Regulation of the Anaphase-Promoting Complex Examined at the Single Cell Level

Cell cycle transitions are driven by oscillations of cyclin-cyclin dependent kinase (CDK) activity and associated cyclin degradation, mediated by ubiquitylation by the anaphase-promoting complex (APC). In this work, I analyzed the regulation of the APC by its activator Cdh1 in budding yeast in single cells. Inactivation of APC-Cdh1 is an important regulatory transition leading to mitotic entry. I developed and characterized a fluorescent biosensor to measure the dynamics of APC-Cdh1 activity in single cells by quantitative time-lapse microscopy. I found that APC-Cdh1 is inactivated with very reliable timing, in contrast with other cell cycle events that occur with considerable variability in timing. The activity of APCCdh1 is restrained by multisite phosphorylation by early cyclin-CDKs. Complete removal of phosphorylation control of Cdh1 results in cell cycle arrest before mitotic entry, because persistent APC-Cdh1 activity prevents mitotic cyclin gene expression and accumulation of mitotic cyclins. I show that partial phosphorylation of Cdh1 allows for partial inactivation of APC-Cdh1. Interestingly, incompletely restrained APC-Cdh1 activity causes a variable phenotype in cell cycle progression on the single cell level. This partially penetrant phenotype, caused by incomplete inactivation of APC-Cdh1, is highly complex; even though some of the cells arrest in the cell cycle, they occasionally complete later cell cycle events with delay and in incorrect order. I show that Cdh1 can be phosphorylated by multiple cyclin-CDKs, and that additional mechanisms of APCCdh1 inactivation besides phosphorylation also contribute to robust inactivation. In the last part of the thesis, I examine the global cell cycle-associated transcriptional program and its regulation by cyclin-dependent kinase activity

Effect of Hydrophobic Mutations in the H2-H3 Subdomain of Prion Protein on Stability and Conversion In Vitro and In Vivo

Prion diseases are fatal neurodegenerative diseases, which can be acquired, sporadic or genetic, the latter being linked to mutations in the gene encoding prion protein. We have recently described the importance of subdomain separation in the conversion of prion protein (PrP). The goal of the present study was to investigate the effect of increasing the hydrophobic interactions within the H2-H3 subdomain on PrP conversion. Three hydrophobic mutations were introduced into PrP. The mutation V209I associated with human prion disease did not alter protein stability or in vitro fibrillization propensity of PrP. The designed mutations V175I and T187I on the other hand increased protein thermal stability. V175I mutant fibrillized faster than wild-type PrP. Conversion delay of T187I was slightly longer, but fluorescence intensity of amyloid specific dye thioflavin T was significantly higher. Surprisingly, cells expressing V209I variant exhibited inefficient proteinase K resistant PrP formation upon infection with 22L strain, which is in contrast to cell lines expressing wild-type, V175I and T187I mPrPs. In agreement with increased ThT fluorescence at the plateau T187I expressing cell lines accumulated an increased amount of the proteinase K-resistant prion protein. We showed that T187I induces formation of thin fibrils, which are absent from other samples. We propose that larger solvent accessibility of I187 in comparison to wild-type and other mutants may interfere with lateral annealing of filaments and may be the underlying reason for increased conversion efficiency

Decoupling of Nuclear Division Cycles and Cell Size during the Coenocytic Growth of the Ichthyosporean Sphaeroforma arctica

Coordination of the cell division cycle with the growth of the cell is critical to achieve cell size homeostasis [1]. Mechanisms coupling the cell division cycle with cell growth have been described across diverse eukaryotic taxa [2, 3, 4], but little is known about how these processes are coordinated in organisms that undergo more complex life cycles, such as coenocytic growth. Coenocytes (multinucleate cells formed by sequential nuclear divisions without cytokinesis) are commonly found across the eukaryotic kingdom, including in animal and plant tissues and several lineages of unicellular eukaryotes [5]. Among the organisms that form coenocytes are ichthyosporeans, a lineage of unicellular holozoans that are of significant interest due to their phylogenetic placement as one of the closest relatives of animals [6]. Here, we characterize the coenocytic cell division cycle in the ichthyosporean Sphaeroforma arctica. We observe that, in laboratory conditions, S. arctica cells undergo a uniform and easily synchronizable coenocytic cell cycle, reaching up to 128 nuclei per cell before cellularization and release of daughter cells. Cycles of nuclear division occur synchronously within the coenocyte and in regular time intervals (11–12 hr). We find that the growth of cell volume is dependent on concentration of nutrients in the media; in contrast, the rate of nuclear division cycles is constant over a range of nutrient concentrations. Together, the results suggest that nuclear division cycles in the coenocytic growth of S. arctica are driven by a timer, which ensures periodic and synchronous nuclear cycles independent of the cell size and growth.This work was funded by a European Research Council Consolidator Grant ( ERC-2012-Co-616960 ) and a grant ( BFU2014-57779-P ) from Ministerio de Economía y Competitividad (MINECO) , co-funded by the European Regional Development Fund (fondos FEDER) , to I.R.-T. We also acknowledge financial support from Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya (Project 2014 SGR 619 ). A.O. was supported by a Marie Sklodowska-Curie individual fellowship ( MSCA-IF 747086 ). O.D. was supported by a Swiss National Science Foundation Early PostDoc Mobility fellowship ( P2LAP3_171815 ).Peer reviewe

An APC/C-Cdh1 Biosensor Reveals the Dynamics of Cdh1 Inactivation at the G1/S Transition

<div><p>B-type cyclin-dependent kinase activity must be turned off for mitotic exit and G1 stabilization. B-type cyclin degradation is mediated by the anaphase-promoting complex/cyclosome (APC/C); during and after mitotic exit, APC/C is dependent on Cdh1. Cdh1 is in turn phosphorylated and inactivated by cyclin-CDK at the Start transition of the new cell cycle. We developed a biosensor to assess the cell cycle dynamics of APC/C-Cdh1. Nuclear exit of the G1 transcriptional repressor Whi5 is a known marker of Start; APC/C-Cdh1 is inactivated 12 min after Whi5 nuclear exit with little measurable cell-to-cell timing variability. Multiple phosphorylation sites on Cdh1 act in a redundant manner to repress its activity. Reducing the number of phosphorylation sites on Cdh1 can to some extent be tolerated for cell viability, but it increases variability in timing of APC/C-Cdh1 inactivation. Mutants with minimal subsets of phosphorylation sites required for viability exhibit striking stochasticity in multiple responses including budding, nuclear division, and APC/C-Cdh1 activity itself. Multiple cyclin-CDK complexes, as well as the stoichiometric inhibitor Acm1, contribute to APC/C-Cdh1 inactivation; this redundant control is likely to promote rapid and reliable APC/C-Cdh1 inactivation immediately following the Start transition.</p></div

Timing of cell cycle intervals.

<p>A) A histogram of times from Whi5 nuclear exit to APC/C-Cdh1 inactivation. B) A histogram of times from budding to APC/C-Cdh1 inactivation. C) A histogram of times from Whi5 nuclear exit to budding. D) A histogram of times from APC/C-Cdh1 inactivation to subsequent cytokinesis. E) A histogram of times from budding to subsequent cytokinesis. F) A histogram of times from <i>CLB2</i> promoter turn-on to subsequent cytokinesis. In A-F, APC/C-Cdh1 inactivation and <i>CLB2</i> promoter turn on were determined from smoothing spline fits as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159166#pone.0159166.s002" target="_blank">S2A Fig</a>, and cell cycles in which the measurement was sensitive to noise were omitted. Both mother and daughter cells were pooled together. G) A cumulative distribution plot of APC/C-Cdh1 inactivation times with respect to bud emergence in wild type and <i>clb5</i>,<i>6</i> cells. n (wild type) = 55; n (<i>clb5</i>,<i>6</i>) = 47.</p

Biosensor for APC/C-Cdh1 activity.

<p>A) A scheme of the APC/C-Cdh1 biosensor construct. B) Images from a time lapse movie of exponentially growing wild type cells containing the biosensor (yellow) and a bud neck marker Myo1-mCherry (red) at indicated times. The biosensor was also used as a marker for nuclear division. Scale bar– 2 microns. C) Quantification of mean fluorescence in the cell body of a representative mother cell (smoothing spline fit, solid line; raw data, circles). D) Quantification of total nuclear fluorescence of two representative mother and daughter cell pairs (smoothing spline fit). n.d.—nuclear division.</p

Cdh1 inactivation timing in <i>CDH1-1</i>:<i>3P</i> cells.

<p>A,B) Representative traces of biosensor fluorescence for wild type (A) and <i>CDH1-1</i>:<i>3P</i> (B). Each plot shows six representative traces, two each for 10 percentile, median and 90 percentile of the timing distribution population. C,D) A histogram of timing of Cdh1 inactivation with respect to Whi5 exit of the noise-filtered population for wild type (C; same data as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159166#pone.0159166.g003" target="_blank">Fig 3A</a>) and <i>CDH1-1</i>:<i>3P</i> (D).</p

Time-lapse experiments of cells bearing <i>CDH1-2</i>,<i>3P</i> phosphomutant alleles.

<p>A) Fraction of cells that completed the first cell cycle. Completing the cell cycle was scored as completing cytokinesis before rebudding. The cells that did not score as completing the cell cycle typically grew elongated buds for an extended period of time and/or budded multiple times without performing cytokinesis. n (wild type) = 25; n (<i>CDH1-m11</i>) = 14; n (<i>CDH1-2</i>,<i>3P</i>) = 20. B) Fraction of cells that carried out at least one nuclear division during arrest. n (wild type) = 25; n (<i>CDH1-m11</i>) = 14; n (<i>CDH1-2</i>,<i>3P</i>) = 21. In A-B, cell cycle events were determined from time-lapse experiments, where cells were shifted from GAL-ACM1 on to off. We count the first cell cycle as the first cycle that a cell initiated as an unbudded cell in glucose to ensure complete removal of ectopic Acm1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159166#sec012" target="_blank">materials and methods</a>). C) Representative traces of biosensor fluorescence in <i>CDH1-2</i>,<i>3P</i> cells during arrest (smoothing spline fit). D) Quantification of mean cell fluorescence, averaged across the time period of arrest. Each data point represents average fluorescence throughout the period of arrest for one cell. Error bars represent standard error of the mean n (wild type) = 9; n (<i>CDH1-m11</i>) = 14; n (<i>CDH1-2</i>,<i>3P</i>) = 16.</p

Multisite phosphorylation of Cdh1.

<p>A) Position of CDK consensus phosphorylation sites on Cdh1. B) tenfold serial dilutions of strains bearing indicated partially phosphorylatable <i>CDH1</i> alleles. The alleles are named by the sequential numbers of the phosphorylation site counting from the N-terminus that are retained (e.g. <i>CDH1-1</i>:<i>3P</i> contains the sites 1,2 and 3 but lacks the subsequent eight sites; <i>CDH1-1</i>,<i>3P</i> contains the sites 1 and 3 but lacks site 2 and sites 4–11.). C) DIC images of cells bearing the phosphorylation mutant alleles. Scale bar– 5 microns.</p