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Publications (268)
The cell division cycle is a fundamental physiological process displaying a great degree of plasticity during the course of multicellular development. This plasticity is evident in the transition from rapid and stringently-timed divisions of the early embryo to subsequent size-controlled mitotic cycles. Later in development, cells may pause and res...
In most situations, eukaryotic cells proceed irreversibly through the cell division cycle (G1-S-G2-M) in order to produce two daughter cells with the same number and identity of chromosomes of their progenitor. The integrity of this process is maintained by checkpoints that hold a cell at particular transition points of the cycle until all requisit...
Cell growth, DNA replication, mitosis and division are the fundamental processes by which life is passed on from one generation of eukaryotic cells to the next. The eukaryotic cell cycle is intrinsically a periodic process but not so much a ‘clock’ as a ‘copy machine’, making new daughter cells as warranted. Cells growing under ideal conditions div...
In 1996, Kim Nasmyth¹ proposed that the eukaryotic cell cycle is an alternating sequence of transitions from G1 to S-G2-M and back again. These two phases correlate to high activity of cyclin-dependent kinases (CDKs) that trigger S-G2-M events and CDK antagonists that stabilize G1 phase. We associated these “alternative phases” with the coexistence...
As cells pass through each replication–division cycle, they must be able to postpone further progression if they detect any threats to genome integrity, such as DNA damage or misaligned chromosomes. Once a ‘decision’ is made to proceed, the cell unequivocally enters into a qualitatively different biochemical state, which makes the transitions from...
Typically cells replicate their genome only once per division cycle, but under some circumstances, both natural and unnatural, cells synthesize an overabundance of DNA, either in a disorganized fashion (‘over-replication’) or by a systematic doubling of chromosome number (‘endoreplication’). These variations on the theme of DNA replication and divi...
Two mitotic cyclin types, cyclin A and B, exist in higher eukaryotes, but their specialised functions in mitosis are incompletely understood. Using degron tags for rapid inducible protein removal, we analyse how acute depletion of these proteins affects mitosis. Loss of cyclin A in G2-phase prevents mitotic entry. Cells lacking cyclin B can enter m...
The driving passion of molecular cell biologists is to understand the molecular mechanisms that control important aspects of cell physiology, but this ambition is often limited by the wealth of molecular details currently known about these mechanisms. Their complexity overwhelms our intuitive notions of how molecular regulatory networks might respo...
Most eukaryotic cells execute binary division after each mass doubling in order to maintain size homeostasis by coordinating cell growth and division. By contrast, the photosynthetic green alga Chlamydomonas can grow more than 8-fold during daytime and then, at night, undergo rapid cycles of DNA replication, mitosis, and cell division, producing up...
The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation....
Proliferating cells need to coordinate cell division and growth to maintain size homeostasis. Any systematic deviation from a balance between growth and division results in progressive changes of cell size over subsequent generations. While most eukaryotic cells execute binary division after a mass doubling, the photosynthetic green alga Chlamydomo...
The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. Loops and TADs are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Curr...
Spindle checkpoint signaling is initiated by recruitment of the kinase MPS1 to unattached kinetochores during mitosis. We show that CDK1-CCNB1 and a counteracting phosphatase PP2A-B55 regulate the engagement of human MPS1 with unattached kinetochores by controlling the phosphorylation status of S281 in the kinetochore-binding domain. This regulatio...
Two mitotic Cyclins, A and B, exist in higher eukaryotes, but their specialised functions in mitosis are poorly understood. Using degron tags we analyse how acute depletion of these proteins affects mitosis. Loss of Cyclin A in G2-phase prevents the initial activation of Cdk1. Cells lacking Cyclin B can enter mitosis and phosphorylate most mitotic...
Video S4. Mitosis in Gwl siRNA-Transfected, MK1775-Treated, and Gwl siRNA/STLC-Treated Cells, Relating to Figures 4F and 4G
As in Video S3. Left Panes: Cells transfected with Gwl siRNA, Middle Panels: Cells treated with 1 μM MK1775. Right Panel: Cells transfected with Gwl siRNAand treated with 5 μM STLC before initiating the imaging sequence.
Video S1. Synchronous Mitotic Entry and Exit in HeLa cdk1as Cells, Related to Figure 2B
HeLa cdk1as cells were incubated for 20 hours with 2 μM 1NM-PP1 and 1 μM sirDNA. Inhibitor-containing medium was removed by washing the cells five times in drug-free medium. Finally, cells were taken up in medium containing 25 μM MG132 and 1 μM sirDNA and image...
Video S2. Examples of Cells after Re-addition of 1NM-PP1 following Release, Related to Figure 4B
HeLa cdk1as cells with endogenously tagged CyclinB1-GFP were released from 1NM-PP1 arrest as in Video S1 and retreated with 1 μM 1NM-PP1 25 minutes after initial release. Left panels show sirDNA (Far-red), middle panels show Cyclin B1-GFP and right pan...
Video S3. Mitosis in Ctr siRNA-Transfected and Gwl siRNA-Transfected HeLa Cells after Wee1 Inhibition, Related to Figures 4F and 4G
Left Panels: HeLa cells transfected with Ctr siRNA were imaged in the presence of sirDNA 48 hours later. Right Panels: HeLa cells transfected with Gwl siRNA and treated with 1 μM MK1775.
Distinct protein phosphorylation levels in interphase and M phase require tight regulation of Cdk1 activity [1, 2]. A bistable switch, based on positive feedback in the Cdk1 activation loop, has been proposed to generate different thresholds for transitions between these cell-cycle states [3, 4, 5]. Recently, the activity of the major Cdk1-countera...
The size of a cell sets the scale for all biochemical processes within it, thereby affecting cellular fitness and survival. Hence, cell size needs to be kept within certain limits and relatively constant over multiple generations. However, how cells measure their size and use this information to regulate growth and division remains controversial. H...
Model of size-dependent and size-independent protein expression.
(TXT)
Related to Fig 3.
(A) Amount of Whi5 and Cln3 (upper panels) and cell volume (lower panels) in haploid cells with one WHI5 copy (left), diploid cells with one WHI5 copy (middle) and diploid cells with two WHI5 copies (right). Note the increase in Whi5 synthesis (increased slope during synthesis period) and cell volume in the latter case. (B, C) Sam...
Differences between inhibitor-dilution and titration model.
(DOCX)
Model for increasing SBF concentration.
(DOCX)
Parameters used in both size-control models.
(DOCX)
Non-zero initial conditions for both models.
(DOCX)
Related to Fig 1.
Expression patterns of genes with different equilibrium constants for TM binding, ranging from high (size-independent) to low (size-dependent) affinity. (A) Gene occupation by TM in dependence on cell volume. (B) Relative protein concentration (normalised to initial concentration) in dependence on time in a growing cell. (C) Prote...
Related to Figs 2 and 4.
(A) Scheme of SBF inhibition in the inhibitor-dilution model. Whi5 strongly binds to SBF in a concentration-based manner, causing SBF inhibition. Both free and complexed Whi5 can be phosphorylated by Cln3, Cln1/2 and Clb1/2. Phosphorylation of SBF:Whi5 complexes leads to their dissociation, which activates SBF. (B) Scheme o...
Related to Fig 5.
(A) Duration of the indicated cell cycle phase or the whole cycle with respect to volume at the beginning of the phase for the simulations in Fig 5. Note the logarithmic scaling of the x-axis. (B) Same as in Fig 5B, except that the amount of Whi5 at cell birth was manually set to a constant, birth-size-independent value. This resu...
Titration-of-nuclear-sites model for budding yeast size control.
(TXT)
Related to Fig 4.
(A) Amount of Whi5:SBF, Whi5:SBF:Cln3 and active SBF (upper panels), and cell volume (lower panels) in haploid (left) and diploid (right) cells with one WHI5 copy in the titration model. Note the increase in cell volume for diploid cells due to the presence of twice the number of SBF complexes on binding sites (sum of the three sp...
Parameters specific to the titration-of-nuclear-sites model.
(DOCX)
Inhibitor-dilution model for budding yeast size control.
(TXT)
(A) Schematic of the SBF-increase model. In early G1, Whi5 outnumbers SBF and prevents its activation. A fraction of Whi5 is phosphorylated by Cln3 and does not participate in inhibition. As cells grow, the SBF concentration increases such that SBF is able to overcome inhibition and induce Cln1 and Cln2 synthesis. Whi5 phosphorylation then liberate...
Parameters specific to the inhibitor-dilution model.
(DOCX)
Parameters changes for ploidy mutants.
(DOCX)
The size of a cell sets the scale for all biochemical processes within it, thereby affecting cellular fitness and survival. Hence, cell size needs to be kept within certain limits and relatively constant over multiple generations. However, how cells measure their size and use this information to regulate growth and division remains controversial. H...
Significance
Controlled transitions of human cells between proliferating and nonproliferating states are essential for normal development and tissue homeostasis. To understand how the decision to proliferate is made in response to positive input from growth factors and negative input from the DNA damage response, we have built a mathematical model...
Ostreococcus tauri is the smallest free-living unicellular organism with one copy of each core cell cycle genes in its genome. There is a growing interest in this green algae due to its evolutionary origin. Since O. tauri is diverged early in the green lineage, relatively close to the ancestral eukaryotic cell, it might hold a key phylogenetic posi...
Well-nourished cells in a favorable environment (well-supplied with growth factors, cytokines, and/or hormones and free from stresses, ionizing radiation, etc.) will grow, replicate their genome, and divide into two daughter cells, fully prepared to repeat the process. This cycle of DNA replication and division underlies all aspects of biological g...
The cell division cycle is the process by which eukaryotic cells replicate their chromosomes and partition them to two daughter cells. To maintain the integrity of the genome, proliferating cells must be able to block progression through the division cycle at key transition points (called "checkpoints") if there have been problems in the replicatio...
The cell division cycle is the process by which eukaryotic cells replicate their chromosomes and partition them to two daughter cells. To maintain the integrity of the genome, proliferating cells must be able to block progression through the division cycle at key transition points (called 'checkpoints'), if there have been problems in the replicati...
The transitions between phases of the cell cycle have evolved to be robust and switch-like, which ensures temporal separation of DNA replication, sister chromatid separation, and cell division. Mathematical models describing the biochemical interaction networks of cell cycle regulators attribute these properties to underlying bistable switches, whi...
In mammalian females, germ cells remain arrested as primordial follicles. Resumption of meiosis is heralded by germinal vesicle breakdown, condensation of chromosomes, and their eventual alignment on metaphase plates. At the first meiotic division, anaphase-promoting complex/cyclosome associated with Cdc20 (APC/CCdc20) activates separase and thereb...
Following DNA damage caused by exogenous sources, such as ionizing radiation, the tumour suppressor p53 mediates cell cycle arrest via expression of the CDK inhibitor, p21. However, the role of p21 in maintaining genomic stability in the absence of exogenous DNA-damaging agents is unclear. Here, using live single-cell measurements of p21 protein in...
Unperturbed, asynchronous hTert-RPE1 mRubyPCNA p21-GFP expressing cells. mRuby-PCNA is in red and p21-GFP is in green. p21-GFP levels vary both between individual cells and within individual cells as they cycle.
Unperturbed hTert-RPE1 p21-GFP mRuby-53BP1 expressing cells. mRuby-53BP1 is in red and p21-GFP is in green. A single G2 cell is shown at the start of the movie that expresses a low level of p21- GFP. The cell divides, giving rise to two daughter cells that display different characteristics. The left hand daughter cell displays 53BP1 foci shortly af...
Code for deterministic and stochastic models of the p21 control network. "Barr2017_DynamicsOfP21_SBtoolbox": Deterministic version of the mathematical model for the Systems Biology Toolbox 2 for MatLab. "Barr2017_DynamicsOfP21_XPP": Deterministic version of the mathematical model for XPP-Aut. "Barr2017_DynamicsOfP21_SBML": Deterministic version of...
hTert-RPE1 mRuby-PCNA p21-GFP expressing cells treated with DMSO (Movie 2) or 5 ìM Nutlin-3 (Movie 3). mRubyPCNA is in red and p21-GFP is in green. Drugs are added at time zero. DMSO-treated cells continue to cycle and display periodic expression of p21-GFP. In cells treated with Nutlin-3, p21-GFP expression starts to increase immediately in G1 and...
hTert-RPE1 mRuby-PCNA p21-GFP expressing cells treated with DMSO (Movie 2) or 5 ìM Nutlin-3 (Movie 3). mRubyPCNA is in red and p21-GFP is in green. Drugs are added at time zero. DMSO-treated cells continue to cycle and display periodic expression of p21-GFP. In cells treated with Nutlin-3, p21-GFP expression starts to increase immediately in G1 and...
hTert-RPE1 mRuby-PCNA p21-GFP expressing cells treated with Cdt2 siRNA. mRuby-PCNA is in red and p21-GFP is in green. At the start of the movie, the cell second from the top on the extreme right of the frame is in G1. At 4h20, this cell enters S-phase and subsequently undergoes alternating cycles of DNA replication (indicated by the presence of red...
Custom Matlab scripts for cell segmentation and tracking, cell cycle phase identification and fluorescence quantification over time.
Document S1. Supplemental Experimental Procedures, Figures S1–S4, and Tables S1 and S2
The abrupt and irreversible transition from interphase to M phase is essential to separate DNA replication from chromosome segregation. This transition requires the switch-like phosphorylation of hundreds of proteins by the cyclin-dependent kinase 1 (Cdk1):cyclin B (CycB) complex. Previous studies have ascribed these switch-like phosphorylations to...
PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 s...
The transition from G1 into DNA replication (S phase) is an emergent behavior resulting from dynamic and complex interactions between cyclin-dependent kinases (Cdks), Cdk inhibitors (CKIs), and the anaphase-promoting complex/cyclosome (APC/C). Understanding the cellular decision to commit to S phase requires a quantitative description of these inte...
Movie S3. CyclinA2-GFP/LSS2-mKate PCNA Expressing HeLa Cells, Related to Figure 1E
Movie S1. p27Kip1-GFP/LSS2-mKate PCNA Expressing HeLa Cells, Related to Figure 1A
Movie S2. CyclinE1-GFP/LSS2-mKate PCNA Expressing HeLa Cells, Related to Figure 1B
Movie S6. GFP-PCNA-Expressing HeLa Cells Treated with Emi1 siRNA, Related to Figure S6B
Proliferating cells adjust their cell size depending on the nutritional environment. Cells are large in rich media and small in poor media. This physiological response has been demonstrated in both unicellular and multicellular organisms. Here we show that the greatwall-endosulfine (Ppk18-Igo1 in fission yeast) pathway couples the nutritional envir...
Sister chromatid cohesion, mediated by the cohesin complex, is essential for faithful mitosis. Nevertheless, evidence suggests that the surveillance mechanism that governs mitotic fidelity, the spindle assembly checkpoint (SAC), is not robust enough to halt cell division when cohesion loss occurs prematurely. The mechanism behind this poor response...
In this essay we illustrate some general principles of mathematical modeling in biology by our experiences in studying the molecular regulatory network underlying eukaryotic cell division. We discuss how and why the models moved from simple, parsimonious cartoons to more complex, detailed mechanisms with many kinetic parameters. We describe how the...
Ring-shaped cohesin keeps sister chromatids paired until cleavage of its Scc1/Rad21 subunit by separase triggers chromosome segregation in anaphase. Vertebrate separase is held inactive by mutually exclusive binding to securin or Cdk1-cyclin B1 and becomes unleashed only upon ubiquitin-dependent degradation of these regulators. Although most separa...
For proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles (bi-orientation). To establish bi-orientation, aberrant kinetochore-microtubule attachments are disrupted (error correction) by aurora B kinase (Ipl1 in budding yeast). Paradoxically, during this disruption, new attachments are still f...
To facilitate rapid accumulation of Cdk1-phosphorylated substrate proteins, the Cdk1 counter-acting phosphatase, PP2A-B55 is inhibited during M phase by stoichiometric inhibitors (ENSA and Arpp19). These inhibitors are activated when phosphorylated by Cdk1-activated Greatwall-kinase. Recent experiments show that ENSA is dephosphorylated and inactiv...
In present-day eukaryotes, the cell division cycle is controlled by a complex network of inter-acting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk) families, and the Anaphase Promoting Complex (APC). Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions...
Mathematical modeling is a powerful tool for unraveling the complexities of the molecular regulatory networks underlying all aspects of cell physiology. To support this claim, we review our experiences modeling the cyclin-dependent kinase (CDK) network that controls events of the eukaryotic cell cycle. The model was derived from classic experiments...
By way of surface receptor molecules and internal surveillance mechanisms, the living cell receives information about its external environment and internal state. In light of this information, the cell must determine its most appropriate course of action under the circumstances and initiate the relevant response pathways. Typical responses include...
Activation of anaphase-promoting complex/cyclosome (APC/C(Cdc20)) by Cdc20 is delayed by the spindle assembly checkpoint (SAC). When all kinetochores come under tension, the SAC is turned off and APC/C(Cdc20) degrades cyclin B and securin, which activates separase [1]. The latter then cleaves cohesin holding sister chromatids together [2]. Because...
