1. 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?

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

3. Herpes virus in cultured neuron

4. Why do cargos need multiple motors? Many intercellular distances are longer than 1 micron

5. 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

6. 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

7. 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

8. 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

9. 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?

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

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

12. 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

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

14. 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

15. 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:

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

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

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

19. Analytic Mean-field theory of average cargo travel carried by two motors Klumpp and Lipowsky, PNAS, 2005 Xu et al, Traffic, 2012 Velocity: crucial initial condition  : binding rate (1/s)  : unbinding rate (1/s) d=v*(1/  v/ 

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

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

22. Experiment: difficult to interpret for more motors ?

23. ?

24. ?

25. ?

26. Experiment: modify surface chemistry for two-motor

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

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

30. 1mM ATP 20  M ATP 10  M ATP Velocity (  m/s)Travel (  m) Experiment: one-motor travel d

31. 1mM ATP 20  M ATP 10  M ATP Velocity (  m/s)Travel (  m) Experiment: two-motor travel D

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

33. 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

34. 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.

35. 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.

36. 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)