Molecular Motors

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.htmlhttp://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

9. Tubulin polymerization is self-organizing but requires some help getting started • Scaffolding proteins serve as microtubule organizing centers - centrioles are only one example

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. MicrotubulesHighways 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.htmlhttp://programs.northlandcollege.edu/biology/Biology1111/animations/flagellum.html

22. Other uses for motorsDNA unwinding and packaging • When stretched out to its full extent, the DNA is around 10µm 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 5µm 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 • Surfaces of sarcomeres are covered by sacroplasmic reticulum • Each sarcomere is capped by a transverse tubule (t-tubule), an extension of sarcolemmal membrane

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 FilamentsMyosin - 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