PubMed Health⌕ Search

Biomedical subjects

Duane A Compton

Publications and source records attributed to Duane A Compton.

18 recordsLinked to original sources
PubMed2024

Cyclin A/Cdk1 promotes chromosome alignment and timely mitotic progression.

To ensure genomic fidelity, a series of spatially and temporally coordinated events is executed during prometaphase of mitosis, including bipolar spindle formation, chromosome attachment to spindle microtubules at kinetochores, the correction of erroneous kinetochore-microtubule (k-MT) attachments, and chromosome congression to the spindle equator. Cyclin A/Cdk1 kinase plays a key role in destabilizing k-MT attachments during prometaphase to promote correction of erroneous k-MT attachments. However, it is unknown whether Cyclin A/Cdk1 kinase regulates other events during prometaphase. Here, we investigate additional roles of Cyclin A/Cdk1 in prometaphase by using an siRNA knockdown strategy to deplete endogenous Cyclin A from human cells. We find that depleting Cyclin A significantly extends mitotic duration, specifically prometaphase, because chromosome alignment is delayed. Unaligned chromosomes display erroneous monotelic, syntelic, or lateral k-MT attachments suggesting that bioriented k-MT attachment formation is delayed in the absence of Cyclin A. Mechanistically, chromosome alignment is likely impaired because the localization of the kinetochore proteins BUB1 kinase, KNL1, and MPS1 kinase are reduced in Cyclin A-depleted cells. Moreover, we find that Cyclin A promotes BUB1 kinetochore localization independently of its role in destabilizing k-MT attachments. Thus, Cyclin A/Cdk1 facilitates chromosome alignment during prometaphase to support timely mitotic progression.

Humans↗
PubMed2023

Cyclin A/Cdk1 promotes chromosome alignment and timely mitotic progression.

To ensure genomic fidelity a series of spatially and temporally coordinated events are executed during prometaphase of mitosis, including bipolar spindle formation, chromosome attachment to spindle microtubules at kinetochores, the correction of erroneous kinetochore-microtubule (k-MT) attachments, and chromosome congression to the spindle equator. Cyclin A/Cdk1 kinase plays a key role in destabilizing k-MT attachments during prometaphase to promote correction of erroneous k-MT attachments. However, it is unknown if Cyclin A/Cdk1 kinase regulates other events during prometaphase. Here, we investigate additional roles of Cyclin A/Cdk1 in prometaphase by using an siRNA knockdown strategy to deplete endogenous Cyclin A from human cells. We find that depleting Cyclin A significantly extends mitotic duration, specifically prometaphase, because chromosome alignment is delayed. Unaligned chromosomes display erroneous monotelic, syntelic, or lateral k-MT attachments suggesting that bioriented k-MT attachment formation is delayed in the absence of Cyclin A. Mechanistically, chromosome alignment is likely impaired because the localization of the kinetochore proteins BUB1 kinase, KNL1, and MPS1 kinase are reduced in Cyclin A-depleted cells. Moreover, we find that Cyclin A promotes BUB1 kinetochore localization independently of its role in destabilizing k-MT attachments. Thus, Cyclin A/Cdk1 facilitates chromosome alignment during prometaphase to support timely mitotic progression.

Preprint↗
PubMed2006

Mitosis: disorderly conduct at the kinetochore.

Conventional models posit that microtubules bound to kinetochores act coordinately during chromosome movement. Such models need to be revised in the light of new data demonstrating uncoordinated behavior among kinetochore-associated microtubules.

Animals↗
PubMed2006

Functional roles of poleward microtubule flux during mitosis.

Poleward microtubule flux is a conserved process during mitosis and meiosis in metazoan cells and is defined as the translocation of spindle microtubules toward spindle poles coupled to the depolymerization of their minus-ends. In some cell types, the rate of poleward microtubule flux matches that of poleward chromatid movement during anaphase A, suggesting that it pulls chromatids poleward. However, in other cell types, the rate of poleward microtubule flux is significantly slower than chromatid movement during anaphase A, suggesting that it makes little contribution to chromatid movement. This discrepancy led to speculation that flux is maintained in these cells to fulfill other functional roles aside from contributing to anaphase A chromatid movement. These roles include contributing to chromosome alignment, regulating spindle size and microtubule turnover, and correcting errors in chromosome attachment to spindle microtubules. Here, we discuss recent data that begin to pinpoint the functional roles of poleward microtubule flux during mitosis and meiosis.

Animals↗
PubMed2005

Efficient mitosis in human cells lacking poleward microtubule flux.

Chromosome segregation relies on the dynamic properties of spindle microtubules (MTs). Poleward MT flux contributes to spindle dynamics through the disassembly of MT minus ends at spindle poles coupled to the continuous poleward transport of spindle MTs. Despite being conserved in metazoan cells, the function of flux remains controversial because flux rates differ widely in different cell types. In meiotic systems, the rate of flux nearly matches that of chromosome movement, but in mitotic systems, flux is significantly slower than chromosome movement. Here, we show that spindles in human mitotic cells depleted of the kinesin-13 proteins Kif2a and MCAK lack detectable flux and that such cells frequently fail to segregate all chromosomes appropriately at anaphase. Elimination of flux reduces poleward chromosome velocity approximately 20%, but does not hinder bipolar spindle assembly, chromosome alignment, or mitotic progression. Thus, mitosis proceeds efficiently in human cells lacking detectable poleward MT flux. These data demonstrate that in human cultured cells, kinetochores are sufficient to effectively power chromosome movement, leading us to speculate that flux is maintained in these cells to fulfill other functional roles such as error correction or kinetochore regulation.

Biological Transport↗
PubMed2005

Regulation of mitosis by poly(ADP-ribosyl)ation.

The spindle is a dynamic, microtubule-based structure responsible for chromosome segregation during cell division. Spindles in mammalian cells contain several thousand microtubules that are arranged into highly symmetric bipolar arrays by the actions of numerous microtubule-associated motor and non-motor proteins. In addition to these protein constituents, recent work has demonstrated that poly(ADP-ribose) is a key spindle component. Of the multitude of poly(ADP-ribose) polymerase proteins encoded in the genome, tankyrase 1 appears to be the primary enzyme responsible for building poly(ADP-ribose) in spindles during mitosis. In this issue of the Biochemical Journal, Susan Smith and co-workers show that the primary target of tankyrase 1 in dividing cells is NuMA (nuclear mitotic apparatus protein), a protein that cross-links microtubule ends at spindle poles. The impact of poly(ADP-ribosyl)ation on the biochemical function of NuMA remains murky at this time, but these new results represent the first step to clearing the view as to how poly(ADP-ribosyl)ation regulates cell division.

Animals↗
PubMed2005

Mitosis: PARty time in the spindle.

Poly(ADP-ribose), a post-translational protein modification known to affect chromatin structure, has now been shown to regulate microtubule organization during mitosis. These findings alter conventional views of the mechanisms of spindle assembly and function.

Cell Cycle Proteins↗
PubMed2004

Multiple mechanisms regulate NuMA dynamics at spindle poles.

The large coiled-coil protein NuMA plays an essential role in organizing microtubule minus ends at spindle poles in vertebrate cells. Here, we use both in vivo and in vitro methods to examine NuMA dynamics at mitotic spindle poles. Using fluorescence recovery after photobleaching, we show that an exogenously expressed green-fluorescent-protein/NuMA fusion undergoes continuous exchange between soluble and spindle-associated pools in living cells. These dynamics require cellular energy and display an average half-time for fluorescence recovery of approximately 3 minutes. To explore how NuMA dynamics at spindle poles is regulated, we exploited the association of NuMA with microtubule asters formed in mammalian mitotic extracts. Using a monoclonal antibody specific for human NuMA, we followed the fate of human NuMA associated with microtubule asters upon dilution with a hamster mitotic extract. Consistent with in vivo data, this assay shows that NuMA can be displaced from the core of pre-assembled asters into the soluble pool. The half-time of NuMA displacement from asters under these conditions is approximately 5 minutes. Using this assay, we show that protein kinase activity and the NuMA-binding protein LGN regulate the dynamic exchange of NuMA on microtubule asters. Thus, the dynamic properties of NuMA are regulated by multiple mechanisms including protein phosphorylation and binding to the LGN protein, and the rate of exchange between soluble and microtubule-associated pools suggests that NuMA associates with an insoluble matrix at spindle poles.

Animals↗
PubMed2004

The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK.

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore-microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle bipolarity. These treatments failed to restore bipolarity to cells lacking the activity of the kinesin Eg5. Thus, two independent pathways contribute to spindle bipolarity, with the Eg5-dependent pathway using motor force to drive spindle bipolarity and the Kif2a-dependent pathway relying on microtubule polymer dynamics to generate force for spindle bipolarity.

Animals↗
PubMed2004

A mechanistic model for the organization of microtubule asters by motor and non-motor proteins in a mammalian mitotic extract.

We used computer simulation to understand the functional relationships between motor (dynein, HSET, and Eg5) and non-motor (NuMA) proteins involved in microtubule aster organization. The simulation accurately predicted microtubule organization under all combinations of motor and non-motor proteins, provided that microtubule cross-links at minus-ends were dynamic, and dynein and HSET were restricted to cross-linking microtubules in parallel orientation only. A mechanistic model was derived from these data in which a combination of two aggregate properties, Net Minus-end-directed Force and microtubule Cross-linking Orientation Bias, determine microtubule organization. This model uses motor and non-motor proteins, accounts for motor antagonism, and predicts that alterations in microtubule Cross-linking Orientation Bias should compensate for imbalances in motor force during microtubule aster formation. We tested this prediction in the mammalian mitotic extract and, consistent with the model, found that increasing the contribution of microtubule cross-linking by NuMA compensated for the loss of Eg5 motor activity. Thus, this model proposes a precise mechanism of action of each noncentrosomal protein during microtubule aster organization and suggests that microtubule organization in spindles involves both motile forces from motors and static forces from non-motor cross-linking proteins.

Antigens, Nuclear↗
PubMed2004

Human enhancer of invasion-cluster, a coiled-coil protein required for passage through mitosis.

In a cross-species overexpression approach, we used the pseudohyphal transition of Saccharomyces cerevisiae as a model screening system to identify human genes that regulate cell morphology and the cell cycle. Human enhancer of invasion-cluster (HEI-C), encoding a novel evolutionarily conserved coiled-coil protein, was isolated in a screen for human genes that induce agar invasion in S. cerevisiae. In human cells, HEI-C is primarily localized to the spindle during mitosis. Depletion of HEI-C in vivo with short interfering RNAs results in severe mitotic defects. Analysis by immunofluorescence, flow cytometry analysis, and videomicroscopy indicates that HEI-C-depleted cells form metaphase plates with normal timing after G(2)/M transition, although in many cases cells have disorganized mitotic spindles. Subsequently, severe defects occur at the metaphase-anaphase transition, characterized by a significant delay at this stage or, more commonly, cellular disintegration accompanied by the display of classic biochemical markers of apoptosis. These mitotic defects occur in spite of the fact that HEI-C-depleted cells retain functional cell cycle checkpoints, as these cells arrest normally following nocodazole or hydroxyurea treatment. These results place HEI-C as a novel regulator of spindle function and integrity during the metaphase-anaphase transition.

Amino Acid Sequence↗
PubMed2003

A functional relationship between NuMA and kid is involved in both spindle organization and chromosome alignment in vertebrate cells.

We examined spindle morphology and chromosome alignment in vertebrate cells after simultaneous perturbation of the chromokinesin Kid and either NuMA, CENP-E, or HSET. Spindle morphology and chromosome alignment after simultaneous perturbation of Kid and either HSET or CENP-E were no different from when either HSET or CENP-E was perturbed alone. However, short bipolar spindles with organized poles formed after perturbation of both Kid and NuMA in stark contrast to splayed spindle poles observed after perturbation of NuMA alone. Spindles were disorganized if Kid, NuMA, and HSET were perturbed, indicating that HSET is sufficient for spindle organization in the absence of Kid and NuMA function. In addition, chromosomes failed to align efficiently at the spindle equator after simultaneous perturbation of Kid and NuMA despite appropriate kinetochore-microtubule interactions that generated chromosome movement at normal velocities. These data indicate that a functional relationship between the chromokinesin Kid and the spindle pole organizing protein NuMA influences spindle morphology, and we propose that this occurs because NuMA forms functional linkages between kinetochore and nonkinetochore microtubules at spindle poles. In addition, these data show that both Kid and NuMA contribute to chromosome alignment in mammalian cells.

Antigens, Nuclear↗
PubMed2003

Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis.

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living alpha-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.

Animals↗
PubMed2002

hTPX2 is required for normal spindle morphology and centrosome integrity during vertebrate cell division.

Bipolar spindle formation is essential for the accurate segregation of genetic material during cell division. Although centrosomes influence the number of spindle poles during mitosis, motor and non-motor microtubule-associated proteins (MAPs) also play key roles in determining spindle morphology. TPX2 is a novel MAP also characterized in Xenopus cell-free extracts. To examine hTPX2 (human TPX2) function in human cells, we used siRNA to knock-down its expression and found that cells lacking hTPX2 arrest in mitosis with multipolar spindles. NuMA, gamma-tubulin, and centrin localize to each pole, and nocodazole treatment of cells lacking hTPX2 demonstrates that the localization of gamma-tubulin to multiple spindle poles requires intact microtubules. Furthermore, we show that the formation of monopolar microtubule arrays in human cell extracts does not require hTPX2, demonstrating that the mechanism by which hTPX2 promotes spindle bipolarity is independent of activities focusing microtubule minus ends at spindle poles. Finally, inhibition of the kinesin Eg5 in hTPX2-depleted cells leads to monopolar spindles, indicating that Eg5 function is necessary for multipolar spindle formation in the absence of hTPX2. Our observations reveal a structural role for hTPX2 in spindles and provide evidence for a balance between microtubule-based motor forces and structural spindle components.

Animals↗
PubMed2002

LGN blocks the ability of NuMA to bind and stabilize microtubules. A mechanism for mitotic spindle assembly regulation.

LGN is closely related to a Drosophila protein, Partner of inscuteable (Pins), which is required for polarity establishment and asymmetric cell divisions during embryonic development. In mammalian cells, LGN binds with high affinity to the C-terminal tail of NuMA, a large nuclear protein that is required for spindle organization, and accumulates at the spindle poles during mitosis. LGN also regulates spindle organization, possibly through inhibition of NuMA function, but the mechanism of this effect has not yet been understood. Using mammalian cells, frog egg extracts, and in vitro assays, we now show that a small domain within the C terminus of NuMA stabilizes microtubules (MTs), and that LGN blocks stabilization. The nuclear localization signal adjacent to this domain is not involved in stabilization. NuMA can interact directly with MTs, and the MT binding domain on NuMA overlaps by ten amino acid residues with the LGN binding domain. We therefore propose that a simple steric exclusion model can explain the inhibitory effect of LGN on NuMA-dependent mitotic spindle organization.

Animals↗
PubMed2002

Chromosome segregation: pulling from the poles.

Dual wavelength video microscopy has been used to evaluate how chromatids move poleward upon chromosome separation at anaphase. The data reveal that poleward microtubule flux provides the dominant force for separating chromatids in Drosophila embryos during anaphase A.

Anaphase↗
PubMed2002

Searching for the middle ground: mechanisms of chromosome alignment during mitosis.

The contributions of key molecules predicted to align chromosomes at the center of the mitotic spindle have been recently examined. New results dictate that models for how chromosomes align during the early stages of mitosis must be revised to integrate properties of microtubule-based motor proteins as well as microtubule dynamics.

Animals↗