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

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 12

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

18. 18 Step Detection Techniques—individual rapid steps  Force Clamp  High Sampling x B.C. Carter, et al “A Comparison of Step-Detection Methods: How Well Can You Do?” Biophys. J., 94(1):306-19, (2008) Chi-squared Minimization Method

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. KinesinMyosin-VDynein Head (ATPase) 1 4 3 5 c 6 2 Head (ATPase) Lever (?) Stalk PiPi PiPi KAPP KHC KLC KR2 KR3 Cargo Ca 2+ MR2 MR1 Cargo KR1 Dynactin binding MT binding Three families of molecular motors

24. Processivity: porters vs rowers Processive (porter) Non-processive (rower) 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 longer than 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… For single motor, use Binding/moving fraction ≤ 0.3 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

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

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 Increasing Kinesins per bead Bf~0.3 Bf~0.7 Bf~1.0

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

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  Cargos can move long distances  Cargos can reverse course, move bi-directionally  Cargo transport can be regulated We have three ‘systems’ level questions to understand:

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 1.First motor detaches at t 0. How long to rebind? T(rebind) ~ 1/Kon =1/5 sec. 2. Does second motor detach before first rebinds? 3.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 4.Off rate: 1/avg duration = Koff= 1/1.5 5.Prob of second motor detaching is Koff*(rebinding time)=(1/1.5)*(1/5)= 0.1333 6.i.e. ~13% chance of failing to make it though cycle. 7.Adjust this to 26% (ignored second motor detaching right before first motor) 8.On avg make it through ~ 4 cycles.

45. Regulation: how? From expression = 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 Klumpp and Lipowsky, PNAS, 2005 Velocity: crucial initial condition  : binding rate (1/s)  : unbinding rate (1/s) d=v*(1/  v/ 

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. ?

51. ?

52. ?

53. Experiment: modify surface chemistry for two-motor

55. Experiment: force to further require two-motor Position Time Position Time

56. Experiment: clean one- vs. two-motor system! Goal: Test Strategy: reduce ATP to slow down motor Dd

57. 1mM ATP 20  M ATP 10  M ATP Velocity (  m/s)Travel (  m) Experiment: one-motor travel d

58. 1mM ATP 20  M ATP 10  M ATP Velocity (  m/s)Travel (  m) Experiment: two-motor travel D

59. Velocity (  m/s)Travel (  m) 1mM ATP Velocity (  m/s)Travel (  m) Rogers et al., Phys. Chem. Chem. Phys, 2009 D=1.7d dD

60. Velocity (  m/s)Travel (  m) 1mM ATP 20  M ATP 10  M ATP 1mM ATP 20  M ATP 10  M ATP Velocity (  m/s)Travel (  m) Velocity tunes travel distance for two-motor system dD

61. Motors work in small ensemble in cells We establish velocity as a control for ensemble travel May be particularly important, as beautiful work by Joanny (Campas, et al, Biophys. J., 2008) suggests a limited number of motors (~ 9 max) can be active.

62. Hw #2 : Model two kinesin motors functioning together, and then investigate velocity effects. In Hw #1 you developed a simulation for 1 motor. Here, stick two such motors together. Assume initially that the motors here have the same properties as in the previous hw. The main goal here is to get the simulation working, and compare its results for a few different choices of ‘on’ rates and ‘off’ rates to the order-of-magnitude theory developed in class. How similar are the two sets of predictions? For a single motor with processivity of 1.2 microns, what is your prediction for the mean travel of a cargo with two such motors, assuming an ‘on’ rate of 2/sec or 5/sec. Do this assuming a velocity of 800 nm/sec, and a velocity of 100 nm/sec.

63. Velocity: the link between temporal and spatial of individual motor unbinding from microtubule Slower velocity buys more time for additional motor to bind before the current bound one detaches. (see Xu et al, Traffic, 2012)