Biology subjects

Dumont, S.

Publications and source records attributed to Dumont, S..

4 recordsLinked to original sources

bioRxiv2017

NuMA Targets Dynein to Microtubule Minus-Ends at Mitosis

To build the spindle at mitosis, motors exert spatially regulated forces on microtubules. We know that dynein pulls on mammalian spindle microtubule minus-ends, and this localized activity at ends is predicted to allow dynein to cluster microtubules into poles. How dynein becomes enriched at minus-ends is not known. Here, we use quantitative imaging and laser ablation to show that NuMA targets dynactin to minus-ends, localizing dynein activity there. NuMA is recruited to new minus-ends independently of dynein and more quickly than dynactin, and both NuMA and dynactin display specific, steady-state binding at minus-ends. NuMA localization to minus-ends requires a C-terminal region outside NuMAs canonical microtubule binding domain, and it is independent of direct minus-end binders {gamma}-TuRC, CAMSAP1, and KANSL1/3. Both NuMAs minus-end-binding and dynein-dynactin-binding modules are required to rescue focused, bipolar spindle organization. Thus, NuMA may serve as a mitosis-specific minus-end cargo adaptor, targeting dynein activity to minus-ends to cluster spindle microtubules into poles.

cell biology↗

bioRxiv2017

Spindle assembly checkpoint satisfaction occurs through end-on but not lateral kinetochore-microtubule interactions under tension

To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) prevents anaphase until all kinetochores attach to the spindle. What signals the SAC monitors remains unclear. We do not know the contributions of different microtubule attachment features, or tension from biorientation, to SAC satisfaction in normal mitosis - or how these possible cues change during attachment. Here, we quantify concurrent Mad1 intensity, reporting on SAC silencing, and real-time attachment geometry, occupancy, and tension at individual mammalian kinetochores. We show that Mad1 loss from the kinetochore occurs in switch-like events with robust kinetics, and that metaphase-like tension across sister kinetochores is established just before Mad1 loss events at the first sister. We demonstrate that CenpE-mediated lateral attachment of the second sister can persistently generate this metaphase-like tension prior to biorientation, likely stabilizing sister end-on attachment, yet cannot induce Mad1 loss from that kinetochore. Instead, Mad1 loss begins after several end-on microtubules attach. Thus, end-on attachment provides geometry-specific molecular cues, or force on specific kinetochore linkages, that other attachment geometries cannot provide.\n\nSummaryThe spindle assembly checkpoint (SAC) delays anaphase until kinetochores are properly attached to the spindle. The authors demonstrate that the SAC monitors geometry-specific molecular cues, or force on specific kinetochore linkages, that \"end-on\" but not \"lateral\" attachments generating persistent tension can provide.

cell biology↗

bioRxiv2017

The mammalian kinetochore independently regulates its passive and active force-generating interfaces with microtubules

The kinetochore links chromosomes to dynamic spindle microtubules and drives both chromosome congression and segregation. To do so, the kinetochore must hold on to depolymerizing and polymerizing microtubules. At metaphase, one sister kinetochore couples to depolymerizing microtubules, pulling its sister along polymerizing microtubules [1,2]. Distinct kinetochore-microtubule interfaces mediate these behaviors: active interfaces transduce microtubule depolymerization into mechanical work, and passive interfaces generate friction as the kinetochore slides along microtubules [3,4]. We do not know the physical and molecular nature [5-7] of these interfaces, or how they are regulated to support diverse mitotic functions in mammalian cells. To address this question, we focus on the mechanical role of the essential load-bearing protein Hec1 [8-11]. Hec1s affinity for microtubules is regulated by Aurora B phosphorylation on its N-terminal tail [12-15], but its role at the passive and active interfaces remains unclear. Here, we use laser ablation to trigger cellular pulling on mutant kinetochores and decouple sisters in vivo, and thereby separately probe Hec1s role as it moves on polymerizing versus depolymerizing microtubules. We show that Hec1 phosphorylation tunes passive friction along polymerizing microtubules, modulating both the magnitude and timescale of responses to force. In contrast, we find that Hec1 phosphorylation does not affect the kinetochores ability to grip depolymerizing microtubules, or switch to this active force-generating state. Together, the data suggest that different kinetochore interfaces engage with growing and shrinking microtubules, and that passive friction can be regulated without disrupting active force generation. Through this mechanism, the kinetochore can modulate its grip on microtubules as its functional needs change during mitosis, and yet retain its ability to couple to microtubules powering chromosome movement.

cell biology↗

bioRxiv2017

Mapping load-bearing in the mammalian spindle reveals local kinetochore-fiber anchorage that provides mechanical isolation and redundancy

Active forces generated at kinetochores move chromosomes, and the dynamic spindle must robustly anchor kinetochore-fibers (k-fibers) to bear this load. We know that the mammalian spindle body can bear the load of chromosome movement far from poles, but do not know where and how - physically and molecularly - this load is distributed across the spindle. In part, this is because perturbing and reading out spindle mechanics in live cells is difficult. Yet, answering this question is key to understanding how the spindle generates and responds to force, and performs its diverse mechanical functions. Here, we map load-bearing across the mammalian spindle in space-time, and dissect local anchorage mechanics and mechanism. To do so, we laser ablate single k-fibers at different spindle locations, and in different molecular backgrounds, and quantify at high time resolution the immediate relaxation of chromosomes, k-fibers, and microtubule speckles. We find that load redistribution is locally confined in all directions: along the first 3-4 m from kinetochores, scaling with k-fiber length, and laterally within ~2 m of k-fiber sides, without neighboring k-fibers sharing load. A phenomenological model constrains the mechanistic underpinnings of these data: it suggests that dense, transient crosslinks to the spindle along k-fibers bear the load of chromosome movement, but that these connections do not limit the timescale of spindle reorganization. The microtubule crosslinker NuMA is needed for the local load-bearing observed, while Eg5 and PRC1 are not, suggesting specialization in mechanical function and a novel function for NuMA throughout the spindle body. Together, the data and model suggest that widespread NuMA-mediated crosslinks locally bear load, providing mechanical isolation and redundancy while allowing spindle fluidity. These features are well-suited to support robust chromosome segregation.

cell biology↗