L8 Muscle Structure and Function PDF

BS31004 Biochemistry and Cell Biology Muscle and muscle proteins Alan Prescott 1. Structure of muscle 2. Actin and myosin 3. The force generating machinery of muscle 4. Regulatory proteins 5. Placing the sarcomere in a functional cell 1. Structure of muscle  T...

BS31004 Biochemistry and Cell Biology Muscle and muscle proteins Alan Prescott 1. Structure of muscle 2. Actin and myosin 3. The force generating machinery of muscle 4. Regulatory proteins 5. Placing the sarcomere in a functional cell 1. Structure of muscle  Three types of muscle: 1. Striated (skeletal) 2. Cardiac (heart muscle) 3. Smooth (blood vessels, intestine, skin)  Striated skeletal muscle seen in tissue sections by H&E staining LS TS  M Striated muscle cell (muscle fibre) a muscle cell seen by electron microscopy Contains many parallel myofibrils  Z M I A Z a sarcomere Cardiac muscle Differs from skeletal by: branching fibres, central nuclei, intercalated discs  smooth striated Different muscle types, seen by electron microscopy cardiac M Figure 16-73a Molecular Biology of the Cell (© Garland Science 2008) Figure 16-73b Molecular Biology of the Cell (© Garland Science 2008) Organisation of a muscle fibre (muscle cell) Skeletal muscle Muscle cells are myofibres or muscle fibres, with many parallel myofibrils in the cytoplasm Filament symmetry in muscle  Organization in a muscle cells a highly ordered structure in 3 dimensions Muscle structure = myofibre (cell), myofibrils, sarcomeres (repeat units in myofibrils) Sarcomere - A band, I band The A band is anisotropic, i.e. it is birefringent because of the intrinsic order of the thick filament. The thin filaments are seen in the isotropic or I-band. Thick filaments = myosin, thin filaments = actin Additional structures for co-ordination of filament movement 2. Actin and myosin Figure 16-74 Molecular Biology of the Cell (© Garland Science 2008) Skeletal muscle muscle cells are myofibers, with many parallel myofibrils in the cytoplasm  Actin Ubiquitous protein: all cells, all animals Basis of motility in all cell types 5-30% of protein in any cell 43 kDa molecular weight At least 6 isoforms Highly conserved sequence  Actin isoforms are tissue- specific :  skeletal skeletal muscle, tongue  cardiac heart  vascular smooth muscle  enteric smooth muscle of viscera  cytoplasmic mostly non-muscle  cytoplasmic mostly non-muscle 80% sequence homology  Actin filaments are the THIN FILAMENTS – Actin filament is polar - one end distinguishable from the other: Barbed end (plus, +, net subunit addition) and Pointed end (minus, -) – The barbed (+) end is imbedded into the Z-band Actin orientation in the sarcomere: plus ends at the Z line  Myosins - motor proteins Myosin proteins all have : – A highly conserved head domain – A motor domain or catalytic domain – A unique, cargo-binding tail domain 16 classes of myosin motor proteins now known – Up to 15 in one cell Myosin families  Myosin families cont’d VIII - Plant Unknown. IX Signal transduction Unknown. Pleckstrin homology may indicate signal X transduction role XI - Plant Vesicle transport XII Unknown. XIII - Plant Unknown. XIV Unknown. Toxoplasma and Plasmodium species only XV Auditory? XVI Neuronal cell migration? Unknown. Located in the plasma membrane. Pyricularia XVII and Emiricella species only.  THICK FILAMENTS of muscle are made of sarcomeric myosin II Protein domains of head, hinge and tail. The tail forms an -helix, and then a coiled-coil with another myosin monomer. Muscle myosin Thick filament structure Figure 16-76 Molecular Biology of the Cell (© Garland Science 2008)  THICK FILAMENTS of muscle are made of myosin II Protein domains of head, hinge and tail. The tail forms an -helix, and then a coiled-coil with another myosin monomer. The thick filament is a polymer of myosin and is BIPOLAR Thick filaments (myosin) interact with thin filaments (actin), but ALWAYS IN THE SAME ORIENTATION. Muscle contraction involves the shortening of the I-band but not the A- band. 3. The force generating machinery of muscle Function of myosin subdomains examined by fragmentation with proteases  Myosin S1 : has it got the strength ? Experiments by Kishino and Yanagida. 1988. Flexible needles calibrated in terms of force per displacement. Attach actin filament to one end of the rod and the other end of actin filament to another needle. – Actin made visible by the phalloidin - a fungal toxin. First measured the force required to break an actin filament, and showed that the presence or absence of tropomyosin made no difference. Next experiment: one end attached to the flexible needle and the other to a surface which has been coated in myosin. Provide some ATP and watch the needle bend. Force measured was approx 0.2 pN. Much less than that measured by optical tweezers (= 3-4 pN) Myosin not orientated is a possible explanation  Motility assays Initial experiment looked at the movement of beads on an orientated actin filament. Coated latex beads with myosin and observed the movement on the parallel actin arrays found in the alga, Nitella. -but these experiments did not give any measure of the force which could be exerted by a single myosin. Experiments looking at the sliding of actin filaments on myosin coated glass cover slips showed that you only needed one head - not two. Good evidence that non-muscle myosins with single heads would be functional  Function of Myosin Domains Myosin S1 portion defines the motor Importance of the hinge region for movement Proteolytically produced myosin fragments, along with the development of in vitro motility assays, identified function of myosin subdomains Coiled tail domain required for arrangement in the thick filament Figure 16-56 Molecular Biology of the Cell (© Garland Science 2008) Cross-bridge cycle for myosin II Using an optical trap to measure myosin force generation  Matching actin and myosin periodicity Since actin subunits are helically arranged, the myosin “stride” must equal the actin helical repeat for it to move in a straight line.  Myosin V as a model for studying myosin Myosin V increasingly used as a model for studying myosin function Found in many tissues; abundant in brain and nervous system Involved in vesicle transport Head domain of myosin V is about twice as long as that of muscle myosin Myosin V heads move processively along actin filaments Myosin head orientation changes from the leading to the trailing head Computer enhanced images of actin binding myosin V show that head domain changes in orientation between “ leading ” and “ lagging” position. Motor domains are conserved and their bionding mechanism is common to all myosins. See Matthew et al., Nature, 405, 804-807 (2000). Scale bar 20nm. 4. Regulatory proteins Muscle force generation requires calcium Ca2+ influx triggers muscle contraction Troponin changes its conformation, allowing tropomyosin to shift its position on actin filament when calcium is present, permitting the interaction with myosin M Muscle contraction depends on calcium: Calcium enters rapidly through T tubules and sarcoplasmic reticulum Movie sequence of myosin moving on actin First identify the different molecules Determining the position of the myofilaments Myosin thick filament is bipolar - means there are regions where parallel and antiparallel interactions are necessary Other structural proteins position thick filaments and pattern the sarcomere  1. The Z disc 2. Molecular rulers The Z disc (Z line)  Actin filaments attach to the Z disc with Cap Z and -actinin accessory proteins  Tropomodulin and CapZ - thin filament capping proteins in muscle Titin and Nebulin  Titin and nebulin : “molecular rulers” ? Titin - 3.5MDa protein, largest known protein Single molecule extends from the Z-band to the M band. Template for myosin assembly in centre of sarcomere – based on  -structure domains Elastic domain between end of thick filament and Z line – provides passive tension, keeps thick filament centred Extends by unfolding -structure domain of polypeptide – A hitherto unrecognized protein behaviour?  Titin and nebulin : “molecular rulers” ? Nebulin - 776kDa protein. – 35 residue repeat - stretch over 38.5nm. – Coincident with the location of the troponin - tropomyosin complexes associated with the thin filaments. Again a template hypothesis suggested, but yet to be proven. Myosins in non-muscle cells Myosin families  Actin-myosin contraction in non-muscle cells  The myosin tail specifies function Figure 16-57 Molecular Biology of the Cell (© Garland Science 2008) Do myosins all work the same way? Different length tails should mean different length displacement on power stroke  Myosin V transports cargo in cells With a longer lever arm it takes bigger steps  Myosin-linked disorders and mutants Familial Hypertrophic Cardiomyopathy. Many genetic defects occur in myosin but also other muscle specific proteins such as troponin T, tropomyosin and the myosin binding protein, protein C. FMC - a disease of the sarcomere. In the case of the myosin, all but one mutation so far described resides in the head domain / head-rod junction. Myosin VI and VII linked to deafness Myosin VII causes USHER’S SYNDROME due to the presence of this myosin both in the cochlea and the retinal pigmented epithelial cells. Individuals are blind and deaf. Myosin V - mutations basis of the mouse mutant “dilute” because of the involvement in the transport of melanosomes. Another mouse mutant - “dilute lethal” implicate myosin V in neuronal vesicular transport.