Experiments: how do we know what single motors do?

1. Experiments: how do we know what single motors do?

2. Optical tweezers

3. Optical trapping • Is simply a TM-00 (Gausian cross section) laser beam focussed to a diffraction-limited spot. • Can use it to grab, and manipulate, small dielectric objects • Vesicles, lipid droplets, cell membranes, small glass or plastic spheres are all small dielectric objects

4. Optical trap • Can position beads anywhere • Easy to see motor-microtubule binding events

5. How much force can the motor exert: Optical tweezers as a spring

6. Methods For Force calibration in Optical Tweezers(If interested, please see Dr. Yonggun Jun who has just spent a lot of time thinking about OT calibration!!)

8. Equipartition Theorem

9. Equipartition Theorem

10. Stalling Force measurements

11. How far can a single motor move a cargo? • Vesicle transport motors such as kinesin and Myosin-V are “processive” enzymes • Processive: go through repeated complete enzymatic cycles, while remaining bound to the substrate (in this case the MT or AF)

12. Use of Optical trap to characterize kinesin

13. Microtubule motors are uni-directional Single Kinesin motor moving a bead in vitro A single motor moves ~ 1 m

14. Single motor bead assays: processivity Histogram Individual Traces

15. Does it take discrete steps, or move continuously? If steps, what size? • MT built of repeating subunits (dimers). Each dimer is 8 nm in length • If moves along a single protofilament, expect steps that are some multiple of 8nm. If lateral steps possible, could take smaller steps.

16. Single-molecule driven beads move in 8 nm steps at low [ATP], low load

17. Extraction of Step size from displacement records Pairwise Distance Function analysis Step Size ? Position Time You expect to see regular peaks in a histogram of such pairwise distance at multiples of the Step size

18. Step Detection Techniques—individual rapid steps AOD • Force Clamp • High Sampling Feedback Halogen Lamp L5 PSD 980nm IR Laser M1 DM1 BFPC L1 Condenser NA=1.4 XYZ Piezo-Stage 100x Objective NA=1.3 BFPO L2 DM2 L6 M2 CCD camera L4 L3 M3 Chi-squared Minimization Method B.C. Carter, et al “A Comparison of Step-Detection Methods: How Well Can You Do?”Biophys. J., 94(1):306-19, (2008) x

19. Step Size Measurements Kinesin

20. Label one head Selvin, Science

21. Tracks:

22. Distribution of steps: The average step-size is 17.3 ±3.3 nm; uncertanty of mean (SEM) is 0.27 nm

23. Kinesin Myosin-V Dynein Cargo Cargo KLC Dynactin binding KR2 KR1 Pi Ca2+ MR2 Head (ATPase) Stalk Pi 1 2 c KAPP 3 6 4 5 KR3 MR1 Lever (?) KHC Head (ATPase) MT binding Three families of molecular motors

24. Processivity: porters vs rowers Non-processive (rower) Processive (porter) Images: MCRI Molecular motors group

25. Kinesin is Processive; Myosin II (muscle) is not. Why? • A processive motor doesn’t let go of the substrate (MT or AF) so the cargo doesn’t diffuse away • Many processive motors could get in each others way--all bound to the filament at the same time • A non-processive motor lets go of the filament at some point in its enzymatic cycle. Thus, multiple motors don’t get in each others way--not active at exactly the same time • The ‘duty ratio’ is the ratio of (time bound to substrate)/(complete time for enzymatic cycle) • Duty ratio=1 for processive motor

26. Summary of single-molecule experiments Motor proteins: • Are uni-directional, and move along straight filaments • Exert 1-6 pN force • Typically go ~ 1m before detaching • Kinesin motors take 8 nm steps, Dynein takes a variety of step sizes, Myosins take 36 nm steps • Move between 0.1 and 2 m/s Is this how transport functions inside cells?

27. How do we go from single-molecule characterization to in vivo function?

28. Herpes virus in cultured neuron

29. Why do cargos need multiple motors?Many intercellular distances are longerthan 1 micron

30. Motion in cells is different from what might be expected based on single-molecule properties Cargos can move long distances Maybe multiple motors? Bead moved by multiple kinesin motors

31. So, multiple motors can move a cargo long distances. Now, lets look more carefully… Start to build complexity in a controlled environment, i.e. in vitro, and understand how motors work together

32. Poisson statistics: Getting down to the single molecule limit… • Catch Dynein- or kinesin-coated beads, bring in contact with MT • Find probability for Binding/motion (Bind fraction) • Repeat at different motor:Bead ratios • Plot the Bind fraction Vs motor:Bead ratio • Stay where probability for “doubles” is negligible For single motor, use Binding/moving fraction ≤ 0.3

33. Motor - polystyrene bead assays Kinesin I: single motor 30% or less of beads bind to MTs Force production Run Length (Processivity) Peak center ± SEM : 4.8±0.06 pN Decay constant ± SEM : 1.46±0.16 µm

34. Poisson statistics: Getting down to the single molecule limit…and then back to multiple motors • Catch motor-coated beads, bring in contact with MT • Find probability for Binding/motion (Bind fraction) • Repeat at different motor:Bead ratios • Plot the Bind fraction Vs motor:Bead ratio • Now, use concentration where probability for “doubles” is high: mixed population Mixed bead population--> How do we know how many motors are moving a specific bead?

35. What we think is going on Bf~1.0 Bf~0.7 Bf~0.3 Bf~1.0 Increasing Kinesins per bead

36. Evolution of force production with increasing kinesins per bead 1-2 motor Mostly single motor (Bf ~1) Single motor (Bf ~0.3)

37. Conclusion: for multiple-motor driven transport, binding fraction cannot tell you how many motors engaged. Stalling forces are additive at low motor number; use this as a readout of the number of instantaneously engaged motors

38. Motor - polystyrene bead assays Kinesin I: ~two motors driving polystyrene bead Run Length Force production

39. Summary for ~2 engaged Kinesins:* Velocities unchanged (not shown) * Stall forces ~ additive* Cargo travel lengths very long, but this is not really correct (see next)>> Similar results for cytoplasmic dynein (see Mallik et al, Curr. Bio, 2005)More: see website bioweb.bio.uci.edu/sgross

40. Conclusion: motion in cells is different from what might be expected based on single-molecule properties We have three ‘systems’ level questions to understand: Cargos can move long distances Cargos can reverse course, move bi-directionally Cargo transport can be regulated

41. What single-molecule properties are particularly important for how multiple motors function together?

42. Cartoon of processive motion of a cargo moved by two motors

43. From cartoon… On-rate Off-rate Overall number of motors

44. Back of the envelope calculation for how far 2 motors will go on average…. Assume single-motor processivity of 1200 nm, velocity of 800 nm/sec, on rate of 5/sec First motor detaches at t0 . How long to rebind? T(rebind) ~ 1/Kon =1/5 sec. Does second motor detach before first rebinds? What is off rate? Processivity (mean travel): 1200 nm, vel 800 nm/sec avg duration of run: 1200 nm/800 nm/sec=1.5 sec Off rate: 1/avg duration = Koff= 1/1.5 Prob of second motor detaching is Koff*(rebinding time)=(1/1.5)*(1/5)= 0.1333 i.e. ~13% chance of failing to make it though cycle. Adjust this to 26% (ignored second motor detaching right before first motor) On avg make it through ~ 4 cycles.

45. Regulation: how? From expression <X>= ½*(D/N)*(Kon/Koff)N-1 Kon: On-rate, i.e. rate at which single motor binds MT Koff: Off-rate in time, i.e. rate at which single motor detaches from MT D=processivity, i.e. mean travel (distance) before detaching. Note that Koff and D are NOT independent: Koff = V/D V=Motor velocity  can tune mean travel by altering N, Kon, or Koff (V or D, or both).

46. Analytic Mean-field theory of average cargo travel carried by two motors Velocity: crucial initial condition p: binding rate (1/s) e: unbinding rate (1/s) d=v*(1/ e)=v/ e Klumpp and Lipowsky, PNAS, 2005

47. Experiment: established for single-motor study Valentine et al., Nat. Cell Bio., 2006

48. Experiment: established for single-motor study Valentine et al., Nat. Cell Bio., 2006

49. Experiment: difficult to interpret for more motors ?

50. Experiment: difficult to interpret for more motors ?