Modeling Dual Pathways for the Metazoan Spindle Assembly Checkpoint

Using computational modelling, we investigate mechanisms of signal transduction focusing on the spindle assembly checkpoint where a single unattached kinetochore is able to signal to prevent cell cycle progression. This inhibitory signal switches off rapidly once spindle microtubules have attached to all kinetochores. This requirement tightly constrains the possible mechanisms. Here we investigate two possible mechanisms for spindle checkpoint operation in metazoan cells, both supported by recent experiments. The first involves the free diffusion and sequestration of cell-cycle regulators. This mechanism is severely constrained both by experimental fluorescence recovery data and also by the large volumes involved in open mitosis in metazoan cells. Using a simple mathematical analysis and computer simulation, we find that this mechanism can generate the inhibition found in experiment but likely requires a two stage signal amplification cascade. The second mechanism involves spatial gradients of a short-lived inhibitory signal that propagates first by diffusion but then primarily via active transport along spindle microtubules. We propose that both mechanisms may be operative in the metazoan spindle assembly checkpoint, with either able to trigger anaphase onset even without support from the other pathway.Comment: 9 pages, 2 figure

Securin Is Not Required for Chromosomal Stability in Human Cells

Abnormalities of chromosome number are frequently observed in cancers. The mechanisms regulating chromosome segregation in human cells are therefore of great interest. Recently it has been reported that human cells without an hSecurin gene lose chromosomes at a high frequency. Here we show that, after hSecurin knockout through homologous recombination, chromosome losses are only a short, transient effect. After a few passages hSecurin(−/−) cells became chromosomally stable and executed mitoses normally. This was unexpected, as the securin loss resulted in a persisting reduction of the sister-separating protease separase and inefficient cleavage of the cohesin subunit Scc1. Our data demonstrate that securin is dispensable for chromosomal stability in human cells. We propose that human cells possess efficient mechanisms to compensate for the loss of genes involved in chromosome segregation

SMC is recruited to oriC by ParB and promotes chromosome segregation in Streptococcus pneumoniae

Segregation of replicated chromosomes is an essential process in all organisms. How bacteria, such as the oval-shaped human pathogen Streptococcus pneumoniae, efficiently segregate their chromosomes is poorly understood. Here we show that the pneumococcal homologue of the DNA-binding protein ParB recruits S. pneumoniae condensin (SMC) to centromere-like DNA sequences (parS) that are located near the origin of replication, in a similar fashion as was shown for the rod-shaped model bacterium Bacillus subtilis. In contrast to B. subtilis, smc is not essential in S. pneumoniae, and Δsmc cells do not show an increased sensitivity to gyrase inhibitors or high temperatures. However, deletion of smc and/or parB results in a mild chromosome segregation defect. Our results show that S. pneumoniae contains a functional chromosome segregation machine that promotes efficient chromosome segregation by recruitment of SMC via ParB. Intriguingly, the data indicate that other, as of yet unknown mechanisms, are at play to ensure proper chromosome segregation in this organism.

Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity

© 2009 Maresca and Salmon. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 License. The definitive version was published in Journal of Cell Biology 184 (2009): 373-381, doi:10.1083/jcb.200808130.Cells have evolved a signaling pathway called the spindle assembly checkpoint (SAC) to increase the fidelity of chromosome segregation by generating a "wait anaphase" signal until all chromosomes are properly aligned within the mitotic spindle. It has been proposed that tension generated by the stretch of the centromeric chromatin of bioriented chromosomes stabilizes kinetochore microtubule attachments and turns off SAC activity. Although biorientation clearly causes stretching of the centromeric chromatin, it is unclear whether the kinetochore is also stretched. To test whether intrakinetochore stretch occurs and is involved in SAC regulation, we developed a Drosophila melanogaster S2 cell line expressing centromere identifier–mCherry and Ndc80–green fluorescent protein to mark the inner and outer kinetochore domains, respectively. We observed stretching within kinetochores of bioriented chromosomes by monitoring both inter- and intrakinetochore distances in live cell assays. This intrakinetochore stretch is largely independent of a 30-fold variation in centromere stretch. Furthermore, loss of intrakinetochore stretch is associated with enhancement of 3F3/2 phosphorylation and SAC activation.This work was supported by the American Cancer Society (grant PF0711401 to T.J. Maresca) and the National Institutes of Health (grant GM24364 to E.D. Salmon)

Molecular basis for SMC rod formation and its dissolution upon DNA binding.

SMC condensin complexes are central modulators of chromosome superstructure in all branches of life. Their SMC subunits form a long intramolecular coiled coil, which connects a constitutive "hinge" dimerization domain with an ATP-regulated "head" dimerization module. Here, we address the structural arrangement of the long coiled coils in SMC complexes. We unequivocally show that prokaryotic Smc-ScpAB, eukaryotic condensin, and possibly also cohesin form rod-like structures, with their coiled coils being closely juxtaposed and accurately anchored to the hinge. Upon ATP-induced binding of DNA to the hinge, however, Smc switches to a more open configuration. Our data suggest that a long-distance structural transition is transmitted from the Smc head domains to regulate Smc-ScpAB's association with DNA. These findings uncover a conserved architectural theme in SMC complexes, provide a mechanistic basis for Smc's dynamic engagement with chromosomes, and offer a molecular explanation for defects in Cornelia de Lange syndrome

Identification of genes induced by BRCA1 in breast cancer cells

Cataloged from PDF version of article.Inherited mutations of the BRCA1 gene predispose to breast, ovarian, and other cancers. The role of the BRCA1 gene in the maintenance of chromosomal integrity is linked to a number of biological properties of its protein product, including transcriptional regulation. In the present study, we have used suppression subtractive hybridisation (SSH) to identify genes induced by BRCA1 by comparing control MCF7 breast carcinoma cells (driver) with MCF7 cells ectopically expressing BRCA1 (tester) and generated a forward subtracted cDNA library. We screened 500 putative positive clones from this library. Two hundred and ten of these clones were positive by differential screening with forward and reverse subtracted probes and the 65 cDNA clones which showed more than fivefold increase were selected for sequencing analysis. We clustered 46 different genes that share high homology with sequences in the GenBank/EMBL databases. Among these, 30 were genes whose function had been previously identified while the remaining 16 clones were genes with,unknown functions. Of particular interest, BRCA1 gene induces the expression of genes encoding DNA repair proteins RAD21 and MSH2, ERBB2/HER2 interacting protein ERBIN, meningioma-associated protein MAC30, and a candidate ovarian tumour-suppressor OVCA1. Northern and Western blot analyses confirmed that the expression of these five genes are up-regulated following BRCA1 overexpression in MCF7 and UBR60-bcl2 cells. This is the first study reporting a set of BRCA1-induced genes in breast carcinoma cells by the SSH technique. We suggest that some known genes identified in this study may provide new insights into the tumour-suppressor function of BRCA1. (C) 2002 Elsevier Science (USA). All rights reserved

Mechanism of metabolic control. Target of rapamycin signaling links nitrogen quality to the activity of the Rtg1 and Rtg3 transcription factors.

De novo biosynthesis of amino acids uses intermediates provided by the TCA cycle that must be replenished by anaplerotic reactions to maintain the respiratory competency of the cell. Genome-wide expression analyses in Saccharomyces cerevisiae reveal that many of the genes involved in these reactions are repressed in the presence of the preferred nitrogen sources glutamine or glutamate. Expression of these genes in media containing urea or ammonia as a sole nitrogen source requires the heterodimeric bZip transcription factors Rtg1 and Rtg3 and correlates with a redistribution of the Rtg1p/Rtg3 complex from a predominantly cytoplasmic to a predominantly nuclear location. Nuclear import of the complex requires the cytoplasmic protein Rtg2, a previously identified upstream regulator of Rtg1 and Rtg3, whereas export requires the importin-beta-family member Msn5. Remarkably, nuclear accumulation of Rtg1/Rtg3, as well as expression of their target genes, is induced by addition of rapamycin, a specific inhibitor of the target of rapamycin (TOR) kinases. We demonstrate further that Rtg3 is a phosphoprotein and that its phosphorylation state changes after rapamycin treatment. Taken together, these results demonstrate that target of rapamycin signaling regulates specific anaplerotic reactions by coupling nitrogen quality to the activity and subcellular localization of distinct transcription factors

Principles of meiotic chromosome assembly revealed in S. cerevisiae

During meiotic prophase, chromosomes organise into a series of chromatin loops emanating from a proteinaceous axis, but the mechanisms of assembly remain unclear. Here we use Saccharomyces cerevisiae to explore how this elaborate three-dimensional chromosome organisation is linked to genomic sequence. As cells enter meiosis, we observe that strong cohesin-dependent grid-like Hi-C interaction patterns emerge, reminiscent of mammalian interphase organisation, but with distinct regulation. Meiotic patterns agree with simulations of loop extrusion with growth limited by barriers, in which a heterogeneous population of expanding loops develop along the chromosome. Importantly, CTCF, the factor that imposes similar features in mammalian interphase, is absent in S. cerevisiae, suggesting alternative mechanisms of barrier formation. While grid-like interactions emerge independently of meiotic chromosome synapsis, synapsis itself generates additional compaction that matures differentially according to telomere proximity and chromosome size. Collectively, our results elucidate fundamental principles of chromosome assembly and demonstrate the essential role of cohesin within this evolutionarily conserved process