1. Ron Rock University of Chicago The Role of the Proximal Tail in the Large Steps of Myosin VI

2. Myosin II and F-Actin Architecture Catalytic Domain RLC ELC Coiled Coil Myosin II: Hexamer of 2 Heavy Chains & 4 Light Chains F-Actin: Polymer of actin monomers Pointed EndBarbed End 36 nm

3. The Myosin II Chemomechanical Cycle

4. Step Size and the Lever Arm D The light chain binding domain is believed to rotate upon binding to actin Result: Small structural changes in the catalytic domain are amplified

5. Step Size Correlates to Lever Arm Length for Myosin II Velocities in gliding filament assays correlate Uyeda et al. PNAS 93 4459 (1996) Step sizes correlate Warshaw et al. JBC 275 37167 (2000) Ruff et al. Nat. Str. Biol. 8 226 (2001)

6. Myosin Classes

7. DirectionProcessiveStep Myosin IIBarbedNo 5 nm Lever Arm Myosin VBarbedYes 36 nm Lever Arm Myosin VIPointedYes 30-36 nm Myosin Properties

8. Total Internal Reflection Microscopy

9. Myosins V and VI are Processive V VI

10. A Hand-Over-Hand Model Myosin V and VI walk… … in a hand-over-hand manner … … using two catalytic heads

11. rate-limiting P D T A Hand-Over-Hand Model

12. Hand-Over-Hand Motility Matthew L. Walker, Stan A. Burgess, James R. Sellers, Fei Wang, John A. Hammer III, John Trinick & Peter J. Knight. Nature, 405, 804-807 (2000).

13. Myosin V Can Cross Actin Filaments Two Actin Tracks

14. How Does Myosin V Cross Filaments? Side View Top View Diffusive Search? Flexibility here?

15. Optical Trap Design Brightfield Trap Steering

16. Dual Bead Force Clamp

17. 2 mM ATP Myosin V Stepping in the Trap 5.455.5 5.55 Time [s] 543210 6 480 400 320 240 160 80 0 Time (s) Displacement (nm)

18. Myosin VI Stepping in the Trap VI V

19. Myosin V and VI Takes Large Steps VI V Mean step is near the actin helical repeat (VI) Large steps, much larger than expected from lever arm model (VI) Distribution very broad (30 ± 12 nm) (VI) Many backsteps (toward barbed end) (-13 ± 8 nm)

20. Myosin V and VI Stepping Model Coiled-coil unfolds

21. How does myosin VI take such large steps? VI V

22. The two heads of myosin VI can separate 27 ± 6 nm (SD)

23. The proximal tail is not predicted to be a coiled-coil

24. Less than half of the processive stepsize is generated by the working stroke Similar to Myosin V: Veigel et al., Nat. Struct. Bio. 4 59 (2002)

25. The proximal tail does not act as a rigid lever arm N = 195 11.9 ± 1.2 nm (SE)

26. The proximal tail does not act as a rigid lever arm X

27. The proximal tail is exposed and sensitive to proteolysis by V8 protease Solid arrows indicate 97 kD band. Uncut Myosin VI is 146 kD. Actin:myosin is at 6:1 mol ratio and nucleotides are at 2 mM unless indicated.

28. The proximal tail allows separation of the heads to produce a large step M6-2hepzip V858 to S888 -> GCN4 19 ± 2 nm (SD)

29. The proximal tail allows separation of the heads to produce a large step

30. Myosin VI and 2hepzip stepping model

31. Myosin VI stepping model

32. 80 AA => 28.8 nm (contour length) each stiffness k = 0.3 pN/nm (WLC, Lp = 0.9 nm, constant over these ranges) First passage time under zero ext. load (26 nm) is ~6 ms Under 2 pN load, first passage time is 3 s Dock proximal tail along the actin filament Alpha helical proximal tail

33. Acknowledgments Protein production, EM, kinetics Bhagavathi Ramamurthy Sara Beccafico Carl Morris Clara Franzini-Armstrong H. Lee Sweeney Optical Trapping, proteolysis Alex Dunn Ben Spink Bhadresh Rami Jim Spudich The Helen Hay Whitney Foundation The Burroughs Wellcome Fund

35. Full-length myosin VI is a monomer A form of motor regulation like Unc104?

36. EM of Myosin VI Decorated Actin Shows Evidence of Left Handed Rotation Pointed End Barbed End ADPRigor Wells et al. Nature 401 505 (1999)

37. Load and the Diffusive Search 30 nm F = 1.7 pN 30 nm F = 1.7 pN 30 nm F = 1.7 pN 50 pNnm = 200,000x slower 0 pNnm 25 pNnm = 400x slower

38. ADP ADP Release is the Rate Limiting Transition 10 µM ATP ( = K m ) k 0 = 9 s -1, k 1 =17 s -1 2 mM ATP, 400 µM ADP k 0 = 6.4 s -1, k 1 =161 s -1

39. Adapted from Tokunaga BBRC 235 47 (1997) Single Fluorophore Detection Nd:YAG 532 nm HeNe 633 nm Ar Ion 488 nm