Cell size-dependent regulation of Wee1 localization by Cdr2 cortical nodes

AbstractCell size control requires mechanisms that link cell growth with Cdk1 activity. In fission yeast, the protein kinase Cdr2 forms cortical nodes that include the Cdk1 inhibitor Wee1, along with the Wee1-inhibitory kinase Cdr1. We investigated how nodes inhibit Wee1 during cell growth. Biochemical fractionation revealed that Cdr2 nodes were megadalton structures enriched for activated Cdr2, which increases in level during interphase growth. In live-cell TIRF movies, Cdr2 and Cdr1 remained constant at nodes over time, but Wee1 localized to nodes in short bursts. Recruitment of Wee1 to nodes required Cdr2 kinase activity and the noncatalytic N-terminus of Wee1. Bursts of Wee1 localization to nodes increased 20-fold as cells doubled in size throughout G2. Size-dependent signaling was due in part to the Cdr2 inhibitor Pom1, which suppressed Wee1 node bursts in small cells. Thus, increasing Cdr2 activity during cell growth promotes Wee1 localization to nodes, where inhibitory phosphorylation of Wee1 by Cdr1 and Cdr2 kinases promotes mitotic entry.SummaryCells turn off the mitotic inhibitor Wee1 to enter into mitosis. This study shows how cell growth progressively inhibits fission yeast Wee1 through dynamic bursts of localization to cortical node structures that contain Wee1 inhibitory kinases.

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Cell size–dependent regulation of Wee1 localization by Cdr2 cortical nodes

The Journal of Cell Biology ◽

10.1083/jcb.201709171 ◽

2018 ◽

Vol 217 (5) ◽

pp. 1589-1599 ◽

Cited By ~ 16

Author(s):

Corey A.H. Allard ◽

Hannah E. Opalko ◽

Ko-Wei Liu ◽

Uche Medoh ◽

James B. Moseley

Keyword(s):

Cell Growth ◽

Cell Size ◽

Total Internal Reflection ◽

Kinase Activity ◽

Size Control ◽

Small Cells ◽

Mitotic Entry ◽

Size Dependent ◽

N Terminus ◽

Biochemical Fractionation

Cell size control requires mechanisms that link cell growth with Cdk1 activity. In fission yeast, the protein kinase Cdr2 forms cortical nodes that include the Cdk1 inhibitor Wee1 along with the Wee1-inhibitory kinase Cdr1. We investigated how nodes inhibit Wee1 during cell growth. Biochemical fractionation revealed that Cdr2 nodes were megadalton structures enriched for activated Cdr2, which increases in level during interphase growth. In live-cell total internal reflection fluorescence microscopy videos, Cdr2 and Cdr1 remained constant at nodes over time, but Wee1 localized to nodes in short bursts. Recruitment of Wee1 to nodes required Cdr2 kinase activity and the noncatalytic N terminus of Wee1. Bursts of Wee1 localization to nodes increased 20-fold as cells doubled in size throughout G2. Size-dependent signaling was caused in part by the Cdr2 inhibitor Pom1, which suppressed Wee1 node bursts in small cells. Thus, increasing Cdr2 activity during cell growth promotes Wee1 localization to nodes, where inhibitory phosphorylation of Wee1 by Cdr1 and Cdr2 kinases promotes mitotic entry.

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Size-Dependent Expression of the Mitotic Activator Cdc25 as a Mechanism of Size Control in Fission Yeast

10.1101/078592 ◽

2016 ◽

Author(s):

Daniel Keifenheim ◽

Xi-Ming Sun ◽

Edridge D'Souza ◽

Makoto J. Ohira ◽

Mira Magner ◽

...

Keyword(s):

Fission Yeast ◽

Cell Size ◽

Budding Yeast ◽

Size Control ◽

Population Level ◽

Threshold Concentration ◽

Fixed Amount ◽

Size Dependent ◽

Population Average ◽

The Subject

SummaryProper cell size is essential for cellular function (Hall et al., 2004). Nonetheless, despite more than 100 years of work on the subject, the mechanisms that maintain cell size homeostasis are largely mysterious (Marshall et al., 2012). Cells in growing populations maintain cell size within a narrow range by coordinating growth and division. Bacterial and eukaryotic cells both demonstrate homeostatic size control, which maintains population-level variation in cell size within a certain range, and returns the population average to that range if it is perturbed (Marshall et al., 2012; Turner et al., 2012; Amodeo and Skotheim, 2015). Recent work has proposed two different strategies for size control: budding yeast has been proposed to use an inhibitor-dilution strategy to regulate size at the G1/S transition (Schmoller et al., 2015), while bacteria appear to use an adder strategy, in which a fixed amount of growth each generation causes cell size to converge on a stable average, a mechanism also suggested for budding yeast (Campos et al., 2014; Jun and Taheri-Araghi, 2015; Taheri-Araghi et al., 2015; Tanouchi et al., 2015; Soifer et al., 2016). Here we present evidence that cell size in the fission yeast Schizosaccharomyces pombe is regulated by a third strategy: the size dependent expression of the mitotic activator Cdc25. The cdc25 transcript levels are regulated such that smaller cells express less Cdc25 and larger cells express more Cdc25, creating an increasing concentration of Cdc25 as cell grow and providing a mechanism for cell to trigger cell division when they reach a threshold concentration of Cdc25. Since regulation of mitotic entry by Cdc25 is well conserved, this mechanism may provide a wide spread solution to the problem of size control in eukaryotes.

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The reverse, but coordinated, roles of Tor2 (TORC1) and Tor1 (TORC2) kinases for growth, cell cycle and separase-mediated mitosis in Schizosaccharomyces pombe

Open Biology ◽

10.1098/rsob.110007 ◽

2011 ◽

Vol 1 (3) ◽

pp. 110007 ◽

Cited By ~ 43

Author(s):

Nobuyasu Ikai ◽

Norihiko Nakazawa ◽

Takeshi Hayashi ◽

Mitsuhiro Yanagida

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Schizosaccharomyces Pombe ◽

Cell Size ◽

Chromosome Segregation ◽

Kinase Activity ◽

Size Control ◽

Phosphatidyl Inositol ◽

Nutrient Utilization ◽

A Cell

Target of rapamycin complexes (TORCs), which are vital for nutrient utilization, contain a catalytic subunit with the phosphatidyl inositol kinase-related kinase (PIKK) motif. TORC1 is required for cell growth, while the functions of TORC2 are less well understood. We show here that the fission yeast Schizosaccharomyces pombe TORC2 has a cell cycle role through determining the proper timing of Cdc2 Tyr15 dephosphorylation and the cell size under limited glucose, whereas TORC1 restrains mitosis and opposes securin–separase, which are essential for chromosome segregation. These results were obtained using the previously isolated TORC1 mutant tor2-L2048S in the phosphatidyl inositol kinase (PIK) domain and a new TORC2 mutant tor1-L2045D , which harbours a mutation in the same site. While mutated TORC1 and TORC2 displayed diminished kinase activity and FKBP12/Fkh1-dependent rapamycin sensitivity, their phenotypes were nearly opposite in mitosis. Premature mitosis and the G2–M delay occurred in TORC1 and TORC2 mutants, respectively. Surprisingly, separase/cut1—securin/cut2 mutants were rescued by TORC1/ tor2-L2048S mutation or rapamycin addition or even Fkh1 deletion, whereas these mutants showed synthetic defect with TORC2/ tor1-L2045D . TORC1 and TORC2 coordinate growth, mitosis and cell size control, such as Wee1 and Cdc25 do for the entry into mitosis.

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Phase transitions and size scaling of membrane-less organelles

The Journal of Cell Biology ◽

10.1083/jcb.201308087 ◽

2013 ◽

Vol 203 (6) ◽

pp. 875-881 ◽

Cited By ~ 226

Author(s):

Clifford P. Brangwynne

Keyword(s):

Phase Transitions ◽

Cell Growth ◽

Embryonic Development ◽

Cell Size ◽

Size Control ◽

Size Dependent ◽

Size Scaling ◽

Rnp Granules ◽

Condensed Liquid ◽

Shed Light

The coordinated growth of cells and their organelles is a fundamental and poorly understood problem, with implications for processes ranging from embryonic development to oncogenesis. Recent experiments have shed light on the cell size–dependent assembly of membrane-less cytoplasmic and nucleoplasmic structures, including ribonucleoprotein (RNP) granules and other intracellular bodies. Many of these structures behave as condensed liquid-like phases of the cytoplasm/nucleoplasm. The phase transitions that appear to govern their assembly exhibit an intrinsic dependence on cell size, and may explain the size scaling reported for a number of structures. This size scaling could, in turn, play a role in cell growth and size control.

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Large cells activate global protein degradation to maintain cell size homeostasis

10.1101/2021.11.09.467936 ◽

2021 ◽

Author(s):

Shixuan Liu ◽

Ceryl Tan ◽

Chloe Melo-Gavin ◽

Kevin G. Mark ◽

Miriam Bracha Ginzberg ◽

...

Keyword(s):

Cell Cycle ◽

Protein Synthesis ◽

Protein Degradation ◽

Cell Size ◽

Cell Cycle Progression ◽

Mapk Pathway ◽

Cellular Growth ◽

Small Cells ◽

Animal Cells ◽

Size Dependent

Proliferating animal cells maintain a stable size distribution over generations despite fluctuations in cell growth and division size. This tight control of cell size involves both cell size checkpoints (e.g., delaying cell cycle progression for small cells) and size-dependent compensation in rates of mass accumulation (e.g., slowdown of cellular growth in large cells). We previously identified that the mammalian cell size checkpoint is mediated by a selective activation of the p38 MAPK pathway in small cells. However, mechanisms underlying the size-dependent compensation of cellular growth remain unknown. In this study, we quantified global rates of protein synthesis and degradation in naturally large and small cells, as well as in conditions that trigger a size-dependent compensation in cellular growth. Rates of protein synthesis increase proportionally with cell size in both perturbed and unperturbed conditions, as well as across cell cycle stages. Additionally, large cells exhibit elevated rates of global protein degradation and increased levels of activated proteasomes. Conditions that trigger a large-size-induced slowdown of cellular growth also promote proteasome-mediated global protein degradation, which initiates only after growth rate compensation occurs. Interestingly, the elevated rates of global protein degradation in large cells were disproportionately higher than the increase in size, suggesting activation of protein degradation pathways. Large cells at the G1/S transition show hyperactivated levels of protein degradation, even higher than similarly sized or larger cells in S or G2, coinciding with the timing of the most stringent size control in animal cells. Together, these findings suggest that large cells maintain cell size homeostasis by activating global protein degradation to induce a compensatory slowdown of growth.

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PP2ARts1 is a master regulator of pathways that control cell size

The Journal of Cell Biology ◽

10.1083/jcb.201309119 ◽

2014 ◽

Vol 204 (3) ◽

pp. 359-376 ◽

Cited By ~ 47

Author(s):

Jessica Zapata ◽

Noah Dephoure ◽

Tracy MacDonough ◽

Yaxin Yu ◽

Emily J. Parnell ◽

...

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Cell Size ◽

Control Cell ◽

Size Control ◽

Regulatory Subunit ◽

Delay Cell ◽

Cell Cycle Entry ◽

G1 Cyclin

Cell size checkpoints ensure that passage through G1 and mitosis occurs only when sufficient growth has occurred. The mechanisms by which these checkpoints work are largely unknown. PP2A associated with the Rts1 regulatory subunit (PP2ARts1) is required for cell size control in budding yeast, but the relevant targets are unknown. In this paper, we used quantitative proteome-wide mass spectrometry to identify proteins controlled by PP2ARts1. This revealed that PP2ARts1 controls the two key checkpoint pathways thought to regulate the cell cycle in response to cell growth. To investigate the role of PP2ARts1 in these pathways, we focused on the Ace2 transcription factor, which is thought to delay cell cycle entry by repressing transcription of the G1 cyclin CLN3. Diverse experiments suggest that PP2ARts1 promotes cell cycle entry by inhibiting the repressor functions of Ace2. We hypothesize that control of Ace2 by PP2ARts1 plays a role in mechanisms that link G1 cyclin accumulation to cell growth.

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Compartmentalized nodes control mitotic entry signaling in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e13-02-0104 ◽

2013 ◽

Vol 24 (12) ◽

pp. 1872-1881 ◽

Cited By ~ 22

Author(s):

Lin Deng ◽

James B. Moseley

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Fission Yeast ◽

Cell Polarity ◽

Cell Cycle Progression ◽

Interaction Network ◽

Yeast Cells ◽

Cell Cortex ◽

Cycle Progression ◽

Mitotic Entry

Cell cycle progression is coupled to cell growth, but the mechanisms that generate growth-dependent cell cycle progression remain unclear. Fission yeast cells enter into mitosis at a defined size due to the conserved cell cycle kinases Cdr1 and Cdr2, which localize to a set of cortical nodes in the cell middle. Cdr2 is regulated by the cell polarity kinase Pom1, suggesting that interactions between cell polarity proteins and the Cdr1-Cdr2 module might underlie the coordination of cell growth and division. To identify the molecular connections between Cdr1/2 and cell polarity, we performed a comprehensive pairwise yeast two-hybrid screen. From the resulting interaction network, we found that the protein Skb1 interacted with both Cdr1 and the Cdr1 inhibitory target Wee1. Skb1 inhibited mitotic entry through negative regulation of Cdr1 and localized to both the cytoplasm and a novel set of cortical nodes. Skb1 nodes were distinct structures from Cdr1/2 nodes, and artificial targeting of Skb1 to Cdr1/2 nodes delayed entry into mitosis. We propose that the formation of distinct node structures in the cell cortex controls signaling pathways to link cell growth and division.

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Fission Yeast Cells Grow Approximately Exponentially

10.1101/489708 ◽

2018 ◽

Author(s):

Mary Pickering ◽

Lauren Nicole Hollis ◽

Edridge D’Souza ◽

Nicholas Rhind

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Fission Yeast ◽

Cell Size ◽

Exponential Growth ◽

Cell Biology ◽

Growth Models ◽

Growth Period ◽

Yeast Cells ◽

Yeast Growth

ABSTRACTHow the rate of cell growth is influenced by cell size is a fundamental question of cell biology. The simple model that cell growth is proportional to cell size, based on the proposition that larger cells have proportionally greater synthetic capacity than smaller cells, leads to the predication that the rate of cell growth increases exponentially with cell size. However, other modes of cell growth, including bilinear growth, have been reported. The distinction between exponential and bilinear growth has been explored in particular detail in the fission yeast Schizosaccharomyces pombe. We have revisited the mode of fission yeast cell growth using high-resolution time-lapse microscopy and find, as previously reported, that these two growth models are difficult to distinguish both because of the similarity in shapes between exponential and bilinear curves over the two-fold change in length of a normal cell cycle and because of the substantial biological and experimental noise inherent to these experiments. Therefore, we contrived to have cells grow more than two fold, by holding them in G2 for up to eight hours. Over this extended growth period, in which cells grow up to 5.5-fold, the two growth models diverge to the point that we can confidently exclude bilinear growth as a general model for fission yeast growth. Although the growth we observe is clearly more complicated than predicted by simple exponential growth, we find that exponential growth is a robust approximation of fission yeast growth, both during an unperturbed cell cycle and during extended periods of growth.

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Characterizing non-exponential growth and bimodal cell size distributions in Schizosaccharomyces pombe: an analytical approach

10.1101/2021.06.10.447927 ◽

2021 ◽

Author(s):

Chen Jia ◽

Abhyudai Singh ◽

Ramon Grima

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Cell Size ◽

Control Strategy ◽

Size Control ◽

Birth Size ◽

Size Distributions ◽

Cell Size Distribution ◽

A Cell ◽

Cell Size Control

Unlike many single-celled organisms, the growth of fission yeast cells within a cell cycle is not exponential. It is rather characterized by three distinct phases (elongation, septation and fission), each with a different growth rate. Experiments also show that the distribution of cell size in a lineage is often bimodal, unlike the unimodal distributions measured for the bacterium Escherichia coli. Here we construct a detailed stochastic model of cell size dynamics in fission yeast. The theory leads to analytic expressions for the cell size and the birth size distributions, and explains the origin of bimodality seen in experiments. In particular our theory shows that the left peak in the bimodal distribution is associated with cells in the elongation phase while the right peak is due to cells in the septation and fission phases. We show that the size control strategy, the variability in the added size during a cell cycle and the fraction of time spent in each of the three cell growth phases have a strong bearing on the shape of the cell size distribution. Furthermore we infer all the parameters of our model by matching the theoretical cell size and birth size distributions to those from experimental single cell time-course data for seven different growth conditions. Our method provides a much more accurate means of determining the cell size control strategy (timer, adder or sizer) than the standard method based on the slope of the best linear fit between the birth and division sizes. We also show that the variability in added size and the strength of cell size control of fission yeast depend weakly on the temperature but strongly on the culture medium.

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