University of Copenhagen Dias 1 Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro Megan T. Valentine, Polly M. Fordyce, Troy C. Krzysiak, Susan P. Gilbert and Steven M. Block, 2006, Nature Cell Biology 8: Jeg hedder Anders

University of Copenhagen Dias 2 Eg5 is interesting because: -Member of the kinesin-5 family. -Speculated to move nonprocessively, unlike regular Kinesin. -Also speculated to move exactly like kinesin (i.e. walk with ~8 nm steps) ‏ -Human Eg5 is not easily purified from cells. Clearly we need single molecule biophysics!

University of Copenhagen Dias 3 Experimental setup Notes: Recombinant human Eg His from E.coli PLL-PEG-biotin = biotinylated poly-(L-lysine)-graft-polyethylene polymers Video tracking, data sampling at 2 kHz; Fixed position optical trap.

University of Copenhagen Dias 4 How many dimers does it take to move a bead?

University of Copenhagen Dias 5 Steps vs. time a, b, d: Load = –4 pN and c(APT) = 8 µM c:Load = –4 pN and c(APT) = 2 mM e: Load = –4 pN and c(APT) = 31 µM f: Load = +4 pN and c(APT) = 2 mM Notes: c(ATP) in human cells is in the range of 1-10 mM says Wikipedia. Step size is identical to that of kinesin (8.1 ± 0.1 nm) ‏

University of Copenhagen Dias 6 Average run length Average run length of Eg His: 67± 7 nm or 8.3 steps Average run length of regular kinesin is more than 100 steps per run. The authors discard the first three bins to obtain a good exponential fit. Eg His dissociates easily compared to kinesin.

University of Copenhagen Dias 7 Sammenligning med ensemble værdier Steady-state solution kinetics: K M = 8 ± 2 µM (for Eg5-513) ‏ k cat = 0.48 ± 0.02 s -1 (for Eg5-513) ‏ k cat = 0.45 ± 0.04 s -1 (for Eg His) ‏ Single-molecule velocities at zero force: K M = 10 ± 2 µM (for Eg His) ‏ k cat = 11.9 ± 0.2 s -1 (for Eg His) ‏ Possible explanation: The k cat underestimation in steady- state solution kinetics is caused by frequent detachments from microtubles.

University of Copenhagen Dias 8 Velocity vs. ATP concentration/force Michaelis-Menten kinetics gives us: Looks fine, but does velocity really increase with positive force?

University of Copenhagen Dias 9 Rate limiting steps/mechanisms a: F = –4 pN; APT concentration is clearly rate limiting at low concentrations. b: At saturated ATP concentrations, the load force acts slightly rate limiting.  At least two rate limiting steps/mechanisms are involved.

University of Copenhagen Dias 10 Another point of interest Stall force: No load force greater than –5 pN was recorded. because Eg His is dissociates from microtubles at this force. –7 pN has been recorded in another experiment. It’s like a strong car with too little traction.

University of Copenhagen Dias 11 Speculations Eg5: Dissociates at high loads Walks roughly 8.3 steps V ~ 100 nm s -1 Kinesin: Slows down at high loads Walks >100 steps V ~ 6-8 times faster The authors have lots of speculations on why these characteristics may be practical in vivo, but very few references (one).