Browse
Submit Data
Databases
API
Help

Dataset Information

48 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Direct role of dynein motor in stable kinetochore-microtubule attachment, orientation, and alignment.

ABSTRACT: Cytoplasmic dynein has been implicated in diverse mitotic functions, several involving its association with kinetochores. Much of the supporting evidence comes from inhibition of dynein regulatory factors. To obtain direct insight into kinetochore dynein function, we expressed a series of dynein tail fragments, which we find displace motor-containing dynein heavy chain (HC) from kinetochores without affecting other subunits, regulatory factors, or microtubule binding proteins. Cells with bipolar mitotic spindles progress to late prometaphase-metaphase at normal rates. However, the dynein tail, dynactin, Mad1, and BubR1 persist at the aligned kinetochores, which is consistent with a role for dynein in self-removal and spindle assembly checkpoint inactivation. Kinetochore pairs also show evidence of misorientation relative to the spindle equator and abnormal oscillatory behavior. Further, kinetochore microtubule bundles are severely destabilized at reduced temperatures. Dynein HC RNAi and injection of anti-dynein antibody in MG132-arrested metaphase cells produced similar effects. These results identify a novel function for the dynein motor in stable microtubule attachment and maintenance of kinetochore orientation during metaphase chromosome alignment.

SUBMITTER: Varma D

PROVIDER: S-EPMC2542467 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

The microtubule catastrophe promoter Sentin delays stable kinetochore-microtubule attachment in oocytes.

Project description:The critical step in meiosis is to attach homologous chromosomes to the opposite poles. In mouse oocytes, stable microtubule end-on attachments to kinetochores are not established until hours after spindle assembly, and phosphorylation of kinetochore proteins by Aurora B/C is responsible for the delay. Here we demonstrated that microtubule ends are actively prevented from stable attachment to kinetochores until well after spindle formation in Drosophila melanogaster oocytes. We identified the microtubule catastrophe-promoting complex Sentin-EB1 as a major factor responsible for this delay. Without this activity, microtubule ends precociously form robust attachments to kinetochores in oocytes, leading to a high proportion of homologous kinetochores stably attached to the same pole. Therefore, regulation of microtubule ends provides an alternative novel mechanism to delay stable kinetochore-microtubule attachment in oocytes.

| S-EPMC4687879 | biostudies-literature

Stable kinetochore-microtubule attachment requires loop-dependent Ndc80-Ndc80 binding.

Project description:During cell division, kinetochores link chromosomes to spindle microtubules. The Ndc80 complex, a crucial microtubule binder, populates each kinetochore with dozens of copies. Whether adjacent Ndc80 complexes cooperate to promote microtubule binding remains unclear. Here we demonstrate that the Ndc80 loop, a short sequence that interrupts the Ndc80 coiled-coil at a conserved position, folds into a more rigid structure than previously assumed and promotes direct interactions between full-length Ndc80 complexes on microtubules. Mutations in the loop impair these Ndc80-Ndc80 interactions, prevent the formation of force-resistant kinetochore-microtubule attachments, and cause cells to arrest in mitosis for hours. This arrest is not due to an inability to recruit the kinetochore-microtubule stabilizing SKA complex and cannot be overridden by mutations in the Ndc80 tail that strengthen microtubule attachment. Thus, loop-mediated organization of adjacent Ndc80 complexes is crucial for stable end-on kinetochore-microtubule attachment and spindle assembly checkpoint satisfaction.

| S-EPMC10308368 | biostudies-literature

Stable kinetochore-microtubule attachment requires loop-dependent Ndc80-Ndc80 binding

Project description:During cell division, kinetochores link chromosomes to spindle microtubules. The Ndc80 complex, a crucial microtubule binder, populates each kinetochore with dozens of copies. Whether adjacent Ndc80 complexes cooperate to promote microtubule binding remains unclear. Here we demonstrate that the Ndc80 loop, a short sequence that interrupts the Ndc80 coiled-coil at a conserved position, folds into a more rigid structure than previously assumed and promotes direct interactions between full-length Ndc80 complexes on microtubules. Mutations in the loop impair these Ndc80-Ndc80 interactions, prevent the formation of force-resistant kinetochore-microtubule attachments, and cause cells to arrest in mitosis for hours. This arrest is not due to an inability to recruit the kinetochore-microtubule stabilizing SKA complex and cannot be overridden by mutations in the Ndc80 tail that strengthen microtubule attachment. Thus, loop-mediated organisation of adjacent Ndc80 complexes is crucial for stable end-on kinetochore-microtubule attachment and spindle assembly checkpoint satisfaction.

| S-SCDT-10_15252-EMBJ_2022112504 | biostudies-other

MAST/Orbit has a role in microtubule-kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity.

Project description:Multiple asters (MAST)/Orbit is a member of a new family of nonmotor microtubule-associated proteins that has been previously shown to be required for the organization of the mitotic spindle. Here we provide evidence that MAST/Orbit is required for functional kinetochore attachment, chromosome congression, and the maintenance of spindle bipolarity. In vivo analysis of Drosophila mast mutant embryos undergoing early mitotic divisions revealed that chromosomes are unable to reach a stable metaphase alignment and that bipolar spindles collapse as centrosomes move progressively closer toward the cell center and eventually organize into a monopolar configuration. Similarly, soon after depletion of MAST/Orbit in Drosophila S2 cells by double-stranded RNA interference, cells are unable to form a metaphase plate and instead assemble monopolar spindles with chromosomes localized close to the center of the aster. In these cells, kinetochores either fail to achieve end-on attachment or are associated with short microtubules. Remarkably, when microtubule dynamics is suppressed in MAST-depleted cells, chromosomes localize at the periphery of the monopolar aster associated with the plus ends of well-defined microtubule bundles. Furthermore, in these cells, dynein and ZW10 accumulate at kinetochores and fail to transfer to microtubules. However, loss of MAST/Orbit does not affect the kinetochore localization of D-CLIP-190. Together, these results strongly support the conclusion that MAST/Orbit is required for microtubules to form functional attachments to kinetochores and to maintain spindle bipolarity.

| S-EPMC2173411 | biostudies-literature

Polyploids require Bik1 for kinetochore-microtubule attachment.

Project description:The attachment of kinetochores to spindle microtubules (MTs) is essential for maintaining constant ploidy in eukaryotic cells. Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface. Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids. The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton. In polyploid cells, Bik1 is required before anaphase to maintain kinetochore separation and therefore contributes to the force that opposes the elastic recoil of attached sister chromatids. The role of Bik1 in kinetochore separation appears to be independent of the role of Bik1 in regulating MT dynamics. The finding that a protein involved in kinetochore-MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics.

| S-EPMC2199317 | biostudies-literature

Dynein prevents erroneous kinetochore-microtubule attachments in mitosis.

Project description:Equal distribution of the genetic material during cell division relies on efficient congression of chromosomes to the metaphase plate. Prior to their alignment, the Dynein motor recruited to kinetochores transports a fraction of laterally-attached chromosomes along microtubules toward the spindle poles. By doing that, Dynein not only contributes to chromosome movements, but also prevents premature stabilization of end-on kinetochore-microtubule attachments. This is achieved by 2 parallel mechanisms: 1) Dynein-mediated poleward movement of chromosomes counteracts opposite polar-ejection forces (PEFs) on chromosome arms by the microtubule plus-end-directed motors chromokinesins. Otherwise, they could stabilize erroneous syntelic kinetochore-microtubule attachments and lead to the random ejection of chromosomes away from the spindle poles; and 2) By transporting chromosomes to the spindle poles, Dynein brings the former to the zone of highest Aurora A kinase activity, further destabilizing kinetochore-microtubule attachments. Thus, Dynein plays an important role in keeping chromosome segregation error-free by preventing premature stabilization of kinetochore-microtubule attachments near the spindle poles.

| S-EPMC4825607 | biostudies-literature

Stable kinetochore-microtubule attachment is sufficient to silence the spindle assembly checkpoint in human cells.

Project description:During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. In the absence of stable kinetochore-microtubule attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore-microtubule attachments in human cells using a non-phosphorylatable version of the protein Hec1, a core component of the attachment machinery. We find that stable attachments are sufficient to silence the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal.

| S-EPMC4686653 | biostudies-literature

Molecular architecture of a kinetochore-microtubule attachment site.

Project description:Kinetochore attachment to spindle microtubule plus-ends is necessary for accurate chromosome segregation during cell division in all eukaryotes. The centromeric DNA of each chromosome is linked to microtubule plus-ends by eight structural-protein complexes. Knowing the copy number of each of these complexes at one kinetochore-microtubule attachment site is necessary to understand the molecular architecture of the complex, and to elucidate the mechanisms underlying kinetochore function. We have counted, with molecular accuracy, the number of structural protein complexes in a single kinetochore-microtubule attachment using quantitative fluorescence microscopy of GFP-tagged kinetochore proteins in the budding yeast Saccharomyces cerevisiae. We find that relative to the two Cse4p molecules in the centromeric histone, the copy number ranges from one or two for inner kinetochore proteins such as Mif2p, to 16 for the DAM-DASH complex at the kinetochore-microtubule interface. These counts allow us to visualize the overall arrangement of a kinetochore-microtubule attachment. As most of the budding yeast kinetochore proteins have homologues in higher eukaryotes, including humans, this molecular arrangement is likely to be replicated in more complex kinetochores that have multiple microtubule attachments.

| S-EPMC2867088 | biostudies-literature

Insights from an erroneous kinetochore-microtubule attachment state.

Project description:Faithful distribution of the genome requires that sister kinetochores, which assemble on each chromatid during cell division, interact with dynamic microtubules from opposite spindle poles in a configuration called chromosome biorientation. Biorientation produces tension that increases the affinity of kinetochores for microtubules via ill-defined mechanisms. Non-bioriented kinetochore-microtubule (kt-MT) interactions are prevalent but short-lived due to an error correction pathway that reduces the affinity of kinetochores for microtubules. Interestingly, incorrect kt-MT interactions can be stabilized by experimentally applying force to misoriented chromosomes. Here, a live-cell force assay is utilized to characterize the molecular composition of a common type of improper kt-MT attachment. Our force-related studies are also discussed in the context of current models for tension-dependent stabilization of kt-MT interactions.

| S-EPMC3782542 | biostudies-literature

hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells.

Project description:Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore-microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.

| S-EPMC2173110 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data