The cytoskeleton. The cytoskeleton, the motor proteins, the muscle and its regulation. The cytoskeleton. The cytoskeleton.

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1 , the motor proteins, the muscle and its regulation Dept. of Biophysics, University of Pécs Zoltán Ujfalusi January-February 2012 Dynamic framework of the Eukaryotes Three main filament-class: 1. Intermedier filaments 2. Microtubules 3. Microfilaments Subcellular-, cellular-level movements Requires ATP (energy) Intracellular movements Formation and degradation of filaments (microfilaments and microtubules) Provide road for the motor proteins actin rich cortex retraction pseudopodium substrate focal contacts movement of the nonpolymerized actin actin polymerization and protrusion of pseudopodium further growth of the pseudopodium The actin and the contractile system consist of three main components: microtubules, building from tubulin proteins with ~24 nm diameter microfilaments, building from actin and other associated proteins with ~6 nm diameter cell specific intermedier filaments, with high variation of quality (~10 nm diameter) Subunit: globular (G-) actin MW: 42,3 kda, 375 amino acid, 1 molecule adenosine nucleotide bound (ATP or ADP) 4 Subdomains 4 3 nucleotide 2 1 1

2 The actin The actin cytoskeleton The microtubules Intermedier filaments Subunit: tubulin MW: ~50 kda, a- and b-tubulin -> heterodimer 1 molecule guanosine nucleotide bound (GTP or GDP); exchangeable (b), or not exchangeable (a) ~24 nm diameter, cavernous 13 protofilaments right-screw, short-screw helix Left-screw, long-screw helix stiff polymer chain (persistence length: few mm!) structure polarization: +end: polymerization is fast, -end: polymerization is slow GTP-cap a b Tissue-specific types of intermedier filaments Nuclear lamins A, B, C lamins (65-75 kda) Vimentin type Vimentin (54 kda) Desmin (53 kda) Peripherin (66 kda) Keratins type I (acid) (40-70 kda) type II (neutral/basic) (40-70 kda) Neuronal IF neurofilament proteins ( kda) Fibrous monomer (not globular, as the monomers of actin or tubulin). The subunit of the intermedier filament: coiled-coil dimer Polymerization of the intermedier filaments Completely polymerized in cells (not in dynamic equilibrium) Central rods (a-helix) hydrophobe-hydrophobe interaction -> colied-coil dimer 2 dimer -> tetramer (antiparallel arrangement, structure apolarity) protofilament Longitudinal series of tetramers -> protofilament filament Ribbon diagram of a vimentin dimer 8 protofilament -> filament 2

3 The motor proteins Common properties of motor proteins 1. Connect to special cytoskeletal filaments 2. They move along the filament and evolve force 3. Hydrolyze ATP N 1. Structure N-terminal globular head: motor domain, binds and hydrolyzes nucleotide specific binding site for the appropriate cytoskeletal polymer C-terminal: binding site that is responsible for functionality C 2. Mechanics, operation Principle: cyclic operation Motor -> binding to the polymer -> pulling -> dissociation -> relaxation 1 molecule ATP hydrolyzes in 1 mechanical cycle. In the mechanical cycle there can be movements (isotonic conditions) or force evolvements (isometric conditions). Types of motor proteins The myosin protein superfamily 1. Actin-based: myosin protein family. Conventional (myosin II) and non-conventional myosins Myosin I-XVIII classes 2. Microtubule-based a. Dynein Ciliar (flagellar) and cytoplasmic dyneins. MW ~500 kda Move towards the negative end on microtubules b. Kinesin Responsible for axonal vesicle transport in neurons Kinesin protein family: conventional kinesins + isoforms. MW ~110 kda Move towards the positive end on microtubules 3. Nucleic acid based DNA and RNA polymerases Move along the DNA strand and evolve force Actin-myosin interactions Kinesin and Dynein Head motor domain Connection to filaments (microtubule) dimer ATP-binding region Tail Cargo-binding domain Pulling cycle of the dynein. The motor protein binds two neighboring microtubules and performs the relative movement of the microtubules (axonemal dinein). A flexible connecting protein, the nexin transforms the relative movement into deflection. Central linker domain Myosin moves towards the plus-end of an actin filament. 3

4 Force Why molecular motors are needed? The structure of the striated muscle, the molecular basis of muscle function and regulation endocytosis exocytosis transport chromosomes positioning during cell division vesicles transport inside the cytoplasm (ingest food, discard waste, deliver proteins) The muscle is an ordered tissue of cytoskeletal filaments and motor proteins which transforms chemical energy into mechanical work with high efficiency. Skeletal muscle (striated) Heart muscle Smooth muscle Length: can be more than 10 cm Thickness: μm Multinuclear (syntitia) Striped appearance Types of muscles Length: 100 µm Thickness: 10 µm Mononuclear network of myocytes Functional syntitium Striped appearance Length: μm Thickness: 2-10 μm Mononuclear spool-shaped cells No myofibrils, only myofilaments no stripes Voluntary Vegetative Vegetative The structure of the striated muscle Striated muscle Muscle fibers Myofilaments Muscle fiber Thick filaments Thin filaments Myofibril Sarcomere Sarcomere The structural and functional unit of the skeletal muscle. The operation of the striated muscle Muscle contraction Electric stimulation of muscle Contraction, relaxation Incomplete tetanus Complete tetanus Muscle trepidation Electron microscope image Time (ms) Periodic stimulation 4

5 Isometric contraction Isotonic contraction Length of the muscle is constant Force is constant Muscle contracts till the evolved force is equal to the weight of the load. Force tetanus W force + - trepidity length Time W Time Sliding filament theory Relaxed Sarcomere A-band is unchanged, while the I-band is shorten. The lengths of actin and myosin filaments do not change. Z-disc H-zone I-band A-band Z-disc Hugh E. Huxley and Andrew F. Huxley were made this theory independently from each other: When each end of the myosin thick filament moves along the actin filament with which it overlaps, the two actin filaments are drawn closer together. Thus, the ends of the sarcomere are drawn in and the sarcomere shortens. (A.F. H-zone shortens I-band Shortens A-band unchanged Huxley and R. Niedergerke (1954), H.E. Huxley and J. Hanson (1954)) More is the overlap, higher is the tension as well. Contracted Shortened sarcomere Thick filament Thin filament The power of the striated muscle Muscle power: The regulation of the work of muscle Force-speed diagram of the muscle P=F*v Max. evolved force (1,7pN/1 myosin cross bridge): The energy of the chemical bounds between actin and myosin gives the limit Max. speed( 6000nm/s): Correlates with the maximal speed of ATP hydrolyzation Max. power: At the 1/3 of the speed The invested chemical energy is utilized with more than 50 % by the muscle! In skeletal and heart muscle: 1. Tropomyosin 2. Troponin complex 3. Ca 2+ In smooth muscle: Phosphorylation of the light chain In scallop muscle: Calcium bound to myosin 5

6 Tropomyosin The troponin complex One troponin complex interacts with a protein complex consist of 7 actin monomers and one tropomyosin. Troponin T - MW 37 kda binds tropomyosin and the other troponin proteins, stabilizes the protein system. Tropomyosins consist of nearly 100 % α-helix and assemble into parallel dimeric coiled-coils. Each dimer is interacted with 7 actin protomers. Tropomyosin dimers take place along the surface of the whole actin filament by following each other in a tandem way. Troponin I - MW 22 kda inhibits the actin-myosin interaction. Troponin C- MW 18 kda if binds calcium than its structure is changed -> key step of the regulation of muscle. The much of the troponin complex takes place at the middle of the tropomyosin dimer. The regulation of the work of muscle To activate a muscle there is a need for calcium. Under neuronal control calcium releases from the sarcoplasmic reticule and the cytoplasmic [Ca 2+ ] increases more than 1 μm. 1. TnC binds Ca 2. Conformation changes 3. Affinity of TnC increases to TnI 4. TnI separates from the surface of actin 5. Myosin binding site is not covered by tropomyosin 6. Myosin can bind to actin Further steps of the regulation Rigor state +ATP -Ca 2+ Relaxed state +Ca 2+ Activated state, poor interaction P i dissociates Activated state, force generation ADP dissociates Rigor state Thank you for your attention! 6