Fig. 1. FCPT induced tight binding of Eg5 to microtubules. (A) The structures of kinesin-5 inhibitors: 2-[1-(4-fluorophenyl)cyclopropyl]-4-(pyridin-4-yl)thiazole (FCPT), GSK246053, monastrol and S-Trityl-L-cysteine (STLC). (B) FCPT, similar to AMP-PNP, induced the binding of the recombinant kinesin-5 motor domain to microtubules (10 µM) in the presence of 1 mM ATP. The best-fit line of the dose-response curve identified the EC₅₀ of AMP-PNP as 116 µM (±17 µM) and FCPT as 65 µM (±10 µM). Note that the concentration of FCPT required to bind 100% of the kinesin-5 motor domain to microtubules (1-2 mM) was above the solubility concentration for FCPT (approximately 500-600 µM). Error bars, s.e.m. (C) Coomassie-stained gels of lysates isolated from supernatants (S) and pellets (P) of either a DMSO (control)-, FCPT- or AMP-PNP-treated kinesin-5 motor domain/microtubule mix (in the presence of 1 mM ATP), showing kinesin-5 and tubulin. (D) FCPT enhanced the binding of kinesin-5 to microtubules. The best-fit line identified the apparent K_d of AMP-PNP (grey line) as 6.3 µM (±1.96 µM) and FCPT (black line) as 1.7 µM (±0.437 µM) – both in the presence of 1 mM ATP, 8 µM kinesin-5 and either 10 µM FCPT or 10 µM AMP-PNP. The fraction of kinesin-5 co-sedimenting with microtubules is scaled to reflect maximum binding as 1.0. Error bars, s.e.m. (E) Immunoblots show that FCPT (compared with DMSO as a control) enhanced the binding of kinesin-5 onto pelleted taxol-polymerized microtubules in clarified Xenopus egg extracts (approximately threefold); binding of either XCTK2 or MCAK to microtubules was unaffected. Tubulin is a loading control. (F) FCPT inhibited kinesin-5 motility on Xenopus egg-extract spindles. The kymographs (measured from the red broken line) of image sequences taken over 3.5 minutes (with one image every 3 seconds) show that X-rhodamine-labeled kinesin-5 is not motile when FCPT (right; 200 µM) was present (shown as straight lines). In the control (left; same time frame as the FCPT condition), kinesin-5 was dynamic, with speckles appearing and disappearing over time. The spindle is outlined with a white line. Scale bars: 5 µm.