Kip3, the yeast kinesin-8, is required for clustering of kinetochores at metaphase

In Saccharomyces cerevisiae, chromosome congression clusters kinetochores on either side of the spindle equator at metaphase. Many organisms require one or more kinesin-8 molecular motors to achieve chromosome alignment. The yeast kinesin-8, Kip3, has been well studied in vitro but a role in chromosome congression has not beenreported. We investigated Kip3's role in this process using semi-automated, quantitative fluorescence microscopy and time-lapse imaging and found that Kip3 is required for congression. Deletion of KIP3 increases inter-kinetochore distances and increases the variability in the position of sister kinetochores along the spindle axis during metaphase. Kip3 does not regulate spindle length and is not required for kinetochore-microtubule attachment. Instead, Kip3 clusters kinetochores on the metaphase spindle by tightly regulating kinetochore microtubule lengths.     

Plus end-specific depolymerase activity of Kip3, a kinesin-8 protein, explains its role in positioning the yeast mitotic spindle

The budding yeast protein Kip3p is a member of the conserved kinesin-8 family of microtubule motors, which are required for microtubule-cortical interactions, normal spindle assembly and kinetochore dynamics. Here, we demonstrate that Kip3p is both a plus end-directed motor and a plus end-specific depolymerase--a unique combination of activities not found in other kinesins. The ATPase activity of Kip3p was activated by both microtubules and unpolymerized tubulin. Furthermore, Kip3p in the ATP-bound state formed a complex with unpolymerized tubulin. Thus, motile kinesin-8s may depolymerize microtubules by a mechanism that is similar to that used by non-motile kinesin-13 proteins. Fluorescent speckle analysis established that, in vivo, Kip3p moved toward and accumulated on the plus ends of growing microtubules, suggesting that motor activity brings Kip3p to its site of action. Globally, and more dramatically on cortical contact, Kip3p promoted catastrophes and pausing, and inhibited microtubule growth. These findings explain the role of Kip3p in positioning the mitotic spindle in budding yeast and potentially other processes controlled by kinesin-8 family members.     

Tension-dependent regulation of microtubule dynamics at kinetochores can explain metaphase congression in yeast

During metaphase in budding yeast mitosis, sister kinetochores are tethered to opposite poles and separated, stretching their intervening chromatin, by singly attached kinetochore microtubules (kMTs). Kinetochore movements are coupled to single microtubule plus-end polymerization/depolymerization at kinetochore attachment sites. Here, we use computer modeling to test possible mechanisms controlling chromosome alignment during yeast metaphase by simulating experiments that determine the 1) mean positions of kinetochore Cse4-GFP, 2) extent of oscillation of kinetochores during metaphase as measured by fluorescence recovery after photobleaching (FRAP) of kinetochore Cse4-GFP, 3) dynamics of kMTs as measured by FRAP of GFP-tubulin, and 4) mean positions of unreplicated chromosome kinetochores that lack pulling forces from a sister kinetochore. We rule out a number of possible models and find the best fit between theory and experiment when it is assumed that kinetochores sense both a spatial gradient that suppresses kMT catastrophe near the poles and attachment site tension that promotes kMT rescue at higher amounts of chromatin stretch.     

Mechanisms underlying the dual-mode regulation of microtubule dynamics by Kip3/kinesin-8

The kinesin-8 family of microtubule motors plays?a critical role in microtubule length control in cells. These motors have complex effects on microtubule dynamics: they destabilize growing microtubules yet stabilize shrinking microtubules. The budding yeast kinesin-8, Kip3, accumulates on plus ends of growing but not shrinking microtubules. Here we identify an essential role of the tail domain of Kip3 in mediating both its destabilizing and its stabilizing activities. The Kip3 tail promotes Kip3's accumulation at the plus ends and facilitates the destabilizing effect of Kip3. However, the Kip3 tail also inhibits microtubule shrinkage and is required for promoting microtubule rescue by Kip3. These effects of the tail domain are likely to be mediated by the tubulin- and microtubule-binding activities that we describe. We propose a concentration-dependent model for the coordination of the destabilizing and stabilizing activities of Kip3 and discuss its relevance to cellular microtubule organization.     

The highly processive kinesin-8, Kip3, switches microtubule protofilaments with a bias toward the left

Kinesin-1 motor proteins walk parallel to the protofilament axes of microtubules as they step from one tubulin dimer to the next. Is protofilament tracking an inherent property of processive kinesin motors, like kinesin-1, and what are the structural determinants underlying protofilament tracking? To address these questions, we investigated the tracking properties of the processive kinesin-8, Kip3. Using in vitro gliding motility assays, we found that Kip3 rotates microtubules counterclockwise around their longitudinal axes with periodicities of ∼1 μm. These rotations indicate that the motors switch protofilaments with a bias toward the left. Molecular modeling suggests 1), that the protofilament switching may be due to kinesin-8 having a longer neck linker than kinesin-1, and 2), that the leftward bias is due the asymmetric geometry of the motor neck linker complex.The founding member of the kinesin superfamily, the cargo-transporting kinesin-1, has been studied in great detail. Dimeric kinesin-1 constructs 1) are mechanical processive, taking ∼100 of 8-nm steps in a hand-over-hand fashion without detaching from the microtubule; and 2), walk parallel to the axis of microtubule protofilaments as they step from one tubulin dimer to the next. The latter was inferred from gliding motility assays, where microtubules propelled by motors bound to a planar substrate surface rotated around their longitudinal axis with periodicities corresponding to the helical course of the protofilaments in supertwisted microtubules (1,2). Interestingly, protofilament tracking of kinesin-1 is lost in nonprocessive, monomeric constructs (3). There, and also for other nonprocessive microtubule motors such as the kinesin-14 Ncd (4) or axonemal dynein (5), significantly shorter pitches of microtubule rotations in gliding motility assays were observed. As suggested previously (6) this may indicate that protofilament tracking is an inherent property of processive microtubule motors.To explore this idea further, we investigated the rotations of 14-protofilament microtubules (left-handed helical pitch of ∼8 μm (2)) in gliding motility assays using kinesin-8 that has been observed to perform ≈12 μm-long processive runs in vitro (7). Streptavidin-coated quantum dots (QDs), sparsely bound to the microtubules, served as reporters of microtubule rotations (Fig. 1 A). Information on the three-dimensional paths of the QDs—and thus on microtubule rotations—were obtained from 1), two-dimensional tracking of the QDs with nanometer precision in x and y (8), in combination with 2), z information derived from fluorescence-interference contrast (FLIC) (2) (Fig. 1, B–D). FLIC originates from destructive and constructive interference effects close to reflecting surfaces and gives rise to a modulation of the detected intensity of a fluorescent object depending on its height above the surface. Specifically, the microtubule-attached QDs appear dark when they are in close proximity to the surface (i.e., when being located between the microtubule and the surface) but brighten up significantly when being further away (i.e., when on the microtubule lattice pointing away from the surface). In our experiments, we observed counterclockwise rotations (looking from the trailing microtubule plus-end in the direction toward the leading minus-end) with an average pitch of 0.93 μm ± 0.20 μm (mean ± SD, N = 75; N is the number of complete rotations obtained from 15 gliding microtubules). Considering the geometry of the assay, the counterclockwise directionality of the rotations corresponds to the motors stepping with a perpetual bias (∼1 protofilament switch event per forward movement over 10 tubulin dimers) toward the left.Open in a separate windowFigure 1Monitoring Kip3-driven microtubule rotations in gliding motility assays. (A) Schematic of the experimental setup. Imaging is performed on top of a reflective silicon surface using fluorescence interference contrast (FLIC) microscopy (2). (B) Maximum projection of the fluorescence signal of a microtubule-attached quantum dot in the Kip3 gliding motility assay. (C) FLIC intensity (red) and lateral distance from the microtubule path (blue) of the quantum dot shown in panel B versus traveled distance along the microtubule path. The periodic FLIC signal is indicative of repeated up- and down-motion. (D) Schematic of the deduced Kip3 path (red) in comparison to the protofilament axis (green) on a 14-protofilament microtubule.The behavior observed in our experiments is in stark contrast to kinesin-1, for which counterclockwise rotations with an average pitch of 7.9 μm were previously observed using the same experimental technique (2). Consequently, the question arises: which structural determinants decide whether a kinesin acts as a strict protofilament tracker (and—if it does not—from where the directional bias of the off-axis stepping originates)? Assuming motility in a hand-over-hand fashion, it will matter which binding sites on the microtubule lattice are within reach of the forward swinging motor head. This reach is primarily set by the neck linkers, the structural elements that connect the two motor heads to the coiled-coil neck domain. More precisely, the reach is a function of the length of the neck linkers and their three-dimensional path dictated by the volumes that are occupied by the motor heads when bound to the microtubule. Based on primary sequence alignment between Kip3 with other members of the kinesin-8 family and prediction of the start of the coiled-coil dimerization domain with the program PCOILS, we assigned the neck linker region to the amino acids K436–H452 (i.e., 17 amino acids). Accounting for neck linker docking of the rear motor head (K436–Q447) (9), the corresponding length of the neck linkers between both heads, composed of five amino acids from the undocked part of the rear-head neck linker and 17 amino acids from the front-head neck linker, is estimated to be 85 Å (see the Supporting Material).We then modeled all configurations of Kip3 with both heads bound simultaneously to adjacent tubulin dimers (Fig. 2, A and B, and see the Supporting Material). The estimated three-dimensional distances between the positions where the neck linkers protrude from the motor heads, respecting the volumes of the heads (Fig. 2 C, gray column; see also the Supporting Material), are measures for the minimally required neck linker lengths for each two-head bound configuration. Comparison between the three-dimensional distances obtained from the model and the available neck linker length (85 Å) suggests that a forward-swinging Kip3 head can most readily reach the tubulin dimer in the front (53 Å needed) and can switch to the protofilament on the left (79 Å for left and 93 Å for front-left needed), but it has difficulties in stepping to the protofilament on the right, which would require a longer neck-linker than it actually exhibits (103 Å for front-right and 105 Å for right needed). The main reason why these long neck-linker distances are required (i.e., >100 Å) is that, to reach the tubulin dimer on the right (or front-right), the neck linker has to bend over the humpy back of the front head (see Fig. 2 B, right and front-right). On the contrary, to reach the tubulin dimer on the left (or front-left), this detour is avoided (see Fig. 2 B, left and front-left). The model-derived preference for left-stepping over right-stepping is in agreement with our experimental observations.Open in a separate windowFigure 2Virtual three-dimensional reconstruction of Kip3 stepping. (A) Tubulin dimer: composed of alpha-tubulin (α) and beta-tubulin (β) monomers, with the unstructured surface-exposed E-Hooks (e). Kip3 front head: Shown with undocked neck linker (U) and following coiled-coil (cc) dimerization domain. Kip3 rear head: Shown with docked (D) and undocked (U) neck linker parts. (B) Illustration of different Kip3 configurations bound with both heads to adjacent tubulin dimers (first heptad repeat of the coiled coil region is artificially unfolded to illustrate all binding configurations). (C) Estimated three-dimensional distances between the positions where the neck linkers protrude from the motor heads, respecting the volumes of the heads (nomenclature as in panel B). For comparison, the direct distances between the tubulin dimers are given.Modeling as described can be applied for kinesin-1, whose neck linkers are three-amino-acids shorter than the neck linkers of Kip3. Whereas the modeled minimally required neck linker lengths for each two-head-bound configuration are almost identical to the values for Kip3, we estimate an available neck linker length of 63 Å (see the Supporting Material), which explains the strict forward stepping of kinesin-1.In summary, we have shown what to our knowledge is the first example of a highly processive kinesin motor (run length of several μm) switching between protofilaments of microtubules. Our modeling suggests that protofilament switching may be due to kinesin-8 having a longer neck linker than kinesin-1 so that it is able to reach the extra distance required to change protofilaments. The leftward bias cannot be explained by the geometry of the microtubule lattice alone (Fig. 2 C, last column) but follows from the additional consideration of the asymmetric geometry of the motor neck linker complex. A leftward torque component, which may be present in the powerstrokes of the individual heads (3,4), may further promote the leftward bias but is not strictly necessary. While our results were under review, left-handed spiraling along microtubules of beads coated with a modified kinesin-1 (with extended neck linkers (10)) was reported (11); the handedness of the bead rotations is consistent with the handedness of our microtubule rotations and our model.Our results may also provide an alternative explanation for the short-pitch, counterclockwise rotations of microtubules gliding on surfaces coated by dimeric kinesin-5 (Eg5) motors (6). The authors of this report attributed the short pitch to the low processivity of Eg5, arguing that during processive episodes the motor follows the protofilament axis, but when detaching generates an off-axis force leading to microtubule rotation. Considering the structure of Eg5 (neck linker length of 18 amino acids (12)), protofilament switching may, however, also be possible during the processive episodes. For kinesin-2 (neck linker length of 17 amino acids, although reduced in length by ∼5 Å due to proline in cis-conformation at position 13 (12,13)), the propensity to switch protofilaments is controversially discussed and may depend on the stability of the neck domain (11,14).Previously, Kip3, has been found to depolymerize microtubules in a length-dependent manner (7). The underlying mechanism has been described by an antenna model, where Kip3 binds along the entire microtubule lattice and subsequently walks to the microtubule plus-end relying on its high processivity that is ∼20 times the run length of kinesin-1. During such long runs, motors in vivo are expected to frequently encounter obstacles, such as microtubule-associated proteins. In the case of kinesin-1, shown to follow the microtubule''s protofilament axis (1), obstacles cause motor stalling or accelerated detachment. It is exciting to speculate that Kip3 uses protofilament switching to bypass obstacles on the microtubule surface avoiding premature motor release or stalling that could reduce the efficiency of targeting and subsequent depolymerization of the microtubule plus-ends.     

The kinesin-8 Kip3 scales anaphase spindle length by suppression of midzone microtubule polymerization

Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation.     

Factors required for the binding of reassembled yeast kinetochores to microtubules in vitro

Kinetochores are structures that assemble on centromeric DNA and mediate the attachment of chromosomes to the microtubules of the mitotic spindle. The protein components of kinetochores are poorly understood, but the simplicity of the S. cerevisiae kinetochore makes it an attractive candidate for molecular dissection. Mutations in genes encoding CBF1 and CBF3, proteins that bind to yeast centromeres, interfere with chromosome segregation in vivo. To determine the roles played by these factors and by various regions of centromeric DNA in kinetochore function, we have developed a method to partially reassemble kinetochores on exogenous centromeric templates in vitro and to visualize the attachment of these reassembled kinetochore complexes to microtubules. In this assay, single reassembled complexes appear to mediate microtubule binding. We find that CBF3 is absolutely essential for this attachment but, contrary to previous reports (Hyman, A. A., K. Middleton, M. Centola, T.J. Mitchison, and J. Carbon. 1992. Microtubule- motor activity of a yeast centromere-binding protein complex. Nature (Lond.). 359:533-536) is not sufficient. Additional cellular factors interact with CBF3 to form active microtubule-binding complexes. This is mediated primarily by the CDEIII region of centromeric DNA but CDEII plays an essential modulatory role. Thus, the attachment of kinetochores to microtubules appears to involve a hierarchy of interactions by factors that assemble on a core complex consisting of DNA-bound CBF3.     

Fission yeast kinesin-8 Klp5 and Klp6 are interdependent for mitotic nuclear retention and required for proper microtubule dynamics

Fission yeast has two kinesin-8s, Klp5 and Klp6, which associate to form a heterocomplex. Here, we show that Klp5 and Klp6 are mutually dependent on each other for nuclear mitotic localization. During interphase, they are exported to the cytoplasm. In sharp contrast, during mitosis, Klp5 and Klp6 remain in the nucleus, which requires the existence of each counterpart. Canonical nuclear localization signal (NLS) is identified in the nonkinesin C-terminal regions. Intriguingly individual NLS mutants (NLSmut) exhibit loss-of-function phenotypes, suggesting that Klp5 and Klp6 enter the nucleus separately. Indeed, although neither Klp5-NLSmut nor Klp6-NLSmut enters the nucleus, wild-type Klp6 or Klp5, respectively, does so with different kinetics. In the absence of Klp5/6, microtubule catastrophe/rescue frequency and dynamicity are suppressed, whereas growth and shrinkage rates are least affected. Remarkably, chimera strains containing only the N-terminal Klp5 kinesin domains cannot disassemble interphase microtubules during mitosis, leading to the coexistence of cytoplasmic microtubules and nuclear spindles with massive chromosome missegregation. In this strain, a marked reduction of microtubule dynamism, even higher than in klp5/6 deletions, is evident. We propose that Klp5 and Klp6 play a vital role in promoting microtubule dynamics, which is essential for the spatiotemporal control of microtubule morphogenesis.     

Recruitment of Mad1 to metaphase kinetochores is sufficient to reactivate the mitotic checkpoint

The mitotic checkpoint monitors kinetochore–microtubule attachment and prevents anaphase until all kinetochores are stably attached. Checkpoint regulation hinges on the dynamic localization of checkpoint proteins to kinetochores. Unattached, checkpoint-active kinetochores accumulate multiple checkpoint proteins, which are depleted from kinetochores upon stable attachment, allowing checkpoint silencing. Because multiple proteins are recruited simultaneously to unattached kinetochores, it is not known what changes at kinetochores are essential for anaphase promoting complex/cyclosome (APC/C) inhibition. Using chemically induced dimerization to manipulate protein localization with temporal control, we show that recruiting the checkpoint protein Mad1 to metaphase kinetochores is sufficient to reactivate the checkpoint without a concomitant increase in kinetochore levels of Mps1 or BubR1. Furthermore, Mad2 binding is necessary but not sufficient for Mad1 to activate the checkpoint; a conserved C-terminal motif is also required. The results of our checkpoint reactivation assay suggest that Mad1, in addition to converting Mad2 to its active conformation, scaffolds formation of a higher-order mitotic checkpoint complex at kinetochores.     

The fate of metaphase kinetochores is weighed in the balance of SUMOylation during S phase

Genetic evidence suggests that conjugation of Small Ubiquitin-like Modifier proteins (SUMOs) plays an important role in kinetochore function, although the mechanism underlying these observations are poorly defined. we found that depletion of the SUMO protease SENP6 from HeLa cells causes chromosome misalignment, prolonged mitotic arrest and chromosome missegregation. Many inner kinetochore proteins (IKPs) were mis-localized in SENP6-depleted cells. This gross mislocalization of IKPs is due to proteolytic degradation of CENP-I and CENP-H via the SUMO targeted Ubiquitin Ligase (STUbL) pathway. Our findings show that SENP6 is a key regulator of inner kinetochore assembly that antagonizes the cellular STUbL pathway to protect IKPs from degradation during S phase. Here, we will briefly review the implications of our findings and present new data on how SUMOylation during S phase can control chromosome alignment in the subsequent metaphase.Key words: SUMO, kinetochore, mitosis, SENP6, CENP-H, CENP-I     

The fate of metaphase kinetochores is weighed in the balance of SUMOylation during S phase

Genetic evidence suggests that conjugation of Small Ubiquitin-like Modifier proteins (SUMOs) plays an important role in kinetochore function, although the mechanism underlying these observations are poorly defined. We found that depletion of the SUMO protease SENP6 from HeLa cells causes chromosome misalignment, prolonged mitotic arrest and chromosome missegregation. Many inner kinetochore proteins (IKPs) were mis-localized in SENP6-depleted cells. This gross mislocalization of IKPs is due to proteolytic degradation of CENP-I and CENP-H via the SUMO targeted Ubiquitin Ligase (STUbL) pathway. Our findings show that SENP6 is a key regulator of inner kinetochore assembly that antagonizes the cellular STUbL pathway to protect IKPs from degradation during S phase. Here, we will briefly review the implications of our findings and present new data on how SUMOylation during S phase can control chromosome alignment in the subsequent metaphase.     

Analysis of kinesin motor function at budding yeast kinetochores

Accurate chromosome segregation during mitosis requires biorientation of sister chromatids on the microtubules (MT) of the mitotic spindle. Chromosome-MT binding is mediated by kinetochores, which are multiprotein structures that assemble on centromeric (CEN) DNA. The simple CENs of budding yeast are among the best understood, but the roles of kinesin motor proteins at yeast kinetochores have yet to be determined, despite evidence of their importance in higher eukaryotes. We show that all four nuclear kinesins in Saccharomyces cerevisiae localize to kinetochores and function in three distinct processes. Kip1p and Cin8p, which are kinesin-5/BimC family members, cluster kinetochores into their characteristic bilobed metaphase configuration. Kip3p, a kinesin-8,-13/KinI kinesin, synchronizes poleward kinetochore movement during anaphase A. The kinesin-14 motor Kar3p appears to function at the subset of kinetochores that become detached from spindle MTs. These data demonstrate roles for structurally diverse motors in the complex processes of chromosome segregation and reveal important similarities and intriguing differences between higher and lower eukaryotes.     

NOP3 is an essential yeast protein which is required for pre-rRNA processing

The four nucleolar proteins NOP1, SSB1, GAR1, and NSR1 of Saccharomyces cerevisiae share a repetitive domain composed of repeat units rich in glycine and arginine (GAR domain). We have cloned and sequenced a fifth member of this family, NOP3, and shown it to be essential for cell viability. The NOP3 open reading frame encodes a 415 amino acid protein with a predicted molecular mass of 45 kD, containing a GAR domain and an RNA recognition motif. NOP3-specific antibodies recognize a 60-kD protein by SDS-PAGE and decorate the nucleolus and the surrounding nucleoplasm. A conditional lethal mutation, GAL::nop3, was constructed; growth of the mutant strain in glucose medium represses NOP3 expression. In cells depleted of NOP3, production of cytoplasmic ribosomes is impaired. Northern analysis and pulse-chase labeling indicate that pre-rRNA processing is inhibited at the late steps, in which 27SB pre-rRNA is cleaved to 25S rRNA and 20S pre-rRNA to 18S rRNA.     

Polo-like kinase1 is required for recruitment of dynein to kinetochores during mitosis

Kinetochore dynein has been implicated in microtubule capture, correcting inappropriate microtubule attachments, chromosome movement, and checkpoint silencing. It remains unclear how dynein coordinates this diverse set of functions. Phosphorylation is responsible for some dynein heterogeneity (Whyte, J., Bader, J. R., Tauhata, S. B., Raycroft, M., Hornick, J., Pfister, K. K., Lane, W. S., Chan, G. K., Hinchcliffe, E. H., Vaughan, P. S., and Vaughan, K. T. (2008) J. Cell Biol. 183, 819-834), and phosphorylated and dephosphorylated forms of dynein coexist at prometaphase kinetochores. In this study, we measured the impact of inhibiting polo-like kinase 1 (Plk1) on both dynein populations. Phosphorylated dynein was ablated at kinetochores after inhibiting Plk1 with a small molecule inhibitor (5-Cyano-7-nitro-2-(benzothiazolo-N-oxide)-carboxamide) or chemical genetic approaches. The total complement of kinetochore dynein was also reduced but not eliminated, reflecting the presence of some dephosphorylated dynein after Plk1 inhibition. Although Plk1 inhibition had a profound effect on dynein, kinetochore populations of dynactin, spindly, and zw10 were not reduced. Plk1-independent dynein was reduced after p150(Glued) depletion, consistent with the binding of dephosphorylated dynein to dynactin. Plk1 phosphorylated dynein intermediate chains at Thr-89 in vitro and generated the phospho-Thr-89 phospho-epitope on recombinant dynein intermediate chains. Finally, inhibition of Plk1 induced defects in microtubule capture and persistent microtubule attachment, suggesting a role for phosphorylated dynein in these functions during prometaphase. These findings suggest that Plk1 is a dynein kinase required for recruitment of phosphorylated dynein to kinetochores.     

S-phase cyclins are required for a stable arrest at metaphase

A critical DNA damage checkpoint in Saccharomyces cerevisiae is an arrest at the metaphase stage of mitosis. Here we show that the S-phase cyclins Clb5 and Clb6 are required for this arrest. Strains lacking Clb5 and Clb6 are hypersensitive to DNA damage. Furthermore, in the presence of the DNA alkylating agent methyl methanesulfonate (MMS) over 50% of clb5 clb6 mutants by-passed the metaphase checkpoint and arrested instead with separated sister chromatids. Levels of Pds1, an inhibitor of anaphase that accumulates following DNA damage, were similar in the wild-type and mutant strains following MMS treatment. Furthermore, unlike wild-type cells, clb5 clb6 mutants undergo nuclear division despite the presence of nuclear non-degradable Pds1. Our results suggest a novel role for the S-phase cyclins Clb5 and Clb6 in maintaining sister chromatid cohesion during a metaphase arrest, perhaps by regulating Pds1 activity.     

Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast.

It is widely assumed that degradation of mitotic cyclins causes a decrease in mitotic cdc2/CDC28 kinase activity and thereby triggers the metaphase to anaphase transition. Two observations made on the budding yeast Saccharomyces cerevisiae are inconsistent with this scenario: (i) anaphase occurs in the presence of high levels of kinase in cdc15 mutants and (ii) overproduction of a B-type mitotic cyclin causes arrest not in metaphase as previously reported but in telophase. Kinase destruction is therefore implicated in the exit from mitosis rather than the entry into anaphase. The behaviour of esp1 mutants shows in addition that kinase destruction can occur in the absence of anaphase completion. The execution of anaphase and the destruction of CDC28 kinase activity therefore appear to take place independently of one another.     

AlphaIIb's cytoplasmic domain is not required for ligand-induced clustering of integrin alphaIIbbeta3

The platelet integrin alphaIIbbeta3 exhibits bidirectional signaling, in that intracellular messengers enable adhesive macromolecules to bind to its ectodomain, while ligation promotes the association of cytoskeletal proteins with its cytoplasmic domains. In order to understand the linkage between these distant regions, we investigated the effects of receptor occupancy on the solution structure of both full-length recombinant alphaIIbbeta3 and alphaIIbDelta991beta3, an integrin truncation mutant which lacks one cytoplasmic domain. Lysates of (35)S-labeled human A549 cells expressing either full-length alphaIIbbeta3 or alphaIIbDelta991beta3 were examined by sucrose density gradient sedimentation followed by immunoprecipitation to determine the distributions of integrin protomers and oligomers. Recombinant alphaIIbbeta3 exhibited a weight-average sedimentation coefficient, S(w)=11.3+/-1.4 S with 73% sedimenting as protomers/dimers (9.1+/-1.0 S) and 27% as oligomers (15.4+/-0.4 S). Truncation mutant alphaIIbDelta991beta3 exhibited a similar pattern with 65% sedimenting as protomers/dimers. Upon ligation with eptifibatide, both full-length alphaIIbbeta3 and alphaIIbDelta991beta3 sedimented mainly at >14 S, indicating 2-3-fold increased oligomerization. Thus we have demonstrated that alphaIIb's cytoplasmic region is not required for integrin clustering, a key event in outside-in signaling.     

Mps1 phosphorylation of Dam1 couples kinetochores to microtubule plus ends at metaphase

BACKGROUND: Duplicated chromosomes are equally segregated to daughter cells by a bipolar mitotic spindle during cell division. By metaphase, sister chromatids are coupled to microtubule (MT) plus ends from opposite poles of the bipolar spindle via kinetochores. Here we describe a phosphorylation event that promotes the coupling of kinetochores to microtubule plus ends. RESULTS: Dam1 is a kinetochore component that directly binds to microtubules. We identified DAM1-765, a dominant allele of DAM1, in a genetic screen for mutations that increase stress on the spindle pole body (SPB) in Saccharomyces cerevisiae. DAM1-765 contains the single mutation S221F. We show that S221 is one of six Dam1 serines (S13, S49, S217, S218, S221, and S232) phosphorylated by Mps1 in vitro. In cells with single mutations S221F, S218A, or S221A, kinetochores in the metaphase spindle form tight clusters that are closer to the SPBs than in a wild-type cell. Five lines of experimental evidence, including localization of spindle components by fluorescence microscopy, measurement of microtubule dynamics by fluorescence redistribution after photobleaching, and reconstructions of three-dimensional structure by electron tomography, combined with computational modeling of microtubule behavior strongly indicate that, unlike wild-type kinetochores, Dam1-765 kinetochores do not colocalize with an equal number of plus ends. Despite the uncoupling of the kinetochores from the plus ends of MTs, the DAM1-765 cells are viable, complete the cell cycle with the same kinetics as wild-type cells, and biorient their chromosomes as efficiently as wild-type cells. CONCLUSIONS: We conclude that phosphorylation of Dam1 residues S218 and S221 by Mps1 is required for efficient coupling of kinetochores to MT plus ends. We find that efficient plus-end coupling is not required for (1) maintenance of chromosome biorientation, (2) maintenance of tension between sister kinetochores, or (3) chromosome segregation.     

Rec8 cleavage by separase is required for meiotic nuclear divisions in fission yeast

Sister chromatid cohesion in meiosis is established by cohesin complexes, including the Rec8 subunit. During meiosis I, sister chromatid cohesion is destroyed along the chromosome arms to release connections of recombined homologous chromosomes (homologues), whereas centromeric cohesion persists until it is finally destroyed at anaphase II. In fission yeast, as in mammals, distinct cohesin complexes are used depending on the chromosomal region; Rec8 forms a complex with Rec11 (equivalent to SA3) mainly along chromosome arms, while Psc3 (equivalent to SA1 and SA2) forms a complex mainly in the vicinity of the centromeres. Here we show that separase activation and resultant Rec8 cleavage are required for meiotic chromosome segregation in fission yeast. A non-cleavable form of Rec8 blocks disjunction of homologues at meiosis I. However, displacing non-cleavable Rec8 restrictively from the chromosome arm by genetically depleting Rec11 alleviated the blockage of homologue segregation, but not of sister segregation. We propose that the segregation of homologues at meiosis I and of sisters at meiosis II requires the cleavage of Rec8 along chromosome arms and at the centromeres, respectively.