Jan Skotheim

All Publications

• Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division. Science (New York, N.Y.) Zatulovskiy, E., Zhang, S., Berenson, D. F., Topacio, B. R., Skotheim, J. M. 2020; 369 (6502): 466–71

  Abstract

  Cell size is fundamental to cell physiology. For example, cell size determines the spatial scale of organelles and intracellular transport and thereby affects biosynthesis. Although some genes that affect mammalian cell size have been identified, the molecular mechanisms through which cell growth drives cell division have remained elusive. We show that cell growth during the G1 phase of the cell division cycle dilutes the cell cycle inhibitor Retinoblastoma protein (Rb) to trigger division in human cells. RB overexpression increased cell size and G1 duration, whereas RB deletion decreased cell size and removed the inverse correlation between cell size at birth and the duration of the G1 phase. Thus, Rb dilution through cell growth in G1 provides one of the long-sought molecular mechanisms that promotes cell size homeostasis.

  View details for DOI 10.1126/science.aaz6213

  View details for PubMedID 32703881

• PP2ACdc55 dephosphorylates Pds1 and inhibits spindle elongation. Journal of cell science Khondker, S., Kajjo, S., Chandler-Brown, D., Skotheim, J., Rudner, A., Ikui, A. 2020

  Abstract

  PP2ACdc55 regulates cell cycle progression by reversing cyclin-dependent kinase (CDK) and polo-like kinase (Cdc5) dependent phosphorylation events. In S. cerevisiae, Cdk1 phosphorylates securin (Pds1), which facilitates Pds1 binding and inhibiting separase (Esp1). During anaphase, Esp1 cleaves the cohesin subunit Scc1 and promotes spindle elongation. Here, we show that PP2ACdc55 directly dephosphorylates Pds1 both in vivo and in vitro Pds1 hyperphosphorylation in a cdc55 deletion mutant enhanced the Pds1-Esp1 interaction, which played a positive role in Pds1 nuclear accumulation and in spindle elongation. We also show that nuclear PP2ACdc55 played a role during replication stress to inhibit spindle elongation. This pathway acted independently of the known Mec1, Swe1 or Spindle Assembly Checkpoint (SAC) checkpoint pathways. We propose a model where Pds1 dephosphorylation by PP2ACdc55 disrupts the Pds1-Esp1 protein interaction and inhibits Pds1 nuclear accumulation, which prevents spindle elongation, a process that is elevated during replication stress.

  View details for DOI 10.1242/jcs.243766

  View details for PubMedID 32591482

• Long-range single-molecule mapping of chromatin accessibility in eukaryotes. Nature methods Shipony, Z., Marinov, G. K., Swaffer, M. P., Sinnott-Armstrong, N. A., Skotheim, J. M., Kundaje, A., Greenleaf, W. J. 2020

  Abstract

  Mapping open chromatin regions has emerged as a widely used tool for identifying active regulatory elements in eukaryotes. However, existing approaches, limited by reliance on DNA fragmentation and short-read sequencing, cannot provide information about large-scale chromatin states or reveal coordination between the states of distal regulatory elements. We have developed a method for profiling the accessibility of individual chromatin fibers, a single-molecule long-read accessible chromatin mapping sequencing assay (SMAC-seq), enabling the simultaneous, high-resolution, single-molecule assessment of chromatin states at multikilobase length scales. Our strategy is based on combining the preferential methylation of open chromatin regions by DNA methyltransferases with low sequence specificity, in this case EcoGII, an N6-methyladenosine (m6A) methyltransferase, and the ability of nanopore sequencing to directly read DNA modifications. We demonstrate that aggregate SMAC-seq signals match bulk-level accessibility measurements, observe single-molecule nucleosome and transcription factor protection footprints, and quantify the correlation between chromatin states of distal genomic elements.

  View details for DOI 10.1038/s41592-019-0730-2

  View details for PubMedID 32042188

• Integrating Old and New Paradigms of G1/S Control. Molecular cell Rubin, S. M., Sage, J., Skotheim, J. M. 2020

  Abstract

  The Cdk-Rb-E2F pathway integrates external and internal signals to control progression at the G1/S transition of the mammalian cell cycle. Alterations in this pathway are found in most human cancers, and specific cyclin-dependent kinase Cdk4/6 inhibitors are approved or in clinical trials for the treatment of diverse cancers. In the long-standing paradigm for G1/S control, Cdks inactivate the retinoblastoma tumor suppressor protein (Rb) through phosphorylation, which releases E2F transcription factors to drive cell-cycle progression from G1 to S. However, recent observations in the laboratory and clinic challenge central tenets of the current paradigm and demonstrate that our understanding of the Rb pathway and G1/S control is still incomplete. Here, we integrate these new findings with the previous paradigm to synthesize a current molecular and cellular view of the mammalian G1/S transition. A more complete and accurate understanding of G1/S control will lead to improved therapeutic strategies targeting the cell cycle in cancer.

  View details for DOI 10.1016/j.molcel.2020.08.020

  View details for PubMedID 32946743

• On the Molecular Mechanisms Regulating Animal Cell Size Homeostasis. Trends in genetics : TIG Zatulovskiy, E., Skotheim, J. M. 2020; 36 (5): 360–72

  Abstract

  Cell size is fundamental to cell physiology because it sets the scale of intracellular geometry, organelles, and biosynthetic processes. In animal cells, size homeostasis is controlled through two phenomenologically distinct mechanisms. First, size-dependent cell cycle progression ensures that smaller cells delay cell cycle progression to accumulate more biomass than larger cells prior to cell division. Second, size-dependent cell growth ensures that larger and smaller cells grow slower per unit mass than more optimally sized cells. This decade has seen dramatic progress in single-cell technologies establishing the diverse phenomena of cell size control in animal cells. Here, we review this recent progress and suggest pathways forward to determine the underlying molecular mechanisms.

  View details for DOI 10.1016/j.tig.2020.01.011

  View details for PubMedID 32294416

• A G1 Sizer Coordinates Growth and Division in the Mouse Epidermis. Current biology : CB Xie, S., Skotheim, J. M. 2020

  Abstract

  Cell size homeostasis is often achieved by coupling cell-cycle progression to cell growth. Growth has been shown to drive cell-cycle progression in bacteria and yeast through "sizers," wherein cells of varying birth size divide at similar final sizes [1-3], and "adders," wherein cells increase in size a fixed amount per cell cycle [4-6]. Intermediate control phenomena are also observed, and even the same organism can exhibit different control phenomena depending on growth conditions [2, 7, 8]. Although studying unicellular organisms in laboratory conditions may give insight into their growth control in the wild, this is less apparent for studies of mammalian cells growing outside the organism. Sizers, adders, and intermediate phenomena have been observed in vitro [9-12], but it is unclear how this relates to mammalian cell proliferation in vivo. To address this question, we analyzed time-lapse images of the mouse epidermis taken over 1 week during normal tissue turnover [13]. We quantified the 3D volume growth and cell-cycle progression of single cells within the mouse skin. In dividing epidermal stem cells, we found that cell growth is coupled to division through a sizer operating largely in the G1 phase of the cell cycle. Thus, although the majority of tissue culture studies have identified adders, our analysis demonstrates that sizers are important in vivo and highlights the need to determine their underlying molecular origin.

  View details for DOI 10.1016/j.cub.2019.12.062

  View details for PubMedID 32109398

• Constitutive expression of a fluorescent protein reports the size of live human cells. Molecular biology of the cell Berenson, D. F., Zatulovskiy, E., Xie, S., Skotheim, J. M. 2019: mbcE19030171

  Abstract

  Cell size is important for cell physiology because it sets the geometric scale of organelles and biosynthesis. A number of methods exist to measure different aspects of cell size, but each has significant drawbacks. Here, we present an alternative method to measure the size of single human cells using a nuclear localized fluorescent protein expressed from a constitutive promoter. We validate this method by comparing it to several established cell size measurement strategies, including flow cytometry optical scatter, total protein dyes, and quantitative phase microscopy. We directly compare our fluorescent protein measurement to the commonly used measurement of nuclear volume and show that our measurements are more robust and less dependent on image segmentation. We apply our method to examine how cell size impacts the cell division cycle and reaffirm that there is a negative correlation between size at cell birth and G1 duration. Importantly, combining our size reporter with fluorescent labeling of a different protein in a different color channel allows measurement of concentration dynamics using simple wide-field fluorescence imaging. Thus, we expect our method will be of use to researchers interested in how dynamically changing protein concentrations control cell fates. [Media: see text].

  View details for DOI 10.1091/mbc.E19-03-0171

  View details for PubMedID 31599704

• Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein's C-Terminal Helix MOLECULAR CELL Topacio, B. R., Zatulovskiy, E., Cristea, S., Xie, S., Tambo, C. S., Rubin, S. M., Sage, J., Koivomagi, M., Skotheim, J. M. 2019; 74 (4): 758-+

  View details for DOI 10.1016/j.molcel.2019.03.020

  View details for Web of Science ID 000468094300012

• Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9 MOLECULAR CELL Shariati, S., Dominguez, A., Xie, S., Wernig, M., Qi, L. S., Skotheim, J. M. 2019; 74 (3): 622-+

  View details for DOI 10.1016/j.molcel.2019.04.011

  View details for Web of Science ID 000466703900020

• Cell cycle, cell division, cell death MOLECULAR BIOLOGY OF THE CELL Maddox, A., Skotheim, J. M. 2019; 30 (6): 732

  View details for DOI 10.1091/mbc.E18-12-0819

  View details for Web of Science ID 000461742900002

• Cell cycle, cell division, cell death. Molecular biology of the cell Maddox, A. S., Skotheim, J. M. 2019; 30 (6): 732

  View details for PubMedID 30870093

• Multiple Layers of Phospho-Regulation Coordinate Metabolism and the Cell Cycle in Budding Yeast. Frontiers in cell and developmental biology Zhang, L., Winkler, S., Schlottmann, F. P., Kohlbacher, O., Elias, J. E., Skotheim, J. M., Ewald, J. C. 2019; 7: 338

  Abstract

  The coordination of metabolism and growth with cell division is crucial for proliferation. While it has long been known that cell metabolism regulates the cell division cycle, it is becoming increasingly clear that the cell division cycle also regulates metabolism. In budding yeast, we previously showed that over half of all measured metabolites change concentration through the cell cycle indicating that metabolic fluxes are extensively regulated during cell cycle progression. However, how this regulation is achieved still remains poorly understood. Since both the cell cycle and metabolism are regulated to a large extent by protein phosphorylation, we here decided to measure the phosphoproteome through the budding yeast cell cycle. Specifically, we chose a cell cycle synchronization strategy that avoids stress and nutrient-related perturbations of metabolism, and we grew the yeast on ethanol minimal medium to force cells to utilize their full biosynthetic repertoire. Using a tandem-mass-tagging approach, we found over 200 sites on metabolic enzymes and transporters to be phospho-regulated. These sites were distributed among many pathways including carbohydrate catabolism, lipid metabolism, and amino acid synthesis and therefore likely contribute to changing metabolic fluxes through the cell cycle. Among all one thousand sites whose phosphorylation increases through the cell cycle, the CDK consensus motif and an arginine-directed motif were highly enriched. This arginine-directed R-R-x-S motif is associated with protein-kinase A, which regulates metabolism and promotes growth. Finally, we also found over one thousand sites that are dephosphorylated through the G1/S transition. We speculate that the phosphatase Glc7/PP1, known to regulate both the cell cycle and carbon metabolism, may play an important role because its regulatory subunits are phospho-regulated in our data. In summary, our results identify extensive cell cycle dependent phosphorylation and dephosphorylation of metabolic enzymes and suggest multiple mechanisms through which the cell division cycle regulates metabolic signaling pathways to temporally coordinate biosynthesis with distinct phases of the cell division cycle.

  View details for DOI 10.3389/fcell.2019.00338

  View details for PubMedID 31921850

  View details for PubMedCentralID PMC6927922

• Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts ELIFE Bell, J. C., Jukam, D., Teran, N. A., Risca, V. I., Smith, O. K., Johnson, W. L., Skotheim, J. M., Greenleaf, W., Straight, A. F. 2018; 7

  Abstract

  RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here, we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. Using Drosophila cells, we show that ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.

  View details for PubMedID 29648534

• A Precise Cdk Activity Threshold Determines Passage through the Restriction Point. Molecular cell Schwarz, C., Johnson, A., Kõivomägi, M., Zatulovskiy, E., Kravitz, C. J., Doncic, A., Skotheim, J. M. 2018; 69 (2): 253–64.e5

  Abstract

  At the restriction point (R), mammalian cells irreversibly commit to divide. R has been viewed as a point in G1 that is passed when growth factor signaling initiates a positive feedback loop of Cdk activity. However, recent studies have cast doubt on this model by claiming R occurs prior to positive feedback activation in G1 or even before completion of the previous cell cycle. Here we reconcile these results and show that whereas many commonly used cell lines do not exhibit a G1 R, primary fibroblasts have a G1 R that is defined by a precise Cdk activity threshold and the activation of cell-cycle-dependent transcription. A simple threshold model, based solely on Cdk activity, predicted with more than 95% accuracy whether individual cells had passed R. That a single measurement accurately predicted cell fate shows that the state of complex regulatory networks can be assessed using a few critical protein activities.

  View details for PubMedID 29351845

  View details for PubMedCentralID PMC5790185

• Form and function of topologically associating genomic domains in budding yeast PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Eser, U., Chandler-Brown, D., Ay, F., Straight, A. F., Duang, Z., Noble, W. S., Skotheim, J. M. 2017; 114 (15): E3061-E3070

  Abstract

  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.

  View details for DOI 10.1073/pnas.1612256114

  View details for Web of Science ID 000398789800011

  View details for PubMedID 28348222

  View details for PubMedCentralID PMC5393236

• Spatial and temporal signal processing and decision making by MAPK pathways. journal of cell biology Atay, O., Skotheim, J. M. 2017; 216 (2): 317-330

  Abstract

  Mitogen-activated protein kinase (MAPK) pathways are conserved from yeast to man and regulate a variety of cellular processes, including proliferation and differentiation. Recent developments show how MAPK pathways perform exquisite spatial and temporal signal processing and underscores the importance of studying the dynamics of signaling pathways to understand their physiological response. The importance of dynamic mechanisms that process input signals into graded downstream responses has been demonstrated in the pheromone-induced and osmotic stress-induced MAPK pathways in yeast and in the mammalian extracellular signal-regulated kinase MAPK pathway. Particularly, recent studies in the yeast pheromone response have shown how positive feedback generates switches, negative feedback enables gradient detection, and coherent feedforward regulation underlies cellular memory. More generally, a new wave of quantitative single-cell studies has begun to elucidate how signaling dynamics determine cell physiology and represents a paradigm shift from descriptive to predictive biology.

  View details for DOI 10.1083/jcb.201609124

  View details for PubMedID 28043970

  View details for PubMedCentralID PMC5294789

• Zygotic Genome Activation in Vertebrates. Developmental cell Jukam, D., Shariati, S. A., Skotheim, J. M. 2017; 42 (4): 316–32

  Abstract

  The first major developmental transition in vertebrate embryos is the maternal-to-zygotic transition (MZT) when maternal mRNAs are degraded and zygotic transcription begins. During the MZT, the embryo takes charge of gene expression to control cell differentiation and further development. This spectacular organismal transition requires nuclear reprogramming and the initiation of RNAPII at thousands of promoters. Zygotic genome activation (ZGA) is mechanistically coordinated with other embryonic events, including changes in the cell cycle, chromatin state, and nuclear-to-cytoplasmic component ratios. Here, we review progress in understanding vertebrate ZGA dynamics in frogs, fish, mice, and humans to explore differences and emphasize common features.

  View details for PubMedID 28829942

  View details for PubMedCentralID PMC5714289

• The Adder Phenomenon Emerges from Independent Control of Pre- and Post-Start Phases of the Budding Yeast Cell Cycle. Current biology : CB Chandler-Brown, D., Schmoller, K. M., Winetraub, Y., Skotheim, J. M. 2017

  Abstract

  Although it has long been clear that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained poorly understood. In budding yeast, cell size primarily modulates the duration of the cell-division cycle by controlling the G1/S transition known as Start. We have recently shown that the rate of progression through Start increases with cell size, because cell growth dilutes the cell-cycle inhibitor Whi5 in G1. Recent phenomenological studies in yeast and bacteria have shown that these cells add an approximately constant volume during each complete cell cycle, independent of their size at birth. These results seem to be in conflict, as the phenomenological studies suggest that cells measure the amount they grow, rather than their size, and that size control acts over the whole cell cycle, rather than specifically in G1. Here, we propose an integrated model that unifies the adder phenomenology with the molecular mechanism of G1/S cell-size control. We use single-cell microscopy to parameterize a full cell-cycle model based on independent control of pre- and post-Start cell-cycle periods. We find that our model predicts the size-independent amount of cell growth during the full cell cycle. This suggests that the adder phenomenon is an emergent property of the independent regulation of pre- and post-Start cell-cycle periods rather than the consequence of an underlying molecular mechanism measuring a fixed amount of growth.

  View details for PubMedID 28889980

• Switch-like Transitions Insulate Network Motifs to Modularize Biological Networks. Cell systems Atay, O., Doncic, A., Skotheim, J. M. 2016; 3 (2): 121-132

  Abstract

  Cellular decisions are made by complex networks that are difficult to analyze. Although it is common to analyze smaller sub-networks known as network motifs, it is unclear whether this is valid, because these motifs are embedded in complex larger networks. Here, we address the general question of modularity by examining the S. cerevisiae pheromone response. We demonstrate that the feedforward motif controlling the cell-cycle inhibitor Far1 is insulated from cell-cycle dynamics by the positive feedback switch that drives reentry to the cell cycle. Before cells switch on positive feedback, the feedforward motif model predicts the behavior of the larger network. Conversely, after the switch, the feedforward motif is dismantled and has no discernable effect on the cell cycle. When insulation is broken, the feedforward motif no longer predicts network behavior. This work illustrates how, despite the interconnectivity of networks, the activity of motifs can be insulated by switches that generate well-defined cellular states.

  View details for DOI 10.1016/j.cels.2016.06.010

  View details for PubMedID 27453443

  View details for PubMedCentralID PMC5001915

• Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq NATURE Treutlein, B., Lee, Q. Y., Camp, J. G., Mall, M., Koh, W., Shariati, S. A., Sim, S., Neff, N. F., Skotheim, J. M., Wernig, M., Quake, S. R. 2016; 534 (7607): 391-?

  Abstract

  Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts to induced neuronal cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts, suggesting that the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.

  View details for DOI 10.1038/nature18323

  View details for Web of Science ID 000377856800037

  View details for PubMedID 27281220

  View details for PubMedCentralID PMC4928860

• The Yeast Cyclin-Dependent Kinase Routes Carbon Fluxes to Fuel Cell Cycle Progression MOLECULAR CELL Ewald, J. C., Kuehne, A., Zamboni, N., Skotheim, J. M. 2016; 62 (4): 532-545

  Abstract

  Cell division entails a sequence of processes whose specific demands for biosynthetic precursors and energy place dynamic requirements on metabolism. However, little is known about how metabolic fluxes are coordinated with the cell division cycle. Here, we examine budding yeast to show that more than half of all measured metabolites change significantly through the cell division cycle. Cell cycle-dependent changes in central carbon metabolism are controlled by the cyclin-dependent kinase (Cdk1), a major cell cycle regulator, and the metabolic regulator protein kinase A. At the G1/S transition, Cdk1 phosphorylates and activates the enzyme Nth1, which funnels the storage carbohydrate trehalose into central carbon metabolism. Trehalose utilization fuels anabolic processes required to reliably complete cell division. Thus, the cell cycle entrains carbon metabolism to fuel biosynthesis. Because the oscillation of Cdk activity is a conserved feature of the eukaryotic cell cycle, we anticipate its frequent use in dynamically regulating metabolism for efficient proliferation.

  View details for DOI 10.1016/j.molcel.2016.02.017

  View details for Web of Science ID 000376445000008

  View details for PubMedID 27203178

  View details for PubMedCentralID PMC4875507

• Punctuated evolution and transitional hybrid network in an ancestral cell cycle of fungi ELIFE Medina, E. M., Turner, J. J., Gordan, R., Skotheim, J. M., Buchler, N. E. 2016; 5

  Abstract

  Although cell cycle control is an ancient, conserved, and essential process, some core animal and fungal cell cycle regulators share no more sequence identity than non-homologous proteins. Here, we show that evolution along the fungal lineage was punctuated by the early acquisition and entrainment of the SBF transcription factor through horizontal gene transfer. Cell cycle evolution in the fungal ancestor then proceeded through a hybrid network containing both SBF and its ancestral animal counterpart E2F, which is still maintained in many basal fungi. We hypothesize that a virally-derived SBF may have initially hijacked cell cycle control by activating transcription via the cis-regulatory elements targeted by the ancestral cell cycle regulator E2F, much like extant viral oncogenes. Consistent with this hypothesis, we show that SBF can regulate promoters with E2F binding sites in budding yeast.

  View details for DOI 10.7554/eLife.09492

  View details for Web of Science ID 000376602700001

  View details for PubMedID 27162172

  View details for PubMedCentralID PMC4862756

• Cell-Size Control COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Amodeo, A. A., Skotheim, J. M. 2016; 8 (4)

  Abstract

  Cells of a given type maintain a characteristic cell size to function efficiently in their ecological or organismal context. They achieve this through the regulation of growth rates or by actively sensing size and coupling this signal to cell division. We focus this review on potential size-sensing mechanisms, including geometric, external cue, and titration mechanisms. Mechanisms that titrate proteins against DNA are of particular interest because they are consistent with the robust correlation of DNA content and cell size. We review the literature, which suggests that titration mechanisms may underlie cell-size sensing inXenopusembryos, budding yeast, andEscherichia coli, whereas alternative mechanisms may function in fission yeast.

  View details for DOI 10.1101/cshperspect.a019083

  View details for Web of Science ID 000373486900002

  View details for PubMedID 26254313

  View details for PubMedCentralID PMC4744813

• The Biosynthetic Basis of Cell Size Control. Trends in cell biology Schmoller, K. M., Skotheim, J. M. 2015; 25 (12): 793-802

  Abstract

  Cell size is an important physiological trait that sets the scale of all biosynthetic processes. Although physiological studies have revealed that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained unclear. Here we review recent progress in identifying the molecular mechanisms of cell size control. We focus on budding yeast, where cell growth dilutes a cell cycle inhibitor to couple growth and division. We discuss a new model for size control based on the titration of activator and inhibitor molecules whose synthesis rates are differentially dependent on cell size.

  View details for DOI 10.1016/j.tcb.2015.10.006

  View details for PubMedID 26573465

• Mitosis is swell. journal of cell biology Zatulovskiy, E., Skotheim, J. M. 2015; 211 (4): 733-735

  Abstract

  Cell volume and dry mass are typically correlated. However, in this issue, Zlotek-Zlotkiewicz et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505056) and Son et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505058) use new live-cell techniques to show that entry to mitosis coincides with rapid cell swelling, which is reversed before division.

  View details for DOI 10.1083/jcb.201511007

  View details for PubMedID 26598610

  View details for PubMedCentralID PMC4657178

• Dilution of the cell cycle inhibitor Whi5 controls budding-yeast cell size. Nature Schmoller, K. M., Turner, J. J., Kõivomägi, M., Skotheim, J. M. 2015; 526 (7572): 268-272

  Abstract

  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.

  View details for DOI 10.1038/nature14908

  View details for PubMedID 26390151

• A genetically encoded Forster resonance energy transfer sensor for monitoring in vivo trehalose-6-phosphate dynamics ANALYTICAL BIOCHEMISTRY Peroza, E. A., Ewald, J. C., Parakkal, G., Skotheim, J. M., Zamboni, N. 2015; 474: 1-7

  Abstract

  Trehalose-6-phosphate is a pivotal regulator of sugar metabolism, growth, and osmotic equilibrium in bacteria, yeasts, and plants. To directly visualize the intracellular levels of intracellular trehalose-6-phosphate, we developed a series of specific Förster resonance energy transfer (FRET) sensors for in vivo microscopy. We demonstrated real-time monitoring of regulation in the trehalose pathway of Escherichia coli. In Saccharomyces cerevisiae, we could show that the concentration of free trehalose-6-phosphate during growth on glucose is in a range sufficient for inhibition of hexokinase. These findings support the hypothesis of trehalose-6-phosphate as the effector of a negative feedback system, similar to the inhibition of hexokinase by glucose-6-phosphate in mammalian cells and controlling glycolytic flux.

  View details for DOI 10.1016/j.ab.2014.12.019

  View details for Web of Science ID 000352170800001

  View details for PubMedID 25582303

• Compartmentalization of a Bistable Switch Enables Memory to Cross a Feedback-Driven Transition CELL Doncic, A., Atay, O., Valk, E., Grande, A., Bush, A., Vasen, G., Colman-Lerner, A., Loog, M., Skotheim, J. M. 2015; 160 (6): 1182-1195

  Abstract

  Cells make accurate decisions in the face of molecular noise and environmental fluctuations by relying not only on present pathway activity, but also on their memory of past signaling dynamics. Once a decision is made, cellular transitions are often rapid and switch-like due to positive feedback loops in the regulatory network. While positive feedback loops are good at promoting switch-like transitions, they are not expected to retain information to inform subsequent decisions. However, this expectation is based on our current understanding of network motifs that accounts for temporal, but not spatial, dynamics. Here, we show how spatial organization of the feedback-driven yeast G1/S switch enables the transmission of memory of past pheromone exposure across this transition. We expect this to be one of many examples where the exquisite spatial organization of the eukaryotic cell enables previously well-characterized network motifs to perform new and unexpected signal processing functions.

  View details for DOI 10.1016/j.cell.2015.02.032

  View details for Web of Science ID 000351951800017

  View details for PubMedID 25768911

• Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition. Proceedings of the National Academy of Sciences of the United States of America Amodeo, A. A., Jukam, D., Straight, A. F., Skotheim, J. M. 2015; 112 (10): E1086-95

  Abstract

  During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development.

  View details for DOI 10.1073/pnas.1413990112

  View details for PubMedID 25713373

  View details for PubMedCentralID PMC4364222

• Modularity and predictability in cell signaling and decision making MOLECULAR BIOLOGY OF THE CELL Atay, O., Skotheim, J. M. 2014; 25 (22): 3445-3450

  Abstract

  Cells make decisions to differentiate, divide, or apoptose based on multiple signals of internal and external origin. These decisions are discrete outputs from dynamic networks comprised of signaling pathways. Yet the validity of this decomposition of regulatory proteins into distinct pathways is unclear because many regulatory proteins are pleiotropic and interact through cross-talk with components of other pathways. In addition to the deterministic complexity of interconnected networks, there is stochastic complexity arising from the fluctuations in concentrations of regulatory molecules. Even within a genetically identical population of cells grown in the same environment, cell-to-cell variations in mRNA and protein concentrations can be as high as 50% in yeast and even higher in mammalian cells. Thus, if everything is connected and stochastic, what hope could we have for a quantitative understanding of cellular decisions? Here we discuss the implications of recent advances in genomics, single-cell, and single-cell genomics technology for network modularity and cellular decisions. On the basis of these recent advances, we argue that most gene expression stochasticity and pathway interconnectivity is nonfunctional and that cellular decisions are likely much more predictable than previously expected.

  View details for DOI 10.1091/mbc.E14-02-0718

  View details for Web of Science ID 000344236800004

  View details for PubMedCentralID PMC4230600

• Modularity and predictability in cell signaling and decision making. Molecular biology of the cell Atay, O., Skotheim, J. M. 2014; 25 (22): 3445-3450

  Abstract

  Cells make decisions to differentiate, divide, or apoptose based on multiple signals of internal and external origin. These decisions are discrete outputs from dynamic networks comprised of signaling pathways. Yet the validity of this decomposition of regulatory proteins into distinct pathways is unclear because many regulatory proteins are pleiotropic and interact through cross-talk with components of other pathways. In addition to the deterministic complexity of interconnected networks, there is stochastic complexity arising from the fluctuations in concentrations of regulatory molecules. Even within a genetically identical population of cells grown in the same environment, cell-to-cell variations in mRNA and protein concentrations can be as high as 50% in yeast and even higher in mammalian cells. Thus, if everything is connected and stochastic, what hope could we have for a quantitative understanding of cellular decisions? Here we discuss the implications of recent advances in genomics, single-cell, and single-cell genomics technology for network modularity and cellular decisions. On the basis of these recent advances, we argue that most gene expression stochasticity and pathway interconnectivity is nonfunctional and that cellular decisions are likely much more predictable than previously expected.

  View details for DOI 10.1091/mbc.E14-02-0718

  View details for PubMedID 25368418

  View details for PubMedCentralID PMC4230600

• Docking interactions: cell-cycle regulation and beyond. Current biology Kõivomägi, M., Skotheim, J. M. 2014; 24 (14): R647-9

  Abstract

  In budding yeast, the mating pathway activates Far1 to inhibit G1 cyclins in complex with the cyclin-dependent kinase (Cln-Cdk). Yet, the molecular mechanism has remained largely unclear for over 20 years. A recent report helps shed light on this regulation.

  View details for DOI 10.1016/j.cub.2014.05.060

  View details for PubMedID 25050961

• Unravelling the Size Sensing Mechanism in Budding Yeast Schmoller, K. M., Turner, J. J., Skotheim, J. M. CELL PRESS. 2014: 595A

  View details for DOI 10.1016/j.bpj.2013.11.3292

  View details for Web of Science ID 000337000403347

• Start and the restriction point. Current opinion in cell biology Johnson, A., Skotheim, J. M. 2013; 25 (6): 717-723

  Abstract

  Commitment to division requires that cells sense, interpret, and respond appropriately to multiple signals. In most eukaryotes, cells commit to division in G1 before DNA replication. Beyond a point, known as Start in yeast and the restriction point in mammals, cells will proceed through the cell cycle despite changes in upstream signals. In metazoans, misregulated G1 control can lead to developmental problems or disease, so it is important to understand how cells decipher the myriad external and internal signals that contribute to the fundamental all-or-none decision to divide. Extensive study of G1 control in the budding yeast Saccharomyces cerevisiae and mammalian culture systems has revealed highly similar networks regulating commitment. However, protein sequences of functional orthologs often indicate a total lack of conservation suggesting significant evolution of G1 control. Here, we review recent studies defining the conserved and diverged features of G1 control and highlight systems-level aspects that may be common to other biological regulatory networks.

  View details for DOI 10.1016/j.ceb.2013.07.010

  View details for PubMedID 23916770

  View details for PubMedCentralID PMC3836907

• CONSTRAINTS ON THE ADULT-OFFSPRING SIZE RELATIONSHIP IN PROTISTS EVOLUTION Caval-Holme, F., Payne, J., Skotheim, J. M. 2013; 67 (12): 3537-3544

  Abstract

  The relationship between adult and offspring size is an important aspect of reproductive strategy. Although this filial relationship has been extensively examined in plants and animals, we currently lack comparable data for protists, whose strategies may differ due to the distinct ecological and physiological constraints on single-celled organisms. Here, we report measurements of adult and offspring sizes in 3888 species and subspecies of foraminifera, a class of large marine protists. Foraminifera exhibit a wide range of reproductive strategies; species of similar adult size may have offspring whose sizes vary 100-fold. Yet, a robust pattern emerges. The minimum (5th percentile), median, and maximum (95th percentile) offspring sizes exhibit a consistent pattern of increase with adult size independent of environmental change and taxonomic variation over the past 400 million years. The consistency of this pattern may arise from evolutionary optimization of the offspring size-fecundity trade-off and/or from cell-biological constraints that limit the range of reproductive strategies available to single-celled organisms. When compared with plants and animals, foraminifera extend the evidence that offspring size covaries with adult size across an additional five orders of magnitude in organism size.

  View details for DOI 10.1111/evo.12210

  View details for Web of Science ID 000327572400013

  View details for PubMedID 24299406

• Nuclear Repulsion Enables Division Autonomy in a Single Cytoplasm CURRENT BIOLOGY Anderson, C. A., Eser, U., Korndorf, T., Borsuk, M. E., Skotheim, J. M., Gladfelter, A. S. 2013; 23 (20): 1999-2010

  Abstract

  Current models of cell-cycle control, based on classic studies of fused cells, predict that nuclei in a shared cytoplasm respond to the same CDK activities to undergo synchronous cycling. However, synchrony is rarely observed in naturally occurring syncytia, such as the multinucleate fungus Ashbya gossypii. In this system, nuclei divide asynchronously, raising the question of how nuclear timing differences are maintained despite sharing a common milieu.We observe that neighboring nuclei are highly variable in division-cycle duration and that neighbors repel one another to space apart and demarcate their own cytoplasmic territories. The size of these territories increases as a nucleus approaches mitosis and can influence cycling rates. This nonrandom nuclear spacing is regulated by microtubules and is required for nuclear asynchrony, as nuclei that transiently come in very close proximity will partially synchronize. Sister nuclei born of the same mitosis are generally not persistent neighbors over their lifetimes yet remarkably retain similar division cycle times. This indicates that nuclei carry a memory of their birth state that influences their division timing and supports that nuclei subdivide a common cytosol into functionally distinct yet mobile compartments.These findings support that nuclei use cytoplasmic microtubules to establish "cells within cells." Individual compartments appear to push against one another to compete for cytoplasmic territory and insulate the division cycle. This provides a mechanism by which syncytial nuclei can spatially organize cell-cycle signaling and suggests size control can act in a system without physical boundaries.

  View details for DOI 10.1016/j.cub.2013.07.076

  View details for Web of Science ID 000326317300021

  View details for PubMedID 24094857

• Dead-end phosphorylation of Sic1 by Clb5-Cdk1 within the inhibitory complex controls the G1/S switch Venta, R., Doncic, A., Valk, E., Koivomaegi, M., Skotheim, J., Loog, M. WILEY-BLACKWELL. 2013: 195

  View details for Web of Science ID 000327927400418

• Mutually exclusive phosphorylation events control the decision between mating and cell cycle progression Valk, E., Doncic, A., Koivomaegi, M., Venta, R., Iofik, A., Faustova, I., Kivi, R., Siibak, T., Balog, E. M., Rubin, S. M., Skotheim, J., Loog, M. WILEY-BLACKWELL. 2013: 66

  View details for Web of Science ID 000327927400083

• Control of cell cycle transcription during G1 and S phases NATURE REVIEWS MOLECULAR CELL BIOLOGY Bertoli, C., Skotheim, J. M., de Bruin, R. A. 2013; 14 (8): 518-528

  Abstract

  The accurate transition from G1 phase of the cell cycle to S phase is crucial for the control of eukaryotic cell proliferation, and its misregulation promotes oncogenesis. During G1 phase, growth-dependent cyclin-dependent kinase (CDK) activity promotes DNA replication and initiates G1-to-S phase transition. CDK activation initiates a positive feedback loop that further increases CDK activity, and this commits the cell to division by inducing genome-wide transcriptional changes. G1-S transcripts encode proteins that regulate downstream cell cycle events. Recent work is beginning to reveal the complex molecular mechanisms that control the temporal order of transcriptional activation and inactivation, determine distinct functional subgroups of genes and link cell cycle-dependent transcription to DNA replication stress in yeast and mammals.

  View details for DOI 10.1038/nrm3629

  View details for Web of Science ID 000322118200013

  View details for PubMedID 23877564

• Feedforward regulation ensures stability and rapid reversibility of a cellular state. Molecular cell Doncic, A., Skotheim, J. M. 2013; 50 (6): 856-868

  Abstract

  Cellular transitions are important for all life. Such transitions, including cell fate decisions, often employ positive feedback regulation to establish and stabilize new cellular states. However, positive feedback is unlikely to underlie stable cell-cycle arrest in yeast exposed to mating pheromone because the signaling pathway is linear, rather than bistable, over a broad range of extracellular pheromone concentration. We show that the stability of the pheromone-arrested state results from coherent feedforward regulation of the cell-cycle inhibitor Far1. This network motif is effectively isolated from the more complex regulatory network in which it is embedded. Fast regulation of Far1 by phosphorylation allows rapid cell-cycle arrest and reentry, whereas slow Far1 synthesis reinforces arrest. We expect coherent feedforward regulation to be frequently implemented at reversible cellular transitions because this network motif can achieve the ostensibly conflicting aims of arrest stability and rapid reversibility without loss of signaling information.

  View details for DOI 10.1016/j.molcel.2013.04.014

  View details for PubMedID 23685071

• Cell growth and cell cycle control. Molecular biology of the cell Skotheim, J. M. 2013; 24 (6): 678-?

  View details for DOI 10.1091/mbc.E13-01-0002

  View details for PubMedID 23486402

  View details for PubMedCentralID PMC3596238

• A SHIFT IN THE LONG-TERM MODE OF FORAMINIFERAN SIZE EVOLUTION CAUSED BY THE END-PERMIAN MASS EXTINCTION EVOLUTION Payne, J. L., Jost, A. B., Wang, S. C., Skotheim, J. M. 2013; 67 (3): 816-827

  Abstract

  Size is among the most important traits of any organism, yet the factors that control its evolution remain poorly understood. In this study, we investigate controls on the evolution of organismal size using a newly compiled database of nearly 25,000 foraminiferan species and subspecies spanning the past 400 million years. We find a transition in the pattern of foraminiferan size evolution from correlation with atmospheric pO2 during the Paleozoic (400-250 million years ago) to long-term stasis during the post-Paleozoic (250 million years ago to present). Thus, a dramatic shift in the evolutionary mode coincides with the most severe biotic catastrophe of the Phanerozoic (543 million years ago to present). Paleozoic tracking of pO2 was confined to Order Fusulinida, whereas Paleozoic lagenides, miliolids, and textulariids were best described by the stasis model. Stasis continued to best describe miliolids and textulariids during post-Paleozoic time, whereas random walk was the best supported mode for the other diverse orders. The shift in evolutionary dynamics thus appears to have resulted primarily from the selective elimination of fusulinids at the end of the Permian Period. These findings illustrate the potential for mass extinction to alter macroevolutionary dynamics for hundreds of millions of years.

  View details for DOI 10.1111/j.1558-5646.2012.01807.x

  View details for Web of Science ID 000315894800018

  View details for PubMedID 23461330

• An algorithm to automate yeast segmentation and tracking. PloS one Doncic, A., Eser, U., Atay, O., Skotheim, J. M. 2013; 8 (3)

  Abstract

  Our understanding of dynamic cellular processes has been greatly enhanced by rapid advances in quantitative fluorescence microscopy. Imaging single cells has emphasized the prevalence of phenomena that can be difficult to infer from population measurements, such as all-or-none cellular decisions, cell-to-cell variability, and oscillations. Examination of these phenomena requires segmenting and tracking individual cells over long periods of time. However, accurate segmentation and tracking of cells is difficult and is often the rate-limiting step in an experimental pipeline. Here, we present an algorithm that accomplishes fully automated segmentation and tracking of budding yeast cells within growing colonies. The algorithm incorporates prior information of yeast-specific traits, such as immobility and growth rate, to segment an image using a set of threshold values rather than one specific optimized threshold. Results from the entire set of thresholds are then used to perform a robust final segmentation.

  View details for DOI 10.1371/journal.pone.0057970

  View details for PubMedID 23520484

  View details for PubMedCentralID PMC3592893

• LATE PALEOZOIC FUSULINOIDEAN GIGANTISM DRIVEN BY ATMOSPHERIC HYPEROXIA EVOLUTION Payne, J. L., Groves, J. R., Jost, A. B., Thienan Nguyen, T., Moffitt, S. E., Hill, T. M., Skotheim, J. M. 2012; 66 (9): 2929-2939

  Abstract

  Atmospheric hyperoxia, with pO(2) in excess of 30%, has long been hypothesized to account for late Paleozoic (360-250 million years ago) gigantism in numerous higher taxa. However, this hypothesis has not been evaluated statistically because comprehensive size data have not been compiled previously at sufficient temporal resolution to permit quantitative analysis. In this study, we test the hyperoxia-gigantism hypothesis by examining the fossil record of fusulinoidean foraminifers, a dramatic example of protistan gigantism with some individuals exceeding 10 cm in length and exceeding their relatives by six orders of magnitude in biovolume. We assembled and examined comprehensive regional and global, species-level datasets containing 270 and 1823 species, respectively. A statistical model of size evolution forced by atmospheric pO(2) is conclusively favored over alternative models based on random walks or a constant tendency toward size increase. Moreover, the ratios of volume to surface area in the largest fusulinoideans are consistent in magnitude and trend with a mathematical model based on oxygen transport limitation. We further validate the hyperoxia-gigantism model through an examination of modern foraminiferal species living along a measured gradient in oxygen concentration. These findings provide the first quantitative confirmation of a direct connection between Paleozoic gigantism and atmospheric hyperoxia.

  View details for DOI 10.1111/j.1558-5646.2012.01626.x

  View details for Web of Science ID 000308405100020

  View details for PubMedID 22946813

• Cell Size Control in Yeast CURRENT BIOLOGY Turner, J. J., Ewald, J. C., Skotheim, J. M. 2012; 22 (9): R350-R359

  Abstract

  Cell size is an important adaptive trait that influences nearly all aspects of cellular physiology. Despite extensive characterization of the cell-cycle regulatory network, the molecular mechanisms coupling cell growth to division, and thereby controlling cell size, have remained elusive. Recent work in yeast has reinvigorated the size control field and suggested provocative mechanisms for the distinct functions of setting and sensing cell size. Further examination of size-sensing models based on spatial gradients and molecular titration, coupled with elucidation of the pathways responsible for nutrient-modulated target size, may reveal the fundamental principles of eukaryotic cell size control.

  View details for DOI 10.1016/j.cub.2012.02.041

  View details for Web of Science ID 000303967600019

  View details for PubMedID 22575477

  View details for PubMedCentralID PMC3350643

• Evolution of networks and sequences in eukaryotic cell cycle control PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES Cross, F. R., Buchler, N. E., Skotheim, J. M. 2011; 366 (1584): 3532-3544

  Abstract

  The molecular networks regulating the G1-S transition in budding yeast and mammals are strikingly similar in network structure. However, many of the individual proteins performing similar network roles appear to have unrelated amino acid sequences, suggesting either extremely rapid sequence evolution, or true polyphyly of proteins carrying out identical network roles. A yeast/mammal comparison suggests that network topology, and its associated dynamic properties, rather than regulatory proteins themselves may be the most important elements conserved through evolution. However, recent deep phylogenetic studies show that fungal and animal lineages are relatively closely related in the opisthokont branch of eukaryotes. The presence in plants of cell cycle regulators such as Rb, E2F and cyclins A and D, that appear lost in yeast, suggests cell cycle control in the last common ancestor of the eukaryotes was implemented with this set of regulatory proteins. Forward genetics in non-opisthokonts, such as plants or their green algal relatives, will provide direct information on cell cycle control in these organisms, and may elucidate the potentially more complex cell cycle control network of the last common eukaryotic ancestor.

  View details for DOI 10.1098/rstb.2011.0078

  View details for Web of Science ID 000296981900006

  View details for PubMedID 22084380

  View details for PubMedCentralID PMC3203458

• Commitment to a Cellular Transition Precedes Genome-wide Transcriptional Change MOLECULAR CELL Eser, U., Falleur-Fettig, M., Johnson, A., Skotheim, J. M. 2011; 43 (4): 515-527

  Abstract

  In budding yeast, commitment to cell division corresponds to activating the positive feedback loop of G1 cyclins controlled by the transcription factors SBF and MBF. This pair of transcription factors has over 200 targets, implying that cell-cycle commitment coincides with genome-wide changes in transcription. Here, we find that genes within this regulon have a well-defined distribution of transcriptional activation times. Combinatorial use of SBF and MBF results in a logical OR function for gene expression and partially explains activation timing. Activation of G1 cyclin expression precedes the activation of the bulk of the G1/S regulon, ensuring that commitment to cell division occurs before large-scale changes in transcription. Furthermore, we find similar positive feedback-first regulation in the yeasts S. bayanus and S. cerevisiae, as well as human cells. The widespread use of the feedback-first motif in eukaryotic cell-cycle control, implemented by nonorthologous proteins, suggests its frequent deployment at cellular transitions.

  View details for DOI 10.1016/j.molcel.2011.06.024

  View details for Web of Science ID 000294151000005

  View details for PubMedID 21855792

  View details for PubMedCentralID PMC3160620

• Distinct Interactions Select and Maintain a Specific Cell Fate MOLECULAR CELL Doncic, A., Falleur-Fettig, M., Skotheim, J. M. 2011; 43 (4): 528-539

  Abstract

  The ability to specify and maintain discrete cell fates is essential for development. However, the dynamics underlying selection and stability of distinct cell types remain poorly understood. Here, we provide a quantitative single-cell analysis of commitment dynamics during the mating-mitosis switch in budding yeast. Commitment to division corresponds precisely to activating the G1 cyclin positive feedback loop in competition with the cyclin inhibitor Far1. Cyclin-dependent phosphorylation and inhibition of the mating pathway scaffold Ste5 are required to ensure exclusive expression of the mitotic transcriptional program after cell cycle commitment. Failure to commit exclusively results in coexpression of both cell cycle and pheromone-induced genes, and a morphologically mixed inviable cell fate. Thus, specification and maintenance of a cellular state are performed by distinct interactions, which are likely a consequence of disparate reaction rates and may be a general feature of the interlinked regulatory networks responsible for selecting cell fates.

  View details for DOI 10.1016/j.molcel.2011.06.025

  View details for Web of Science ID 000294151000006

  View details for PubMedID 21855793

  View details for PubMedCentralID PMC3160603

• Daughter-Specific Transcription Factors Regulate Cell Size Control in Budding Yeast PLOS BIOLOGY Di Talia, S., Wang, H., Skotheim, J. M., Rosebrock, A. P., Futcher, B., Cross, F. R. 2009; 7 (10)

  Abstract

  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.

  View details for DOI 10.1371/journal.pbio.1000221

  View details for Web of Science ID 000272031800011

  View details for PubMedID 19841732

  View details for PubMedCentralID PMC2756959

• Cell signaling. To divide or not to divide. Science Skotheim, J. M. 2009; 324 (5926): 476-477

  View details for DOI 10.1126/science.1173769

  View details for PubMedID 19390035

• Positive feedback of G1 cyclins ensures coherent cell cycle entry NATURE Skotheim, J. M., Di Talia, S., Siggia, E. D., Cross, F. R. 2008; 454 (7202): 291-U12

  Abstract

  In budding yeast, Saccharomyces cerevisiae, the Start checkpoint integrates multiple internal and external signals into an all-or-none decision to enter the cell cycle. Here we show that Start behaves like a switch due to systems-level feedback in the regulatory network. In contrast to current models proposing a linear cascade of Start activation, transcriptional positive feedback of the G1 cyclins Cln1 and Cln2 induces the near-simultaneous expression of the approximately 200-gene G1/S regulon. Nuclear Cln2 drives coherent regulon expression, whereas cytoplasmic Cln2 drives efficient budding. Cells with the CLN1 and CLN2 genes deleted frequently arrest as unbudded cells, incurring a large fluctuation-induced fitness penalty due to both the lack of cytoplasmic Cln2 and insufficient G1/S regulon expression. Thus, positive-feedback-amplified expression of Cln1 and Cln2 simultaneously drives robust budding and rapid, coherent regulon expression. A similar G1/S regulatory network in mammalian cells, comprised of non-orthologous genes, suggests either conservation of regulatory architecture or convergent evolution.

  View details for DOI 10.1038/nature07118

  View details for Web of Science ID 000257665300029

  View details for PubMedID 18633409

  View details for PubMedCentralID PMC2606905

• Settling and swimming of flexible fluid-lubricated foils PHYSICAL REVIEW LETTERS Argentina, M., Skotheim, J., Mahadevan, L. 2007; 99 (22)

  Abstract

  We study the dynamics of a flexible foil immersed in a fluid and moving close to a rigid wall. Lubrication theory allows us to derive equations of motion for the foil and thus examine the passive settling and the active swimming of a foil. This also allows us to partly answer the long-standing question in cartoon physics--can carpets fly? Our analysis suggests a region in parameter space where one may realize this dream and move the virtual towards reality.

  View details for DOI 10.1103/PhysRevLett.99.224503

  View details for Web of Science ID 000251327500029

  View details for PubMedID 18233292

• The effects of molecular noise and size control on variability in the budding yeast cell cycle NATURE Di Talia, S., Skotheim, J. M., Bean, J. M., Siggia, E. D., Cross, F. R. 2007; 448 (7156): 947-U12

  Abstract

  Molecular noise in gene expression can generate substantial variability in protein concentration. However, its effect on the precision of a natural eukaryotic circuit such as the control of cell cycle remains unclear. We use single-cell imaging of fluorescently labelled budding yeast to measure times from division to budding (G1) and from budding to the next division. The variability in G1 decreases with the square root of the ploidy through a 1N/2N/4N ploidy series, consistent with simple stochastic models for molecular noise. Also, increasing the gene dosage of G1 cyclins decreases the variability in G1. A new single-cell reporter for cell protein content allows us to determine the contribution to temporal G1 variability of deterministic size control (that is, smaller cells extending G1). Cell size control contributes significantly to G1 variability in daughter cells but not in mother cells. However, even in daughters, size-independent noise is the largest quantitative contributor to G1 variability. Exit of the transcriptional repressor Whi5 from the nucleus partitions G1 into two temporally uncorrelated and functionally distinct steps. The first step, which depends on the G1 cyclin gene CLN3, corresponds to noisy size control that extends G1 in small daughters, but is of negligible duration in mothers. The second step, whose variability decreases with increasing CLN2 gene dosage, is similar in mothers and daughters. This analysis decomposes the regulatory dynamics of the Start transition into two independent modules, a size sensing module and a timing module, each of which is predominantly controlled by a different G1 cyclin.

  View details for DOI 10.1038/nature06072

  View details for Web of Science ID 000248912900051

  View details for PubMedID 17713537

• Red blood cells and other nonspherical capsules in shear flow: Oscillatory dynamics and the tank-treading-to-tumbling transition PHYSICAL REVIEW LETTERS Skotheim, J. M., Secomb, T. W. 2007; 98 (7)

  Abstract

  We consider the motion of red blood cells and other nonspherical microcapsules dilutely suspended in a simple shear flow. Our analysis indicates that depending on the viscosity, membrane elasticity, geometry, and shear rate, the particle exhibits either tumbling, tank-treading of the membrane about the viscous interior with periodic oscillations of the orientation angle, or intermittent behavior in which the two modes occur alternately. For red blood cells, we compute the complete phase diagram and identify a novel tank-treading-to-tumbling transition as the shear rate decreases. Observations of such motions coupled with our theoretical framework may provide a sensitive means of assessing capsule properties.

  View details for DOI 10.1103/PhysRevLett.98.078301

  View details for Web of Science ID 000244250300069

  View details for PubMedID 17359066

• Gravitational collapse of colloidal gels PHYSICAL REVIEW LETTERS Manley, S., Skotheim, J. M., Mahadevan, L., Weitz, D. A. 2005; 94 (21)

  Abstract

  We present a unified framework for understanding the compaction of colloidal gels under their own weight. The dynamics of the collapse are determined by the value of the gravitational stress sigma(g), as compared to the yield stress sigma(Y) of the network. For sigma(g)sigma(Y), the network eventually yields, leading to rapid settling. In both cases, the rate of collapse is backflow limited, while its overall magnitude is determined by a balance between gravitational stress and network elastic stress.

  View details for DOI 10.1103/PhysRevLett.94.218302

  View details for Web of Science ID 000229543900053

  View details for PubMedID 16090356

• Physical limits and design principles for plant and fungal movements SCIENCE Skotheim, J. M., Mahadevan, L. 2005; 308 (5726): 1308-1310

  Abstract

  The typical scales for plant and fungal movements vary over many orders of magnitude in time and length, but they are ultimately based on hydraulics and mechanics. We show that quantification of the length and time scales involved in plant and fungal motions leads to a natural classification, whose physical basis can be understood through an analysis of the mechanics of water transport through an elastic tissue. Our study also suggests a design principle for nonmuscular hydraulically actuated structures: Rapid actuation requires either small size or the enhancement of motion on large scales via elastic instabilities.

  View details for DOI 10.1126/science.1107976

  View details for Web of Science ID 000229482300047

  View details for PubMedID 15919993

• How the Venus flytrap snaps NATURE Forterre, Y., Skotheim, J. M., Dumais, J., Mahadevan, L. 2005; 433 (7024): 421-425

  Abstract

  The rapid closure of the Venus flytrap (Dionaea muscipula) leaf in about 100 ms is one of the fastest movements in the plant kingdom. This led Darwin to describe the plant as "one of the most wonderful in the world". The trap closure is initiated by the mechanical stimulation of trigger hairs. Previous studies have focused on the biochemical response of the trigger hairs to stimuli and quantified the propagation of action potentials in the leaves. Here we complement these studies by considering the post-stimulation mechanical aspects of Venus flytrap closure. Using high-speed video imaging, non-invasive microscopy techniques and a simple theoretical model, we show that the fast closure of the trap results from a snap-buckling instability, the onset of which is controlled actively by the plant. Our study identifies an ingenious solution to scaling up movements in non-muscular engines and provides a general framework for understanding nastic motion in plants.

  View details for DOI 10.1038/nature03185

  View details for Web of Science ID 000226546200044

  View details for PubMedID 15674293

• Soft elastohydrodynamic contacts Phys. Fluids J, S. M., L, M. 2005; 17
• Soft lubrication PHYSICAL REVIEW LETTERS Skotheim, J. M., Mahadevan, L. 2004; 92 (24)

  Abstract

  We consider some basic principles of fluid-induced lubrication at soft interfaces. In particular, we quantify how a soft substrate changes the geometry of and the forces between surfaces sliding past each other. By considering the model problem of a symmetric nonconforming contact moving tangentially to a thin elastic layer, we determine the normal force in the small and large deflection limit, and show that there is an optimal combination of material and geometric properties which maximizes the normal force. Our results can be generalized to a variety of other geometries which show the same qualitative behavior. Thus, they are relevant in the elastohydrodynamic lubrication of soft elastic and poroelastic gels and shells, and in the context of biolubrication in cartilaginous joints.

  View details for DOI 10.1103/PhysRevLett.92.245509

  View details for Web of Science ID 000222112900030

  View details for PubMedID 15245101

• Dynamics of poroelastic filaments Proc. R. Soc. London Ser. A J, S. M., L, M. 2004; 460: 1995-2020
• On the instability of a falling film due to localized heating J. Fluid Mech., J, S. M., U, T., B, S. 2003; 475: 1-19
• Evaporatively driven convection in a draining soap film Phys. Fluids, J, S. M., JWM, B. 2000; S1