Architecture of the Dam1 kinetochore ring complex and implications for microtubule-driven assembly and force-coupling mechanisms

The Dam1 kinetochore complex is essential for chromosome segregation in budding yeast. This ten-protein complex self-assembles around microtubules, forming ring-like structures that move with depolymerizing microtubule ends, a mechanism with implications for cellular function. Here we used EM-based single-particle and helical analyses to define the architecture of the Dam1 complex at 30-A resolution and the self-assembly mechanism. Ring oligomerization seems to be facilitated by a conformational change upon binding to microtubules, suggesting that the Dam1 ring is not preformed, but self-assembles around kinetochore microtubules. The C terminus of the Dam1p protein, where most of the Aurora kinase Ipl1 phosphorylation sites reside, is in a strategic location to affect oligomerization and interactions with the microtubule. One of Ipl1's roles might be to fine-tune the coupling of the microtubule interaction with the conformational change required for oligomerization, with phosphorylation resulting in ring breakdown.     

Phospho‐regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore

Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer kinetochore, assembling into 3 MDa‐sized microtubule‐embracing rings, but how ring assembly is specifically initiated in vivo remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi‐orientation. We show that Dam1c and the general microtubule plus end‐associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c‐Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c‐Bim1 binding by relieving an intramolecular inhibition of the Dam1 C‐terminus. In addition, Bim1 recruits Bik1/CLIP‐170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end‐on attachments are formed during the process of attachment error correction.     

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

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.     

Subunit organization in the Dam1 kinetochore complex and its ring around microtubules

All eukaryotic cells must segregate their chromosomes equally between two daughter cells at each division. This process needs to be robust, as errors in the form of loss or gain of genetic material have catastrophic effects on viability. Chromosomes are captured, aligned, and segregated to daughter cells via interaction with spindle microtubules mediated by the kinetochore. In Saccharomyces cerevisiae one microtubule attaches to each kinetochore, requiring extreme processivity from this single connection. The yeast Dam1 complex, an essential component of the outer kinetochore, forms rings around microtubules and in vitro recapitulates much of the functionality of a kinetochore-microtubule attachment. To understand the mechanism of the Dam1 complex at the kinetochore, we must know how it binds to microtubules, how it assembles into rings, and how assembly is regulated. We used electron microscopy to map several subunits within the structure of the Dam1 complex and identify the organization of Dam1 complexes within the ring. Of importance, new data strongly support a more passive role for the microtubule in Dam1 ring formation. Integrating this information with previously published data, we generated a structural model for the Dam1 complex assembly that advances our understanding of its function and will direct future experiments.     

Molecular architecture of the Dam1 complex–microtubule interaction

Mitosis is a highly regulated process that allows the equal distribution of the genetic material to the daughter cells. Chromosome segregation requires the formation of a bipolar mitotic spindle and assembly of a multi-protein structure termed the kinetochore to mediate attachments between condensed chromosomes and spindle microtubules. In budding yeast, a single microtubule attaches to each kinetochore, necessitating robustness and processivity of this kinetochore–microtubule attachment. The yeast kinetochore-localized Dam1 complex forms a direct interaction with the spindle microtubule. In vitro, the Dam1 complex assembles as a ring around microtubules and couples microtubule depolymerization with cargo movement. However, the subunit organization within the Dam1 complex, its higher-order oligomerization and how it interacts with microtubules remain under debate. Here, we used chemical cross-linking and mass spectrometry to define the architecture and subunit organization of the Dam1 complex. This work reveals that both the C termini of Duo1 and Dam1 subunits interact with the microtubule and are critical for microtubule binding of the Dam1 complex, placing Duo1 and Dam1 on the inside of the ring structure. Integrating this information with available structural data, we provide a coherent model for how the Dam1 complex self-assembles around microtubules.     

The requirement for the Dam1 complex is dependent upon the number of kinetochore proteins and microtubules

The Dam1 complex attaches the kinetochore to spindle microtubules and is a processivity factor in vitro. In Saccharomyces cerevisiae, which has point centromeres that attach to a single microtubule, deletion of any Dam1 complex member results in chromosome segregation failures and cell death. In Schizosaccharomyces pombe, which has epigenetically defined regional centromeres that each attach to 3-5 kinetochore microtubules, Dam1 complex homologs are not essential. To determine why the complex is essential in some organisms and not in others, we used Candida albicans, a multimorphic yeast with regional centromeres that attach to a single microtubule. Interestingly, the Dam1 complex was essential in C. albicans, suggesting that the number of microtubules per centromere is critical for its requirement. Importantly, by increasing CENP-A expression levels, more kinetochore proteins and microtubules were recruited to the centromeres, which remained fully functional. Furthermore, Dam1 complex members became less crucial for growth in cells with extra kinetochore proteins and microtubules. Thus, the requirement for the Dam1 complex is not due to the DNA-specific nature of point centromeres. Rather, the Dam1 complex is less critical when chromosomes have multiple kinetochore complexes and microtubules per centromere, implying that it functions as a processivity factor in vivo as well as in vitro.     

Aurora B switches relative strength of kinetochore–microtubule attachment modes for error correction

To establish chromosome biorientation, aberrant kinetochore–microtubule interaction must be resolved (error correction) by Aurora B kinase. Aurora B differentially regulates kinetochore attachment to the microtubule plus end and its lateral side (end-on and lateral attachment, respectively). However, it is still unclear how kinetochore–microtubule interactions are exchanged during error correction. Here, we reconstituted the budding yeast kinetochore–microtubule interface in vitro by attaching the Ndc80 complexes to nanobeads. These Ndc80C nanobeads recapitulated in vitro the lateral and end-on attachments of authentic kinetochores on dynamic microtubules loaded with the Dam1 complex. This in vitro assay enabled the direct comparison of lateral and end-on attachment strength and showed that Dam1 phosphorylation by Aurora B makes the end-on attachment weaker than the lateral attachment. Similar reconstitutions with purified kinetochore particles were used for comparison. We suggest the Dam1 phosphorylation weakens interaction with the Ndc80 complex, disrupts the end-on attachment, and promotes the exchange to a new lateral attachment, leading to error correction.     

Molecular mechanisms of microtubule-dependent kinetochore transport toward spindle poles

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.     

Phosphoregulation and depolymerization-driven movement of the Dam1 complex do not require ring formation

During mitosis, kinetochores form persistent attachments to microtubule tips and undergo corrective detachment in response to phosphorylation by Ipl1 (Aurora B) kinase. The Dam1 complex is required to establish and maintain bi-oriented attachment to microtubule tips in vivo, and it contains multiple sites phosphorylated by Ipl1 (Refs 2, 3, 4, 5, 6, 7, 8, 9, 10). Moreover, a number of kinetochore-like functions can be reconstituted in vitro with pure Dam1 complex. These functions are believed to derive from the ability of the complex to self-assemble into rings. Here we show that rings are not necessary for dynamic microtubule attachment, Ipl1-dependent modulation of microtubule affinity or the ability of Dam1 to move processively with disassembling microtubule tips. Using two fluorescence-based assays, we found that the complex exhibited a high affinity for microtubules (Kd of approximately 6 nM) that was reduced by phosphorylation at Ser 20, a single Ipl1 target residue in Dam1. Moreover, individual complexes underwent one-dimensional diffusion along microtubules and detached 2.5-fold more frequently after phosphorylation by Ipl1. Particles consisting of one to four Dam1 complexes - too few to surround a microtubule - were captured and carried by disassembling tips. Thus, even a small number of binding elements could provide a dynamic, phosphoregulated microtubule attachment and thereby facilitate accurate chromosome segregation.     

Rings, bracelets, sleeves, and chevrons: new structures of kinetochore proteins

Electron microscopy has recently revealed striking structural orderliness in kinetochore proteins and protein complexes that associate with microtubules. In addition to their astonishing appearance and intrinsic beauty, the structures are functionally informative. The Dam1 and Ndc80 complexes bind to the microtubule lattice as rings and chevrons, respectively. These structures give insight into how the kinetochore couples to dynamic microtubules, a process crucial to the accurate segregation of chromosomes. HURP and kinesin-13 arrange tubulin into sleeves and bracelets surrounding the microtubule lattice. These structures might reflect the ability of these proteins to modulate microtubule dynamics by interacting with specialized tubulin configurations. In this review, we compare and contrast the structure of these proteins and their interactions with microtubules to illustrate how they attach to and modulate the dynamics of microtubules.     

The essentiality of the fungus-specific Dam1 complex is correlated with a one-kinetochore-one-microtubule interaction present throughout the cell cycle, independent of the nature of a centromere

A fungus-specific outer kinetochore complex, the Dam1 complex, is essential in Saccharomyces cerevisiae, nonessential in fission yeast, and absent from metazoans. The reason for the reductive evolution of the functionality of this complex remains unknown. Both Candida albicans and Schizosaccharomyces pombe have regional centromeres as opposed to the short-point centromeres of S. cerevisiae. The interaction of one microtubule per kinetochore is established both in S. cerevisiae and C. albicans early during the cell cycle, which is in contrast to the multiple microtubules that bind to a kinetochore only during mitosis in S. pombe. Moreover, the Dam1 complex is associated with the kinetochore throughout the cell cycle in S. cerevisiae and C. albicans but only during mitosis in S. pombe. Here, we show that the Dam1 complex is essential for viability and indispensable for proper mitotic chromosome segregation in C. albicans. The kinetochore localization of the Dam1 complex is independent of the kinetochore-microtubule interaction, but the function of this complex is monitored by a spindle assembly checkpoint. Strikingly, the Dam1 complex is required to prevent precocious spindle elongation in premitotic phases. Thus, constitutive kinetochore localization associated with a one-microtubule-one kinetochore type of interaction, but not the length of a centromere, is correlated with the essentiality of the Dam1 complex.     

Protein arms in the kinetochore-microtubule interface of the yeast DASH complex

The yeast DASH complex is a heterodecameric component of the kinetochore necessary for accurate chromosome segregation. DASH forms closed rings around microtubules with a large gap between the DASH ring and the microtubule cylinder. We characterized the microtubule-binding properties of limited proteolysis products and subcomplexes of DASH, thus identifying candidate polypeptide extensions involved in establishing the DASH-microtubule interface. The acidic C-terminal extensions of tubulin subunits are not essential for DASH binding. We also measured the molecular mass of DASH rings on microtubules with scanning transmission electron microscopy and found that approximately 25 DASH heterodecamers assemble to form each ring. Dynamic association and relocation of multiple flexible appendages of DASH may allow the kinetochore to translate along the microtubule surface.     

The Dam1 complex confers microtubule plus end–tracking activity to the Ndc80 kinetochore complex

Kinetochores must remain associated with microtubule ends, as they undergo rapid transitions between growth and shrinkage. The molecular basis for this essential activity that ensures correct chromosome segregation is unclear. In this study, we have used reconstitution of dynamic microtubules and total internal reflection fluorescence microscopy to define the functional relationship between two important budding yeast kinetochore complexes. We find that the Dam1 complex is an autonomous plus end–tracking complex. The Ndc80 complex, despite being structurally related to the general tip tracker EB1, fails to recognize growing ends efficiently. Dam1 oligomers are necessary and sufficient to recruit Ndc80 to dynamic microtubule ends, where both complexes remain continuously associated. The interaction occurs specifically in the presence of microtubules and is subject to regulation by Ipl1 phosphorylation. These findings can explain how the force harvested by Dam1 is transmitted to the rest of the kinetochore via the Ndc80 complex.     

Kinetochore protein interactions and their regulation by the Aurora kinase Ipl1p

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.     

Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B

The coupling of kinetochores to dynamic spindle microtubules is crucial for chromosome positioning and segregation, error correction, and cell cycle progression. How these fundamental attachments are made and persist under tensile forces from the spindle remain important questions. As microtubule-binding elements, the budding yeast Ndc80 and Dam1 kinetochore complexes are essential and not redundant, but their distinct contributions are unknown. In this study, we show that the Dam1 complex is a processivity factor for the Ndc80 complex, enhancing the ability of the Ndc80 complex to form load-bearing attachments to and track with dynamic microtubule tips in vitro. Moreover, the interaction between the Ndc80 and Dam1 complexes is abolished when the Dam1 complex is phosphorylated by the yeast aurora B kinase Ipl1. This provides evidence for a mechanism by which aurora B resets aberrant kinetochore–microtubule attachments. We propose that the action of the Dam1 complex as a processivity factor in kinetochore–microtubule attachment is regulated by conserved signals for error correction.     

Sister kinetochore recapture in fission yeast occurs by two distinct mechanisms, both requiring Dam1 and Klp2

In eukaryotic cells, proper formation of the spindle is necessary for successful cell division. We have studied chromosome recapture in the fission yeast Schizosaccharomyces pombe. We show by live cell analysis that lost kinetochores interact laterally with intranuclear microtubules (INMs) and that both microtubule depolymerization (end-on pulling) and minus-end-directed movement (microtubule sliding) contribute to chromosome retrieval to the spindle pole body (SPB). We find that the minus-end-directed motor Klp2 colocalizes with the kinetochore during its transport to the SPB and contributes to the effectiveness of retrieval by affecting both end-on pulling and lateral sliding. Furthermore, we provide in vivo evidence that Dam1, a component of the DASH complex, also colocalizes with the kinetochore during its transport and is essential for its retrieval by either of these mechanisms. Finally, we find that the position of the unattached kinetochore correlates with the size and orientation of the INMs, suggesting that chromosome recapture may not be a random process.     

Chmp4c is required for stable kinetochore-microtubule attachments

Formation of stable kinetochore-microtubule attachments is essential for accurate chromosome segregation in human cells and depends on the NDC80 complex. We recently showed that Chmp4c, an endosomal sorting complex required for transport protein involved in membrane remodelling, localises to prometaphase kinetochores and promotes cold-stable kinetochore microtubules, faithful chromosome alignment and segregation. In the present study, we show that Chmp4c associates with the NDC80 components Hec1 and Nuf2 and is required for optimal NDC80 stability and Hec1-Nuf2 localisation to kinetochores in prometaphase. However, Chmp4c-depletion does not cause a gross disassembly of outer or inner kinetochore complexes. Conversely, Nuf2 is required for Chmp4c kinetochore targeting. Constitutive Chmp4c kinetochore tethering partially rescues cold-stable microtubule polymers in cells depleted of the endogenous Nuf2, showing that Chmp4c also contributes to kinetochore-microtubule stability independently of regulating Hec1 and Nuf2 localisation. Chmp4c interacts with tubulin in cell extracts, and binds and bundles microtubules in vitro through its highly basic N-terminal region (amino acids 1–77). Furthermore, the N-terminal region of Chmp4c is required for cold-stable kinetochore microtubules and efficient chromosome alignment. We propose that Chmp4c promotes stable kinetochore-microtubule attachments by regulating Hec1–Nuf2 localisation to kinetochores in prometaphase and by binding to spindle microtubules. These results identify Chmp4c as a novel protein that regulates kinetochore-microtubule interactions to promote accurate chromosome segregation in human cells.     

Microtubule binding by KNL-1 contributes to spindle checkpoint silencing at the kinetochore

Accurate chromosome segregation requires coordination between microtubule attachment and spindle checkpoint signaling at the kinetochore. The kinetochore-localized KMN (KNL-1/Mis12 complex/Ndc80 complex) network, which mediates microtubule attachment and scaffolds checkpoint signaling, harbors two distinct microtubule-binding activities: the load-bearing activity of the Ndc80 complex and a less well-understood activity in KNL-1. In this paper, we show that KNL-1 microtubule-binding and -bundling activity resides in its extreme N terminus. Selective perturbation of KNL-1 microtubule binding in Caenorhabditis elegans embryos revealed that this activity is dispensable for both load-bearing attachment formation and checkpoint activation but plays a role in checkpoint silencing at the kinetochore. Perturbation of both microtubule binding and protein phosphatase 1 docking at the KNL-1 N terminus additively affected checkpoint silencing, indicating that, despite their proximity in KNL-1, these two activities make independent contributions. We propose that microtubule binding by KNL-1 functions in checkpoint silencing by sensing microtubules attached to kinetochores and relaying their presence to eliminate generation of the checkpoint signal.     

Microtubules: a ring for the depolymerization motor

Newly discovered rings around microtubules, assembled from the Dam1 protein complex, may provide the dynamic linkage at microtubule ends for force generation coupled to microtubule depolymerization and polymerization.     

Microtubule polymerases and depolymerases

The variety of shapes and sizes of the microtubule cytoskeleton is as great as the number of different cell types. This large variety is a consequence of the dynamic properties of microtubules, which allow them to adopt distributions of arbitrary size and form. How is the distribution of microtubule lengths controlled? Recent work suggests that the length distribution is controlled, at least in part, by the activity of microtubule polymerases and depolymerases, which accelerate microtubule growth and shrinkage. Specifically, biochemical and single-molecule studies have shown how MCAK (kinesin-13) and Kip3p (kinesin-8) accelerate depolymerization and how XMAP215 may accelerate growth. Studies on the yeast Dam1 complex have shown how proteins can couple a cellular structure, the kinetochore, to the ends of polymerizing and depolymerizing microtubules.