Probing Mitotic CENP-E Kinesin with the Tethered Cargo Motion Assay and Laser Tweezers

Abstract

Coiled-coil stalks of various kinesins differ significantly in predicted length and structure; this is an adaption that helps these motors carry out their specialized functions. However, little is known about the dynamic stalk configuration in moving motors. To gain insight into the conformational properties of the transporting motors, we developed a theoretical model to predict Brownian motion of a microbead tethered to the tail of a single, freely walking molecule. This approach, which we call the tethered cargo motion (TCM) assay, provides an accurate measure of the mechanical properties of motor-cargo tethering, verified using kinesin-1 conjugated to a microbead via DNA links in vitro. Applying the TCM assay to the mitotic kinesin CENP-E unexpectedly revealed that when walking along a microtubule track, this highly elongated molecule with a contour length of 230 nm formed a 20-nm-long tether. The stalk of a walking CENP-E could not be extended fully by application of sideways force with optical tweezers (up to 4 pN), implying that CENP-E carries its cargo in a compact configuration. Assisting force applied along the microtubule track accelerates CENP-E walking, but this increase does not depend on the presence of the CENP-E stalk. Our results suggest that the unusually large stalk of CENP-E has little role in regulating its function as a transporter. The adjustable stalk configuration may represent a regulatory mechanism for controlling the physical reach between kinetochore-bound CENP-E and spindle microtubules, or it may assist localizing various kinetochore regulators in the immediate vicinity of the kinetochore-embedded microtubule ends. The TCM assay and underlying theoretical framework will provide a general guide for determining the dynamic configurations of various molecular motors moving along their tracks, freely or under force.

Molecular requirements for transition from lateral to end-on microtubule binding and dynamic coupling

“…Full-length CENP-E can walk to MT plus-ends and briefly (< 20 s) maintain MT end-association thanks to a MT-binding domain in its tail region 19 . To determine how Ndc80 effects CENP-E motor motility without the interference of this domain, we used a truncated version of CENP-E that falls off MT tips, but on MT walls it walks and responds to force similarly to the full-length protein 20 . Glass microbeads coated with these motor domains (hereafter referred to as "CENP-E motor") were also coated with purified Ndc80 "Broccoli" complexes containing the wild-type MT-binding domains (Fig.…”

“…Although CENP-E is thought of as a mitotic version of Kinesin-1 35 , the motility characteristics of the two proteins under force are markedly different (Fig. 6d) 20,35,36 . When these Kinesin-1 characteristics were incorporated, the model correctly predicted a low survival probability for MT-bead attachments (Fig.…”

“…Assays were carried out as in 20 . COOH-activated glass beads (0.5 m, Bangs Laboratories, Fishers, IN, USA) were coated with a mixture of Broccoli Ndc80-GFP and truncated CENP-E-GFP using 12-nm DNA links as in 20 . The ratio of these proteins was varied while holding constant the combined total protein concentration (40 nM).…”

“…Solutions were exchanged using a peristaltic pump as in 51 . Taxol-stabilized MTs were prepared and immobilized on the coverslip using anti-tubulin antibodies (Serotec) as in 20 . For experiments with dynamic MTs, digoxigenin (DIG)-labeled GMPCPP-stabilized MT seeds were prepared and immobilized on the coverslip via anti-DIG antibodies (Roche) as in 41 .…”

“…For experiments with dynamic MTs, the imaging buffer was supplemented with 6 M soluble tubulin and 1 mM Mg-GTP. Using the 1064-nm laser beam of our laser trap 34 , a free-floating bead was trapped and brought into contact with the wall of the taxol-stabilized MT 20 , examining 4-12 beads for each Ndc80/CENP-E ratio. In experiments with dynamic MTs, the beads attached spontaneously to the MTs.…”