The Dam1 kinetochore complex harnesses microtubule dynamics to produce force and movement

Abstract

Kinetochores remain attached to microtubule (MT) tips during mitosis even as the tips assemble and disassemble under their grip, allowing filament dynamics to produce force and move chromosomes. The specific proteins that mediate tip attachment are uncertain, and the mechanism of MT-dependent force production is unknown. Recent work suggests that the Dam1 complex, an essential component of kinetochores in yeast, may contribute directly to kinetochore-MT attachment and force production, perhaps by forming a sliding ring encircling the MT. To test these hypotheses, we developed an in vitro motility assay where beads coated with pure recombinant Dam1 complex were bound to the tips of individual dynamic MTs. The Dam1-coated beads remained tip-bound and underwent assembly-and disassembly-driven movement over Ϸ3 m, comparable to chromosome displacements in vivo. Dam1-based attachments to assembling tips were robust, supporting 0.5-3 pN of tension applied with a feedback-controlled optical trap as the MTs lengthened Ϸ1 m. The attachments also harnessed energy from MT disassembly to generate movement against tension. Reversing the direction of force (i.e., switching to compressive force) caused the attachments to disengage the tip and slide over the filament, but sliding was blocked by areas where the MT was anchored to a coverslip, consistent with a coupling structure encircling the filament. Our findings demonstrate how the Dam1 complex may contribute directly to MT-driven chromosome movement.cytoskeleton ͉ DASH ͉ disassembly ͉ mitosis ͉ motility

Tension applied through the Dam1 complex promotes microtubule elongation providing a direct mechanism for length control in mitosis

“…This persistent tip tracking has enabled us to measure the effects of continuous tension on MT dynamic parameters in a reconstituted system for the first time. In our assay, beads coated with the Dam1 complex are attached to the tips of individual dynamic MTs grown from stabilized seeds anchored to a glass coverslip 14,22, 23 (Fig. 1a).…”

“…To overcome these limitations, we relied on a motility assay using the Dam1 complex 11 (also known as DASH, or DDD), a putative load-bearing component of kinetochores in yeast [11][12][13][14]26 . Like kinetochores, pure recombinant Dam1 complex forms robust connections to growing and shortening MT tips that remain attached even when tension is applied 14 .…”

Rings, bracelets, sleeves, and chevrons: new structures of kinetochore proteins

“…It can bind to depolymerizing microtubule ends (13) and stay attached to the end of polymerizing or depolymerizing microtubules even against a force of up to 3 pN (12). The level of force is similar to that generated by ATP-driven motors that move organelles inside cells.…”

“…Rings are difficult to detect in vitro in the absence of microtubules suggesting that microtubules might guide the assembly of these structures. The lack of 1:1 stoichiometry between the number of Dam1 complexes and the number of protofilaments argues that Dam1 does not bind tightly to one part of the tubulin dimer and thus could have a loose grip that allows free movement along the microtubule (12). Recent results suggest that flexible tethers on two of the proteins (Dam1 and Duo1) bind to the microtubule (11).…”

The perpetual movements of anaphase

“…However, while such rings may efficiently couple microtubule depolymerization to chromosome movement in a system where kinetochores bind only to a single microtubule [130,131], they are not essential in fission yeast [132] and orthologues have never been found outside fungi. Additionally, it was subsequently shown that ring formation is not required for microtubule depolymerization-driven motion of the Dam1 complex [133,134].…”