The Molecules of Movement Musc 1 - Professor Michael Ferenczi

The Molecules of Movement Musc 1 - Professor Michael Ferenczi (m.ferenczi@imperial.ac.uk) 1. Appreciate that there are a large number of molecular motors, each with its assigned role. 2. Linear molecular motors are associated with polymeric filaments. Which filaments are myosins and kinesins associated with? 3. Which molecular motor is that found predominantly in muscle cells? 4. Describe the structure of myosin II. 5. Describe the structure of myosin filaments. 6. Describe the structure of actin filaments. 7. What are sarcomeres? Draw a sarcomere in longitudinal section. 8. Activation of the skeletal muscle cell results from a cascade of events: 9. What causes depolarisation of the muscle cell membrane? 10. How does cell membrane depolarisation propagate 11. along the cell surface? 12. into the core of the muscle cell? 13. Describe how calcium ions are released from intracellular calcium stores (sarcoplasmic reticulum). Which membrane-bound proteins are involved in this process? 14. Describe the fate of calcium ions during activation of the myofilaments. 15. How is the thin filament affected by calcium? What are the consequences? 16. What is relaxation? What happens to calcium ions during relaxation? Molecular Motors: Overview Protein assembles which convert chemical energy into mechanical work Energy for the biological motors is usually derived directly from the hydrolysis of ATP or from an ionic gradient (e.g. H+ or Na+) o Behave like molecular engines LINEAR MOTORS: - require protein rails: o actin filaments for myosins o microtubules for dynein o kinesins for NCD o DNA/RNA for helicases and tropoisomerases - other linear motors rely on filament polymerisation o e.g. actin polymerisation in listeria, or microtubule polymerisation ROTARY MOTORS: - Require stators: o E.g. the flagellar motor o E.g. 2. ATP-hydrolysing F1 portion of the F1F0-ATPase OSCILLATORY MOTORS: - Found in cilia and require cross-linked microtubule bundles called AXONEMES - Powered by dynein - Cross-links generate oscillation 1

Microtubule Polymerisation EB-1 protein attached to the positive end of microtubules Polymerise from the centre of the cell to the periphery Another mechanism by which movement is generated E.g. how vesicles are moved around the cell Types of Movement Intracellular - Vesicle transport - Tuning of hair cells in the ear (sensory organs) - Cytoplasmic streaming - Anaphase: segregation of chromosomes in mitosis - Cytokinesis: formation of cleavage furrow - Unwinding of DNA Cellular - Bacterial motility - Cell locomotion - Axon growth - Phagocytosis - Muscle contraction - Chemotaxis Cytokinesis Spindles contain microtubules and dynein Formation of a cleavage furrow by the contraction of a circumferential ring of actin and myosin II at the cell equator Chemotaxis Ability of cells to move or orient themselves according to a chemical gradient o Cells respond to the chemical signal by either moving away or towards the source Directed movement in response to environment Anaphase Spindles contain microtubules and dynein Involves myosin I and II, as well as other proteins Chromosomes are segregated to opposite ends of the diving cell during mitosis Unwinding of/movement along DNA X-ray diffraction can be used to assess the structure of a protein, e.g. HUMAN TROPOSIOMERASE 1 HT1 catalyses the transient breaking of DNA strands and movement along the strand Actin Polymerisation Another type of molecular motor Actin: small protein, forms very long filaments 2

Listeria moves in infected cells by inducing actin polymerisation o Makes use of host cell machinery o The growth of actin filaments forms a COMET TAIL o Tail generates movement, but does not require self motors o Unique mechanism as travels through cells, used to travel through placental membrane to infect foetus Molecular Motors Kinesin Consists of two amino-acid chains, each terminating into a globular ATP-binding head, which interacts with microtubules Tail of proteins form a coiled-coil; vesicles are held at specialised binding sites at the end of tails Kinesin moves along microtubules o Microtubules are polymers of tubulin molecules o This moves vesicles through cells o Requires ATP hydrolysis Dynein Oscillatory motor found in cilia CILIA: o o o Found lining the respiratory tract Also responsible for sperm movement Cilium contains an AXONEME (bundle of microtubules) Between the inner and outer cilium, there are a range of proteins which connect the microtubules: o ELASTIN proteins nexin o MOTOR proteins dynein When ATP is hydrolysed, Dynein bends (and vice versa when ATP is synthesised) o This changes the distance between the inner and outer cilium o This drives the oscillation F1-ATPase/ATP Synthase Found in the inner membrane of mitochondria Acts as a generator Driven by the protein gradient between the outer and inner compartments in the mitochondrion o Result of the electron transport chain 3

The flagellum Rotary motor Rotation driven by either H+ or Na+ gradient MS ring does not rotate Extracellular section allows movement of the entire cell e.g. bacterium E. coli Myosin The molecular motor found in muscle cells Moves along actin filaments Vast number of different classes identified; phylogenetic tree shows evolutionary formation of the different classes o Myosin II conventional Between classes, length of tails and functional domains vary All classes have a motor domain, most have a light chain binding domain Myosin II - 2 amino acid chains coiled around each other - 2 smaller peptides (light chains) coild around other chains - N terminus: globular head (myosin subfragment 1)- actin and ATP binding site - C terminus: polypeptide tail - Thick filament structure: tail diameter 2nm - Backbone diameter: 16.3 nm - Length of filament: 1.6 micrometer Myosin V - Found in brain and involved in vesicular and mrna transport - E.g. melanin vesicle transport into hair along F-actin filaments coloured hair - Binds microtubules - Found in nerve growth cones Organs designed for movement: Skeletal Muscle Myosin filaments Skeletal muscle packed with myosin II Tail diameter: 2 nm 4

Thick filament backbone diameter: 16.3 nm Thick filament and packed myosin heads: 20.3 nm Length of native thick filaments: 1.6 µm Form thick filaments by forming tail to tail junctions o 294 molecules form 1 thick filament Myosin heads form at either end of the BARE ZONE in opposite orientations GLOBULAR HEAD: o Actin binding site consists of Myosin S1 o ATP-binding site o 50kDa cleft between binding sites o Also consists of: Lever arm Converter Pliant region Switch II SH1 helix relay Actin Filament system- Microfilaments Made by polymerisation of G-actin Found in the periphery of cells underlying cell surface E.g. thin filaments of muscle, core of microvillus in brush border When it polymerises, hydrolyses ATP- ADP remains in the centre of the filament- involved in cell surface shape and cell migration Decorated by myosin globular heads (S1) Sarcomere Defined as distance between Z lines Z-line is source of actin Thick filament length: 1.6 µm: Containing 294 myosin molecules (588 heads). 3 myosins/crown, spaced at 14.3 nm interval. Thin filament length: 1.1 µm. Length regulated by NEBULIN and TROPOMODULIN TITIN is a large elastic and extensible protein that links the thick filaments to the Z-line, thus ensuring that the thick filaments remain centred in the sarcomere. Z-band width: 0.052 µm in Type II fibres, 0.101 µm in Type I fibres. Bare zone: 0.15 µm wide 5

Contraction Of permeabilised muscle fibre by photolytic release of ATP from caged ATP STRIATIONS are patterns of repeating sarcomeres The filaments are stiff and largely inextensible During contraction, sarcomeres shorten Muscle shortening is brought about by the interaction of myosin cross-bridges interacting with the thing (actin) filaments, which cause changes in the overlap between the sets of filaments Myosin cross-bridges act as independent force generators T-tubule organisation Each bundle of filament is called a myofibril. There are hundreds of myofibrils in each cell of a skeletal muscle. T-tubules are invaginations of the sarcoplasmic membrane o T= transmembrane o T-tubules go through muscles at intervals corresponding to the sarcomere length o Open to extracellular space, therefore have the same ion composition The sarcoplasmic reticulum is a specialised endoplasmic reticulum, storing calcium. Mitochondria also power contraction, as it requires ATP Motor neuron makes close contact with the plasma membrane of muscle fibres o This is known as a NEUROMUSCULAR JUNCTION o The combination of a single motor neuron, and its corresponding muscle fibre is known as a MOTOR UNIT NEUROMUSCULAR JUNCTION: o Voltage sensitive receptor (DIHYDROPYRIDINE; DHP receptor) in the t-tubule interacts with a calcium release channel in the sarcoplasmic reticulum membrane (RYANODINE receptor) Mechanism of Contraction Action potential propagated along motor neuron to muscle cell T-tubule is depolarised o DHP receptor interacts with ryanodine receptor on sarcoplasmic reticulum membrane o Ryanodine receptor undergoes conformational change intracellular Ca2+ released Ca2+ binds to TROPONIN-C (actin binding protein on actin filament) o Conformational change in Troponin causes TROPOMYOSIN to move away from the myosin binding site o Myosin can then bind to the actin filament Tropomyosin o Long polymer spanning 7 actin monomers o In relaxed muscle, it prevents the interaction of myosin with actin o In presence of Ca2+, it moves into the actin groove, so myosin can interact with the myosin binding sites 6

Relaxation Once the membrane has repolarised, the calcium channels in the sarcoplasmic reticulum close. The sarcoplasmic Ca-ATPase pumps calcium back into the reticulum, causing a decrease in calcium concentration in the cytoplasm. This causes calcium dissociation from troponin, resulting in inactivation of myosin binding sites on actin, and hence muscle relaxation. Molecules working together: Force & Movement Musc 2 - Professor Nancy Curtin (n.curtin@imperial.ac.uk) 1. Appreciate that the myosin cross-bridge cycle is the elementary process resulting in force generation and shortening by all muscle cells. 2. Draw a diagram of the main steps in the cross-bridge cycle, showing the changes in cross-bridge structure and how ATP is involved. 3. List 3 key features of the cycle (ATP used in each cycle, one-way sliding, small step size). 4. For the following factors, that control the amount of force produced in vivo, briefly explain the mechanisms involved. a. recruitment of motor units b. frequency of activation (covered earlier) c. sarcomere length and filament overlap d. speed and direction of movement 5. List the metabolic sources of ATP and briefly outline the conditions in which each is important. 6. Smooth muscle: a. Describe its main structural features. b. Give examples of tissues contain smooth muscle and state the nature of the signal that triggers contraction. c. Describe the intracellular events during activation of contraction and during relaxation. Be able to explain how this is different from the corresponding events in skeletal and cardiac muscle. 7. Cardiac muscle: a. Describe its main structural features b. Be aware that the crossbridge cycle is like that in skeletal muscle. c. Describe the sources of Ca for activation and how this differs from skeletal muscle. Isometric contraction: force without length change Isovelocity contraction: muscle shortens or is stretched at constant velocity Isotonic contraction: constant force during shortening or lengthening Twitch: force and/or movement in response to a single action potential Tetanus: force and/or movement in response to action potentials in quick succession The cross-bridge cycle Steps 1. Attachment - Ca2+ release 7

- Troponin binds to Ca2+ - Tropomyosin displaces from myosin binding site - Myosin head attaches to actin (along with ADP and Pi) 2. Cross-bridge formation and Pi release - Pi released from myosin - ADP remains attached - Myosin forms cross-bridge with actin; increase binding strength from weak to strong 3. Filament sliding and ADP release - ADP release causes change in orientation of myosin head - Myosin filament pulled towards M-line 4. ATP binding - New ATP molecule binds to myosin head 5. ATP hydrolysis and detachment - ATP molecule hydrolysed to ADP and Pi (which both remain attached) - Energy released used to return myosin head to original orientation, and detach from the actin filament Note: Isometric contraction - In isometric contraction, the myosin head changes orientation, but there is NO FILAMENT SLIDING - Force is directed towards the M line Key Features ATP is used in each cycle to provide the energy o RIGOR MORTIS occurs if ATP concentration = 0 Direction of filament sliding is one way o Thin filament moves towards the centre of the sarcomere o Causes sarcomere shortening o However the attached bridge does resist a stretch applied to the ends of the muscle Step size is small o Sliding produced by one cycle is only 1% of sarcomere length o Many cycles in succession needed to cause large movements e.g. running Variation in Force (in vivo) Recruitment of the Motor Units Motor Unit: a motor neuron and all the muscle fibres is innervates (has synapses with) o Functional unit of normal skeletal muscle o Action potential in the motor neuron triggers an action potential in all the muscle fibres in the motor unit Vary the number of motor units that are active and thus vary the force as more muscle fibres are producing force Functional anatomy of motor units: o Motor unit size varies (muscle fibres per motor neurone) o There is a scattered distribution of motor units within the muscle 8

Frequency of stimulation Force increases with the stimulation frequency Force summates, but action potentials do not: o Twitch unfused tetanus force fused tetanus force (increasing force) o Particularly important for getting high forces In vivo Mechanism: o at higher stimulation frequency, more Ca is released into the space around the filaments. o More troponins bind to Ca, therefore more crossbridges attach and produce force Force depends on filament overlap Drawing Sarcomeres: o Align thick (central) filaments first, then draw thin filaments o Max force produced when completely overlapped o Draw cross bridges attached for each different amount of overlap Force depends on velocity and direction of Movement This behaviour cannot be understood in terms of one cross-bridge. It is due to a large number of bridges acting together. Force depends on the: o proportion of bridges that are attached o Force each attached bridge produces. If the velocity is too great, the myosin heads do not have as much time to form cross-bridges, which may result is less force Energy Demand and Supply ATP Supply Phosphocreatine (PCr) + ADP ATP + Creatine (involves CREATING KINASE) o Buffers ATP concentration so that it is constant o Occurs in every contraction, during recovery reaction is reversed o Phosphocreatine dedicated to this reaction only, and is stored in the muscle Glycolysis does not require oxygen o Substrate: glycogen and glucose o Products: lactic acid and/or pyruvate Oxidation requires oxygen o Makes more ATP per carbon atom metabolised than Glycolysis does o Substrates: glucose, glycogen, fatty acids o Products: carbon dioxide 9

Activity Demand Activity Sustained, low intensity Sustained, high intensity Sprint Cardiac Muscle Fuel supply DURING activity ATP, PCr, fatty acids (also glycogen & glucose) ATP, PCr, glycogen, glucose (also fatty acids) ATP, PCr Structure and function Small cells Action potentials spread between cells Some cell types have SPONTANEOUS action potentials Intercalated disks with mechanical links between the fibres Gap junctions, where ionic currents (action potentials) flow between fibres Action potential and Calcium sources Differences from skeletal muscle: Timing of ventricular action potential and isometric force Action potential lasts as long as isometric force Force is already relaxing during membrane s refractory period o Therefore cannot produced fused tetanus (sustained, summated force) o Designed for pumping Normally not enough Ca to bind to all troponins, therefore anything that changes Ca changes force Between action potentials, Voltage-gated Ca channels in sarcoplasmic reticulum closed, therefore Ca is not bound to Troponin in thin filament. Cross bridges not attached, therefore no force During the action potential, Ca enters from the SR AND through voltage gated channels in the muscle membrane during the PLATEAU of the action potential o Some, but NOT ALL cross-bridges attach Smooth Muscle Structure and function Arranged in sheets forming walls of tubular organs o blood vessels, o gastro-intestinal tract o reproductive tract, etc. Cells appear smooth (not-striated) in micrographs. Contain filaments, but they are not arranged in regular arrays. Contractile mechanism (crossbridge cycle) is similar to skeletal and cardiac muscle Activation and Relaxation Different from skeletal and cardiac muscle: involveso Intracellular Ca o Intracellular Cm = CALMODULIN o Intracellular MIck = MYOSIN LIGHT CHAIN KINASE 10

o NO Troponin Contraction - Inactive myosin is not phosphorylated so cannot bind to actin - Action potential depolarisation release of/increase in intracellular Ca - Ca, Cm and MIck combine to form ACTIVE ENZYME o Phosphorylates myosin (hydrolysing a molecule of free ATP) o Myosin binds to actin Relaxation - PHOSPHATASE removes phosphate from myosin - Inactive myosin cannot bind to actin 11