The Dam1 ring binds microtubules strongly enough to be a processive as well as energy-efficient coupler for chromosome motion

Abstract

Accurate chromosome segregation during mitotic division of budding yeast depends on the multiprotein kinetochore complex, Dam1 (also known as DASH). Purified Dam1 heterodecamers encircle microtubules (MTs) to form rings that can function as "couplers," molecular devices that transduce energy from MT disassembly into the motion of a cargo. Here we show that MT depolymerization develops a force against a Dam1 ring that is sixfold larger than the force exerted on a coupler that binds only one side of an MT. Wild-type rings slow depolymerization fourfold, but rings that include a mutant Dam1p with truncated C terminus slow depolymerization less, consistent with the idea that this tail is part of a strong bond between rings and MTs. A molecular-mechanical model for Dam1-MT interaction predicts that binding between this flexible tail and the MT wall should cause a Dam1 ring to wobble, and Fourier analysis of moving, ring-attached beads corroborates this prediction. Comparison of the forces generated against wild-type and mutant complexes confirms the importance of tight Dam1-MT association for processive cargo movement under load.

Fig. 1.

Quantitative analysis of the force-transducing attachments. (A) A schematic of the experimental system. Position of beads bound to the GDP-MT walls was followed with QPD before and after induction of MT disassembly. (B) Unprocessed QPD records of representative signals from a Dam1-coated bead in the presence of soluble Dam1 and a streptavidin-coated bead (Inset) relative to the center of the laser trap. (C) Mean values for the four parameters that describe force signals (see ref. for details). (Left) 0.5-μm streptavidin-coated beads assayed with biotinylated MTs (n = 35). (Right) Wild type Dam1-coated beads with soluble Alexa488–Dam1 (n = 26). (D and E) Force measurements carried out with streptavidin-coated beads: 0.5 μm (n = 45), 1 μm (n = 81; includes data from ref. 9), 2 μm (n = 35). Photos show DIC images of representative beads attached to MT walls and their fluorescence images (red) taken during dissolution of the rhodaminated GMPCPP caps (arrowheads). Graph shows median values for force amplitudes for these beads. Trap stiffness, ≈0.008 pN/nm. (F) Drawing (not to scale) of the forces from MT depolymerization and the laser trap. The fulcrum is at ring's edge on the bead-distal side of the MT; because of the ring's tilt, F_MT is likely to act slightly off the MT axis, so its lever arm r is defined by a range. The bead (not shown) is 15-times larger than the ring, so a relatively small trapping force can stall the MT-disassembly-driven movement of the ring.

Fig. 2.

Force-coupling via Dam1–19 mutant complexes. (A) Typical (Left) and one of the largest (Right) signals obtained with Dam1–19 coated beads in the presence of soluble Dam1–19. Overall features of these signals are similar to those seen with wild-type protein, but the quantitative characteristics are different. (B) Histogram of force amplitudes shows the same range for wild-type and mutant proteins, but most of the Dam1–19 signals were smaller. (C) Average characteristics of Dam1–19 signals. The experiments with different beads were done under similar conditions, including initial tension that was applied to the beads.

Fig. 3.

MT end-tracking by wild-type and mutant Dam1 complexes. (A) Histograms of MT depolymerization rates. (B) Brightness of the tip-tracking Alexa488–Dam1 complexes (soluble concentration 1–2 nM) was normalized with the intensity of a single Alexa488 fluorophore (18) to plot the number of Dam1 subunits vs. their tracking rates. Thermal motion of MTs is the major contributor to this system's noise. (C and D) Lateral diffusion and end-tracking by Alexa488–Dam1–19. The oblique lines on the kymograph in C (arrows) indicate thermal diffusion. (Scale bar, 2 μm.) (E) Typical kinetics of the end tracking and changes in brightness of the tip-associated Dam1–19 complexes. (F) Peak values for the histograms in A and for Dam1–19 rates were determined with Rayleigh fitting (18).

Fig. 4.

Oscillatory motions of the Dam1-ring associated beads. (A) The drawing illustrates the wobbling of the ring and the resulting changes in bead position. (B) Model results for Dam1–tubulin energy 13k_BT. (C and D) Comparisons of theoretical and experimental results for 0.5-μm beads coupled to a Dam1 ring in the presence of soluble Dam1. The rising parts of the force signals (Insets, see also Fig. 1B) are not smooth, as beads pause and even reverse their motions. The spectral characteristics of these irregular oscillations were analyzed after fitting these raw QPD signals with lines whose slopes describe the linear rates of bead movement and then subtracting this component to obtain the variable parts (Upper) and their Fourier transformations (Lower). The exact positions of the peaks and their amplitudes were different for repetitions of the experiments and theoretical calculations, because of the stochasticity of the Dam1 ring–MT system (e.g., compare D, F, and Fig. S2). As a control, similar transformations were done for beads attached to MTs or after their detachment. (E) Full, unprocessed QPD signal (for bead in Movie S1), which shows typical positions of the segments used for spectral analysis. (F) Experimental spectra of movements of another bead attached to a Dam1 ring and a streptavidin-coated bead. Dam1 beads display discrete but highly variable peak frequencies during forced movement parallel to MT, because the attached ring wobbles as it transits stochastically between energetically preferred configurations on MT lattice (19).