Different assemblies of the DAM1 complex follow shortening microtubules by distinct mechanisms.
Ontology highlight
ABSTRACT:
SUBMITTER: Grishchuk EL
PROVIDER: S-EPMC2383981 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Microtubule kinetochore attachments are essential for accurate mitosis, but how these force-generating connections move chromosomes remains poorly understood. Processive motion at shortening microtubule ends can be reconstituted in vitro using microbeads conjugated to the budding yeast kinetochore protein Dam1, which forms microtubule-encircling rings. Here, we report that, when Dam1 is linked to a bead cargo by elongated protein tethers, the maximum force transmitted from a disassembling microtubule increases sixfold compared with a short tether. We interpret this significant improvement with a theory that considers the geometry and mechanics of the microtubule-ring-bead system. Our results show the importance of fibrillar links in tethering microtubule ends to cargo: fibrils enable the cargo to align coaxially with the microtubule, thereby increasing the stability of attachment and the mechanical work that it can do. The force-transducing characteristics of fibril-tethered Dam1 are similar to the analogous properties of purified yeast kinetochores, suggesting that a tethered Dam1 ring comprises the main force-bearing unit of the native attachment.
| S-EPMC3651439 | biostudies-literature
Project description:Kinetochores couple chromosomes to the mitotic spindle to segregate the genome during cell division. An error correction mechanism drives the turnover of kinetochore-microtubule attachments until biorientation is achieved. The structural basis for how kinetochore-mediated chromosome segregation is accomplished and regulated remains an outstanding question. In this work, we describe the cryo-electron microscopy structure of the budding yeast outer kinetochore Ndc80 and Dam1 ring complexes assembled onto microtubules. Complex assembly occurs through multiple interfaces, and a staple within Dam1 aids ring assembly. Perturbation of key interfaces suppresses yeast viability. Force-rupture assays indicated that this is a consequence of impaired kinetochore-microtubule attachment. The presence of error correction phosphorylation sites at Ndc80-Dam1 ring complex interfaces and the Dam1 staple explains how kinetochore-microtubule attachments are destabilized and reset.
| S-EPMC7615550 | biostudies-literature
Project description: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.
| S-EPMC3216659 | biostudies-literature
Project description: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.
| S-EPMC3100407 | biostudies-literature
Project description: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.
| S-EPMC2291439 | biostudies-literature
Project description: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.
| S-EPMC5354518 | biostudies-literature
Project description:A number of biological parameters have been cited as hallmarks of immune aging. However, it is not clear whether these multiple biological changes are the result of common underlying aging processes and follow correlated trajectories, or whether the patterns of change for multiple parameters vary across individuals and reflect heterogeneity in the aging process. Here, we have studied parameters of immune system aging through longitudinal analysis of telomere length, inflammatory cytokines, and antibody titer to cytomegalovirus (CMV) in 465 subjects ranging in age from 21 to 88 years at the first visit, with an average of 13 years (7-19 years) follow-up. We observed a highly variable rate of change in telomere length of PBMCs with a relatively slow average rate of telomere shortening (-16 bp/year). Similarly, there were significant increases with age in vivo in three inflammation-related cytokines (interferon gamma, IL-6, and IL-10) and in anti-CMV IgG titer, which varied widely across individuals as well. We further observed positive correlative changes among different inflammatory cytokines. However, we did not find significant correlations among the rate of changes in telomere length, inflammatory cytokines, and anti-CMV IgG titers. Our findings thus reveal that age-related trajectories of telomere attrition, elevated circulating inflammatory cytokines, and anti-CMV IgG are independent and that aging individuals do not show a uniform pattern of change in these variables. Immune aging processes are complex and vary across individuals, and the use of multiple biomarkers is essential to evaluation of biological aging of the immune system.
| S-EPMC5609584 | biostudies-literature
Project description: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.
| S-EPMC4821239 | biostudies-literature
Project description:Alzheimer's disease (AD) pathology is characterized by the aggregation of beta-amyloid (Aβ) and tau in the form of amyloid plaques and neurofibrillary tangles in the brain. It has been found that a synergistic relationship between these two proteins may contribute to their roles in disease progression. However, how Aβ and tau interact has not been fully characterized. Here, we analyze how tau seeding or aggregation is influenced by different Aβ self-assemblies (fibrils and oligomers). Our cellular assays utilizing tau biosensor cells show that transduction of Aβ oligomers into the cells greatly enhances seeded tau aggregation in a concentration-dependent manner. In contrast, transduced Aβ fibrils slightly reduce tau seeding while untransduced Aβ fibrils promote it. We also observe that the transduction of α-synuclein fibrils, another amyloid protein, has no effect on tau seeding. The enhancement of tau seeding by Aβ oligomers was confirmed using tau fibril seeds derived from both recombinant tau and PS19 mouse brain extracts containing human tau. Our findings highlight the importance of considering the specific form and cellular location of Aβ self-assembly when studying the relationship between Aβ and tau in future AD therapeutic development.
| S-EPMC6856013 | biostudies-literature
Project description:To ensure the faithful inheritance of DNA, a macromolecular protein complex called the kinetochore sustains the connection between chromosomes and force-generating dynamic microtubules during cell division. Defects in this process lead to aneuploidy, a common feature of cancer cells and the cause of many developmental diseases [1-4]. One of the major microtubule-binding activities in the kinetochore is mediated by the conserved Ndc80 complex (Ndc80c) [5-7]. In budding yeast, the retention of kinetochores on dynamic microtubule tips also depends on the essential heterodecameric Dam1 complex (Dam1c) [8-15], which binds to the Ndc80c and is proposed to be a functional ortholog of the metazoan Ska complex [16, 17]. The load-bearing activity of the Dam1c depends on its ability to oligomerize, and the purified complex spontaneously self-assembles into microtubule-encircling oligomeric rings, which are proposed to function as collars that allow kinetochores to processively track the plus-end tips of microtubules and harness the forces generated by disassembling microtubules [10-15, 18-22]. However, it is unknown whether there are specific regulatory events that promote Dam1c oligomerization to ensure accurate segregation. Here, we used a reconstitution system to discover that Cdk1, the major mitotic kinase that drives the cell cycle, phosphorylates the Ask1 component of the Dam1c to increase its residence time on microtubules and enhance kinetochore-microtubule attachment strength. We propose that Cdk1 activity promotes Dam1c oligomerization to ensure that kinetochore-microtubule attachments are stabilized as kinetochores come under tension in mitosis.
| S-EPMC7497780 | biostudies-literature