1. How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross 1,2 1 Department of Developmental and Cell Biology University of California, Irvine; 2 Department of Physics University of California, Irvine; and 3 King’s College, London

2. A Cell Is Like a City Workers Power Plant Roads Trucks Factories Library Recycling center Police Post office Communications Proteins Mitochondria Actin fibers, microtubules Kinesin, dynein, myosin Ribosomes Genome Lysosome Chaperones Golgi apparatus Signaling networks

3. Intracellular Traffic © Scientific American  How is intracellular transport regulated?

4. Microtubules (MT) are like freeways and actin filaments are like local surface streets.

5. Filaments Actin filament 10 nm diameter 2.77 nm rise 26 subunits/74 nm repeat Microtubule  25 nm diameter  13 protofilaments + end- end + end - end

6. Motor proteins Myosin Kinesin

7. Myosin-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

8. Biochemistry, 4ht Ed, 1995 Motor proteins move cargo along filaments Molecular Biology of the Cell, 3rd Ed, 1994

9. Herpes Virus Transport in Neurons Along Microtubules Virus Movie: VirusMov.movVirusMov.mov

10. How does the cell regulate the transport of vesicles? Microtubules (MT) are like freeways and actin filaments are like local surface streets.

11. Model System: Melanophores ( Pigment granule cells) Cells change color by Dispersing or Aggregating pigment granules granules move bi-directionally along microtubules (Kinesin-II and Dynein) Granules also move along actin filaments (Myosin-V)

12. Dispersing Pigment Granules Dispersion: Granules move out from nucleus: Dispersion1.mov

13. Aggregating Pigment Granules Aggregation: Granules move in toward nucleus: Aggregation1.mov

14. Dispersion: MT  Actin Spread cargos throughout the cell Aggregation: Actin  MT Bring cargos back to nucleus

15. Experiment Pigment cells (melanophores) only have actin filaments (no microtubules) Pigment granules (melanosomes) are tracked Record position r vs. time t Plot average vs. time t

16. Experiment: Cargos traveling solely on actin filaments go farther during dispersion than during aggregation. Do cargos go faster during dispersion? No, the velocity of the cargos is the same for aggregation and dispersion.

17. Why do cargos go farther during dispersion than during aggregation? During dispersion cargos go “straight” to the end of the actin filament and do not turn at intersections with other actin filaments. This is good for spreading out pigment granules uniformly. During aggregation cargos have a 50-50 chance of switching to another actin filament at each intersection. So they don’t go as far. Frequent switching is a good way to find a nearby microtubule.

18. Two Types of Theoretical Modeling Confirm This Scenario Langevin solution interpolates between short time ballistic (straight-line) motion and long time diffusive motion. Computer simulations of cargos moving along actin filaments also confirms this picture.

19. Solution to Langevin Equation Langevin solution interpolates between short time straight line motion and long time diffusive motion. Fitting displacement data yields D and  which can be used to obtain the mean free path ℓ (distance traveled before turning). The mean free path is given by

20. Fitting Parameters Fit to Langevin Solution Fit to power law

21. Langevin Fits Yield Mean Free Path Dispersion: = 539 ± 9 nm Aggregation: = 237 ± 12 nm

22. Compare Langevin ℓ with Electron Micrographs of Actin Filaments Dispersion: Langevin ℓ  L/2 where L≈1300 nm is a typical filament length implying cargos go to end of filament Aggregation: Langevin ℓ ≈ 1.5 d where d≈160 nm is the typical distance between filament intersections consistent with cargos switching with 50% probability at intersections

23. The actin filaments appear denser during aggregation which would encourage frequent switching from one filament to another. (Not enough EMs to confirm this.) Electron Micrographs of Actin Filaments

24. Filament Density and Touching Number To quantify the density of filaments, randomly place circles on the EM. Circle diameter = 568 nm = cargo diameter Touching number = number of filaments in contact with circle

25. Touching Number n t Aggregation Dispersion = 7.8 = 4.2

26. Simulations of Cargos Moving on Actin Filaments Distribution of filament lengths taken from EMs (electron micrographs) Vary density of filaments (touching number) Vary switching probability at filament intersections

27. Simulations of Cargos Moving Along Actin Filament Networks Trajectories more localized Trajectories more spread out

28. Switching Probability Aggregation: n t = 7.8, = 237 nm, switch = 50% Dispersion: n t =4.2, =539 nm, switch = 0% - 6 %

29. Result of Simulations of Cargos Moving on Actin Filaments Using the density of filaments taken from EMs and the Langevin mean free path, we find: For aggregration, switching probability is 50% at filament intersections For dispersion, cargos go to end of filament and then attach to a new filament (switching probability is very small ~ 0%) Result: Average mean free paths agree with EMs and Langevin, confirming scenario

30. Simulations of Cargos Moving Along Actin Filament Networks Trajectories more localized Trajectories more spread out

31. Cargo displacement after 30 sec from simulations Cargos are more localized during aggregation Cargos are more evenly spread out during dispersion

32. SUMMARY: We have explained how and why cargos go farther during dispersion than during aggregation During dispersion cargos go “straight” to the end of the filament and do not turn at intersections with other filaments. This is good for spreading out pigment granules uniformly. During aggregation cargos have a 50-50 chance of switching to another filament at each intersection. So they don’t go as far. Frequent switching is a good way to find a microtubule that leads to the nucleus.

33. Possible Way that the Switching Probability Is Regulated During aggregation, there are about 60 motors per cargo, but only one active motor pulls a cargo along a filament. Another motor can attach to a nearby filament and cause a switch to the new filament. Switch probability is 50%. During dispersion, there are about 90 motors per cargo, but only 2 active motors pull a cargo. Another motor may try to attach to another filament but it is not strong enough to cause a switch to a new filament. Switch probability is 0.

34. Actin Collaborators Steven Gross (Cell and Dev. Biology, and Physics and Astron., U.C. Irvine) Joseph Snider (Physics and Astron., U.C. Irvine) (Langevin and simulations) Francis Lin (Physics and Astron., UC Irvine) (data analysis) Neda Zahedi (King’s College London and U. Conn. Health Sci. Ctr.) (experiments) Vladimir Rodionov (U. Conn. Health Sci. Ctr.) (experiments) Snider et al., PNAS 101, 13204 (2004). Website: http://bioweb.bio.uci.edu/sgross/

35. THE END

36. Fraction of cargos that have touched a MT 15 times by time t (from simulations)

37. Collective Motion vs. Single Motor Properties Cargos go further in dispersion due to collective motion rather than the properties of individual molecular motors. Analogy: To understand traffic flow patterns in southern California, you don’t need to know how a car works. Learning about tires and internal combustion won’t tell you why there’s a traffic jam.

38. Actin Filament Length Distribution

39. Quantification of motion Particle tracking: 8nm resolution, 30 Hz Analysis: Displacement vs. time R (t) random motion