Structure of the DASH/Dam1 complex shows its role at the yeast kinetochore-microtubule interface

Simon Jenni; Stephen C. Harrison

doi:10.1126/science.aar6436

Faculty Opinions recommendation of The requirement for the Dam1 complex is dependent upon the number of kinetochore proteins and microtubules.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.10566957.12160054 ◽

2011 ◽

Author(s):

Uttam Surana ◽

Cheng-Gee Koh

Keyword(s):

Dam1 Complex

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Molecular requirements for the formation of a kinetochore–microtubule interface by Dam1 and Ndc80 complexes

The Journal of Cell Biology ◽

10.1083/jcb.201210091 ◽

2012 ◽

Vol 200 (1) ◽

pp. 21-30 ◽

Cited By ~ 49

Author(s):

Fabienne Lampert ◽

Christine Mieck ◽

Gregory M. Alushin ◽

Eva Nogales ◽

Stefan Westermann

Keyword(s):

Chromosome Segregation ◽

Protein Complexes ◽

Structural Elements ◽

Large Protein ◽

Kinetochore Assembly ◽

Sister Chromatids ◽

Dam1 Complex ◽

Shed Light ◽

Kinetochore Function

Kinetochores are large protein complexes that link sister chromatids to the spindle and transduce microtubule dynamics into chromosome movement. In budding yeast, the kinetochore–microtubule interface is formed by the plus end–associated Dam1 complex and the kinetochore-resident Ndc80 complex, but how they work in combination and whether a physical association between them is critical for chromosome segregation is poorly understood. Here, we define structural elements required for the Ndc80–Dam1 interaction and probe their function in vivo. A novel ndc80 allele, selectively impaired in Dam1 binding, displayed growth and chromosome segregation defects. Its combination with an N-terminal truncation resulted in lethality, demonstrating essential but partially redundant roles for the Ndc80 N-tail and Ndc80–Dam1 interface. In contrast, mutations in the calponin homology domain of Ndc80 abrogated kinetochore function and were not compensated by the presence of Dam1. Our experiments shed light on how microtubule couplers cooperate and impose important constraints on structural models for outer kinetochore assembly.

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The Dam1 ring binds to the E-hook of tubulin and diffuses along the microtubule

Molecular Biology of the Cell ◽

10.1091/mbc.e10-10-0841 ◽

2011 ◽

Vol 22 (4) ◽

pp. 457-466 ◽

Cited By ~ 29

Author(s):

Vincent H. Ramey ◽

Hong-Wei Wang ◽

Yuko Nakajima ◽

Amanda Wong ◽

Jian Liu ◽

...

Keyword(s):

Energy Coupling ◽

Mechanical Analysis ◽

Microtubule Depolymerization ◽

Ring Model ◽

Complex Interactions ◽

Functional Aspects ◽

Significant Step ◽

Statistical Mechanical ◽

Biased Diffusion ◽

Dam1 Complex

There has been much effort in recent years aimed at understanding the molecular mechanism by which the Dam1 kinetochore complex is able to couple microtubule depolymerization to poleward movement. Both a biased diffusion and a forced walk model have been proposed, and several key functional aspects of Dam1-microtubule binding are disputed. Here, we investigate the elements involved in tubulin-Dam1 complex interactions and directly visualize Dam1 rings on microtubules in order to infer their dynamic behavior on the microtubule lattice and its likely relevance at the kinetochore. We find that the Dam1 complex has a preference for native tubulin over tubulin that is lacking its acidic C-terminal tail. Statistical mechanical analysis of images of Dam1 rings on microtubules, applied to both the distance between rings and the tilt angle of the rings with respect to the microtubule axis, supports a diffusive ring model. We also present a cryo-EM reconstruction of the Dam1 ring, likely the relevant assembly form of the complex for energy coupling during microtubule depolymerization in budding yeast. The present studies constitute a significant step forward by linking structural and biochemical observations toward a comprehensive understanding of the Dam1 complex.

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Divalent Cations Affect the Stability and Structure of Dad2p, a Subunit of the Candida Albicans Kinetochore Dam1 Complex

Journal of Molecular Biology Research ◽

10.5539/jmbr.v6n1p26 ◽

2016 ◽

Vol 6 (1) ◽

pp. 26

Author(s):

Jennifer Turner Waldo ◽

Tsering Dolma ◽

Emily Rouse

Keyword(s):

Candida Albicans ◽

Mitotic Spindle ◽

Divalent Cations ◽

Dynamic Structure ◽

Biochemical Properties ◽

Differential Scanning Fluorimetry ◽

A Subunit ◽

Dam1 Complex ◽

The Stability ◽

Shed Light

<p class="1Body">The heterodecameric Dam1 complex is involved in establishing and maintaining the connection between the kinetochore and the mitotic spindle during mitosis. Biochemical studies of the reconstituted complex have shed light upon how it interacts with microtubules. However, little information about the biochemical properties of the isolated subunits has been available. This report examines the stability and structure of Dad2p, one of the Dam1 complex subunits isolated from <em>Candida albicans</em>. By employing differential scanning fluorimetry, protease protection and hydrodynamic analyses, we show that Dad2p is specifically responsive to the presence of divalent cations. This observation may be important for understanding the dynamic structure and regulation of the Dam1 complex in fungal cells.</p>

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Kinetochore Protein Interactions and their Regulation by the Aurora Kinase Ipl1p

Molecular Biology of the Cell ◽

10.1091/mbc.e02-11-0765 ◽

2003 ◽

Vol 14 (8) ◽

pp. 3342-3355 ◽

Cited By ~ 88

Author(s):

Ching Shang ◽

Tony R. Hazbun ◽

Iain M. Cheeseman ◽

Jennifer Aranda ◽

Stanley Fields ◽

...

Keyword(s):

Protein Interactions ◽

Aurora Kinase ◽

Open Reading Frames ◽

Binding Assays ◽

A Genome ◽

Physical Interactions ◽

Dam1 Complex ◽

Two Hybrid ◽

Kinetochore Function

Although there has been a recent explosion in the identification of budding yeast kinetochore components, the physical interactions that underlie kinetochore function remain obscure. To better understand how kinetochores attach to microtubules and how this attachment is regulated, we sought to characterize the interactions among kinetochore proteins, especially with respect to the microtubule-binding Dam1 complex. The Dam1 complex plays a crucial role in the chromosome-spindle attachment and is a key target for phospho-regulation of this attachment by the Aurora kinase Ipl1p. To identify protein–protein interactions involving the Dam1 complex, and the effects of Dam1p phosphorylation state on these physical interactions, we conducted both a genome-wide two-hybrid screen and a series of biochemical binding assays for Dam1p. A two-hybrid screen of a library of 6000 yeast open reading frames identified nine kinetochore proteins as Dam1p-interacting partners. From 113 in vitro binding reactions involving all nine subunits of the Dam1 complex and 32 kinetochore proteins, we found at least nine interactions within the Dam1 complex and 19 potential partners for the Dam1 complex. Strikingly, we found that the Dam1p–Ndc80p and Dam1p–Spc34p interactions were weakened by mutations mimicking phosphorylation at Ipl1p sites, allowing us to formulate a model for the effects of phosphoregulation on kinetochore function.

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Molecular mechanisms of microtubule-dependent kinetochore transport toward spindle poles

The Journal of Cell Biology ◽

10.1083/jcb.200702141 ◽

2007 ◽

Vol 178 (2) ◽

pp. 269-281 ◽

Cited By ~ 114

Author(s):

Kozo Tanaka ◽

Etsushi Kitamura ◽

Yoko Kitamura ◽

Tomoyuki U. Tanaka

Keyword(s):

Saccharomyces Cerevisiae ◽

Family Member ◽

Lateral Surface ◽

Molecular Mechanisms ◽

Microtubule Depolymerization ◽

Spindle Poles ◽

Dam1 Complex ◽

Pulling Force

In mitosis, kinetochores are initially captured by the lateral sides of single microtubules and are subsequently transported toward spindle poles. Mechanisms for kinetochore transport are not yet known. We present two mechanisms involved in microtubule-dependent poleward kinetochore transport in Saccharomyces cerevisiae. First, kinetochores slide along the microtubule lateral surface, which is mainly and probably exclusively driven by Kar3, a kinesin-14 family member that localizes at kinetochores. Second, kinetochores are tethered at the microtubule distal ends and pulled poleward as microtubules shrink (end-on pulling). Kinetochore sliding is often converted to end-on pulling, enabling more processive transport, but the opposite conversion is rare. The establishment of end-on pulling is partly hindered by Kar3, and its progression requires the Dam1 complex. We suggest that the Dam1 complexes, which probably encircle a single microtubule, can convert microtubule depolymerization into the poleward kinetochore-pulling force. Thus, microtubule-dependent poleward kinetochore transport is ensured by at least two distinct mechanisms.

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The Ndc80 complex bridges two Dam1 complex rings

eLife ◽

10.7554/elife.21069 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 23

Author(s):

Jae ook Kim ◽

Alex Zelter ◽

Neil T Umbreit ◽

Athena Bollozos ◽

Michael Riffle ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Aurora B ◽

Aurora B Kinase ◽

Microtubule Attachment ◽

Sister Chromatids ◽

Ndc80 Complex ◽

Interaction Regions ◽

Dam1 Complex

Strong kinetochore-microtubule attachments are essential for faithful segregation of sister chromatids during mitosis. The Dam1 and Ndc80 complexes are the main microtubule binding components of the Saccharomyces cerevisiae kinetochore. Cooperation between these two complexes enhances kinetochore-microtubule coupling and is regulated by Aurora B kinase. We show that the Ndc80 complex can simultaneously bind and bridge across two Dam1 complex rings through a tripartite interaction, each component of which is regulated by Aurora B kinase. Mutations in any one of the Ndc80p interaction regions abrogates the Ndc80 complex’s ability to bind two Dam1 rings in vitro, and results in kinetochore biorientation and microtubule attachment defects in vivo. We also show that an extra-long Ndc80 complex, engineered to space the two Dam1 rings further apart, does not support growth. Taken together, our work suggests that each kinetochore in vivo contains two Dam1 rings and that proper spacing between the rings is vital.

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