Publications by authors named "Yves Barral"

90 Publications

Demixing the cell: how cells channel and store signaling information.

Curr Opin Cell Biol 2021 Mar 28. Epub 2021 Mar 28.

Institute of Biochemistry, Department of Biology, ETH Zürich, Switzerland. Electronic address:

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http://dx.doi.org/10.1016/j.ceb.2021.02.012DOI Listing
March 2021

Surface tensiometry of phase separated protein and polymer droplets by the sessile drop method.

Soft Matter 2021 Feb;17(6):1655-1662

Department of Materials, ETH Zürich, Switzerland.

Phase separated macromolecules play essential roles in many biological and synthetic systems. Physical characterization of these systems can be challenging because of limited sample volumes, particularly for phase-separated proteins. Here, we demonstrate that a classic method for measuring the surface tension of liquid droplets, based on the analysis of the shape of a sessile droplet, can be effectively scaled down to measure the interfacial tension between a macromolecule-rich droplet phase and its co-existing macromolecule-poor continuous phase. The connection between droplet shape and surface tension relies on the density difference between the droplet and its surroundings. This can be determined with small sample volumes in the same setup by measuring the droplet sedimentation velocity. An interactive MATLAB script for extracting the capillary length from a droplet image is included in the ESI.
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http://dx.doi.org/10.1039/d0sm01319fDOI Listing
February 2021

Mapping bilayer thickness in the ER membrane.

Sci Adv 2020 Nov 11;6(46). Epub 2020 Nov 11.

Institute of Biochemistry, Department of Biology, ETH Zürich, Otto-Stern-Weg 3, 8093 Zürich, Switzerland.

In the plasma membrane and in synthetic membranes, resident lipids may laterally unmix to form domains of distinct biophysical properties. Whether lipids also drive the lateral organization of intracellular membranes is largely unknown. Here, we describe genetically encoded fluorescent reporters visualizing local variations in bilayer thickness. Using them, we demonstrate that long-chained ceramides promote the formation of discrete domains of increased bilayer thickness in the yeast ER, particularly in the future plane of cleavage and at ER-trans-Golgi contact sites. Thickening of the ER membrane in the cleavage plane contributed to the formation of lateral diffusion barriers, which restricted the passage of short, but not long, protein transmembrane domains between the mother and bud ER compartments. Together, our data establish that the ER membrane is laterally organized and that ceramides drive this process, and provide insights into the physical nature and biophysical mechanisms of the lateral diffusion barriers that compartmentalize the ER.
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http://dx.doi.org/10.1126/sciadv.aba5130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7673731PMC
November 2020

Yeast Sporulation and [SMAUG] Prion: Faster Is Not Always Better.

Mol Cell 2020 01;77(2):203-204

Institute of Biochemistry, Department of Biology, ETH, Zürich, Switzerland. Electronic address:

Chakravarty et al. (2019) and Itakura et al. (2019) report that the yeast RNA-binding protein Vts1 can convert into the [SMAUG] prion state and delay meiosis commitment in response to starvation. It enables budding yeast to optimize their sporulation efficiency depending on how quickly nutrient availability fluctuates in their environment.
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http://dx.doi.org/10.1016/j.molcel.2019.12.017DOI Listing
January 2020

Remote control of microtubule plus-end dynamics and function from the minus-end.

Elife 2019 09 6;8. Epub 2019 Sep 6.

Institute of Biochemistry, ETH Zürich, Zurich, Switzerland.

In eukaryotes, the organization and function of the microtubule cytoskeleton depend on the allocation of different roles to individual microtubules. For example, many asymmetrically dividing cells differentially specify microtubule behavior at old and new centrosomes. Here we show that yeast spindle pole bodies (SPBs, yeast centrosomes) differentially control the plus-end dynamics and cargoes of their astral microtubules, remotely from the minus-end. The old SPB recruits the kinesin motor protein Kip2, which then translocates to the plus-end of the emanating microtubules, promotes their extension and delivers dynein into the bud. Kip2 recruitment at the SPB depends on Bub2 and Bfa1, and phosphorylation of cytoplasmic Kip2 prevents random lattice binding. Releasing Kip2 of its control by SPBs equalizes its distribution, the length of microtubules and dynein distribution between the mother cell and its bud. These observations reveal that microtubule organizing centers use minus to plus-end directed remote control to individualize microtubule function.
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http://dx.doi.org/10.7554/eLife.48627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754230PMC
September 2019

Yeast ceramide synthases, Lag1 and Lac1, have distinct substrate specificity.

J Cell Sci 2019 06 24;132(12). Epub 2019 Jun 24.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel

was the first longevity assurance gene discovered in The Lag1 protein is a ceramide synthase and its homolog, Lac1, has a similar enzymatic function but no role in aging. Lag1 and Lac1 lie in an enzymatic branch point of the sphingolipid pathway that is interconnected by the activity of the C4 hydroxylase, Sur2. By uncoupling the enzymatic branch point and using lipidomic mass spectrometry, metabolic labeling and assays we show that Lag1 preferentially synthesizes phyto-sphingolipids. Using photo-bleaching experiments we show that Lag1 is uniquely required for the establishment of a lateral diffusion barrier in the nuclear envelope, which depends on phytoceramide. Given the role of this diffusion barrier in the retention of aging factors in the mother cell, we suggest that the different specificities of the two ceramide synthases, and the specific effect of Lag1 on asymmetrical inheritance, may explain why Δ cells have an increased lifespan while Δ cells do not.
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http://dx.doi.org/10.1242/jcs.228411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602303PMC
June 2019

Modulation of asymmetric cell division as a mechanism to boost CD8 T cell memory.

Sci Immunol 2019 04;4(34)

Institute of Microbiology, ETH Zürich, Vladimir-Prelog-Weg 4, 8093 Zürich, Switzerland.

Asymmetric partitioning of fate determinants is a mechanism that contributes to T cell differentiation. However, it remains unclear whether the ability of T cells to divide asymmetrically is influenced by their differentiation state, as well as whether enforcing asymmetric cell division (ACD) rates would have an impact on T cell differentiation and memory formation. Using the murine LCMV infection model, we established a correlation between cell stemness and the ability of CD8 T cells to undergo ACD. Transient mTOR inhibition was proven to increase ACD rates in naïve and memory cells and to install this ability in exhausted CD8 T cells. Functionally, enforced ACD correlated with increased memory potential, leading to more efficient recall response and viral control upon acute or chronic LCMV infection. Moreover, transient mTOR inhibition also increased ACD rates in human CD8 T cells. Transcriptional profiling revealed that progenies emerging from enforced ACD exhibited more pronounced early memory signatures, which functionally endowed these cells with better survival in the absence of antigen exposure and more robust homing to secondary lymphoid organs, providing critical access to survival niches. Our data provide important insights into how ACD can improve long-term survival and function of T cells and open new perspectives for vaccination and adoptive T cell transfer therapies.
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http://dx.doi.org/10.1126/sciimmunol.aav1730DOI Listing
April 2019

Centromeres License the Mitotic Condensation of Yeast Chromosome Arms.

Cell 2018 10 11;175(3):780-795.e15. Epub 2018 Oct 11.

Institute of Biochemistry, Biology Department, ETH Zurich, 8093 Zurich, Switzerland. Electronic address:

During mitosis, chromatin condensation shapes chromosomes as separate, rigid, and compact sister chromatids to facilitate their segregation. Here, we show that, unlike wild-type yeast chromosomes, non-chromosomal DNA circles and chromosomes lacking a centromere fail to condense during mitosis. The centromere promotes chromosome condensation strictly in cis through recruiting the kinases Aurora B and Bub1, which trigger the autonomous condensation of the entire chromosome. Shugoshin and the deacetylase Hst2 facilitated spreading the condensation signal to the chromosome arms. Targeting Aurora B to DNA circles or centromere-ablated chromosomes or releasing Shugoshin from PP2A-dependent inhibition bypassed the centromere requirement for condensation and enhanced the mitotic stability of DNA circles. Our data indicate that yeast cells license the chromosome-autonomous condensation of their chromatin in a centromere-dependent manner, excluding from this process non-centromeric DNA and thereby inhibiting their propagation.
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http://dx.doi.org/10.1016/j.cell.2018.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197839PMC
October 2018

Asymmetric Segregation of Aged Spindle Pole Bodies During Cell Division: Mechanisms and Relevance Beyond Budding Yeast?

Bioessays 2018 08 5;40(8):e1800038. Epub 2018 Jul 5.

Institute of Biochemistry, ETH Zurich, Otto-Stern-Weg 3, 8093, Zurich, Switzerland.

Asymmetric cell division generates cell diversity and contributes to cellular aging and rejuvenation. Here, we review the molecular mechanisms enabling budding yeast to recognize spindle pole bodies (SPB, centrosome equivalent) based on their age, and guide their non-random mitotic segregation: SPB inheritance requires the distinction of old from new SPBs and is regulated by the SPB-inheritance network (SPIN) and the mitotic exit network (MEN). The SPIN marks the pre-existing SPB as old and the MEN recognizes these marks translating them into spindle orientation. We next revisit other molecules and structures that partition depending on their age rather than their abundance at mitosis as, for example, DNA, centrosomes, mitochondria, and histones in yeast and other systems. The recurrence of this differential behavior suggests a functional significance for numerous cell types, which we then discuss. We conclude that non-random segregation may facilitate asymmetric cell fate determination and thereby indirectly aging and rejuvenation. Also see the video abstract here: https://youtu.be/1sQ4rAomnWY.
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http://dx.doi.org/10.1002/bies.201800038DOI Listing
August 2018

Structure-Function Relationship of the Bik1-Bim1 Complex.

Structure 2018 04 22;26(4):607-618.e4. Epub 2018 Mar 22.

Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland; University of Basel, Biozentrum, 4056 Basel, Switzerland. Electronic address:

In budding yeast, the microtubule plus-end tracking proteins Bik1 (CLIP-170) and Bim1 (EB1) form a complex that interacts with partners involved in spindle positioning, including Stu2 and Kar9. Here, we show that the CAP-Gly and coiled-coil domains of Bik1 interact with the C-terminal ETF peptide of Bim1 and the C-terminal tail region of Stu2, respectively. The crystal structures of the CAP-Gly domain of Bik1 (Bik1CG) alone and in complex with an ETF peptide revealed unique, functionally relevant CAP-Gly elements, establishing Bik1CG as a specific C-terminal phenylalanine recognition domain. Unlike the mammalian CLIP-170-EB1 complex, Bik1-Bim1 forms ternary complexes with the EB1-binding motifs SxIP and LxxPTPh, which are present in diverse proteins, including Kar9. Perturbation of the Bik1-Bim1 interaction in vivo affected Bik1 localization and astral microtubule length. Our results provide insight into the role of the Bik1-Bim1 interaction for cell division, and demonstrate that the CLIP-170-EB1 module is evolutionarily flexible.
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http://dx.doi.org/10.1016/j.str.2018.03.003DOI Listing
April 2018

Heat stress promotes longevity in budding yeast by relaxing the confinement of age-promoting factors in the mother cell.

Elife 2017 12 28;6. Epub 2017 Dec 28.

Institute of Biochemistry, Department of Biology, ETH Zürich, Zürich, Switzerland.

Although individuals of many species inexorably age, a number of observations established that the rate of aging is modulated in response to a variety of mild stresses. Here, we investigated how heat stress promotes longevity in yeast. We show that upon growth at higher temperature, yeast cells relax the retention of DNA circles, which act as aging factors in the mother cell. The enhanced frequency at which circles redistribute to daughter cells was not due to changes of anaphase duration or nuclear shape but solely to the downregulation of the diffusion barrier in the nuclear envelope. This effect depended on the PKA and Tor1 pathways, downstream of stress-response kinase Pkc1. Inhibition of these responses restored barrier function and circle retention and abrogated the effect of heat stress on longevity. Our data indicate that redistribution of aging factors from aged cells to their progeny can be a mechanism for modulating longevity.
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http://dx.doi.org/10.7554/eLife.28329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771669PMC
December 2017

A Droplet to Sense Sugar Drops.

Mol Cell 2017 12;68(6):1017-1019

School of Biological and Chemical Sciences, Queen Mary University of London, London, UK. Electronic address:

Cells need to rewire their metabolic network depending on the available carbon source. Simpson-Lavy et al. (2017) have discovered that Std1, the activator of the yeast AMP kinase Snf1, condensates into granules to tune Snf1 activity.
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http://dx.doi.org/10.1016/j.molcel.2017.12.005DOI Listing
December 2017

Spatial cues and not spindle pole maturation drive the asymmetry of astral microtubules between new and preexisting spindle poles.

Mol Biol Cell 2018 01 15;29(1):10-28. Epub 2017 Nov 15.

Department of Biology, Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland

In many asymmetrically dividing cells, the microtubule-organizing centers (MTOCs; mammalian centrosome and yeast spindle pole body [SPB]) nucleate more astral microtubules on one of the two spindle poles than the other. This differential activity generally correlates with the age of MTOCs and contributes to orienting the mitotic spindle within the cell. The asymmetry might result from the two MTOCs being in distinctive maturation states. We investigated this model in budding yeast. Using fluorophores with different maturation kinetics to label the outer plaque components of the SPB, we found that the Cnm67 protein is mobile, whereas Spc72 is not. However, these two proteins were rapidly as abundant on both SPBs, indicating that SPBs mature more rapidly than anticipated. Superresolution microscopy confirmed this finding for Spc72 and for the γ-tubulin complex. Moreover, astral microtubule number and length correlated with the subcellular localization of SPBs rather than their age. Kar9-dependent orientation of the spindle drove the differential activity of the SPBs in astral microtubule organization rather than intrinsic differences between the spindle poles. Together, our data establish that Kar9 and spatial cues, rather than the kinetics of SPB maturation, control the asymmetry of astral microtubule organization between the preexisting and new SPBs.
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http://dx.doi.org/10.1091/mbc.E16-10-0725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746063PMC
January 2018

Budding yeast Wee1 distinguishes spindle pole bodies to guide their pattern of age-dependent segregation.

Nat Cell Biol 2017 Aug 17;19(8):941-951. Epub 2017 Jul 17.

Institute of Biochemistry, ETH Zurich, Otto-Stern-Weg 3, 8093 Zurich, Switzerland.

Many asymmetrically dividing cells unequally partition cellular structures according to age. Yet, it is unclear how cells differentiate pre-existing from newly synthesized material. Yeast cells segregate the spindle pole body (SPB, centrosome equivalent) inherited from the previous mitosis to the bud, while keeping the new one in the mother cell. Here, we show that the SPB inheritance network (SPIN), comprising the kinases Swe1 (also known as Wee1) and Kin3 (also known as Nek2) and the acetyltransferase NuA4 (also known as Tip60), distinguishes pre-existing from new SPBs. Swe1 phosphorylated Nud1 (orthologous to Centriolin) on young SPBs as they turned into pre-existing ones. The subsequent inactivation of Swe1 protected newly assembling SPBs from being marked. Kin3 and NuA4 maintained age marks on SPBs through following divisions. Downstream of SPIN, the Hippo regulator Bfa1-Bub2 bound the marked SPB, directed the spindle-positioning protein Kar9 towards it and drove its partition to the bud. Thus, coordination of SPIN activity and SPB assembly encodes age onto SPBs to enable their age-dependent segregation.
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http://dx.doi.org/10.1038/ncb3576DOI Listing
August 2017

Aggregation of the Whi3 protein, not loss of heterochromatin, causes sterility in old yeast cells.

Science 2017 03 16;355(6330):1184-1187. Epub 2017 Mar 16.

Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, CA 94720, USA.

In yeast, heterochromatin silencing is reported to decline in aging mother cells, causing sterility in old cells. This process is thought to reflect a decrease in the activity of the NAD (oxidized nicotinamide adenine dinucleotide)-dependent deacetylase Sir2. We tested whether Sir2 becomes nonfunctional gradually or precipitously during aging. Unexpectedly, silencing of the heterochromatic and loci was not lost during aging. Old cells could initiate a mating response; however, they were less sensitive to mating pheromone than were young cells because of age-dependent aggregation of Whi3, an RNA-binding protein controlling S-phase entry. Removing the polyglutamine domain of Whi3 restored the pheromone sensitivity of old cells. We propose that aging phenotypes previously attributed to loss of heterochromatin silencing are instead caused by aggregation of the Whi3 cell cycle regulator.
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http://dx.doi.org/10.1126/science.aaj2103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728107PMC
March 2017

Compartmentalization of ER-Bound Chaperone Confines Protein Deposit Formation to the Aging Yeast Cell.

Curr Biol 2017 Mar 2;27(6):773-783. Epub 2017 Mar 2.

Institute of Biochemistry, ETH Zurich, Otto-Stern-Weg 3, 8093 Zurich, Switzerland. Electronic address:

In order to produce rejuvenated daughters, dividing budding yeast cells confine aging factors, including protein aggregates, to the aging mother cell. The asymmetric inheritance of these protein deposits is mediated by organelle and cytoskeletal attachment and by cell geometry. Yet it remains unclear how deposit formation is restricted to the aging lineage. Here, we show that selective membrane anchoring and the compartmentalization of the endoplasmic reticulum (ER) membrane confine protein deposit formation to aging cells during division. Supporting the idea that the age-dependent deposit forms through coalescence of smaller aggregates, two deposits rapidly merged when placed in the same cell by cell-cell fusion. The deposits localized to the ER membrane, primarily to the nuclear envelope (NE). Strikingly, weakening the diffusion barriers that separate the ER membrane into mother and bud compartments caused premature formation of deposits in the daughter cells. Detachment of the Hsp40 protein Ydj1 from the ER membrane elicited a similar phenotype, suggesting that the diffusion barriers and farnesylated Ydj1 functioned together to confine protein deposit formation to mother cells during division. Accordingly, fluorescence correlation spectroscopy measurements in dividing cells indicated that a slow-diffusing, possibly client-bound Ydj1 fraction was asymmetrically enriched in the mother compartment. This asymmetric distribution depended on Ydj1 farnesylation and intact diffusion barriers. Taking these findings together, we propose that ER-anchored Ydj1 binds deposit precursors and prevents them from spreading into daughter cells during division by subjecting them to the ER diffusion barriers. This ensures that the coalescence of precursors into a single deposit is restricted to the aging lineage.
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http://dx.doi.org/10.1016/j.cub.2017.01.069DOI Listing
March 2017

Molecular basis of Kar9-Bim1 complex function during mating and spindle positioning.

Mol Biol Cell 2016 Sep 28. Epub 2016 Sep 28.

Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, Villigen PSI, Switzerland

The Kar9 pathway promotes nuclear fusion during mating and spindle alignment during metaphase in budding yeast. How Kar9 supports the different outcome of these two divergent processes is an open question. Here, we show that three sites in the C-terminal disordered domain of Kar9 mediate tight Kar9 interaction with the C-terminal dimerization domain of Bim1 (EB1 orthologue). Site1 and Site2 contain SxIP motifs; however, Site3 defines a novel type of EB1-binding site. Whereas Site2 and Site3 mediate Kar9 recruitment to microtubule tips, nuclear movement and karyogamy, solely Site2 functions in spindle positioning during metaphase. Site1 in turn plays an inhibitory role during mating. Additionally, the Kar9-Bim1 complex is involved in microtubule-independent activities during mating. Together, our data reveal how multiple and partially redundant EB1-binding sites provide a microtubule-associated protein with the means to modulate its biochemical properties to promote different molecular processes during cell proliferation and differentiation.
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http://dx.doi.org/10.1091/mbc.E16-07-0552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5170556PMC
September 2016

Compartmentalization of the endoplasmic reticulum in the early C. elegans embryos.

J Cell Biol 2016 Sep 5;214(6):665-76. Epub 2016 Sep 5.

Department of Biology, Institute of Biochemistry, Swiss Federal Institute of Technology Zürich, CH-8093 Zürich, Switzerland

The one-cell Caenorhabditis elegans embryo is polarized to partition fate determinants between the cell lineages generated during its first division. Using fluorescence loss in photobleaching, we find that the endoplasmic reticulum (ER) of the C. elegans embryo is physically continuous throughout the cell, but its membrane is compartmentalized shortly before nuclear envelope breakdown into an anterior and a posterior domain, indicating that a diffusion barrier forms in the ER membrane between these two domains. Using mutants with disorganized ER, we show that ER compartmentalization is independent of the morphological transition that the ER undergoes in mitosis. In contrast, compartmentalization takes place at the position of the future cleavage plane in a par-3-dependent manner. Together, our data indicate that the ER membrane is compartmentalized in cells as diverse as budding yeast, mouse neural stem cells, and the early C. elegans embryo.
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http://dx.doi.org/10.1083/jcb.201601047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5021094PMC
September 2016

Asymmetric partitioning of transfected DNA during mammalian cell division.

Proc Natl Acad Sci U S A 2016 06 13;113(26):7177-82. Epub 2016 Jun 13.

Institute of Biochemistry, Department of Biology, Swiss Federal Institute of Technology Zurich (ETH Zurich), CH-8093 Zurich, Switzerland,

Foreign DNA molecules and chromosomal fragments are generally eliminated from proliferating cells, but we know little about how mammalian cells prevent their propagation. Here, we show that dividing human and canine cells partition transfected plasmid DNA asymmetrically, preferentially into the daughter cell harboring the young centrosome. Independently of how they entered the cell, most plasmids clustered in the cytoplasm. Unlike polystyrene beads of similar size, these clusters remained relatively immobile and physically associated to endoplasmic reticulum-derived membranes, as revealed by live cell and electron microscopy imaging. At entry of mitosis, most clusters localized near the centrosomes. As the two centrosomes split to assemble the bipolar spindle, predominantly the old centrosome migrated away, biasing the partition of the plasmid cluster toward the young centrosome. Down-regulation of the centrosomal proteins Ninein and adenomatous polyposis coli abolished this bias. Thus, we suggest that DNA clustering, cluster immobilization through association to the endoplasmic reticulum membrane, initial proximity between the cluster and centrosomes, and subsequent differential behavior of the two centrosomes together bias the partition of plasmid DNA during mitosis. This process leads to their progressive elimination from the proliferating population and might apply to any kind of foreign DNA molecule in mammalian cells. Furthermore, the functional difference of the centrosomes might also promote the asymmetric partitioning of other cellular components in other mammalian and possibly stem cells.
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http://dx.doi.org/10.1073/pnas.1606091113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4932973PMC
June 2016

Protein aggregation as a mechanism of adaptive cellular responses.

Curr Genet 2016 Nov 31;62(4):711-724. Epub 2016 Mar 31.

ETH Zurich, Institute of Biochemistry, Otto-Stern-Weg 3, 8093, Zurich, Switzerland.

Coalescence of proteins into different types of intracellular bodies has surfaced as a widespread adaptive mechanism to re-organize cells and cellular functions in response to specific cues. These structures, composed of proteins or protein-mRNA-complexes, regulate cellular processes through modulating enzymatic activities, gene expression or shielding macromolecules from damage. Accordingly, such bodies are associated with a wide-range of processes, including meiosis, memory-encoding, host-pathogen interactions, cancer, stress responses, as well as protein quality control, DNA replication stress and aneuploidy. Importantly, these distinct coalescence responses are controlled, and in many cases regulated by chaperone proteins. While cells can tolerate and proficiently coordinate numerous distinct types of protein bodies, some of them are also intimately linked to diseases or the adverse effects of aging. Several protein bodies that differ in composition, packing, dynamics, size, and localization were originally discovered in budding yeast. Here, we provide a concise and comparative review of their nature and nomenclature.
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http://dx.doi.org/10.1007/s00294-016-0596-0DOI Listing
November 2016

Posttranslational Regulation: A Way to Evolve.

Curr Biol 2016 Feb;26(3):R119-21

Institute of Biochemistry, Department of Biology, ETH Zürich, Otto-Stern-Weg 3, 8093 Zürich, Switzerland. Electronic address:

A new study shows that differences in the regulation of lipin can account for the different strategies of nuclear division in two closely related fission yeast species.
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http://dx.doi.org/10.1016/j.cub.2015.12.027DOI Listing
February 2016

Axial contraction and short-range compaction of chromatin synergistically promote mitotic chromosome condensation.

Elife 2015 Nov 28;4:e1039. Epub 2015 Nov 28.

Institute of Biochemistry, Department of Biology, Eidgenössische Technische Hochschule Zürich, Zürich, Switzerland.

The segregation of eukaryotic chromosomes during mitosis requires their extensive folding into units of manageable size for the mitotic spindle. Here, we report on how phosphorylation at serine 10 of histone H3 (H3 S10) contributes to this process. Using a fluorescence-based assay to study local compaction of the chromatin fiber in living yeast cells, we show that chromosome condensation entails two temporally and mechanistically distinct processes. Initially, nucleosome-nucleosome interaction triggered by H3 S10 phosphorylation and deacetylation of histone H4 promote short-range compaction of chromatin during early anaphase. Independently, condensin mediates the axial contraction of chromosome arms, a process peaking later in anaphase. Whereas defects in chromatin compaction have no observable effect on axial contraction and condensin inactivation does not affect short-range chromatin compaction, inactivation of both pathways causes synergistic defects in chromosome segregation and cell viability. Furthermore, both pathways rely at least partially on the deacetylase Hst2, suggesting that this protein helps coordinating chromatin compaction and axial contraction to properly shape mitotic chromosomes.
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http://dx.doi.org/10.7554/eLife.10396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755758PMC
November 2015

Protein aggregates are associated with replicative aging without compromising protein quality control.

Elife 2015 Nov 6;4. Epub 2015 Nov 6.

Institute of Biochemistry, Eidgenössische Technische Hochschule Zürich, Zürich, Switzerland.

Differentiation of cellular lineages is facilitated by asymmetric segregation of fate determinants between dividing cells. In budding yeast, various aging factors segregate to the aging (mother)-lineage, with poorly understood consequences. In this study, we show that yeast mother cells form a protein aggregate during early replicative aging that is maintained as a single, asymmetrically inherited deposit over the remaining lifespan. Surprisingly, deposit formation was not associated with stress or general decline in proteostasis. Rather, the deposit-containing cells displayed enhanced degradation of cytosolic proteasome substrates and unimpaired clearance of stress-induced protein aggregates. Deposit formation was dependent on Hsp42, which collected non-random client proteins of the Hsp104/Hsp70-refolding machinery, including the prion Sup35. Importantly, loss of Hsp42 resulted in symmetric inheritance of its constituents and prolonged the lifespan of the mother cell. Together, these data suggest that protein aggregation is an early aging-associated differentiation event in yeast, having a two-faceted role in organismal fitness.
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http://dx.doi.org/10.7554/eLife.06197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4635334PMC
November 2015

Fluorescence Recovery After Photo-Bleaching (FRAP) and Fluorescence Loss in Photo-Bleaching (FLIP) Experiments to Study Protein Dynamics During Budding Yeast Cell Division.

Methods Mol Biol 2016 ;1369:25-44

ETH Zürich, Institute of Biochemistry, Otto-Stern-Weg 3, Zürich, 8093, Switzerland.

The easiness of tagging any protein of interest with a fluorescent marker together with the advance of fluorescence microscopy techniques enable researchers to study in great detail the dynamic behavior of proteins both in time and space in living cells. Two commonly used techniques are FRAP (Fluorescent Recovery After Photo-bleaching) and FLIP (Fluorescence Loss In Photo-bleaching). Upon single bleaching (FRAP) or constant bleaching (FLIP) of the fluorescent signal in a specific area of the cell, the intensity of the fluorophore is monitored over time in the bleached area and in surrounding regions; information is then derived about the diffusion speed of the tagged molecule, the amount of mobile versus immobile molecules as well as the kinetics with which they exchange between different parts of the cell. Thereby, FRAP and FLIP are very informative about the kinetics with which the different organelles of the cell separate into mother- and daughter-specific compartments during cell division. Here, we describe protocols for both FRAP and FLIP and explain how they can be used to study protein dynamics during cell division in the budding yeast Saccharomyces cerevisiae. These techniques are easily adaptable to other model organisms.
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http://dx.doi.org/10.1007/978-1-4939-3145-3_3DOI Listing
May 2016

A mechanism for the segregation of age in mammalian neural stem cells.

Science 2015 09;349(6254):1334-8

Brain Research Institute, Faculty of Medicine and Science, University of Zürich, 8057 Zürich, Switzerland.

Throughout life, neural stem cells (NSCs) generate neurons in the mammalian brain. Using photobleaching experiments, we found that during cell division in vitro and within the developing mouse forebrain, NSCs generate a lateral diffusion barrier in the membrane of the endoplasmic reticulum, thereby promoting asymmetric segregation of cellular components. The diffusion barrier weakens with age and in response to impairment of lamin-associated nuclear envelope constituents. Weakening of the diffusion barrier disrupts asymmetric segregation of damaged proteins, a product of aging. Damaged proteins are asymmetrically inherited by the nonstem daughter cell in embryonic and young adult NSC divisions, whereas in the older adult brain, damaged proteins are more symmetrically distributed between progeny. Thus, these data identify a mechanism of how damage that accumulates with age is asymmetrically distributed during somatic stem cell division.
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http://dx.doi.org/10.1126/science.aac9868DOI Listing
September 2015

Rho1- and Pkc1-dependent phosphorylation of the F-BAR protein Syp1 contributes to septin ring assembly.

Mol Biol Cell 2015 Sep 15;26(18):3245-62. Epub 2015 Jul 15.

Centre de Recherche en Biochimie Macromoléculaire, 34293 Montpellier, France

In many cell types, septins assemble into filaments and rings at the neck of cellular appendages and/or at the cleavage furrow to help compartmentalize the plasma membrane and support cytokinesis. How septin ring assembly is coordinated with membrane remodeling and controlled by mechanical stress at these sites is unclear. Through a genetic screen, we uncovered an unanticipated link between the conserved Rho1 GTPase and its effector protein kinase C (Pkc1) with septin ring stability in yeast. Both Rho1 and Pkc1 stabilize the septin ring, at least partly through phosphorylation of the membrane-associated F-BAR protein Syp1, which colocalizes asymmetrically with the septin ring at the bud neck. Syp1 is displaced from the bud neck upon Pkc1-dependent phosphorylation at two serines, thereby affecting the rigidity of the new-forming septin ring. We propose that Rho1 and Pkc1 coordinate septin ring assembly with membrane and cell wall remodeling partly by controlling Syp1 residence at the bud neck.
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http://dx.doi.org/10.1091/mbc.E15-06-0366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569315PMC
September 2015

Asymmetry of the budding yeast Tem1 GTPase at spindle poles is required for spindle positioning but not for mitotic exit.

PLoS Genet 2015 Feb 6;11(2):e1004938. Epub 2015 Feb 6.

Centre de Recherche en Biochimie Macromoléculaire, Montpellier, France; Dipartimento di Biotecnologie e Bioscienze Università degli Studi di Milano-Bicocca, Milano, Italy.

The asymmetrically dividing yeast S. cerevisiae assembles a bipolar spindle well after establishing the future site of cell division (i.e., the bud neck) and the division axis (i.e., the mother-bud axis). A surveillance mechanism called spindle position checkpoint (SPOC) delays mitotic exit and cytokinesis until the spindle is properly positioned relative to the mother-bud axis, thereby ensuring the correct ploidy of the progeny. SPOC relies on the heterodimeric GTPase-activating protein Bub2/Bfa1 that inhibits the small GTPase Tem1, in turn essential for activating the mitotic exit network (MEN) kinase cascade and cytokinesis. The Bub2/Bfa1 GAP and the Tem1 GTPase form a complex at spindle poles that undergoes a remarkable asymmetry during mitosis when the spindle is properly positioned, with the complex accumulating on the bud-directed old spindle pole. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. The mechanism driving asymmetry of Bub2/Bfa1/Tem1 in mitosis is unclear. Furthermore, whether asymmetry is involved in timely mitotic exit is controversial. We investigated the mechanism by which the GAP Bub2/Bfa1 controls GTP hydrolysis on Tem1 and generated a series of mutants leading to constitutive Tem1 activation. These mutants are SPOC-defective and invariably lead to symmetrical localization of Bub2/Bfa1/Tem1 at spindle poles, indicating that GTP hydrolysis is essential for asymmetry. Constitutive tethering of Bub2 or Bfa1 to both spindle poles impairs SPOC response but does not impair mitotic exit. Rather, it facilitates mitotic exit of MEN mutants, likely by increasing the residence time of Tem1 at spindle poles where it gets active. Surprisingly, all mutant or chimeric proteins leading to symmetrical localization of Bub2/Bfa1/Tem1 lead to increased symmetry at spindle poles of the Kar9 protein that mediates spindle positioning and cause spindle misalignment. Thus, asymmetry of the Bub2/Bfa1/Tem1 complex is crucial to control Kar9 distribution and spindle positioning during mitosis.
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http://dx.doi.org/10.1371/journal.pgen.1004938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450052PMC
February 2015

Role of SAGA in the asymmetric segregation of DNA circles during yeast ageing.

Elife 2014 Nov 17;3. Epub 2014 Nov 17.

Institute of Biochemistry, Department of Biology, ETH Zürich, Zürich, Switzerland.

In eukaryotes, intra-chromosomal recombination generates DNA circles, but little is known about how cells react to them. In yeast, partitioning of such circles to the mother cell at mitosis ensures their loss from the population but promotes replicative ageing. Nevertheless, the mechanisms of partitioning are debated. In this study, we show that the SAGA complex mediates the interaction of non-chromosomal DNA circles with nuclear pore complexes (NPCs) and thereby promotes their confinement in the mother cell. Reciprocally, this causes retention and accumulation of NPCs, which affects the organization of ageing nuclei. Thus, SAGA prevents the spreading of DNA circles by linking them to NPCs, but unavoidably causes accumulation of circles and NPCs in the mother cell, and thereby promotes ageing. Together, our data provide a unifying model for the asymmetric segregation of DNA circles and how age affects nuclear organization.
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http://dx.doi.org/10.7554/eLife.03790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4232608PMC
November 2014

A sphingolipid-dependent diffusion barrier confines ER stress to the yeast mother cell.

Elife 2014 May 6;3:e01883. Epub 2014 May 6.

Institute of Biochemistry, Department of Biology, ETH Zürich, Zürich, Switzerland

In many cell types, lateral diffusion barriers compartmentalize the plasma membrane and, at least in budding yeast, the endoplasmic reticulum (ER). However, the molecular nature of these barriers, their mode of action and their cellular functions are unclear. Here, we show that misfolded proteins of the ER remain confined into the mother compartment of budding yeast cells. Confinement required the formation of a lateral diffusion barrier in the form of a distinct domain of the ER-membrane at the bud neck, in a septin-, Bud1 GTPase- and sphingolipid-dependent manner. The sphingolipids, but not Bud1, also contributed to barrier formation in the outer membrane of the dividing nucleus. Barrier-dependent confinement of ER stress into the mother cell promoted aging. Together, our data clarify the physical nature of lateral diffusion barriers in the ER and establish the role of such barriers in the asymmetric segregation of proteotoxic misfolded proteins during cell division and aging.DOI: http://dx.doi.org/10.7554/eLife.01883.001.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4009826PMC
http://dx.doi.org/10.7554/eLife.01883DOI Listing
May 2014