1. Lecture 2 Outline: Brief overview of a long history Sarcomere structure and function Myosin Regulation of contraction Paper: A large protein required for sarcomere stability in flight muscle Muscle, myosin

2. 1660muscle dissected into fibers 1682striations seen in skeletal muscle fibers 1700-1900metabolism – lactic acid, heat production 18641 st muscle prep – actomyosin – salt extraction of tissue 1939ATPase activity of actomyosin demonstrated 1943actin and myosin separated – different viscosity properties 1950sEM, X-ray diffraction structural studies A brief history 1954 sliding filament model proposed (H. Huxley)

4. striated muscle multinucleate cells 10-100  m thick up to 40 mm long light micrograph 

5. Sarcomere = contractile unit banding pattern due to partial overlap of two types of filaments thick filaments = myosin thin filaments = actin EM:

6. EM cross section: hexagonal lattice of thin filaments surrounding thick filaments

8. Sliding Filament Model: thick and thin filaments slide past one another Question: How does muscle contract? Evidence: 1) EM of sarcomeres at different stages during contractile process shows decreased width of banding pattern 2) both filament systems maintain constant length, region of overlap increases

9. relaxed contracted

10. How does sliding of filaments occur? 1960s Higher resolution EM - cross bridges, individual filaments

11. myosin thick filaments: bipolar

12. actin thin filaments: uniform polarity barbed (+) ends pointed (-) ends barbed (+) ends

13. model by ~1970molecular details still controversial

14. 1980s-present reductionist approach in vitro reconstitutions: simplifed motility assays X-ray crystallography and EM reconstructions single molecule measurements

15. primitive contractility assay superprecipitation: combine actomyosin with ATP in beaker, see what happens

16. modern motility assay 1) Adsorb myosin molecules on glass coverslip in chamber 2) Perfuse in rhodamine-labeled actin filaments and ATP 3) Observe by fluorescence video microscopy

17. + + - - muscle myosin ~4.5  m/sec

18. Myosin - the most studied of all proteins (!?) large family of myosin-related proteins ~14 in human heavy chain: 1) large globular head: contains actin-binding and ATPase domains 2)  -helical neck region - binds light chains common features: one or two heavy chains and several light chains 3) tail domain - for oligomerization or cargo binding light chains: 1) calcium-binding proteins, sometimes calmodulin 2) regulate myosin activity

19. myosin II

20. muscle, stress fibers vesicles, organelles vesicles, organelles

21. Myosin II mechanism ATPase activity stimulated by actin: from 4/hour to 20/second ATP binding, hydrolysis and dissociation of ADP-Pi produce a series of allosteric changes in myosin conformation Energy release is coupled to movement

22. cross bridge cycle

23. Myosin II crystal structure (S1 fragment)

24. catalytic head neck domain = lever arm superimpose structures in two different nucleotide states

25. Other evidence for lever arm model Spudich lab (1996): replace endogenous Dicteostelium myosin II gene with neck domain mutants - longer or shorter purify and measure velocity in motility assay velocity = step size/time bound to actin WT (2)103 light chain binding sites

26. # of light chains velocity  m/sec 1230 1 2 3 4 motility assay

27. Current Issues/Questions How is the large conformational change of lever arm generated during phosphate release? How many steps are taken per ATP hydrolyzed? What is the step size? Approaches: single molecule assays, optical traps and high resolution fluorescence analyses

29. Regulation of muscle contraction motor nerve  action potential muscle cell plasma membrane depolarized T-tubules (invaginations) carry signal throughout myofibril sarcoplasmic reticulum releases calcium contraction occurs calcium pumped back in over in 30 milliseconds

31. Striated muscle: Calcium regulation of contraction occurs through thin filament accessory proteins: tropomyosin and troponin when calcium binds troponin, tropomyosin shifts to allow actin-myosin interaction

33. Variations: Smooth muscle gut - slower, sustained contractions less ordered myofibrils - no striations less extensive sarcoplasmic reticulum regulation also through myosin, still calcium dependent: change in light chain conformation phosphorylation of light chain by MLCK regulation through thin filament dependent on caldesmon

34. Maintenance of sarcomere structure Why are thick and thin filaments of fixed length? actin capping proteins: Tropomodulin, Cap Z

35. What gives muscle its elasticity? stretch muscle beyond overlap of thick and thin filaments and it resumes resting length when released

36. Giant Muscle Proteins Titin: 3rd most abundant muscle protein M.W. 2,700,000! 25k amino acids. extends from Z-disk to M-line Ig and fibronectin-like domains “super repeats” - myosin binding sites PEVK domains - elastic? Nebulin: M.W. 800,000 helical, wrapped around thin filament repeats that bind actin nebulin length correlates with thin filament length

37. Titin and Nebulin are thought to provide compliance to muscle, and may serve as sarcomere “rulers” determining the length of thick and thin filaments

38. Dystrophin: M.W. 400,000 largest gene - 2 megabases links actin to p. membrane mutation - muscular dystrophy