Microtubule sliding and ATPase activity of Eg5 homodimers. (A) SDS–polyacrylamide gel electrophoresis of double-headed (left) and single-headed (right) Eg5. (B) Eg5-driven MT sliding velocities. Squares, single-headed Eg5; circles, double-headed Eg5. (C) MT-activated ATPase activities. Activities are expressed in terms of per second per head. MT, microtubule.

Model of Eg5 homotetramer molecules in spindles. The orange shading indicates active molecules. Individual molecules contribute intermittent, single-headed force-generating interactions and then revert to the low-friction Eg5•ADP state. The collective of Eg5 molecules maintains active crosslinking of the MTs and exert consistent sliding force that drives the MTs to slide apart. Individual Eg5 molecules within the collective cycle between sticking (force-generating and force-holding) and slipping (low-friction, Eg5•ADP) states. Processive events (8 nm steps by an alternate-heads mechanism) can occur, but are rare (top left). MT, microtubule.

Force generation with negative intramolecular cooperativity. The orange shading indicates active molecules. In any one molecule, only one of the two heads is involved in force generation, whereas the other head is redundant for the duration of the force-generating event. In subsequent cycles, the heads might re-assort and the redundant head might be active instead. For each active head, the cycle of force generation begins with ADP release, which converts the head to stable strong binding, and continues with ATP binding and P_i release, which returns the head to a weak binding Eg5•ADP state. Weakly bound Eg5•ADP heads (left column) remain attached to the MT in a low-friction state that can be readily slid along the MT by forces developed by other, active molecules in the array. D, ADP; MT, microtubule.

Microtubule sliding by kinesin-1–Eg5 heterodimers. (A) SDS–polyacrylamide gel electrophoresis of kinesin-1–Eg5 heterodimers. Two heavy chains are present in equal amounts. (B) Histograms of MT sliding velocities driven by kinesin-1–Eg5 heterodimer in the absence (top) and presence (bottom) of 1 mM monastrol. (C) MT sliding velocities driven by kinesin-1–Eg5 heterodimer (squares), kinesin-1 homodimer (triangles) and Eg5 homodimer (circles). Monastrol inhibited MT sliding by the Eg5 homodimer. A fit (solid line) to a hyperbolic inhibition equation V=Intcpt−(amplitude × [monastrol]/(K_50%+[monastrol])) indicated that 50% inhibition occurs at 256 μM monastrol. The predicted velocity of kinesin-1–Eg5 heterodimer on a model with strict alternation of the kinesin-1 and Eg5 heads, and a monastrol-dependent dwell time for the Eg5 component is shown by the dotted line. This line was generated by calculating V_predicted=2Vk Ve/(Vk+Ve) for each monastrol concentration, where Vk is the measured MT sliding velocity for kinesin-1 and Ve is that for Eg5. The dotted line is the fit of the above inhibition equation to these calculated points. The actual behaviour (squares) is different, emphasizing that a scheme with strict alternation does not apply. MT, microtubule.