The yeast DASH complex forms closed rings on microtubules

The Saccharomyces cerevisiae DASH complex is an essential microtubule-binding component of the kinetochore. We coexpressed all ten subunits of this assembly in Escherichia coli and purified a single complex, a approximately 210-kDa heterodecamer with an apparent stoichiometry of one copy of each subunit. The hydrodynamic properties of the recombinant assembly are indistinguishable from those of the native complex in yeast extracts. The structure of DASH alone and bound to microtubules was visualized by EM. The free heterodecamer is relatively globular. In the presence of microtubules, DASH oligomerizes to form rings and paired helices that encircle the microtubules. We discuss potential roles for such collar-like structures in maintaining microtubule attachment and spindle integrity during chromosome segregation.     

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.     

Formation of a dynamic kinetochore- microtubule interface through assembly of the Dam1 ring complex

How kinetochore proteins form a dynamic interface with microtubules is largely unknown. In budding yeast, the 10-protein Dam1 complex is an Aurora kinase target that plays essential roles maintaining the integrity of the mitotic spindle and regulating interactions with the kinetochore. Here, we investigated the biochemical properties of purified Dam1 complex. The complex oligomerized into rings around microtubules. Ring formation was facilitated by microtubules but could occur in their absence. Mutant alleles led to partially assembled complexes or reduced microtubule binding. The interaction between rings and microtubules is mediated by the C termini of both Dam1 and alphabeta-tubulin. Ring formation promotes microtubule assembly, stabilizes against disassembly, and promotes bundling. A GTP-tubulin lattice is the preferred binding partner for the complex, and Dam1 rings can exhibit lateral mobility on microtubules. These observations suggest a mechanism by which the kinetochore can recognize and stay attached to the plus ends of microtubules.     

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.     

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.     

Meiosis in Drosophila melanogaster. II. The prometaphase-I kinetochore microtubule bundle and kinetochore orientation in males

Fourteen prometaphase kinetochore microtubule bundles have been examined in electron micrographs of serial sections. The majority (54%) of the microtubules extended from the polar region towards the kinetochore but do not end in the kinetochore proper. Rather, they stop short of the kinetochore (21%), graze the kinetochore (19%), or pass through the kinetochore (9%), displaying a free end distal to the pole. Other microtubules that make up the kinetochore bundle include: kinetochore-to-pole microtubules (24%), chromosome-to-pole microtubules (5%), pieces with two free ends (14%), and those microtubules with one end in the kinetochore and a free end distal to the kinetochore (9%). We conclude that the majority of the microtubules in the kinetochore bundle are most likely of polar origin rather than having been nucleated at the kinetochore. Prometaphase-I kinetochores can display any one of four patterns of microtubule connections with the poles, but the pattern of microtubule connections is not always correlated with kinetochore position. For instance, a kinetochore directly facing one pole may have microtubule connections with both poles while a kinetochore positioned 90 degrees to the spindle axis may have microtubules running towards one pole only.     

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.     

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.     

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.     

Microtubules,chromosome movement,and reorientation after chromosomes are detached from the spindle by micromanipulation

The relationship between chromosome movement and mirotubules was explored by combining micromanipulation of living grasshopper spermatocytes with electron microscopy. We detached chromosomes from the spindle and placed them far out in the cytoplasm. Soon, the chromosomes began to move back toward the spindle and the cells were fixed at a chosen moment. The microtubules seen in three-dimensional reconstructions were correlated with the chromosome movement just prior to fixation. Before movement began, detached chromosomes had no kinetochore microtubules or a single one at most. Renewed movement was always accompanied by the reappearance of kinetochore microtubules; a single kinetochore microtubule appeared to suffice. Chromosome movements and kinetochore microtubule arrangements were unusual after reattachment, but their relationship was not: poleward forces, parallel to the kinetochore microtubule axis (as in normal anaphase), would explain the movement, however odd. The initial arrangement of kinetochore microtubules would have led to aberrant chromosome distribution if it persisted, but instead, reorientation to the appropriate arrangement always followed. Observations on living cells permitted us to place in sequence the kinetochore microtubule arrangements seen in fixed cells, revealing the microtubule transformations during reorientation. From the sequence of events we conclude that chromosome movement can cause reorientation to begin and that in the changes which follow, an unstable attachment of kinetochore microtubules to the spindle plays a major role.     

Polarity of kinetochore microtubules in Chinese hamster ovary cells after recovery from a colcemid block

The polarity of kinetochore microtubules was determined in a system for which kinetochore-initiated microtubule assembly has been demonstrated. Chinese hamster ovary cells were treated with 0.3 micrograms/ml colcemid for 8 h and then released from the block. Prior to recovery, microtubules were completely absent from the cells. The recovery was monitored using light and electron microscopy to establish that the cells progress through anaphase and that the kinetochore fibers are fully functional. Since early stages of recovery are characterized by short microtubule segments that terminate in the kinetochore fibrous corona rather than on the outer disk, microtubule polarity was determined at later stages of recovery when longer kinetochore bundles had formed, allowing us to establish unambiguously the spatial relationship between microtubules, kinetochores, and chromosomes. The cells were lysed in a detergent mixture containing bovine brain tubulin under conditions that allowed the formation of polarity-revealing hooks. 20 kinetochore bundles were assayed for microtubule polarity in either thick or thin serial sections. We found that 95% of the decorated kinetochore microtubules had the same polarity and that, according to the hook curvature, the plus ends of the microtubules were at the kinetochores. Hence, the polarity of kinetochore microtubules in Chinese hamster ovary cells recovering from a colcemid block is the same as in normal untreated cells. This result suggests that microtubule polarity is likely to be important for spindle function since kinetochore microtubules show the same polarity, regardless of the pattern of spindle formation.     

Cell division and the microtubular cytoskeleton]

Kinetochore microtubules result from an interaction between astral microtubules and the kinetochore of the chromosomes after breakdown of the nuclear envelope at the end of prophase. In this process, the end of a microtubule projecting from one of the polar regions contacts the primary constriction of a chromosome. The latter then undergoes rapid poleward movement. Concerning the mechanism of anaphase chromosome movement, the motive force for the chromosome-to-pole movement appears to be generated at the kinetochore or in the region very close to it. It has not been determined whether chromosomes propel themselves along stationary kinetochore microtubules by a motor at the kinetochore, or they are pulled poleward by a traction fiber consisting of kinetochore microtubules and associated motors. As chromosomes move poleward coordinate disassembly of kinetochore microtubules might occur from their kinetochore ends. In diatom and yeast spindles, elongation of the spindle in anaphase (anaphase B) may be explained by microtubule assembly at polar microtubule ends in the spindle mid-zone and sliding of the antiparallel microtubules from the opposite poles. The sliding force appears to be generated through an ATP-dependent microtubule motor. In isolated sea urchin spindles, the microtubule assembly at the equator alone might provide the force for spindle elongation, although, in addition, involvement of microtubule sliding by a GTP-requiring mechanochemical enzyme cannot be excluded. Discussions were made on possible participation in anaphase chromosome movement of such microtubule motors as dynein, kinesin, dynamin and the claret segregation protein.     

Microtubule dynamics in the chromosomal spindle fiber: analysis by fluorescence and high-resolution polarization microscopy

We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24 degrees C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24 degrees C. In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into "rods" composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.     

Microtubule-dependent Changes in Assembly of Microtubule Motor Proteins and Mitotic Spindle Checkpoint Proteins at PtK1 Kinetochores

The ability of kinetochores to recruit microtubules, generate force, and activate the mitotic spindle checkpoint may all depend on microtubule- and/or tension-dependent changes in kinetochore assembly. With the use of quantitative digital imaging and immunofluorescence microscopy of PtK1 tissue cells, we find that the outer domain of the kinetochore, but not the CREST-stained inner core, exhibits three microtubule-dependent assembly states, not directly dependent on tension. First, prometaphase kinetochores with few or no kinetochore microtubules have abundant punctate or oblate fluorescence morphology when stained for outer domain motor proteins CENP-E and cytoplasmic dynein and checkpoint proteins BubR1 and Mad2. Second, microtubule depolymerization induces expansion of the kinetochore outer domain into crescent and ring morphologies around the centromere. This expansion may enhance recruitment of kinetochore microtubules, and occurs with more than a 20- to 100-fold increase in dynein and relatively little change in CENP-E, BubR1, and Mad2 in comparison to prometaphase kinetochores. Crescents disappear and dynein decreases substantially upon microtubule reassembly. Third, when kinetochores acquire their full metaphase complement of kinetochore microtubules, levels of CENP-E, dynein, and BubR1 decrease by three- to sixfold in comparison to unattached prometaphase kinetochores, but remain detectable. In contrast, Mad2 decreases by 100-fold and becomes undetectable, consistent with Mad2 being a key factor for the "wait-anaphase" signal produced by unattached kinetochores. Like previously found for Mad2, the average amounts of CENP-E, dynein, or BubR1 at metaphase kinetochores did not change with the loss of tension induced by taxol stabilization of microtubules.     

Tension,microtubule rearrangements,and the proper distribution of chromosomes in mitosis

The basis for stable versus unstable kinetochore orientation was investigated by a correlated living-cell/ultrastructural study of grasshopper spermatocytes. Mal-oriented bivalents having both kinetochores oriented to one spindle pole were induced by micromanipulation. Such malorientations are stable while the bivalent is subject to tension applied by micromanipulation but unstable after tension is released. Unstable bivalents always reorient with movement of one kinetochore toward the opposite pole. Microtubules associated with stably oriented bivalents, whether they are mal-oriented or in normal bipolar orientation, are arranged in orderly parallel bundles running from each kinetochore toward the pole. Similar orderly kinetochore microtubule arrangements characterize mal-oriented bivalents fixed just after release of tension. A significantly different microtubule arrangement is found only some time after tension release, when kinetochore movement is evident. The microtubules of a reorienting kinetochore always include a small number of microtubules running toward the pole toward which the kinetochore was moving at the time of fixation. All other microtubules associated with such a moving kinetochore appear to have lost their anchorage to the original pole and to be dragged passively as the kinetochore proceeds to the other pole. Thus, the stable anchorage of kinetochore microtubules to the spindle is associated with tension force and unstable anchorage with the absence of tension. The effect of tension is readily explained if force production and anchorage are both produced by mitotic motors, which link microtubules to the spindle as they generate tension forces.     

The total length of spindle microtubules depends on the number of chromosomes present

We extracted chromosomes by micromanipulation from Melanoplus differentialis spermatocytes, producing metaphase spindles with only one or a few chromosomes instead of the usual complement of 23. Cells with various numbers of chromosomes were prepared for electron microscopy, and spindle microtubule length was measured. A constant increment of microtubule length was lost upon the removal of each chromosome; we estimate that only approximately 40% of the original length would remain in the total absence of chromosomes. Unexpectedly, kinetochore microtubules were not the only ones affected when chromosomes were removed: nonkinetochore microtubules accounted for a substantial fraction of the total length lost. No compensatory increase in microtubule length outside the spindle was found. Studies by others show that the kinetochore microtubules of extracted chromosomes are left behind in the cell and dissassemble. The resulting increase in subunit concentration would be expected from in vitro studies to drive microtubule assembly until the original total microtubule length was restored, but that did not happen in these living cells. We conclude that the assembly of a certain, large fraction of microtubule subunits into stable microtubules is dependent on the presence of chromosomes. Possible explanations include (a) limits on microtubule length that prevent any net assembly of the subunits released after chromosomes are removed or (b) a promotion of microtubule assembly by chromosomes, which therefore is reduced in their absence. Chromosome-dependent regulation of microtubule length may account for some features of normal mitosis.     

Kinetochore microtubule dynamics and attachment stability are regulated by Hec1

Mitotic cells face the challenging tasks of linking kinetochores to growing and shortening microtubules and actively regulating these dynamic attachments to produce accurate chromosome segregation. We report here that Ndc80/Hec1 functions in regulating kinetochore microtubule plus-end dynamics and attachment stability. Microinjection of an antibody to the N terminus of Hec1 suppresses both microtubule detachment and microtubule plus-end polymerization and depolymerization at kinetochores of PtK1 cells. Centromeres become hyperstretched, kinetochore fibers shorten from spindle poles, kinetochore microtubule attachment errors increase, and chromosomes severely mis-segregate. The N terminus of Hec1 is phosphorylated by Aurora B kinase in vitro, and cells expressing N-terminal nonphosphorylatable mutants of Hec1 exhibit an increase in merotelic attachments, hyperstretching of centromeres, and errors in chromosome segregation. These findings reveal a key role for the Hec1 N terminus in controlling dynamic behavior of kinetochore microtubules.     

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.     

Spindle microtubules and their mechanical associations after micromanipulation in anaphase

Micromanipulation of living grasshopper spermatocytes in anaphase has been combined with electron microscopy to reveal otherwise obscure features of spindle organization. A chromosome is pushed laterally outside the spindle and stretched, and the cell is fixed with a novel, agar-treated glutaraldehyde solution. Two- and three-dimensional reconstructions from serial sections of seven cells show that kinetochore microtubules of the manipulated chromosome are shifted outside the confusing thicket of spindle microtubules and mechanical associations among microtubules are revealed by bent or shifted microtubules. These are the chief results: (a) The disposition of microtubules invariably is consistent with a skeletal role for spindle microtubules. (b) The kinetochore microtubule bundle is composed of short and long microtubules, with weak but recognizable mechanical associations among them. Some kinetochore microtubules are more tightly linked to one other microtubule within the bundle. (c) Microtubules of the kinetochore microtubule bundle are firmly connected to other spindle microtubules only near the pole, although some nonkinetochore microtubules of uncertain significance enter the bundle nearer to the kinetochore. (d) The kinetochore microtubules of adjacent chromosomes are mechanically linked, which provides an explanation for interdependent chromosome movement in "hinge anaphases." In the region of the spindle open to analysis after chromosome micromanipulation, microtubules may be linked mechanically by embedment in a gel, rather than by dynein or other specific, cross-bridging molecules.