Slide 1. Slide 2. Slide 3. Muscles general information. Muscles - introduction. Microtubule Function

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1 Slide 1 Muscles general information Vertebrates and many invertebrates have three main classes of muscle Skeletal muscle connect bones are are used for complex coordianted activities. Smooth muscles surround internal organs such as the large and small intestines, the uterus, and large blood vessels The contraction and relaxation of smooth muscles controls the diameter of blood vessels and also propels food along the gastrointestinal tract. Compared with skeletal muscles, smooth muscle cells contract and relax slowly, and they can create and maintain tension for long periods of time. Cardiac muscle: Striated muscle of the heart. Slide 2 Muscles - introduction B. Skeletal muscle from the neck of a hamster C. Heart muscle from a rat D. Smooth muscle from the urinary bladder of a guinea pig E. Myoepithelial cells in a secretory alveolus from a lactating rat mammary gland Slide 3 Microtubule Function Move subcellular components Use motor proteins kinesin and dynein e.g., Rapid change in skin color

2 Slide 4 Microtubules Show Dynamic Instability Balance between growth and shrinkage Factors Local concentrations of tubulin Dynamic instability Microtubule-associated proteins (MAPs) Temperature Chemicals can disrupt the dynamics (e.g., plant poisons) Slide 5 Movement Along Microtubules Direction is determined by polarity and the type of motor protein Kinesin move in + direction Dynein moves in direction Fueled by ATP Rate of movement is determined by the ATPase domain of the protein and regulatory proteins Dynein is larger than kinesin and moves 5-times faster Slide 6 Cilia and Flagella Cilia numerous, wavelike motion Flagella single or in pairs, whiplike movement Composed of microtubules Arranged into axoneme Movement results from asymmetric activation of dynein

3 Slide 7 Microtubules and Physiology Slide 8 Microfilaments Other type of cytoskeletal fiber Polymers composed of the protein actin Often use the motor protein myosin Found in all eukaryotic cells Movement arises from Actin polymerization Sliding filament model using myosin (more common) Slide 9 Microfilament Structure and Growth Polymers of G-actin called F-actin Spontaneous growth (6-10X faster at + end) Treadmilling when length is constant Capping proteins increase length by stabilizing minus end

4 Slide 10 Microfilament Arrangement Slide 11 Actin Polymerization Amoeboid movement Two types Filapodia are rodlike extensions Neural connections Microvilli of digestive epithelia Lamellapodia resemble pseudopodia Leukocytes Macrophages Slide 12 Skeletal muscle (striated muscle) Skeletal muscle cells are one of the largest cells in the body Are multinucleate formed by the fusion of myoblasts Diameters range from 50 to 150 microns with lengths ranging from mm to cm Muscle fibers contract in response to an electrical signal ie depolarization The signal is generated at the synapse (the neuromuscular junction) and propagated through an action potential via the muscle fiber membrane The membrane of the cell has specialized invaginations called Transverse-tubules (T-tubules) that enter into the cell (at every 1-2 microns) The action potential can be rapidly transmitted deep into the interior of the cell resulting a delay of only 3-5 msec between the depolarization at the synapse and the first muscle fiber tension. The T-tubule network is so extensive that 50-80% of the plasma membrane is in the T-tubules.

5 Slide 13 Neuromuscular junction things to remember Each muscle fiber is innervated by a motorneuron One motorneuron can innervate one or multiple fibers Each motorneuron plus its complement of muscle fibers is called a motor unit as well all contract in concert. The synpase between the motorneuron and the muscle fiber is called a neuromuscular junction(nmj) Nerve terminal contains many mitochondria and vesicles which can be seen lined up in double rows along side the voltage-gated Ca 2+ channels attached to presynaptic membrane => active zone. The vesicles of the NMJ have very high concentrations of neurotransmitter (2,000 to 10,000 molecules of ACH per vesicle) Excess of neurotransmitter is released to ensure that the resulting post-synaptic depolarization is strong enough to generate an action potential - "safety margin" Slide 14 Neuromuscular junction things to know Slide 15 The nachr -again The receptor is made up of 4 different transmembrane proteins one of which (the alpha subunit) is repeated to give 5 subunits to create the ion channel ACH binds to the alpha subunit and thus it takes two molecules of acetylcholine to open the channel One nachr opens and allow 1.5 x 10 4 Na + ions/msec of open time The channel opens on average 1 msec. 1 vesicle contains enough neurotransmitter to open ~3000 receptors (wow!) and because two molecules of Ach is needed to open one receptor there must be a minimum of ~6,000 molecules Ach per vesicle. Studies have shown that the amount of neurotransmitter contained in one vesicle causes an post-synaptic potential of ~ 1 mv. If the average depolarization generated at a NMJ of a muscle fiber is 40 mv then there must be at least 40 vesicles released and in the order of 120,000 receptors activated at the NMJ. WOW!

6 Slide 16 Striated muscle channels and action potentials Slide 17 Striated muscle channels and action potentials Pumps and transporters 1) Na + /K + ATPase pump - to establish the electrochemical gradients of Na + and K + 2) Ca 2+ ATPase pump - uses energy from ATP to remove 2 Ca 2+ from the inside to the outside of the cell to ensure that internal Ca 2+ concentrations remain low (10-7 mm internal) 3) Na + /Ca 2+ cotransporter - to also remove Ca 2+ from the inside of the cell and uses the energy from the cotransport of 3 Na + molecules to export 1 Ca 2+ 4) Muscle Ca 2+ ATPase pump - a different pump from number 2 above. Found highly concentrated on the sarcoplasmic reticulum (SR) (constitutes 80% of the protein found in the SR membranes). The muscle Ca 2+ ATPase pumps 2 Ca 2+ into the SR to lower cytosolic Ca 2+ and to concentrate Ca 2+ into internal stores. Slide 18 Striated muscle channels and action potentials Channels - muscle cells share many of the same ion channels as neurons 1) leak channels - besides the leak K + channel, skeletal muscle cells have a high concentration of Cl - leak channels so much so that the resting membrane potential is usually the same as the Nernst potential for Cl - (around -80 mv in these cells). The high permeability to Cl - helps repolarize the membrane after an action potential 2) Voltage-gated Na+ channels - Voltage-gated Na + channel (very much like the neuronal voltage-gated Na + channel) Responsible for the production of the action potential. Remember the regenerative cycle of these channels ie during an Action potential 3) Voltage-gated K+ channel -the delayed rectifier K + Has a high threshold and needs a strong depolarization to open and works to bring the membrane back to resting potential 4) Voltage gated Ca 2+ channels - high threshold Ca 2+ channels Needs a strong depolarization to open and very slowly Concentrated in the T-tubules.

7 Slide 19 Skeletal muscle (striated muscle) Terminology Muscle cell: Muscle fiber Myofibrils: Main intracellular structures in striated muscles. Are bundles of contractile and elastic proteins Sarcolemma: Cell membrane of a muscle cell Cytoplasm: Sarcoplasm Sarcoplasmic reticulum: wraps around each myofibril like a piece of lace. Release and sequester Ca 2+ ions Slide 20 Skeletal muscle (striated muscle) The sarcoplasmic reticulum (SR) regulates the cytosolic Ca 2+ levels in skeletal muscle Myofibril: A long bundle of actin, myosin and associated proteins in muscle cell. Transverse (T) tubules: invaginations of the plasma membrane, enter myofibers at the Z disks, where they come in close contact with the terminal cisternae of the SR Terminal cisternae: store Ca 2+ ions and connect with the lacelike network of SR tubules that overlie the A band. Slide 21 T-Tubules and SRs Transverse tubules Sarcolemmal invaginations Enhance action potential penetration More developed in larger, faster twitching muscles Sarcoplasmic reticulum (SR) Stores Ca 2+ Terminal cisternae - storage

8 Slide 22 Triads and Sarcoplasmic reticulum The link between depolarization and Ca 2+ release or excitationcontraction coupling occurs at the junctions between the T-tubule and the sarcoplasmic reticulum 80% of the T-tubules membrane is associated with the sarcoplasmic reticulum at triads The voltage-gated Ca 2+ channels are concentrated in the T-tubules in the triads The Ca 2+ release channel found in the sarcoplasmic reticulum membrane is associated with the voltage-gated Ca 2+ channel at this point This close association allows for the rapid signaling from action potential to Ca 2+ release. Slide 23 General sequence of events Resting [Ca 2+ ]i = 0.1 µm AP propagation along Sarcolemma and into T- tubules Depolarization opens the voltage-gated Ca 2+ channels at triad junctions This results in a release of Ca 2+ through the Ca 2+ release channels from the SR Cytosolic [Ca 2+ ]i reaches 1-10 µm Diffusion and binding of Ca 2+ to TnC Contraction events [Ca 2+ ] to resting levels: 1. After the action potential is passed and the voltage-gated Ca 2+ channels close, the Ca 2+ release channels close 2. Ca 2+ is recycled back into the SR through the Ca 2+ ATPases 3. Ca 2+ binds to calsequesterin Slide 24 Ca 2+ channels and its release Release of Ca 2+ stores mediated by ryanodine receptors (RYRs) in skeletal muscle Voltage sensing dihydropyridine (DHP) receptors in the plasma membrane contact ryanodine receptors located in the membrane of the SR In response to a change in voltage, the dihydropyridine receptors undergo a conformational change This produces a conformational change in the associated RYRs, opening them so that Ca 2+ ions can exit into the cytosol. The voltage-gated Ca 2+ channel is either closely localized to or makes a physical connection to the Ca 2+ release channels in the SR Cont..

9 Slide 25 Ca 2+ channels and its release Not all Ca 2+ release channels are associated with voltage-gated Ca 2+ channels These non-associated channels are thought to be opened solely by Ca 2 influx into the cytosol from the voltage-gated Ca 2+ channels. The Ca 2+ release channel in the SR of most muscle cells (smooth, cardiac, skeletal) is a Ca 2+ activated Ca 2+ channel The Ca 2+ release channel is stimulated to open at low concentrations of Ca 2+ ( up to 0.1 mm) in the cytosol but inhibited by high concentrations of Ca 2+ in the cytosol (0.5 mm and higher for cardiac cells) So as Ca 2+ is released from the SR it starts to inhibit the Ca 2+ release channel. Slide 26 Depolarization induced Ca 2+ release Slide 27 Ca 2+ induced Ca 2+ release

10 Slide 28 Experiment Slide 29 Sarcomeres Skeletal muscle is made up of bundles of multinucleate muscle cells (myofibers) Each cell contains myofibrils that are composed of repeated units of actin and myosin called sacromeres Thick and thin filaments arranged into sarcomeres Repeated in parallel and in series Features Z-disk A-band I-band M-lines Slide 30 Sarcomeres Electron micrograph of a longitudinal section through a skeletal muscle cell of a rabbit Schematic diagram of a single sarcomere Z discs: At each end of the sarcomere Attachment sites for the plus ends of actin filaments (thin filaments) M line: Midline. Location of proteins that link adjacent myosin II filaments (thick filaments) to one another Dark bands: mark the location of the thick filaments = A bands Light bands: which contain only thin filaments and therefore have a lower density of protein = I bands.

11 Slide 31 Sarcomeres Slide 32 Myofibril A single continuous stretch of interconnected sarcomeres Runs the length of the muscle cell More myofibrils in parallel more force Slide 33 Striated Muscle Cell Structure Composed of thick and thin filaments Thick: myosin 300 myosin II hexamers Thin: actin Capped by tropomodulin (-) and CapZ (+) to stabilize Decorated by troponin and tropomyosin Globular protein (G-actin) Form long chains called F-actin In skeletal muscle 2 F- actin polymers twist together

12 Slide 34 Actin and Myosin Function Slide 35 Myosin Motor protein used by actin Sliding filament model Most common type of movement Myosin is an ATPase Converts E released from ATP to mechanical E 17 classes of myosin with multiple isoforms Similar structure Head, tail, and neck Slide 36 Sliding Filament Model Analogous to pulling yourself along a rope Actin: the rope Myosin: your arm

13 Slide 37 Sliding Filament Model Two processes Chemical Myosin binds to actin (Cross-bridge) Structural Myosin bends (Power stroke) Cross-bridge cycle Formation of crossbridge, power stroke, and release Need ATP to attach and release No ATP rigor mortis Slide 38 Sliding filament model Slide 39 Sliding Filament Model, Cont. Myosin is bound to actin in the absence of ATP and this is the "rigor" state i.e. gives rigidity to the muscle ATP binds to the myosin causing the head domain to dissociate from actin ATP is then hydrolyzed causing a conformational change in the mysoin head to move it to a new position and bind to actin Pi is released causing the myosin head to change conformation again and it is this movement that moves the actin ADP is released

14 Slide 40 Ca 2+ Allows Myosin to Bind to Actin Ca 2+ binds to TnC Reorganization of troponin-tropomyosin Expose myosin-binding site on actin Slide 41 Ca2+ Allows Myosin to Bind to Actin * * Ca 2+ levels increase in cytosol Ca 2+ binds to troponin C Troponin-Ca 2+ complex pulls tropomyosin away form G-actin binging site Myosin binds to actin and completes power stroke Actin filament moves * * * Slide 42 Sliding Filament Model

15 Slide 43 Contractile Force Depends on sarcomere length: distance between the Z-disks Number of myofibrils Number of cells (recruitment) Slide 44 Isotonic and isometric contraction Slide 45 Regulation of Contraction Excitation-contraction coupling Depolarization of the muscle plasma membrane (sarcolemma) Elevation of intracellular Ca 2+ Contraction Relaxation when the sarcolemma repolarizes and Ca 2+ returns to resting levels

16 Slide 46 Excitation contraction coupling ACH released at the NMJ Net entry of Na + initiates a muscle action potential AP in T- tubule alters conformation of DHP receptor DHP receptor opens Ca 2+ release channels in SR and Ca 2+ enters the cytoplasm Ca 2+ binds to troponin C Troponin-Ca 2+ complex pulls tropomyosin away form G-actin binging site Myosin binds to actin and completes power stroke Actin filament moves Slide 47 Time Course of Depolarization Slide 48 Time Course of Depolarization

17 Slide 49 Cause of Depolarization Myogenic Spontaneous e.g., Vertebrate heart Pacemaker Cells that depolarize fastest Unstable resting membrane potential Neurogenic Excited by neurotransmitters e.g., Vertebrate skeletal muscle Can have multiple (tonic) or single (twitch) innervation sites Slide 50 Relaxation Repolarization Reestablish Ca 2+ gradients Extracellular Ca 2+ ATPase Na + /Ca 2+ exchanger (NaCaX) in reverse Intracellular Ca 2+ ATPase (SERCA) Parvalbumin cytosolicca 2+ buffer Slide 51 The Z disk The Z disk is complex of proteins Responsible for anchoring the actin filaments to ensure that the sacromere will shorten during contraction Actin filaments are capped at both ends to ensure that the actin will not depolymerize Titin-nebulin filament system stabilizes the alignment of thick and thin filaments in skeletal muscle Thick filaments are connected at both ends to Z disks through titin Nebulin is associated with a thin filament from its (+) end at the Z disk to its other end

18 Slide 52 Accessory proteins in a sarcomere Each giant titin molecule extends from the Z disc to the M line Part of each titin molecule is closely associated with a myosin thick filament The rest of the titin molecule is elastic and changes length as the sarcomere contracts and relaxes Each nebulin molecule is exactly the length of a thin filament The actin filaments are also coated with tropomyosin and troponin and are capped at both ends. Tropomodulin caps the minus end of the actin filaments, and CapZ anchors the plus end at the Z disc, which also contains a-actinin. Slide 53 Skeletal muscle metabolism Muscles require a large source of ATP to allow for contraction and for transport of Ca 2+ ATP requirements are normally met by glycolysis or respiration Skeletal muscles contain large glycogen stores Skeletal muscles contain creatine phosphate that generates ATP: creatine phosphate + ADP = creatine + ATP Muscles have lots of mitochondria which extend through out the myofibrils and are red coloured due to a large blood flow and myoglobin (which stores oxygen) The breakdown of glycogen stores in muscles can be stimulated by both Ca 2+ and epinephrine Slide 54 Asynchronous Insect Flight Muscles Wing beats: 250 to 1000 Hz Fastest vertebrate contraction: 100 Hz (toadfish sonic muscle) Asynchronous because nervous stimulation is not synchronized to contraction Due to stretch-activation Contracted: Ca 2+ insensitive Stretched: Ca 2+ sensitive

19 Slide 55 Trans-Differentiation Cells with novel properties Heater organs Electric organs Slide 56 HAVE A GREAT WEEKEND!