Summary Kinetochores of mitotic chromosomes are coupled to spindle microtubules in ways that allow the energy from tubulin dynamics to drive chromosome motion. Most kinetochore-associated microtubule ends display curving “protofilaments,” strands of tubulin dimers that bend away from the microtubule axis. Both a kinetochore “plate” and an encircling, ring-shaped protein complex have been proposed to link protofilament bending to poleward chromosome motion. Here we show by electron tomography that slender fibrils connect curved protofilaments directly to the inner kinetochore. Fibril-protofilament associations correlate with a local straightening of the flared protofilaments. Theoretical analysis reveals that protofilament-fibril connections would be efficient couplers for chromosome motion, and experimental work on two very different kinetochore components suggests that filamentous proteins can couple shortening microtubules to cargo movements. These analyses define a ring-independent mechanism for harnessing microtubule dynamics directly to chromosome movement.
Presence of multiple copies of the microtubule-binding NDC80 complex is an evolutionary conserved feature of kinetochores, points of attachment of chromosomes to spindle microtubules. This may enable multivalent attachments to microtubules, with implications that remain unexplored. Using recombinant human kinetochore components, we show that while single NDC80 complexes do not track depolymerizing microtubules, reconstituted particles containing the NDC80 receptor CENP-T bound to three or more NDC80 complexes do so effectively, as expected for a kinetochore force coupler. To study multivalency systematically, we engineered modules allowing incremental addition of NDC80 complexes. The modules’ residence time on microtubules increased exponentially with the number of NDC80 complexes. Modules with two or more complexes tracked depolymerizing microtubules with increasing efficiencies, and stalled and rescued microtubule depolymerization in a force-dependent manner when conjugated to cargo. Our observations indicate that NDC80, rather than through biased diffusion, tracks depolymerizing microtubules by harnessing force generated during microtubule disassembly.
Mitotic chromosomes segregate at the ends of shortening spindle microtubules (MTs). In budding yeast, the Dam1 multiprotein complex supports this dynamic attachment, thereby contributing to accurate chromosome segregation. Purified Dam1 will track the end of a depolymerizing MT and can couple it to microbead transport in vitro. The processivity of such motions has been thought to depend on rings that the Dam1 complex can form around MTs, but the possibility that alternative coupling geometries contribute to these motilities has not been considered. Here, we demonstrate that both rings and nonencircling Dam1 oligomers can track MT ends and enable processive cargo movement in vitro. The coupling properties of these two assemblies are, however, quite different, so each may make a distinct contribution to chromosome motility.chromosome motions ͉ kinetochore-microtubule interactions ͉ microtubule end tracking M itotic chromosomes are attached to spindle fibers by a multiprotein complex, the kinetochore (1-3). Kinetochores bind strongly to the plus ends of spindle microtubules (MTs), but tubulin subunits can still be added or lost from these MT ends as the chromosomes move. Kinetochores are also sites where forces can be generated to move chromosomes either toward or away from the pole that a kinetochore faces, so this interface provides multiple important mitotic functions. Kinetochores include MT-dependent motors and several protein complexes that lack motor activity but can still bind to a MT wall or end. These nonmotor complexes too may be important for chromosome motion, because depolymerizing MTs can generate enough force to move chromosomes without the help of motors, both in vivo and in vitro (4-7).The mechanism by which a kinetochore is coupled to the end of a shortening or lengthening MT is still unknown, but a 10-subunit protein complex from budding yeast kinetochores, called Dam1 or DASH, is important for chromosome-spindle attachments and spindle integrity in this organism (3). Purified Dam1 heterodecamers assemble around MTs into rings of 16-25 subunits (8, 9). At higher protein concentrations, both multiple rings and helices of Dam1 will form (10, 11).In yeast spindles, Dam1 is found primarily at kinetochores, although some of it associates with spindle MTs (12). A careful estimate of the number of Dam1 subunits at metaphase kinetochores in budding yeast gave 16-20 heterodecamers (12), enough to form a single ring. The behavior of kinetochore-associated Dam1 fluorescence suggests that these subunits are stably attached to kinetochore MTs, showing little or no turnover. However, a second population of Dam1, which is dimly localized to spindle MTs, shows faster turnover, indicating the presence of a dynamic pool of complexes (12).Oligomers of Dam1 can travel with a shortening MT in vitro (8), suggesting that this complex may be the primary MT-chromosome coupler in yeast. A bead coated with Dam1 will also associate with a MT in vitro and follow its end during either assembly or disassembly (13, 14), proper...
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