The Ndc80 Kinetochore Complex Forms Load-Bearing Attachments to Dynamic Microtubule Tips via Biased Diffusion

The generation of pulling and pushing forces is one of the important functions of microtubules, which are dynamic and polarized structures. The ends of dynamic microtubules are able to form relatively stable links to cellular structures, so that when a microtubule grows it can exert a pushing force and when it shrinks it can exert a pulling force. Microtubule growth and shrinkage are tightly regulated by microtubule-associated proteins (MAPs) that bind to microtubule ends. Given their localization, MAPs may be exposed to compressive and tensile forces. The effect of such forces on MAP function, however, is poorly understood. Here we show that beads coated with the microtubule polymerizing protein XMAP215, the Xenopus homolog of Dis1 and chTOG, are able to link stably to the plus ends of microtubules, even when the ends are growing or shrinking; at growing ends, the beads increase the polymerization rate. Using optical tweezers, we found that tensile force further increased the microtubule polymerization rate. These results show that physical forces can regulate the activity of MAPs. Furthermore, our results show that XMAP215 can be used as a handle to sense and mechanically manipulate the dynamics of the microtubule tip.microtubule polymerase | microtubule dynamics | optical trap X MAP215 is a microtubule plus-end binding protein that alters microtubule dynamics (1-4) by promoting microtubule growth. It was identified as a factor increasing microtubule polymerization rates in Xenopus egg extracts (5). In vivo studies have shown that disruption of XMAP215 function leads to short interphase microtubules, reduced microtubule growth rate, and increased frequency of microtubule catastrophe and pause events (6-11). Depletion of XMAP215 also results in short, abnormally formed mitotic spindles and short astral microtubules (7,12,13). In vitro studies have demonstrated that XMAP215 binds preferentially to the microtubule plus end and follows the growth and shrinkage of the microtubule tip, catalyzing microtubule growth (14-16). In addition to localization at the microtubule plus end and centrosomes (17-19), proteins of the XMAP215/Dis1 family are found at the kinetochores (9, 20, 21), which are sites of high force during cell division. Microtubules themselves can exert pulling and pushing forces (22)(23)(24)(25)(26)(27)(28)(29)(30). Therefore, the localization of XMAP215 at microtubule ends suggests that XMAP215 can function under load. However, the effect of forces on XMAP215 activity has not been directly analyzed. In this study, we investigated the influence of forces-directed toward the microtubule plus and minus end-on XMAP215 activity in vitro. Microtubule growth and shrinkage were monitored with high spatial and temporal resolution by visualizing the position of XMAP215-coated polystyrene microspheres bound to the tips of dynamic microtubules. The microspheres, so-called "beads," served as handles to apply forces on XMAP215 molecules using optical tweezers. Results XMAP215-Coated Beads Remain Attached to Growing ...

Section: Discussionsupporting

confidence: 56%

The growth speed of microtubules with XMAP215-coated beads coupled to their ends is increased by tensile force

Trushko

Schäffer

Howard

2013

“…The property to track shrinking cytomotive filament ends to couple pulling forces to DNA has so far only been described for the eukaryotic kinetochore complex (30). In vitro, kinetochores processively track depolymerizing microtubules using biased diffusion and multiple binding sites (31), and they also reduce shrinking rates (32).…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of a prokaryotic minus end-tracking system using TubRC centromeric complexes and tubulin-like protein TubZ filaments

Fink

Löwe

2015

Segregation of DNA is a fundamental process during cell division. The mechanism of prokaryotic DNA segregation is largely unknown, but several low-copy-number plasmids encode cytomotive filament systems of the actin type and tubulin type important for plasmid inheritance. Of these cytomotive filaments, only actin-like systems are mechanistically well characterized. In contrast, the mechanism by which filaments of tubulin-like TubZ protein mediate DNA motility is unknown. To understand polymer-driven DNA transport, we reconstituted the filaments of TubZ protein (TubZ filaments) from Bacillus thuringiensis pBtoxis plasmid with their centromeric TubRC complexes containing adaptor protein TubR and tubC DNA. TubZ alone assembled into polar filaments, which annealed laterally and treadmilled. Using single-molecule imaging, we show that TubRC complexes were not pushed by filament polymerization; instead, they processively tracked shrinking, depolymerizing minus ends. Additionally, the TubRC complex nucleated TubZ filaments and allowed for treadmilling. Overall, our results indicate a pulling mechanism for DNA transport by the TubZRC system. The discovered minus end-tracking property of the TubRC complex expands the mechanistic diversity of the prokaryotic cytoskeleton.cytoskeleton | tubulin homologue | DNA segregation

“…When conjugated to microbeads, several kinetochore proteins can sustain processive motions at the ends of dynamic MTs (reviewed in ref. 6), but only a few have so far been reported to form force-transducing attachments (7)(8)(9)(10)(11). The average force that these proteins captured from MT disassembly was <5 pN.…”

mentioning

confidence: 99%

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

Volkov¹,

Zaytsev

Gudimchuk

et al. 2013

Microtubule kinetochore attachments are essential for accurate mitosis, but how these force-generating connections move chromosomes remains poorly understood. Processive motion at shortening microtubule ends can be reconstituted in vitro using microbeads conjugated to the budding yeast kinetochore protein Dam1, which forms microtubule-encircling rings. Here, we report that, when Dam1 is linked to a bead cargo by elongated protein tethers, the maximum force transmitted from a disassembling microtubule increases sixfold compared with a short tether. We interpret this significant improvement with a theory that considers the geometry and mechanics of the microtubule-ring-bead system. Our results show the importance of fibrillar links in tethering microtubule ends to cargo: fibrils enable the cargo to align coaxially with the microtubule, thereby increasing the stability of attachment and the mechanical work that it can do. The force-transducing characteristics of fibril-tethered Dam1 are similar to the analogous properties of purified yeast kinetochores, suggesting that a tethered Dam1 ring comprises the main force-bearing unit of the native attachment.laser tweezers | mathematical modeling | microtubule depolymerization | anaphase | forced walk