Microtubule Assembly Dynamics at the Nanoscale

1. Microtubule Assembly Dynamics at the Nanoscale METHODS, MEASUREMENTS, AND IMPLICATIONS FOR UNDERSTANDING MICROTUBULE DYNAMIC INSTABILITY Henry T. Schek, III European Molecular Biology Laboratory Melissa K. Gardner David Odde University of Minnesota Jun Cheng Alan J. Hunt University of Michigan

3. Microtubules

4. Dynamic Instability From Fygenson et al, Phys Rev E, 1994

5. Microtubule Polymerization in Cells • Works with actin to guide an axonal growth cone • Kinetochore attachments • Push on mitotic chromosomes arms

6. Why Study Polymerization Under Load? • Quantify forces • Study microtubule dynamics with nanometer resolution • Improve models of dynamic instability

7. Optical Tweezers • Optical Tweezers Features • Extremely stable (<1 nm drift/min) • Multiple Independently Maneuverable Traps • Sub-Nanometer Detection and Manipulation • Forces from Less than one pN to greater than 100 pN • Servo-control for Force Clamp or Position Clamp Cellular & Molecular Biomechanics Lab

8. Experimental Strategy Tightly Focused Trapping Laser Trapped Particle Attached Microtubule Seed Barrier on cover glass

9. Barrier Design • No interference • No image degradation • Constrain MT • Short MT • Vertex • Undercut • Laser footprint • LOR, SU-8

10. Barrier Fabrication Primary structure and undercut fabrication are independent

11. Barrier Results Scale bar=14 mm Scale bar=2 mm

12. What an Experiment Looks Like

13. Stationary Trap Results • Microtubule growth is highly variable, and exhibits pauses over a broad range of forces, even in a single microtubule • F-V relation is complex

14. Stationary trap underestimates filament displacement

15. Force Clamping

16. Force Clamp-MT length change Clamped Force (pN) 1.6 1.4 1.3 1.1 0.9 0.7

17. Large Growth Rate Variability Microtubule Length Change (nm) { Close to zero = 3% (speed < 1 nm/s) Growth= 55% Shortening=41% Growth rates

18. Variability on Longer Scales Microtubule Length Change (nm)

19. Microtubule Length Change (nm) Nano-Shortening Events 9/minute, do not lead to rapid shortening

20. Summary so far • Forces greater than 1 pN - plenty to influence chromosome movements* • Large growth rate variability • Frequent shortening events > 20 nm • Persistent velocities at longer times scales *Joglekar & Hunt. Biophys. J. (2002) Marshall et al. Curr. Biol.(2001)

21. Grows with GTP-cap Occasionally loses cap Rapid Shortening results GTP-Cap Hypothesis

22. Trouble for the GTP cap? It has been widely argued that the GTP-cap is at most one layer thick1, or slightly larger2 (e.g. lateral cap hypothesis) • Bayley et al., FEBS Lett., 1989; Bayley et al., J. Cell Sci., 1990; O'Brien et al., Biochemistry, 1987; Panda et al., Biochemistry, 2002; Stewart et al., Biochemistry, 1990 • Vandecandelaere et al., Biochemistry, 1999; Voter et al., Cell Motil. Cyto., 1991

23. If the GTP-cap is one layer thick, then: A microtubule that shortens sufficiently to lose more than one layer of tubulin subunits (~ 8 nm) will transition to rapid shortening. This is contradicted by our results.

24. Can these results be explained by a mechanochemical model? VanBuren, Cassimeris & Odde, Biophys. J. 2005

25. This… Or this?

26. No Yes

29. Ave. = –9.7 nm

30. Ave. = –6.4 nm < -9.7 nm (p < 10-6)

31. Resolution is sufficient to detect addition of individual subunits. In contrast to the conclusions of Kerssemakers et al*, no evidence of oligomer addition *Nature, 2006

32. Step-like events when data is processed in a manner similar to Kerssemakers et al

33. What’s a step? + XMAP215 Displacement Time

34. Growth rate depends more on the evolving tip structure than force. * *S.E. < 0.1 nm/sec

35. Conclusions • At the nanoscale, microtubule growth is highly variable • Frequent shortening events: as large as 80 nm, > 30 nm (2 layers) @ 8/min, > 40 nm @ 1/min ] • Oligomer addition occurs rarely, if ever • Average growth rate is weakly dependent on force, strongly dependent on tip structure • Shortening excursions are smaller at higher forces

36. Conclusions II • Finding consistent with a physically simple mechanochemical model, which explains: • Unexpected growth-phase shortening • Smaller shortening excursions at higher force • Weak force dependence of average growth rate • Reject other models for microtubule polymerization • Small cap induced hydrolysis models (i.e. “lateral cap”) • “Coupled hydrolysis” models (Flyvbjerg et al, Phys. Rev. Lett., 1994; Phys. Rev. E, 1996)

37. Acknowledgements • NSF • Whitaker Foundation (to H. Schek) • Burroughs Wellcome Fund