200 nm 200 nm 200 nm 200 nm 300 nm 100 nm 100 nm I-band titin myosin titin wrapping
“end- filament”
Kellermayer et al. J.Struct.Biol 2018
Overnight incubation at 150 mM KCl Myosin splaying at filament tip
M-line complex 200 nm
Globular head at the C-terminus (M-line)
Tskhovrebova, L., and Trinick,
Tskhovrebova, L., and Trinick,
Electron microscopy (metal shadowing) Atomic force microscopy
section
200 nm
Dominik Sziklai
AFM cantilever Titin M-line complex
(~1 nN)
cantilever laterally
long thin strands
There is a reservoir of elastic strands in the M- line complex
200 nm
section
Relaxed titin
Overstretched M-line-complex strands
is a tunable telescope; the globular domains are shock-absorbers.
continuous extensibility and an apparently linear force response - accelerometer.