Biomedical subjects

Tarun M Kapoor

Publications and source records attributed to Tarun M Kapoor.

At least 19 recordsLinked to original sources

PubMed2006

Microtubule flux: drivers wanted.

While the metaphase spindle maintains a constant shape and size during cell division, its major component microtubules are continuously being polymerized, depolymerized and transported towards the two spindle poles in a process called microtubule poleward flux. This process has been observed in all metazoan cells. Recent studies have indicated that Kinesin-5s, which can drive the relative sliding of microtubules, and kinesin-13s, which regulate microtubule polymerization, are directly involved in microtubule poleward flux. The availability of molecular and chemical tools to perturb protein functions together with improvements in imaging and analytical methods have allowed the examination of these two kinesins' roles in poleward flux at high temporal and spatial resolution. These advances have shed some light on the molecular mechanisms that drive microtubule poleward flux.

Animals↗

PubMed2006

Probing cell-division phenotype space and Polo-like kinase function using small molecules.

Cell-permeable small molecules that inhibit their targets on fast timescales are powerful probes of cell-division mechanisms. Such inhibitors have been identified using phenotype-based screens with chemical libraries. However, the characteristics of compound libraries needed to effectively span cell-division phenotype space, to find probes that target different mechanisms, are not known. Here we show that a small collection of 100 diaminopyrimidines (DAPs) yields a range of cell-division phenotypes, including changes in spindle geometry, chromosome positioning and mitotic index. Monopolar mitotic spindles are induced by four inhibitors, including one that targets Polo-like kinases (Plks), evolutionarily conserved serine/threonine kinases. Using chemical inhibitors and high-resolution live-cell microscopy, we found that Plk activity is needed for the assembly and maintenance of bipolar mitotic spindles. Plk inhibition destabilizes kinetochore microtubules while stabilizing other spindle microtubules, leading to monopolar spindles. Further testing of compounds based on 'privileged scaffolds', such as the DAP scaffold, could lead to new cell-division probes and antimitotic agents.

Animals↗

PubMed2006

Targeting protein stability with a small molecule.

Both genetic approaches and small-molecule inhibitors can be used to perturb protein function. Banaszynski et al. (2006) now describe a strategy to regulate the stability of a target protein, in which specificity is provided by a genetically encoded tag and temporal control is imparted by a small molecule.

Animals↗

PubMed2006

Examining how the spatial organization of chromatin signals influences metaphase spindle assembly.

During cell division, the proper assembly of a microtubule-based bipolar spindle depends on signals from chromatin. However, it is unknown how the spatial organization of chromatin signals affects spindle morphology. Here, we use paramagnetic chromatin beads, and magnetic fields for their alignment in cell-free extracts, to examine the spatial components of signals that regulate spindle assembly. We find that for linear chromatin-bead arrays that vary by eightfold in length, metaphase spindle size and shape are constant. Our findings indicate that, although chromatin provides cues for microtubule formation, metaphase spindle organization, which is controlled by microtubule-based motors, is robust to changes in the shape of chromatin signals.

Animals↗

PubMed2006

Allosteric inhibition of kinesin-5 modulates its processive directional motility.

Small-molecule inhibitors of kinesin-5 (refs. 1-3), a protein essential for eukaryotic cell division, represent alternatives to antimitotic agents that target tubulin. While tubulin is needed for multiple intracellular processes, the known functions of kinesin-5 are limited to dividing cells, making it likely that kinesin-5 inhibitors would have fewer side effects than do tubulin-targeting drugs. Kinesin-5 inhibitors, such as monastrol, act through poorly understood allosteric mechanisms, not competing with ATP binding. Moreover, the microscopic mechanism of full-length kinesin-5 motility is not known. Here we characterize the motile properties and allosteric inhibition of Eg5, a vertebrate kinesin-5, using a GFP fusion protein in single-molecule fluorescence assays. We find that Eg5 is a processive kinesin whose motility includes, in addition to ATP-dependent directional motion, a diffusive component not requiring ATP hydrolysis. Monastrol suppresses the directional processive motility of microtubule-bound Eg5. These data on Eg5's allosteric inhibition will impact these inhibitors' use as probes and development as chemotherapeutic agents.

Allosteric Regulation↗

PubMed2006

Chromosomes can congress to the metaphase plate before biorientation.

The stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that congression requires that chromosomes are connected to the opposite poles of the bipolar spindle ("bioriented"). In mammalian cells, we found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy, and correlative electron microscopy, we found that monooriented chromosomes could glide toward the spindle equator alongside kinetochore fibers attached to other already bioriented chromosomes. This congression mechanism depended on the kinetochore-associated, plus end-directed microtubule motor CENP-E (kinesin-7).

Animals↗

PubMed2006

Unraveling cell division mechanisms with small-molecule inhibitors.

Cell division is the process by which a cell creates two genetically identical daughter cells. To maintain genomic integrity, a complex and highly regulated sequence of events ensures that the replicated chromosomes are equally partitioned between the daughter cells. For more than 50 years, strategies designed around small-molecule inhibitors have been critical in advancing our understanding of this essential process. Here we introduce a series of questions on the biology of cell division and illustrate how small molecules have been used to design experiments to address these questions. Because of the highly dynamic nature of cell division, the temporal control over protein function that is possible with small molecules has been particularly valuable in dissecting biological mechanisms.

Animals↗

PubMed2005

The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks.

During cell division, mitotic spindles are assembled by microtubule-based motor proteins. The bipolar organization of spindles is essential for proper segregation of chromosomes, and requires plus-end-directed homotetrameric motor proteins of the widely conserved kinesin-5 (BimC) family. Hypotheses for bipolar spindle formation include the 'push-pull mitotic muscle' model, in which kinesin-5 and opposing motor proteins act between overlapping microtubules. However, the precise roles of kinesin-5 during this process are unknown. Here we show that the vertebrate kinesin-5 Eg5 drives the sliding of microtubules depending on their relative orientation. We found in controlled in vitro assays that Eg5 has the remarkable capability of simultaneously moving at approximately 20 nm s(-1) towards the plus-ends of each of the two microtubules it crosslinks. For anti-parallel microtubules, this results in relative sliding at approximately 40 nm s(-1), comparable to spindle pole separation rates in vivo. Furthermore, we found that Eg5 can tether microtubule plus-ends, suggesting an additional microtubule-binding mode for Eg5. Our results demonstrate how members of the kinesin-5 family are likely to function in mitosis, pushing apart interpolar microtubules as well as recruiting microtubules into bundles that are subsequently polarized by relative sliding.

Animals↗

PubMed2005

Monastrol inhibition of the mitotic kinesin Eg5.

Monastrol is a small, cell-permeable molecule that arrests cells in mitosis by specifically inhibiting Eg5, a member of the Kinesin-5 family. We have used steady-state and presteady-state kinetics as well as equilibrium binding approaches to define the mechanistic basis of S-monastrol inhibition of monomeric human Eg5/KSP. In the absence of microtubules (Mts), the basal ATPase activity is inhibited through slowed product release. In the presence of microtubules, the ATPase activity is also reduced with weakened binding of Eg5 to microtubules during steady-state ATP turnover. Monastrol-treated Eg5 also shows a decreased relative affinity for microtubules under equilibrium conditions. The Mt.Eg5 presteady-state kinetics of ATP binding and the subsequent ATP-dependent isomerization are unaffected during the first ATP turnover. However, monastrol appears to stabilize a conformation that allows for reversals at the ATP hydrolysis step. Monastrol promotes a dramatic decrease in the observed rate of Eg5 association with microtubules, and ADP release is slowed without trapping the Mt.Eg5.ADP intermediate. We propose that S-monastrol binding to Eg5 induces a stable conformational change in the motor domain that favors ATP re-synthesis after ATP hydrolysis. The aberrant interactions with the microtubule and the reversals at the ATP hydrolysis step alter the ability of Eg5 to generate force, thereby yielding a nonproductive Mt.Eg5 complex that cannot establish or maintain the bipolar spindle.

Adenosine Diphosphate↗

PubMed2005

Mitotic kinesin inhibitors induce mitotic arrest and cell death in Taxol-resistant and -sensitive cancer cells.

Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.

Antineoplastic Agents↗

PubMed2004

Chromosome segregation: correcting improper attachment.

Accurate chromosome segregation requires that the two sister kinetochores attach to microtubules from opposite spindle poles. New work reveals how a kinetochore can segregate properly while remaining improperly attached to two spindle poles.

Cell Cycle↗

PubMed2004

The human mitotic checkpoint protein BubR1 regulates chromosome-spindle attachments.

Loss or gain of whole chromosomes, the form of chromosomal instability (CIN) most commonly associated with human cancers, is expected to arise from the failure to accurately segregate chromosomes in mitosis. The mitotic checkpoint is one pathway that prevents segregation errors by blocking the onset of anaphase until all chromosomes make proper attachments to the spindle. Another process that prevents errors is stabilization and destabilization of connections between chromosomes and spindle microtubules. An outstanding question is how these two pathways are coordinated to ensure accurate chromosome segregation. Here we show that in human cells depleted of BubR1 - a critical component of the mitotic checkpoint that can directly regulate the onset of anaphase - chromosomes do not form stable attachments to spindle microtubules. Attachments in these cells are restored by inhibition of Aurora kinase, which is known to stabilize kinetochore-microtubule attachments. Loss of BubR1 function thus perturbs regulation of attachments rather than the ability of kinetochores to bind to microtubules. Consistent with this finding, depletion of BubR1 increases phosphorylation of CENP-A, a kinetochore-specific Aurora kinase substrate. We propose that BubR1 links regulation of chromosome-spindle attachment to mitotic checkpoint signalling.

Aurora Kinases↗

PubMed2004

Par6alpha signaling controls glial-guided neuronal migration.

Neuronal migrations along glial fibers provide a primary pathway for the formation of cortical laminae. To examine the mechanisms underlying glial-guided migration, we analyzed the dynamics of cytoskeletal and signaling components in living neurons. Migration involves the coordinated two-stroke movement of a perinuclear tubulin 'cage' and the centrosome, with the centrosome moving forward before nuclear translocation. Overexpression of mPar6alpha disrupts the perinuclear tubulin cage, retargets PKCzeta and gamma-tubulin away from the centrosome, and inhibits centrosomal motion and neuronal migration. Thus, we propose that during neuronal migration the centrosome acts to coordinate cytoskeletal dynamics in response to mPar6alpha-mediated signaling.

Animals↗

PubMed2004

The rate of bipolar spindle assembly depends on the microtubule-gliding velocity of the mitotic kinesin Eg5.

During early embryonic cycles, the time required for mitotic spindle assembly must match the autonomous cell cycle oscillations because a lack of coordination between these two processes will result in chromosome segregation errors. Members of the widely conserved BimC kinesin family are essential for spindle formation in all eukaryotes, and complete loss of BimC function results in monopolar spindles that have two spindle poles that are not separated. However, the precise roles of BimC motor activity in the spindle assembly process are not known. To examine the contribution of BimC kinesin's motor activity to spindle assembly, we generated and characterized mutants of Eg5, a vertebrate BimC kinesin, with reduced in vitro microtubule-gliding velocities. In Xenopus egg extracts, we replaced endogenous Eg5 with recombinant wild-type or mutant motor proteins. By using centrosome-dependent and centrosome-independent spindle assembly assays, we found that mechanisms that determine spindle size and shape were robust to approximately 6-fold reductions in Eg5 motility. However, the spindle assembly process was slower when Eg5 motor function was impaired. This role of Eg5 was independent of its contribution to centrosome separation. We provide evidence that Eg5 is a rate-limiting component of the cellular machinery that drives spindle assembly in vertebrates.

Animals↗

PubMed2004

Dynein/dynactin regulate metaphase spindle length by targeting depolymerizing activities to spindle poles.

During cell division metaphase spindles maintain constant length, whereas spindle microtubules continuously flux polewards, requiring addition of tubulin subunits at microtubule plus-ends, polewards translocation of the microtubule lattice, and removal of tubulin subunits from microtubule minus-ends near spindle poles. How these processes are coordinated is unknown. Here, we show that dynein/dynactin, a multi-subunit microtubule minus-end-directed motor complex, and NuMA, a microtubule cross-linker, regulate spindle length. Fluorescent speckle microscopy reveals that dynactin or NuMA inhibition suppresses microtubule disassembly at spindle poles without affecting polewards microtubule sliding. The observed uncoupling of these two components of flux indicates that microtubule depolymerization is not required for the microtubule transport associated with polewards flux. Inhibition of Kif2a, a KinI kinesin known to depolymerize microtubules in vitro, results in increased spindle microtubule length. We find that dynein/dynactin contribute to the targeting of Kif2a to spindle poles, suggesting a model in which dynein/dynactin regulate spindle length and coordinate flux by maintaining microtubule depolymerizing activities at spindle poles.

Animals↗

PubMed2004

Mechanistic analysis of the mitotic kinesin Eg5.

Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: k(cat) = 5.5 s(-1), K(1/2,Mt) = 0.7 microm, and K(m,ATP) = 25 microm; and for Eg5-437: k(cat) = 2.9 s(-1), K(1/2,Mt) = 4.5 microm, and K(m,ATP) = 19 microm. 2'(3')-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 microm(-1)s(-1), followed immediately by ATP hydrolysis at 15 s(-1). ATP-dependent Mt.Eg5 dissociation was relatively slow and rate-limiting at 8 s(-1) with mantADP release at 40 s(-1). Surprisingly, Eg5-367 binds microtubules more effectively (11 microm(-1)s(-1)) than Eg5-437 (0.7 microm(-1)s(-1)), consistent with the steady-state K(1/2,Mt) and the mantADP release K(1/2,Mt). These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover.

Adenosine Diphosphate↗

PubMed2004

Correcting improper chromosome-spindle attachments during cell division.

For accurate segregation of chromosomes during cell division, microtubule fibres must attach sister kinetochores to opposite poles of the mitotic spindle (bi-orientation). Aurora kinases are linked to oncogenesis and have been implicated in the regulation of chromosome-microtubule attachments. Although loss of Aurora kinase activity causes an accumulation of mal-orientated chromosomes in dividing cells, it is not known how the active kinase corrects improper chromosome attachments. The use of reversible small-molecule inhibitors allows activation of protein function in living vertebrate cells with temporal control. Here we show that by removal of small-molecule inhibitors, controlled activation of Aurora kinase during mitosis can correct chromosome attachment errors by selective disassembly of kinetochore-microtubule fibres, rather than by alternative mechanisms involving initial release of microtubules from either kinetochores or spindle poles. Observation of chromosomes and microtubule dynamics with real-time high-resolution microscopy showed that mal-orientated, but not bi-orientated, chromosomes move to the spindle pole as both kinetochore-microtubule fibres shorten, followed by alignment at the metaphase plate. Our results provide direct evidence for a mechanism required for the maintenance of genome integrity during cell division.

Animals↗

PubMed2004

New probes for microtubule dynamics.

A phenotype-based screen identifies a purine analog, named diminutol, that perturbs the microtubule cytoskeleton in cells. An affinity-based approach identifies a protein target of this small molecule and leads to the characterization of a new pathway that may regulate cytoskeleton dynamics.

Animals↗