In search of an optimal ring to couple microtubule depolymerization to processive chromosome motions

Abstract

Mitotic chromosome motions are driven by microtubules (MTs) and associated proteins that couple kinetochores to MT ends. A good coupler should ensure a high stability of attachment, even when the chromosome changes direction or experiences a large opposing force. The optimal coupler is also expected to be efficient in converting the energy of MT depolymerization into chromosome motility. As was shown years ago, a ''sleeve''-based, chromosomeassociated structure could, in principle, couple MT dynamics to chromosome motion. A recently identified kinetochore complex from yeast, the ''Dam1'' or ''DASH'' complex, may function as an encircling coupler in vivo. Some features of the Dam1 ring differ from those of the ''sleeve,'' but whether these differences are significant has not been examined. Here, we analyze theoretically the biomechanical properties of encircling couplers that have properties of the Dam1/DASH complex, such as its large diameter and inward-directed extensions. We demonstrate that, if the coupler is modeled as a wide ring with links that bind the MT wall, its optimal performance is achieved when the linkers are flexible and their binding to tubulin dimers is strong. The diffusive movement of such a coupler is limited, but MT depolymerization can drive its motion via a ''forced walk,'' whose features differ significantly from those of the mechanisms based on biased diffusion. Our analysis identifies key experimental parameters whose values should determine whether the Dam1/DASH ring moves via diffusion or a forced walk.biased diffusion ͉ kinetochore ͉ mathematical model ͉ power stroke ͉ energy-transducing coupler C hromosome segregation during cell division depends on the activities of microtubules (MTs), which are cylindrical arrangements of 13 linear polymers of ␣␤-tubulin dimers called protofilaments (PFs) (1). Tubulin complexed with GTP elongates PF ends, but this GTP is hydrolyzed soon after dimers have added (2). The preferred conformation of the GDP dimers is more bent, so the PFs tend to curve (3, 4). In a growing MT, this tendency is counteracted by a relatively straight ''GTP cap,'' but if the terminal layers are lost, the PFs are no longer restrained (1, 2). As they splay out, the dimers dissociate from their longitudinal neighbors, and the MT shortens (5, 6). Consequently, the ends of depolymerizing MTs in vitro and in vivo display curved PFs (3,[5][6][7][8][9]. Representative images of kinetochore MTs are shown in Fig. 1A; the length of PF flares in anaphase mammalian cells is 53 Ϯ 7 nm, n ϭ 368 PFs (J.R.M., E.L.G., A.E., K. Zhudenkov, M. Morphew, et al., unpublished work).It is well documented that MTs can move chromosomes or microbeads that associate with their shortening ends (9-15). Several hypotheses have explained these force-transducing attachments with the help of a coupler that encircles the MT (16-18). Movement in these models is driven ultimately by the energy released during hydrolysis of tubulin-associated GTP (Ϸ12 k B T; k B , Boltzmann constant). However, the design of a ...

Long tethers provide high-force coupling of the Dam1 ring to shortening microtubules

“…This interpretation predicts that, as the bead repositions under the constant load from the force clamp, the load acting on a Dam1 ring should decrease. Because a Dam1 ring moves faster under a smaller load (17,21), the repositioning should lead to faster ring motion, although the applied trapping load and the solution chemistry are constant (Movie S1). To test this prediction, we analyzed the kinetics of bead motion in the force clamp during phase III of the above experiment (Fig.…”

“…The above experiments and calculations highlighted the mechanical aspects of this system, whereas our previous modeling of the energy and dynamics of Dam1 MT-ring coupling emphasized the importance of the affinity between Dam1 and the MT wall (17). A weakly bound Dam1 ring should diffuse well on the MT wall but is predicted to slip easily from the MT end if flared protofilament extensions are lost (e.g., because of some stochastic event).…”

“…In one, the Dam1 ring was described as a loosely bound coupler that diffused quickly, and the low measured force was suggested to reflect the low depolymerization force captured by the Dam1 ring (16). The other model, proposed by our group, posited that a Dam1 ring was a tightly bound coupler, showing negligible diffusion, which moved with the end of a shortening MT by a diffusion-free forced-walk mechanism (17). Importantly, the forced-walk model predicts that the Dam1 ring can capture up to 30 pN force from the depolymerization of one MT (8).…”

A non-ring-like form of the Dam1 complex modulates microtubule dynamics in fission yeast

“…The ring conformation is considered crucial for Dam1's interaction with MTs-specifically, its capabilities to associate stably with the MT, to track the end of a depolymerizing MT, and to garner the energy released from MT depolymerization (4,7). These properties of a Dam1 ring support a working model for the Dam1 coupler-the "power stroke" or "conformational wave" model, which postulates that the curling of the flaring protofilaments of a depolymerizing MT mechanically pushes the coupler, and thereby the associated chromosome, to move along the MT (4,5,(12)(13)(14).…”

A model for the regulatory network controlling the dynamics of kinetochore microtubule plus-ends and poleward flux in metaphase

“…There are already excellent published models describing the physical origin of the forces underlying chromosome and kMT movements and the detailed mechanism of MT polymerization/depolymerization (15)(16)(17)(18)(19)(20)(21). The focus of our current article is to describe the network of regulatory proteins that determine the behavior of polymerization/ depolymerization dynamics at kMT plus-ends.…”