Decoration of microtubules in solution by the kinesin-14, Ncd

Abstract

The kinesin-14, Ncd, is a cellular motor involved in microtubule spindle assembly and contraction during mitosis and meiosis. Like other members of the kinesin superfamily, Ncd consists of two motor heads connected by a linker and a long cargo-carrying stalk. The motor heads hydrolyze ATP to ADP to provide the power stroke that moves them and the cargo along the microtubule. Whereas conventional kinesins move processively along the sense of the microtubule right-handed helix, Ncd moves in the opposite direction, apparently using a different motive mechanism. According to the current model, the microtubule-binding state of Ncd is bound by one head and then released during the motive cycle. This is distinguished from the binding states of conventional kinesins, in which the motor heads are always bound in the motive cycle with alternating one-head and two-head binding. The objective was to determine the extent of binding, the binding states of Ncd in the presence of an ATP analogue, AMPPNP, and whether the binding is cooperative. Small-angle neutron scattering (SANS) of microtubules decorated with a deuterated Ncd construct, Ncd281, in solution containing 42% D(2)O was used. These conditions render the microtubule `invisible' to SANS, while amplifying the SANS from the Ncd constructs. In the presence of AMPPNP, 75% of Ncd281 was not bound. The remainder was bound cooperatively by one of its motor heads to the microtubule.

Figure 1

SANS differential cross-sections of Ncd281 microtubules in f _D = 0.42 D₂O with AMPPNP and comparison with models. Ncd281 data are shown as circles. Total particle densities for Ncd281: N = 3.92 × 10¹⁶ cm⁻³ motor dimers; N = 5.89 × 10¹⁶ cm⁻³ tubulin dimers. Scattering intensity calculated for models: (a) one-head cooperative binding; (b) one-head random binding with an average of approximately one motor per helical turn; (c) two-head cooperative binding. In each of the cooperative binding models sections of decorated microtubules coexist with sections of undecorated microtubules. Model parameters are given in Table 2 ▸. All values are per tubulin dimer, dσ_T(Q)/dΩ (cm²), scaled and corrected for the fraction of unbound Ncd as described in the text.

Figure 2

Model calculations of microtubules and microtubules decorated with deuterated Ncd281 in f _D = 0.42 D₂O. SANS for models, including instrument resolution smearing, are shown for undecorated 12/3 helical microtubules, microtubules decorated in the one-head binding state and microtubules decorated in the two-head binding state. All values are per tubulin dimer, dσ_T(Q)/dΩ (cm²).