1. Kinesin: How it Waits Between Steps Harvard Biovisions: “The Inner Life of the Cell” http://multimedia.mcb.harvard.edu/ Holly Durst

2. What is Kinesin? dimeric motor protein carries cellular cargo along microtubules by hydrolyzing ATP takes several hundred “steps” along a microtubule without detaching Harvard Biovisions: “The Inner Life of the Cell” http://multimedia.mcb.harvard.edu/

3. 1.ATP binding to leading head initiates neck linker docking and the other head is thrown forward 2.New leading head docks onto binding site after diffusional search, resulting in 80 Å movement of attached cargo 3.This accelerates ADP release and trailing head hydrolyzes ATP to ADP-Pi 4.ATP binds to leading head α β Coiled coil Neck linkers

4. FRET http://bio.physics.uiuc.edu/images/FRET_concept.jpg

5. Objective Use a series of smFRET experiments to detect whether kinesin is bound to its microtubule track by one or two heads in its ‘waiting’ conformation between steps How kinesin waits between steps Teppei Mori, Ronald D. Vale & Michio Tomishige

6. ‘cysteine light’ human ubiquitous kinesin-1 dimer which cysteine residues and/or mutations were introduced into Dye-labelled kinesins were imaged moving along sea-urchin axonemes with a custom-built prism-type laser-illuminated total-internal reflection fluorescence microscope The Players

7. 1.Heterodimer one chain containing single cysteine residue in plus end oriented tip of core (residue 215) one chain containing single cysteine residue in minus-end oriented base of the core (residue 43) Used two FRET Sensors:

8. 2. Homodimer with a cysteine residue in both chains at the beginning of the neck linker (residue 324) The Players

9. Donor dye: Maleimide modified Cy3 Acceptor dye: Maleimide modified Cy5 Molecules that contained both dyes were selected for smFRET observations Testing the Sensors

10. Examined FRET efficiency in presence of non- hydrolyzable nucleotide analog AMP-PNP so that both kinesin heads are bound statically to the microtubule

11. Testing the Sensors Bimodal distribution of low (10%) and high (90%) As expected if two kinesin heads are bound to adjacent tubulin subunits 8 nm apart High peak – 43 dye on leading head and 215 dye on trailing head Low peak – 215 dye on leading head and 43 dye on trailing head Unimodal distribution centered at about 35% Is consistent with a two-head bound state smFRET Efficiencies for 215-43 smFRET Efficiencies for 324-324 Binding along single protofilament supported by experiments with a 149-324 sensor

12. Different Conditions

13. Low ADP concentrations Remember: ADP occupying binding site = weak microtubule binding Different Conditions 215-43 Sensor unimodal at about 30% 324-324 Sensor shift from 35% to 60% kinesin heads come closer together Under these conditions, these distributions reflect a one-head bound state

14. Low ADP plus excess inorganic phosphate Different Conditions Partial occupancy of an ADP·Pi state in tethered head 215-43 Sensor 324-324 Sensor Peaks characteristic of a two-head bound state Similar results for addition of AlF4-

15. Both heads nucleotide free Different Conditions 215-43 Sensor 324-324 Sensor distributions similar to AMP-PNP, but with broader distributions Nucleotide-free kinesin primarily adopts a two-head bound state with partial occupancy of a one-head-bound state

16. FRET efficiency trace of individual axoneme-bound 215-43 heterodimer kinesin

17. Signal of 215-43 in AMP-PNP was fairly constant A subset of molecules with ADP or ADP/Pi or under nucleotide-free conditions underwent abrupt FRET transitions Unbinding and rebinding of kinesin head with microtubule

18. Mutated so only one head could bind to microtubules under all conditions –Y274A/R278A/K281A in loop 12 (L12-triple) 1.215(WT) – 43 (L12) 2.215(L12) – 43 (WT) Mutations

19. Mutations

20. 200 nM ADP 215(WT)-43(L12) and 215(L12)-43(WT) both produced unimodal distributions centered at about 30% Distances between 43-labelled and 215 labelled dyes are similar Similar result for nucleotide-free state Mutations

21. Addition of AMP-PNP 215(WT)-43(L12) – bimodal with primary peak at 80% 215(L12)-43(WT) – major peak shifted in opposite direction toward lower efficiencies movement of L12 triple towards plus-end oriented tip of bound head Mutations

22. 215/342 dyes on wild-type chain to probe neck linker conformations in the bound head Mutations

23. Mutations Translation of unbound head from rear position to forward position is driven by nucleotide-dependent docking of neck-linker

24. Saturating ATP concentration (1 mM) –Can only measure an average Dynamic Measurements

25. 215-43 showed broad distribution centered at about 50% Average of bimodal 10%, 90% FRET distribution of static two-head bound kinesin Different from 30% value of one-head bound kinesin Dynamic Measurements

26. 324-324 unimodal distribution centered at about 30% Kinesin spends most of the time bound with two heads to the microtubule when moving at saturating ATP concentrations Dynamic Measurements

27. Subsaturating ATP concentration (2 μM) Dynamic Measurements

28. 215-43 shifted to about 30% 324-324 shifted to about 60% More similar to 200 nM ADP (one-head bound) Suggests that kinesin waits primarily as a one- head bound intermediate when ATP binding becomes the rate-limiting step in the ATPase cycle Dynamic Measurements

29. Longer dwell times at low ATP concentration Dynamic Measurements

30. Spent most time in a roughly 30% FRET state (one-head bound) with brief spikes towards higher (80%) FRET values Higher values represent transient two-head bound intermediate state Transitions from 30% to lower FRET state should also occur Difficult to distinguish from noise Dynamic Measurements

31. Dwell-time histogram best fitted by a convolution of two exponentials Two rate-limiting ATP binding events occur between the two high-FRET spikes Dynamic Measurements

32. Mean dwell time (140 ms) is comparable to predicted dwell time Total number of spikes divided by displacement of these molecules yielded an average distance of about 17 nm per spike Close to double kinesin step size Dynamic Measurements

33. A kinesin step at low ATP concentrations involves a short-lived, two-head bound state, which then undergoes a transition to a longer-lived, one-head bound state Dynamic Measurements

34. At high ATP concentration (the rate-limiting step is the detachment of the trailing head triggered by ATP hydrolysis/phosphate release), kinesin moves quickly from one two-head bound state to the next At low ATP concentration (ATP binding to the leading head is rate-limiting), the trailing head releases its P i and detaches from the microtubule, producing a long-lived one-head bound state Summary

35. Summary

36. Kinesin waits as either a one-head bound or two-head bound intermediate, depending on ATP concentration and the rate-limiting step Discussion

37. ATPase cycles in the two kinesin heads are coordinated during processive motion Gating model proposes that detached head waits in front of bound head and is in a conformation that prevents it from binding tubulin But, transient interactions with the microtubule are seen Additional mechanism must keep detached head from progressing through ATPase cycle until its partner binds ATP Discussion

38. Detached head will not release ADP when it is interacting with rear tubulin-binding site ADP release could occur after the bound head binds ATP and docks the neck-linker, translating the detached head to a forward tubulin-binding site Results are supported by Guydosh and Block who showed that nucleotide dissociation occurs only when a head is in the forward position Position dependence controlled by conformation of neck- linker Discussion

39. How does the conformation of the neck- linker affect transitions in the ATPase cycle? Future Work

40. References Mori, T.; Vale, R. D.; Tomishige, M. Nature 2007, 450, 750-754 Vale, R. D.; Milligan, R. A. Science 2000, 288, 88-95

41. QUESTIONS?