monastrol and hesperadin

The accuracy of chromosome segregation is enhanced by the spindle assembly checkpoint (SAC). The SAC is thought to monitor two distinct events: attachment of kinetochores to microtubules and the stretch of the centromere between the sister kinetochores that arises only when the chromosome becomes properly bioriented. We examined human cells undergoing mitosis with unreplicated genomes (MUG). Kinetochores in these cells are not paired, which implies that the centromere cannot be stretched; however, cells progress through mitosis. A SAC is present during MUG as cells arrest in response to nocodazole, taxol, or monastrol treatments. Mad2 is recruited to unattached MUG kinetochores and released upon their attachment. In contrast, BubR1 remains on attached kinetochores and exhibits a level of phosphorylation consistent with the inability of MUG spindles to establish normal levels of centromere tension. Thus, kinetochore attachment to microtubules is sufficient to satisfy the SAC even in the absence of interkinetochore tension.

Topics: Anaphase; Autoantigens; Calcium-Binding Proteins; Cell Cycle Proteins; Centromere Protein A; Chromatin; Chromosomal Proteins, Non-Histone; DNA Replication; Enzyme Inhibitors; Genome, Human; HeLa Cells; Humans; Hydroxyurea; Indoles; Kinetics; Kinetochores; Mad2 Proteins; Metaphase; Microscopy, Electron; Microtubules; Mitosis; Nocodazole; Paclitaxel; Phosphorylation; Protein Serine-Threonine Kinases; Pyrimidines; Repressor Proteins; Spindle Apparatus; Sulfonamides; Thiones

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).

Topics: Animals; Aurora Kinases; Cell Line; Chromosomal Proteins, Non-Histone; Chromosomes, Mammalian; HeLa Cells; Humans; Indoles; Kinesins; Kinetochores; Metaphase; Microscopy, Confocal; Microscopy, Electron; Microscopy, Interference; Microscopy, Video; Microtubules; Mitosis; Molecular Motor Proteins; Movement; Potoroidae; Protein Serine-Threonine Kinases; Pyrimidines; RNA Interference; RNA, Small Interfering; Spindle Apparatus; Sulfonamides; Thiones

The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling.

Topics: Anaphase; Aneugens; Animals; Aurora Kinase B; Aurora Kinases; Cell Cycle Proteins; Chromosome Segregation; Endopeptidases; Eukaryotic Cells; Genes, cdc; HeLa Cells; Humans; Indoles; Kinetochores; Microtubules; Mitosis; Nocodazole; Paclitaxel; Phenotype; Polyploidy; Protein Kinases; Protein Serine-Threonine Kinases; Pyrimidines; RNA, Small Interfering; Separase; Spindle Apparatus; Sulfonamides; Thiones