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Molecular Motors

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Molecular Motors

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    1. Molecular Motors BL4010 12.07.05

    2. Outline Cytoskeletal components Vesicle movement dynein kinesin Cilia and flagella Muscle contraction tropomyosin regulation by calcium

    3. Actin filaments

    4. Swarming of Dictyostelium

    5. http://www.biochemweb.org/fenteany/research/cell_migration/movement_movies.html University of Illinois, Chicago

    6. Actin polymerization

    7. Tubulin and Microtubules Fundamental components of the eukaryotic cytoskeleton Microtubules are hollow, cylindrical polymers made from tubulin dimers 13 tubulin monomers per turn Dimers add to the "plus" end and dissociate from the "minus" end Microtubules are the basic components of the cytoskeleton and of cilia and flagella Cilia wave; flagella rotate - ATP drives both!

    8. Tubulin is a anisotropic heterodimeric polymer

    10. Polymerization Inhibitors Vinblastine, vincristine inhibit MT polymerization anticancer agents Colchicine, from crocus, inhibits MT polymerization inhibits mitosis (larger plants) impairs white cell movement (gout) Taxol, from yew tree bark, stimulates polymerization but then stabilizes microtubules inhibits tumor growth (esp. breast and ovarian)

    11. Microtubules Highways for "molecular motors� MTs also mediate motion of organelles and vesicles through the cell Typically dyneins move + to - Kinesins move organelles - to +

    13. Dynein Dynein proteins walk along MTs Dynein movement is ATP-driven

    17. Kinesin http://valelab.ucsf.edu/research/res_mec_dynein.html

    18. Microtubules in Cilia & Flagella MTs are the fundamental structural unit in cilia and flagella

    19. The dynein �cargo� in cilia movement is the A-tubule, moves along the B-tubule

    20. Bending of cilia by MT sliding + anchoring

    21. http://programs.northlandcollege.edu/biology/Biology1111/animations/flagellum.html

    22. Other uses for motors DNA unwinding and packaging When stretched out to its full extent, the DNA is around 10�m long, 200 times the size of the capsid This motor can work against loads of up to 57pN on average, making it one of the strongest molecular motors reported to date. Movements of over 5�m are observed, indicating high processivity. Pauses and slips also occur, particularly at higher forces.

    23. Flagella

    25. Morphology of Muscle Four types: skeletal, cardiac, smooth and myoepithelial cells

    26. Morphology of Muscle A fiber bundle contains hundreds of myofibrils that run the length of the fiber Each myofibril is a linear array of sarcomeres

    27. What are t-tubules and SR for? The morphology is all geared to Ca release and uptake! Nerve impulses reaching the muscle produce an "action potential" that spreads over the sarcolemmal membrane and into the fiber along the t-tubule network The signal is passed across the triad junction and induces release of Ca2+ ions from the SR Ca2+ ions bind to sites on the fibers and induce contraction; relaxation involves pumping the Ca2+ back into the SR

    28. Molecular Structure of Muscle Thin filaments are composed of actin polymers F-actin helix is composed of G-actin monomers F-actin helix has a pitch of 72 nm But repeat distance is 36 nm Actin filaments are decorated with tropomyosin heterodimers and troponin complexes Troponin complex consists of: troponin T (TnT), troponin I (TnI), and troponin C (TnC)

    29. Muscle contraction

    30. Muscle fiber

    31. Titin Titin is a giant 3 MDalton muscle protein and a major constituent of the sarcomere in vertebrate striated muscle. It is a multidomain protein which forms filaments approximately 1 micrometre in length spanning half a sarcomere. At low force the whole I-band acts as an entropic spring. At higher forces elasticity is due to the reversible unfolding of individual immunoglobulin domains of the I-band.

    32. Thin filaments are actin + tropomyosin

    33. Structure of Thick Filaments Myosin - 2 heavy chains, 4 light chains Heavy chains - 230 kD Light chains - 2 pairs of different 20 kD chains The "heads" of heavy chains have ATPase activity and hydrolysis here drives contraction Light chains are homologous to calmodulin

    34. Repeating Elements in Myosin 7-residue, 28-residue and 196-residue repeats are responsible for the organization of thick filaments Residues 1 and 4 (a and d) of the seven-residue repeat are hydrophobic; residues 2,3 and 6 (b, c and f) are ionic This repeating pattern favors formation of coiled coil of tails. (with 3.6 - NOT 3.5 - residues per turn, ?-helices will coil!)

    35. Repeating elements in myosin 28-residue repeat (4 x 7) consists of distinct patterns of alternating side-chain charge (+ vs -), and these regions pack with regions of opposite charge on adjacent myosins to stabilize the filament 196-residue repeat (7 x 28) contributes to packing and stability of filaments

    36. Associated proteins of Muscle ?-Actinin, a protein that contains several repeat units, forms dimers and contains actin-binding regions, and is analogous in some ways to dystrophin Dystrophin is the protein product of the first gene to be associated with muscular dystrophy - actually Duchennes MD

    39. Dystrophin Dystrophin is part of a large complex of glycoproteins that bridges the inner cytoskeleton (actin filaments) and the extracellular matrix (via a protein called laminin) Two subcomplexes: dystroglycan and sarcoglycan Defects in these proteins have now been linked to other forms of muscular dystrophy

    40. Intermediate filaments

    42. The Dystrophin Complex Links to disease ?-Dystroglycan - extracellular, binds to merosin (a component of laminin) - mutation in merosin linked to severe congenital muscular dystrophy ?-Dystroglycan - transmembrane protein that binds dystrophin inside Sarcoglycan complex - ?, ?, ? - all transmembrane - defects linked to limb-girdle MD and autosomal recessive MD

    43. The Sliding Filament Model Many contributors! Hugh Huxley and Jean Hanson Andrew Huxley and Ralph Niedergerke Albert Szent-Gyorgyi showed that actin and myosin associate (actomyosin complex) Sarcomeres decrease length during contraction Szent-Gyorgyi also showed that ATP causes the actomyosin complex to dissociate

    45. The Contraction Cycle Cross-bridge formation is followed by power stroke with ADP and Pi release ATP binding causes dissociation of myosin heads and reorientation of myosin head

    46. Ca2+ Controls Contraction Release of Ca2+ from the SR triggers contraction Reuptake of Ca2+ into SR relaxes muscle So how is calcium released in response to nerve impulses? Answer has come from studies of antagonist molecules that block Ca2+ channel activity

    47. http://www.blackwellpublishing.com/matthews/myosin.html

    48. Dihydropyridine Receptor In t-tubules of heart and skeletal muscle Nifedipine and other DHP-like molecules bind to the "DHP receptor" in t-tubules In heart, DHP receptor is a voltage-gated Ca2+ channel In skeletal muscle, DHP receptor is apparently a voltage-sensing protein and probably undergoes voltage-dependent conformational changes

    51. Ryanodine Receptor The "foot structure" in terminal cisternae of SR Foot structure is a Ca2+ channel of unusual design Conformation change or Ca2+ -channel activity of DHP receptor apparently gates the ryanodine receptor, opening and closing Ca2+ channels

    52. The Ryanodine Receptor

    53. Ca 2+ Regulates Contraction Tropomyosin and troponins mediate the effects of Ca2+ In absence of Ca2+, TnI binds to actin to keep myosin off TnI and TnT interact with tropomyosin to keep tropomyosin away from the groove between adjacent actins But Ca2+ binding changes all this!

    54. Ca 2+ Turns on Contraction Binding of Ca2+ to TnC increases binding of TnC to TnI, simultaneously decreasing the interaction of TnI with actin This allows tropomyosin to slide down into the actin groove, exposing myosin-binding sites on actin and initiating contraction Since troponin complex interacts only with every 7th actin, the conformational changes must be cooperative

    56. Binding of Ca 2+ to Troponin C Four sites for Ca2+ on TnC - I, II, III and IV Sites I & II are N-terminal; III and IV on C term Sites III and IV usually have Ca2+ bound Sites I and II are empty in resting state Rise of Ca2+ levels fills sites I and II Conformation change facilitates binding of TnC to TnI

    58. Smooth Muscle Contraction No troponin complex in smooth muscle In smooth muscle, Ca2+ activates myosin light chain kinase (MLCK) which phosphorylates LC2, the regulatory light chain of myosin Ca2+ effect is via calmodulin - a cousin of TnC Hormones regulate contraction - epinephrine, a smooth muscle relaxer, activates adenylyl cyclase, making cAMP, which activates protein kinase, which phosphorylates MLCK, inactivating MLCK and relaxing muscle

    61. Smooth Muscle Effectors Useful drugs Epinephrine (as Primatene) is an over-the-counter asthma drug, but it acts on heart as well as on lungs - a possible problem! Albuterol is a more selective smooth muscle relaxer and acts more on lungs than heart Albuterol is used to prevent premature labor Oxytocin (pitocin) stimulates contraction of uterine smooth muscle, inducing labor

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