Muscle Biophysics Summer School - Budapest, 29 August, 2018 Muscle Cytoskeleton I. Miklós Kellermayer Semmelweis University Department of Biophysics and Radiation Biology
Types of biological motion Collective motion Body motion (“Leap of the century”) Organ motion Wound healing model - collective Autonomous cardiomyocyte fibroblast movement
Types of biological motion Crawling keratinocyte Chemotaxis Moving spermatocytes Axonal (neurite) growth Dividing cell Intracellular movement of pathogenic Listeria bacteria
The cytoskeletal system • Complex intracellular network of interconnected filaments and tubules • The term “cytoskeleton” was first coined, independently, by Nikolai K. Koltsov (1903) and Paul Wintrebert (1931). • Numerous scientists contributed to the discovery of the composition, structure and function of the cytoskeleton. Mary Osborn and Klaus Weber Brunó F. Straub (Labeling methods for intermediate filaments, (Discoverer of actin) • Filaments and tubules are each composed microtubules) of different protein monomers. • Main functions are in motion, mechanics, scaffold for intracellular binding and Actin Microtubules transport processes. • Associated proteins add a multitude of complex functionality. • Until 1992, it was thought that the cytoskeleton is exclusive to the eukaryotic cell. • Prokaryotes express homologues of actin DNA (MreB) and tubulin (FtsZ). • Muscle is a tissue specialized for expressing its cytoskeleton.
The eukaryotic cytoskeleton Microfilaments (Actin) Intermediate filaments Microtubules (rhodamine-phalloidin) (Vimentin, anti-vimentin) (GFP-tubulin) 1. They display polymerization dynamics 2. Their mechanical properties are important 3. They bind a variety of associated proteins which diversify their functions.
Actin in the cell • cortex • stress fibers, • cellular processes (lamellipodia, filopodia, microspikes, focal contacts, invagination) • microvillus Stress fibers cortex filopodium
Actin-dependent cell movement
Microtubular system Filamentous system of eukaryotic cells composed of tubulin and its associated proteins
Functions of the microtubular system 1. “Highways” for motor proteins 2. Senses, monitors and finds the geometric center of the cell. 3. Motility functions (e.g., cell division)
Intermediate filament system • Tissue-specific filamentous protein system composed of 8-10-nm filaments, found in most animal cell types. • Fundamental biological function is providing mechanical stability. Neurofilament tail domain extending from surface (Ueli Aebi, Basel) 100 nm Vimentin, Vic Small Qin et al. J. Biomech. 43, 15, 2010 Epidermolysis bullosa GFP-vimentin, 3T3 cell, R.D. Goldman Anti-keratin, PtK2 cell
Prokaryotic cytoskeleton FtsZ: tubulin homolog • Main component of the Z-ring • Important role in cytokinesis • Dynamic rearrangement MreB: actin homolog • Discovery based on sequence homology • Helical filaments underneath cell membrane • Role in chromosome segregation
Types of muscle myoepithelial cell cardiac myocyte smooth muscle cell skeletal muscle fiber
Skeletal muscle Myofibrils Nucleus Myofibrils: The organelle-level structural and functional units of muscle.
The sarcomere • sarcos: meat (Gr) • mera: unit • the smallest structural and functional unit of striated muscle. A I
Mechanisms of muscle contraction 3 2 4 Phenomenological 5 1 mechanism: Szarkomerhossz ( µ m) Sliding filament theory Andrew F. Huxley, Jean Hanson, Hugh E. Huxley
Theoretical need for an elastic, third filament in the sarcomere The elastic component limits A-band asymmetry Without elastic filament With elastic filament (two-filament sarcomere) (three-filament sarcomere)
Early models of sarcomere structure I A I thin thick M Z Z The C-filament model of Garamvölgyi The two-filament model on which the sliding-filament Miklós Garamvölgyi (Garamvölgyi 1965) theory is founded (A. F. Huxley and Niedergerke 1954; (C-filaments) (1932-1980) H. Huxley and Hanson 1954) The S-filament, extending in between the tips of the thin The T-filament stretching in between the Z-lines (actin) filament (Hanson and Huxley 1956) Ferenc Guba of the sarcomere (McNeill and Hoyle 1967) (Fibrillin) (1919-2000) The gap filament extending between the tips of the The gap filament of Locker and Leet (Locker and Leet thin and thick filaments (Sjöstrand 1962) 1975), which stretches along the core of the thick filament and connects it to one of the Z-lines. Károly Trombitás (C-filaments, titin)
Muscle cytoskeleton • Myofibrils , composed of serially-linked sarcomeres : basic unit of contraction • Intermediate filament (and microtubular) system - links sarcomeres to the membrane and organelles. Special cytoskeletal M-line Z-line structures: • Costamere: links sarcomeres to the extracellular matrix. • Intercalated disc: links cells together (important in cardiac desmin myosin actin myocytes). • Myotendinous junction: links skeletal muscle to tendon.
Titin: giant elastic muscle protein Cardiac myocyte Z-line Z-line Thin filament (actin) Thick filament (myosin) M-line Muscle sacromere AFM structure of a Myomesin single straightened A170 M1 M3 200 nm M-protein M-protein Titin titin molecule M4 Kinase Titin Myomesin M-line complex 7 anti-parallel nm 60 50 40 30 20 10 M ß-strands Immunoglobulin Fibronectin PEVK domain Kinase domain (Ig) domain (FN) domain
Titin function, regulation, pathology Function : Phosphorylation sites in titin: • Elastic element • Template • Mechanosensor Regulation : Hidalgo and Granzier, Trends. Cardiovasc. Med. 2013. • Phosphorylation • Alternative splicing (size isoform expression) Pathology : • Myasthaenia gravis (circulating anti-titin antibodies) • Cardiac failure • Cardiomyopathy Where else is titin? • Smooth muscle (smitin) • Cell nucleus (chromatin organization) • Sarcoplasm (dynamic equilibrium with Diffusible titin pool: Titin-eGFP knockin (MEx6) sarcomere) Embryonic cardiomyocyte T 1/2 ~ 3 h da Silva Lopes et al, JCB 193, 785, 2011.
Titin genetics, titinopathies TTN: 2q31 base-pair region: 179,390,715 - 179,672,149 364 exons - alternative splicing: cardiac- (N2B, N2BA) and skeletal-muscle isoforms Titinopathies : • Hereditary myopathy with early respiratory failure (HMERF): Arg279Trp, postural muscles affected. • Limb-girdle muscular dystrophy type 2J (LGMD2J): limb muscles affected. • Salih myopathy: both cardiac and skeletal muscle affected. • Tibial muscular dystrophy: m. tibialis affected. Frequent in Finnish population. • Familial hypertrophic cardiomyopathy type 9 • Dilated cardiomyopathy type 1G Leinwald et al, Circ. Res. 111, 158, 2012.
Titin gene expression
The canonical titin protein and its truncating vatiants TTN TV - truncating variants DCM - dilated caradiomyopathy ExAC - Exome Aggregation Consortium PSI - percent spliced in Linke Annu Rev Physiol 2018
Binding partners of titin Henderson et al, Compr Physiol. 2017. The titin “interactome” Linke, Cardiovasc. Res. 2008.
How to measure the mechanics of protein filaments? 1. Force deforms shape Rigid body: Polymer chain: Wormlike chain Hooke’s law fluctuations, configurational entropy Force (F) Extension (z) R s Contour length (L c ) Force (F) Force (F) L P = persistence length EI = bending rigidity k B T = thermal energy Extension (z) Extension (z)
2. Force reduces bond lifetime Under thermal activation: Under mechanical load: ω = characteristic time Ea = activation energy Δ x = distance between bound and transition states Unfolded state Conformational space Native Evan A. Evans and David A. Calderwood Science 316, 1148 (2007) state
Methods of single-molecule mechanics Cantilever methods Field methods
Optical tweezers Steven Chu Arthur Ashkin Atom cooling with Developer of optical optical tweezers tweezers (1970) Nobel prize (1997) Escherichia coli bacterium grabbed Tractor beam, Star Trek with optical tweezers Momentum-exchange between photons and refractile particle Laser Light beam P 1 bead in optical trap Objective lens Gradient force Refractile F F bead Refractile EQUILIBRIUM F= Δ P/ Δ t bead P 2 Scatter force Δ P (light pressure)
Force measurement based on light momentum change Incoming laser beam Objective underfilled Latex bead Objective Outgoing underfilled beam displacement Incoming Latex bead Molecule laser beam Micropipette Outgoing beam displacement Micropipette movement → → molecule extension → → bead displacement
Atomic Force Microscope (AFM) 2. Measure the deflection of the cantilever by help of a laser beam reflected off of its back surface 1. Van der Waals interaction between the atoms of the sample and the tip repulsion 3. The sample (or the cantilever) is moved in XYZ directions contact non-contact mode (oscillating) mode attraction
AFM probes AFM tip diameter ~ 10 nm
Contrast mechanisms 500 nm Height contrast Amplitude contrast Phase contrast Phase difference between the sinusoidal Cantilever resonence detuned driving signal and the detected cantilever upon the action of external oscillation forces
AFM imaging 500 nm 50 nm 100 nm 200 nm 50 nm G G G G G 400 nm
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