Different assemblies of the DAM1 complex follow shortening microtubules by distinct mechanisms

Abstract

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...

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

“…Together, these findings show that in fission yeast, Dam1 forms small speckles both on cytoplasmic MTs during interphase and kinetochores during mitosis. Similar small assemblies, called patches, also formed in vitro with recombinant ScDam1 complex (10,15,16). Hereafter, we refer to the small SpDam1 speckles as patches.…”

“…Also noteworthy, a Dam1 patch occasionally separated into smaller spots, followed by merging into one spot later (Fig. S3B), reminiscent of the ScDam1 patches in vitro (16)…”

“…With less than 16 copies of Dam1 complex per oligomer (one to four copies by one account) (15), the small assemblies, called patches, are nonetheless able to oscillate along the MT and track the depolymerizing end of a MT (15,16). Mechanistically, the power-stroke model cannot adequately explain how these small Dam1 oligomers, without an encircling structure to secure the topological association with the MT, are able to track the dynamic end of a shortening MT.…”

“…To link a chromosome to spindle MTs, a coupler should be able to maintain association with the dynamic end of kMT (4,16). To test this on SpDam1 patches, we performed time-lapse microscopy, focusing on the Dam1 associated with the end of the MT.…”

The perpetual movements of anaphase

“…Additionally, it was subsequently shown that ring formation is not required for microtubule depolymerization-driven motion of the Dam1 complex [133,134].…”