The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Andrew W Murray - One of the best experts on this subject based on the ideXlab platform.
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a putative bet hedging strategy buffers Budding Yeast against environmental instability
Current Biology, 2020Co-Authors: Laura E Bagamery, Quincey A Justman, Ethan C Garner, Andrew W MurrayAbstract:Summary To grow and divide, cells must extract resources from dynamic and unpredictable environments. Many organisms use different metabolic strategies for distinct contexts. Budding Yeast can produce ATP from carbon sources by mechanisms that prioritize either speed (fermentation) or yield (respiration). Withdrawing glucose from exponentially growing cells reveals variability in their ability to switch from fermentation to respiration. We observe two subpopulations of glucose-starved cells: recoverers, which rapidly adapt and resume growth, and arresters, which enter a shock state characterized by deformation of many cellular structures, including mitochondria. These states are heritable, and on high glucose, arresters grow and divide faster than recoverers. Recoverers have a fitness advantage during a carbon source shift but are less fit in a constant, high-glucose environment, and we observe natural variation in the frequency of the two states across wild Yeast strains. These experiments suggest that bet hedging has evolved in Budding Yeast.
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bet hedging buffers Budding Yeast against environmental instability
bioRxiv, 2020Co-Authors: Laura E Bagamery, Quincey A Justman, Ethan C Garner, Andrew W MurrayAbstract:To grow and divide, cells must extract resources from dynamic and unpredictable environments. Organisms thus possess redundant metabolic pathways for distinct contexts. In Budding Yeast, ATP can be produced from carbon by mechanisms that prioritize either speed (fermentation) or yield (respiration). We find that in the absence of predictive cues, cells vary in their intrinsic ability to switch metabolic strategies from fermentation to respiration. We observe subpopulations of Yeast cells which either rapidly adapt or enter a shock state characterized by deformation of many cellular structures, including mitochondria. This capacity to adapt is a bimodal and heritable state. We demonstrate that metabolic preparedness confers a fitness advantage during an environmental shift but is costly in a constant, high-glucose environment, and we observe natural variation in the frequency of prepared cells across wild Yeast strains. These experiments suggest that bet-hedging has evolved in Budding Yeast.
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cell size regulation in Budding Yeast does not depend on linear accumulation of whi5
bioRxiv, 2020Co-Authors: Felix Barber, Ariel Amir, Andrew W MurrayAbstract:Abstract Cells must couple cell cycle progress to their growth rate to restrict the spread of cell sizes present throughout a population. Linear, rather than exponential, accumulation of Whi5, was proposed to provide this coordination by causing a higher Whi5 concentration in cells born at smaller size. We tested this model using the inducible GAL1 promoter to make the Whi5 concentration independent of cell size. At an expression level that equalizes the mean cell size with that of wild-type cells, the size distributions of cells with galactose-induced Whi5 expression and wild-type cells are indistinguishable. Fluorescence microscopy confirms that the endogenous and GAL1 promoters produce different relationships between Whi5 concentration and cell volume without diminishing size control in the G1 phase. We also expressed Cln3 from the GAL1 promoter, finding that the spread in cell sizes for an asynchronous population is unaffected by this perturbation. Our findings contradict the previously proposed model for cell size control in Budding Yeast and demonstrate the need for a molecular mechanism that explains how cell size controls passage through Start. Author Contributions FB performed the experiments, data analysis and simulations. All authors designed the experiments and wrote the manuscript. Significance Statement Despite decades of research, the question of how single cells regulate their size remains unclear. Here we demonstrate that a widely supported molecular model for the fundamental origin of size control in Budding Yeast is inconsistent with a set of experiments testing the model’s key prediction. We therefore conclude that the problem of cell size control in Budding Yeast remains unsolved. This work highlights the need for rigorous testing of future models of size control in order to make progress on this fundamental question.
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Reduced Mad2 expression keeps relaxed kinetochores from arresting Budding Yeast in mitosis.
Molecular Biology of the Cell, 2011Co-Authors: Erin L. Barnhart, Andrew W Murray, Russell K. Dorer, Scott C. SchuylerAbstract:Chromosome segregation depends on the spindle checkpoint, which delays anaphase until all chromosomes have bound microtubules and have been placed under tension. The Mad1-Mad2 complex is an essential component of the checkpoint. We studied the consequences of removing one copy of MAD2 in diploid cells of the Budding Yeast, Saccharomyces cerevisiae. Compared to MAD2/MAD2 cells, MAD2/mad2Δ heterozygotes show increased chromosome loss and have different responses to two insults that activate the spindle checkpoint: MAD2/mad2Δ cells respond normally to antimicrotubule drugs but cannot respond to chromosomes that lack tension between sister chromatids. In MAD2/mad2Δ cells with normal sister chromatid cohesion, removing one copy of MAD1 restores the checkpoint and returns chromosome loss to wild-type levels. We conclude that cells need the normal Mad2:Mad1 ratio to respond to chromosomes that are not under tension.
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an in vitro assay for cdc20 dependent mitotic anaphase promoting complex activity from Budding Yeast
Methods of Molecular Biology, 2009Co-Authors: Scott C. Schuyler, Andrew W MurrayAbstract:Cell cycle transitions are controlled, in part, by ubiquitin-dependent proteolysis. In mitosis, the metaphase to anaphase transition is governed by an E3 ubiquitin ligase called the cyclosome or Anaphase-Promoting Complex (APC), and a WD40-repeat protein co-factor called Cdc20. In vitro Cdc20-dependent APC (APC(Cdc20)) assays have been useful in the identification and validation of target substrates, and in the study of APC enzymology and regulation. Many aspects of the regulation of cell cycle progression have been discovered in the Budding Yeast Saccharomyces cerevisiae, and proteins purified from this model organism have been employed in a wide variety of in vitro assays. Here we outline a quantitative in vitro mitotic APC(Cdc20) assay that makes use of a highly active form of the APC that is purified from Budding Yeast cells arrested in mitosis.
Jan M. Skotheim - One of the best experts on this subject based on the ideXlab platform.
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form and function of topologically associating genomic domains in Budding Yeast
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Aaron F Straight, Umut Eser, Devon Chandlerbrown, Zhijun Duan, William Stafford Noble, Jan M. SkotheimAbstract:The genome of metazoan cells is organized into topologically associating domains (TADs) that have similar histone modifications, transcription level, and DNA replication timing. Although similar structures appear to be conserved in fission Yeast, computational modeling and analysis of high-throughput chromosome conformation capture (Hi-C) data have been used to argue that the small, highly constrained Budding Yeast chromosomes could not have these structures. In contrast, herein we analyze Hi-C data for Budding Yeast and identify 200-kb scale TADs, whose boundaries are enriched for transcriptional activity. Furthermore, these boundaries separate regions of similarly timed replication origins connecting the long-known effect of genomic context on replication timing to genome architecture. To investigate the molecular basis of TAD formation, we performed Hi-C experiments on cells depleted for the Forkhead transcription factors, Fkh1 and Fkh2, previously associated with replication timing. Forkhead factors do not regulate TAD formation, but do promote longer-range genomic interactions and control interactions between origins near the centromere. Thus, our work defines spatial organization within the Budding Yeast nucleus, demonstrates the conserved role of genome architecture in regulating DNA replication, and identifies a molecular mechanism specifically regulating interactions between pericentric origins.
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Dilution of the cell cycle inhibitor Whi5 controls Budding-Yeast cell size
Nature, 2015Co-Authors: Kurt M. Schmoller, Mardo Kõivomägi, Jonathan J. Turner, Jan M. SkotheimAbstract:Cell size fundamentally affects all biosynthetic processes by determining the scale of organelles and influencing surface transport. Although extensive studies have identified many mutations affecting cell size, the molecular mechanisms underlying size control have remained elusive. In the Budding Yeast Saccharomyces cerevisiae, size control occurs in G1 phase before Start, the point of irreversible commitment to cell division. It was previously thought that activity of the G1 cyclin Cln3 increased with cell size to trigger Start by initiating the inhibition of the transcriptional inhibitor Whi5 (refs 6-8). Here we show that although Cln3 concentration does modulate the rate at which cells pass Start, its synthesis increases in proportion to cell size so that its total concentration is nearly constant during pre-Start G1. Rather than increasing Cln3 activity, we identify decreasing Whi5 activity--due to the dilution of Whi5 by cell growth--as a molecular mechanism through which cell size controls proliferation. Whi5 is synthesized in S/G2/M phases of the cell cycle in a largely size-independent manner. This results in smaller daughter cells being born with higher Whi5 concentrations that extend their pre-Start G1 phase. Thus, at its most fundamental level, size control in Budding Yeast results from the differential scaling of Cln3 and Whi5 synthesis rates with cell size. More generally, our work shows that differential size-dependency of protein synthesis can provide an elegant mechanism to coordinate cellular functions with growth.
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daughter specific transcription factors regulate cell size control in Budding Yeast
PLOS Biology, 2009Co-Authors: Stefano Di Talia, Jan M. Skotheim, Adam P Rosebrock, Hongyin Wang, Bruce Futcher, Frederick R CrossAbstract:In Budding Yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled Budding Yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle.
Rong Li - One of the best experts on this subject based on the ideXlab platform.
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actin depolymerization drives actomyosin ring contraction during Budding Yeast cytokinesis
Developmental Cell, 2012Co-Authors: Inês Mendes Pinto, Boris Rubinstein, Andrei Kucharavy, Jay R Unruh, Rong LiAbstract:Actin filaments and myosin II are evolutionarily conserved force-generating components of the contractile ring during cytokinesis. Here we show that in Budding Yeast, actin filament depolymerization plays a major role in actomyosin ring constriction. Cofilin mutation or chemically stabilizing actin filaments attenuate actomyosin ring constriction. Deletion of myosin II motor domain or the myosin regulatory light chain reduced the contraction rate and also the rate of actin depolymerization in the ring. We constructed a quantitative microscopic model of actomyosin ring constriction via filament sliding driven by both actin depolymerization and myosin II motor activity. Model simulations based on experimental measurements support the notion that actin depolymerization is the predominant mechanism for ring constriction. The model predicts invariability of total contraction time regardless of the initial ring size, as originally reported for C. elegans embryonic cells. This prediction was validated in Yeast cells of different sizes due to different ploidies.
John J Tyson - One of the best experts on this subject based on the ideXlab platform.
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integrative analysis of cell cycle control in Budding Yeast
Molecular Biology of the Cell, 2004Co-Authors: Katherine C Chen, Attila Csikasznagy, Bela Novak, Frederick R Cross, Laurence Calzone, John J TysonAbstract:The adaptive responses of a living cell to internal and external signals are controlled by networks of proteins whose interactions are so complex that the functional integration of the network cannot be comprehended by intuitive reasoning alone. Mathematical modeling, based on biochemical rate equations, provides a rigorous and reliable tool for unraveling the complexities of molecular regulatory networks. The Budding Yeast cell cycle is a challenging test case for this approach, because the control system is known in exquisite detail and its function is constrained by the phenotypic properties of >100 genetically engineered strains. We show that a mathematical model built on a consensus picture of this control system is largely successful in explaining the phenotypes of mutants described so far. A few inconsistencies between the model and experiments indicate aspects of the mechanism that require revision. In addition, the model allows one to frame and critique hypotheses about how the division cycle is regulated in wild-type and mutant cells, to predict the phenotypes of new mutant combinations, and to estimate the effective values of biochemical rate constants that are difficult to measure directly in vivo.
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kinetic analysis of a molecular model of the Budding Yeast cell cycle
Molecular Biology of the Cell, 2000Co-Authors: Katherine C Chen, Attila Csikasznagy, Bela Gyorffy, Bela Novak, John J TysonAbstract:The molecular machinery of cell cycle control is known in more detail for Budding Yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism. In recent years, many elegant experiments on Budding Yeast have dissected the roles of cyclin molecules (Cln1–3 and Clb1–6) in coordinating the events of DNA synthesis, bud emergence, spindle formation, nuclear division, and cell separation. These experimental clues suggest a mechanism for the principal molecular interactions controlling cyclin synthesis and degradation. Using standard techniques of biochemical kinetics, we convert the mechanism into a set of differential equations, which describe the time courses of three major classes of cyclin-dependent kinase activities. Model in hand, we examine the molecular events controlling “Start” (the commitment step to a new round of chromosome replication, bud formation, and mitosis) and “Finish” (the transition from metaphase to anaphase, when sister chromatids are pulled apart and the bud separates from the mother cell) in wild-type cells and 50 mutants. The model accounts for many details of the physiology, biochemistry, and genetics of cell cycle control in Budding Yeast.
Douglas R Kellogg - One of the best experts on this subject based on the ideXlab platform.
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protein kinase c controls binding of igo ensa proteins to protein phosphatase 2a in Budding Yeast
Journal of Biological Chemistry, 2017Co-Authors: Vu Thai, Steven P Gygi, Noah Dephoure, Amit Weiss, Jacqueline Ferguson, Ricardo Leitao, Douglas R KelloggAbstract:Protein phosphatase 2A (PP2A) plays important roles in controlling mitosis in all eukaryotic cells. The form of PP2A that controls mitosis is associated with a conserved regulatory subunit that is called B55 in vertebrates and Cdc55 in Budding Yeast. The activity of this form of PP2A can be inhibited by binding of conserved Igo/ENSA proteins. Although the mechanisms that activate Igo/ENSA to bind and inhibit PP2A are well understood, little is known about how Igo/Ensa are inactivated. Here, we have analyzed regulation of Igo/ENSA in the context of a checkpoint pathway that links mitotic entry to membrane growth in Budding Yeast. Protein kinase C (Pkc1) relays signals in the pathway by activating PP2ACdc55. We discovered that constitutively active Pkc1 can drive cells through a mitotic checkpoint arrest, which suggests that Pkc1-dependent activation of PP2ACdc55 plays a critical role in checkpoint signaling. We therefore used mass spectrometry to determine how Pkc1 modifies the PP2ACdc55 complex. This revealed that Pkc1 induces changes in the phosphorylation of multiple subunits of the complex, as well as dissociation of Igo/ENSA. Pkc1 directly phosphorylates Cdc55 and Igo/ENSA, and phosphorylation site mapping and mutagenesis indicate that phosphorylation of Cdc55 contributes to Igo/ENSA dissociation. Association of Igo2 with PP2ACdc55 is regulated during the cell cycle, yet mutation of Pkc1-dependent phosphorylation sites on Cdc55 and Igo2 did not cause defects in mitotic progression. Together, the data suggest that Pkc1 controls PP2ACdc55 by multiple overlapping mechanisms.
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conservation of mechanisms controlling entry into mitosis Budding Yeast wee1 delays entry into mitosis and is required for cell size control
Current Biology, 2003Co-Authors: Stacy L Harvey, Douglas R KelloggAbstract:Abstract Background: In fission Yeast, the Wee1 kinase delays entry into mitosis until a critical cell size has been reached; however, a similar role for Wee1-related kinases has not been reported in other organisms. SWE1 , the Budding Yeast homolog of wee1 , is thought to function in a morphogenesis checkpoint that delays entry into mitosis in response to defects in bud morphogenesis. Results: In contrast to previous studies, we found that Budding Yeast swe1Δ cells undergo premature entry into mitosis, leading to birth of abnormally small cells. Additional experiments suggest that conditions that activate the morphogenesis checkpoint may actually be activating a G2/M cell size checkpoint. For example, actin depolymerization is thought to activate the morphogenesis checkpoint by inhibiting bud morphogenesis. However, actin depolymerization also inhibits bud growth, suggesting that it could activate a cell size checkpoint. Consistent with this possibility, we found that actin depolymerization fails to induce a G2/M delay once daughter buds pass a critical size. Other conditions that activate the morphogenesis checkpoint block bud formation, which could also activate a size checkpoint if cell size at G2/M is monitored in the daughter bud. Previous work reported that Swe1 is degraded during G2, which was proposed to account for failure of large-budded cells to arrest in response to actin depolymerization. However, we found that Swe1 is present throughout G2 and undergoes hyperphosphorylation as cells enter mitosis, as found in other organisms. Conclusions: Our results suggest that the mechanisms known to coordinate entry into mitosis in other organisms have been conserved in Budding Yeast.
