Separase Biosensor Reveals that Cohesin Cleavage Timing Depends on Phosphatase PP2ACdc55 Regulation

SummaryIn anaphase, sister chromatids separate abruptly and are then segregated by the mitotic spindle. The protease separase triggers sister separation by cleaving the Scc1/Mcd1 subunit of the cohesin ring that holds sisters together. Polo-kinase phosphorylation of Scc1 promotes its cleavage, but the underlying regulatory circuits are unclear. We developed a separase biosensor in Saccharomyces cerevisiae that provides a quantitative indicator of cohesin cleavage in single cells. Separase is abruptly activated and cleaves most cohesin within 1 min, after which anaphase begins. Cohesin near centromeres and telomeres is cleaved at the same rate and time. Protein phosphatase PP2ACdc55 inhibits cohesin cleavage by counteracting polo-kinase phosphorylation of Scc1. In early anaphase, the previously described separase inhibition of PP2ACdc55 promotes cohesin cleavage. Thus, separase acts directly on Scc1 and also indirectly, through inhibition of PP2ACdc55, to stimulate cohesin cleavage, providing a feedforward loop that may contribute to a robust and timely anaphase

Long-ranged attraction between disordered heterogeneous surfaces

Long-ranged attractions across water between two surfaces that are randomly covered with (mobile) positive and negative charge domains have been attributed to induced correlation of the charges (positive lining up with negative) as the surfaces approach. Here we show, by directly measuring normal forces under a rapid shear field, that these attractions may not in fact be due to such correlations. It is rather the inherent interaction-asymmetry between equally- and between oppositely-charged domains that results in the long-ranged attraction even in the complete absence of any charge correlation

Combination of Two Activating Mutations in One HOG1 Gene Forms Hyperactive Enzymes That Induce Growth Arrest

Mitogen-activated protein kinases (MAPKs) play key roles in differentiation, growth, proliferation, and apoptosis. Although MAPKs have been extensively studied, the precise function, specific substrates, and target genes of each MAPK are not known. These issues could be addressed by sole activation of a given MAPK, e.g., through the use of constitutively active MAPK enzymes. We have recently reported the isolation of eight hyperactive mutants of the Saccharomyces cerevisiae MAPK Hog1, each of which bears a distinct single point mutation. These mutants acquired high intrinsic catalytic activity but did not impose the full biological potential of the Hog1 pathway. Here we describe our attempt to obtain a MAPK that is more active than the previous mutants both catalytically and biologically. We combined two different activating point mutations in the same gene and found that two of the resulting double mutants acquired unusual properties. These alleles, HOG1(D170A,F318L) and HOG1(D170A,F318S), induced a severe growth inhibition and had to be studied through an inducible expression system. This growth inhibition correlated with very high spontaneous (in the absence of any stimulation) catalytic activity and strong induction of Hog1 target genes. Furthermore, analysis of the phosphorylation status of these active alleles shows that their acquired intrinsic activity is independent of either phospho-Thr174 or phospho-Tyr176. Through fluorescence-activated cell sorting analysis, we show that the effect on cell growth inhibition is not a result of cell death. This study provides the first example of a MAPK that is intrinsically activated by mutations and induces a strong biological effect

Gene Transcription as a Limiting Factor in Protein Production and Cell Growth

Cell growth is driven by the synthesis of proteins, genes, and other cellular components. Defining processes that limit biosynthesis rates is fundamental for understanding the determinants of cell physiology. Here, we analyze the consequences of engineering cells to express extremely high levels of mCherry proteins, as a tool to define limiting processes that fail to adapt upon increasing biosynthetic demands. Protein-burdened cells were transcriptionally and phenotypically similar to mutants of the Mediator, a transcription coactivator complex. However, our binding data suggest that the Mediator was not depleted from endogenous promoters. Burdened cells showed an overall increase in the abundance of the majority of endogenous transcripts, except for highly expressed genes. Our results, supported by mathematical modeling, suggest that wild-type cells transcribe highly expressed genes at the maximal possible rate, as defined by the transcription machinery’s physical properties. We discuss the possible cellular benefit of maximal transcription rates to allow a coordinated optimization of cell size and cell growth

Coordinated control of replication and transcription by a SAPK protects genomic integrity

Upon environmental changes or extracellular signals, cells are subjected to marked changes in gene expression 1,2. Dealing with high levels of transcription during replication is critical to prevent collisions between the transcription and replication pathways and avoid recombination events3–5. In response to osmostress, hundreds of stress-responsive genes are rapidly induced by the stress-activated protein kinase (SAPK) Hog1 (ref. 6), even during S phase7. Here we show in Saccharomyces cerevisae that a single signalling molecule, Hog1, coordinates both replication and transcription upon osmostress. Hog1 interacts with and phosphorylates Mrc1, a component of the replication complex8–11. Phosphorylation occurs at different sites to those targeted by Mec1 upon DNA damage8,9. Mrc1 phosphorylation by Hog1 delays early and late origin firing by preventing Cdc45 loading, as well as slowing down replication-complex progression. Regulation of Mrc1 by Hog1 is completely independent of Mec1 and Rad53. Cells carrying a non-phosphorylatable allele of MRC1 (mrc13A) do not delay replication upon stress and show a marked increase in transcription-associated recombination, genomic instability and Rad52 foci. In contrast, mrc13A induces Rad53 and survivalin the presence of hydroxyurea or methylmethanesulphonate. Therefore, Hog1 and Mrc1 define a novel S-phase checkpoint independent of the DNA-damage checkpoint that permits eukaryotic cells to prevent conflicts between DNA replication and transcription, which would otherwise lead to genomic instability when both phenomena are temporally coincident.This work was supported by grants from the Spanish Government (BIO2009-07762 and BFU2012-33503 to F.P., BFU2011-26722 to E.d.N., BFU2010- 16372 to A.A., and Consolider Ingenio 2010 programme CSD2007-0015 to F.P. and A.A.) and FP7 UNICELLSYS grant (no. 201142) and the Fundación Marcelino Botín to F.P. F.P. and E.d.N. are recipients of an ICREA Acadèmia (Generalitat de Catalunya).Peer reviewe