Kegg Pathway: Cell cycle - yeast

Nature. 2008 Jul 17; 454(7202): 291-6
Skotheim JM, Di Talia S, Siggia ED, Cross FR

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.

Cancer Res. 2008 Jul 15; 68(14): 5552-61
Hu Z, Liu Y, Zhang C, Zhao Y, He W, Han L, Yang L, Hopkins KM, Yang X, Lieberman HB, Hang H

The Rad9 gene is evolutionarily conserved from yeast to humans and plays crucial roles in genomic maintenance, DNA repair, and Cell cycle checkpoint controls. However, the function of this gene with respect to tumorigenesis is not well-understood. A Rad9-null mutation in mice causes embryonic lethality. In this study, we created mice in which mouse Rad9, Mrad9, was deleted only in keratinocytes to permit examination of the potential function of the gene in tumor development. Mice with Mrad9(+/-) or Mrad9(-/-) keratinocytes showed no overt, spontaneous morphologic defects and seemed similar to wild-type controls. Painting the carcinogen 7,12-dimethylbenzanthracene (DMBA) onto the skin of the animals caused earlier onset and more frequent formation of tumors and senile skin plaques in Mrad9(-/-) mice, compared with Mrad9(+/-) and Mrad9(+/+) littermates. DNA damage response genes p21, p53, and Mrad9B were expressed at higher levels in Mrad9(-/-) relative to Mrad9(+/+) skin. Keratinocytes isolated from Mrad9(-/-) skin had more spontaneous and DMBA-induced DNA double strand breaks than Mrad9(+/+) keratinocytes, and the levels were reduced by incubation with the antioxidant epigallocatechin gallate. These data suggest that Mrad9 plays an important role in maintaining genomic stability and preventing tumor development in keratinocytes.

Curr Top Microbiol Immunol. 2008; 326: 291-311
Brown JW, Shaw PJ

The nucleolus is a multifunctional compartment of the eukaryotic nucleus. Besides its well-recognised role in transcription and processing of ribosomal RNA and the assembly of ribosomal subunits, the nucleolus has functions in the processing and assembly of a variety of RNPs and is involved in Cell cycle control and senescence and as a sensor of stress. Historically, nucleoli have been tenuously linked to the biogenesis and, in particular, export of mRNAs in yeast and mammalian Cells. Recently, data from plants have extended the functions in which the plant nucleolus is involved to include transcriptional gene silencing as well as mRNA surveillance and nonsense-mediated decay, and mRNA export. The nucleolus in plants may therefore have important roles in the biogenesis and quality control of mRNAs.

Comp Funct Genomics. 2004; 5(5): 419-31
Jones DL, Petty J, Hoyle DC, Hayes A, Oliver SG, Riba-Garcia I, Gaskell SJ, Stateva L

We have used DNA microarray technology and 2-D gel electrophoresis combined with mass spectrometry to investigate the effects of a drastic heat shock from 30 to 50 on a genome-wide scale. This experimental condition is used to differentiate between wild-type Cells and those with a constitutively active cAMP-dependent pathway in Saccharomyces cerevisiae. Whilst more than 50% of the former survive this shock, almost all of the latter lose viability. We compared the transcriptomes of the wildtype and a mutant strain deleted for the gene PDE2, encoding the high-affinity cAMP phosphodiesterase before and after heat shock treatment. We also compared the two heat-shocked samples with one another, allowing us to determine the changes that occur in the pde2Delta mutant which cause such a dramatic loss of viability after heat shock. Several genes involved in ergosterol biosynthesis and carbon source utilization had altered expression levels, suggesting that these processes might be potential factors in heat shock survival. These predictions and also the effect of the different phases of the Cell cycle were confirmed by biochemical and phenotypic analyses. 146 genes of previously unknown function were identified amongst the genes with altered expression levels and deletion mutants in 13 of these genes were found to be highly sensitive to heat shock. Differences in response to heat shock were also observed at the level of the proteome, with a higher level of protein degradation in the mutant, as revealed by comparing 2-D gels of wild-type and mutant heat-shocked samples and mass spectrometry analysis of the differentially produced proteins.

Comp Funct Genomics. 2002; 3(2): 194-204
Wixon J

Schizosaccharomyces pombe, the fission yeast, has long been a crucial model for the study of the eukaryote Cell cycle. We take a look at this important yeast, whose genome has recently been completed, featuring comments from Valerie Wood, Jürg Bähler, Ramsay McFarlane, Susan Forsburg, Iain Hagan and Paul Nurse on the implications of having the complete sequence and future prospects for pombe genomics.

Biochem Biophys Res Commun. 2008 Jul 12;
Park Y, Yoon SK, Yoon JB

The NEDD8 pathway plays an essential role in various physiological processes, such as Cell cycle progression and signal transduction. The conjugation of NEDD8 to target proteins is initiated by the NEDD8-activating enzyme composed of APP-BP1 and Uba3. In the present study, we show that APP-BP1 is degraded by ubiquitin-dependent proteolysis. To study biological functions of TRIP12, a HECT domain-containing E3 ubiquitin ligase, we used the yeast two-hybrid system and identified APP-BP1 as its binding partner. Immunoprecipitation analysis showed that TRIP12 specifically interacts with the APP-BP1 monomer but not with the APP-BP1/Uba3 heterodimer. Overexpression of TRIP12 enhanced the degradation of APP-BP1, whereas knockdown of TRIP12 stabilized it. In vitro ubiquitination assays revealed that TRIP12 functions as an E3 enzyme of APP-BP1 and additionally requires an E4 activity for polyubiquitination of APP-BP1. Moreover, neddylation of endogenous CUL1 was increased in TRIP12 knockdown Cells, while complementation of the knockdown Cells with TRIP12 lowered neddylated CUL1. Our data suggest that that TRIP12 promotes degradation of APP-BP1 by catalyzing its ubiquitination, which in turn modulates the neddylation pathway.

BMC Genomics. 2008 Jul 15; 9(1): 336
Logan MR, Nguyen T, Szapiel N, Knockleby J, Por H, Zadworny M, Neszt M, Harrison P, Bussey H, Mandato CA, Vogel J, Lesage G

ABSTRACT: BACKGROUND: Protein kinases and phosphatases regulate protein phosphorylation, a critical means of modulating protein function, stability and localization. The identification of functional networks for protein phosphatases has been slow due to their redundant nature and the lack of large-scale analyses. We hypothesized that a genome-scale analysis of genetic interactions using the Synthetic Genetic Array could reveal protein phosphatase functional networks. We apply this approach to the conserved type 1 protein phosphatase Glc7, which regulates numerous Cellular processes in budding yeast. RESULTS: We created a novel glc7 catalytic mutant (glc7-E101Q). Phenotypic analysis indicates that this novel allele exhibits slow growth and defects in glucose metabolism but normal Cell cycle progression and chromosome segregation. This suggests that glc7-E101Q is a hypomorphic glc7 mutant. Synthetic Genetic Array analysis of glc7-E101Q revealed a broad network of 245 synthetic sick/lethal interactions reflecting that many processes are required when Glc7 function is compromised such as histone modification, chromosome segregation and cytokinesis, nutrient sensing and DNA damage. In addition, mitochondrial activity and inheritance and lipid metabolism were identified as new processes involved in buffering Glc7 function. An interaction network among 95 genes genetically interacting with GLC7 was constructed by integration of genetic and physical interaction data. The obtained network has a modular architecture, and the inter-connection among the modules reflects the cooperation of the processes buffering Glc7 function. CONCLUSIONS: We found 245 genes required for the normal growth of the glc7-E101Q mutant. Functional grouping of these genes and analysis of their physical and genetic interaction patterns bring new information on Glc7-regulated processes.

PLoS Genet. 2008 Jul; 4(7): e1000120
Niu W, Li Z, Zhan W, Iyer VR, Marcotte EM

Regulation of Cell cycle progression is fundamental to Cell health and reproduction, and failures in this process are associated with many human diseases. Much of our knowledge of Cell cycle regulators derives from loss-of-function studies. To reveal new Cell cycle regulatory genes that are difficult to identify in loss-of-function studies, we performed a near-genome-wide flow cytometry assay of yeast gene overexpression-induced Cell cycle delay phenotypes. We identified 108 genes whose overexpression significantly delayed the progression of the yeast Cell cycle at a specific stage. Many of the genes are newly implicated in Cell cycle progression, for example SKO1, RFA1, and YPR015C. The overexpression of RFA1 or YPR015C delayed the Cell cycle at G2/M phases by disrupting spindle attachment to chromosomes and activating the DNA damage checkpoint, respectively. In contrast, overexpression of the transcription factor SKO1 arrests Cells at G1 phase by activating the pheromone response pathway, revealing new cross-talk between osmotic sensing and mating. More generally, 92%-94% of the genes exhibit distinct phenotypes when overexpressed as compared to their corresponding deletion mutants, supporting the notion that many genes may gain functions upon overexpression. This work thus implicates new genes in Cell cycle progression, complements previous screens, and lays the foundation for future experiments to define more precisely roles for these genes in Cell cycle progression.

J Biol Chem. 2008 Jul 10;
Simoneau M, Boulanger J, Coulombe G, Renaud MA, Duchesne C, Rivard N

SHP-1 is expressed in the nuclei of intestinal epithelial Cells (IECs). Increased SHP-1 expression and phosphatase activity coincide with Cell cycle arrest and differentiation in these Cells. Suspecting the tumor suppressive properties of SHP-1, a yeast two-hybrid screen of an IEC cDNA library was conducted using the full-length SHP-1 as bait. Characterization of many positive clones revealed sequences identical to a segment of the Cdk2 cDNA sequence. Interaction between SHP-1 and Cdk2 was confirmed by co-immunoprecipitations whereby co-precipitated Cdk2 phosphorylated SHP-1 protein. Inhibition of Cdk2 (roscovitine) or proteasome (MG132) was associated with an enhanced nuclear punctuate distribution of SHP-1. Double labeling localization studies with signature proteins of subnuclear domains revealed a co-localization between the splicing factor SC35 and SHP-1 in bright nucleoplasmic foci. Using Western blot analyses with the anti-SHP-1 antibody recognizing the C-terminus, a lower molecular weight species of 45-kDa was observed in addition to the full-length 64-65-kDa SHP-1 protein. Treatment with MG132 led to an increase in expression of the full-length SHP-1 protein while concomitantly leading to a decrease in the levels of the lower 45-kDa molecular species. Further Western blots revealed that the 45-kDa protein corresponds to the C-terminal portion of SHP-1 generated from proteasome activity. Mutational analysis of Y208 and S591 (a Cdk2 phosphorylation site) residues on SHP-1 abolished the expression of the amino-truncated 45-kDa SHP-1 protein. In conclusion, our results indicate that Cdk2-associated complexes, by targeting SHP-1 for proteolysis, likely counteract the ability of SHP-1 to block Cell cycle progression of IECs.

Eur J Cell Biol. 2008 Jul 8;
Cena A, Kozłowska E, Płochocka D, Grynberg M, Ishikawa T, Fronk J, Kurlandzka A

The sister chromatid cohesion complex of Saccharomyces cerevisiae includes chromosomal ATPases Smc1p and Smc3p, the kleisin Mcd1p/Scc1p, and Irr1p/Scc3p, the least studied component. We have created an irr1-1 mutation (F658G substitution) which is lethal in the haploid and semi-dominant in the heterozygous diploid irr1-1/IRR1. The mutated Irr1-1 protein is present in the nucleus, its level is similar to that of wild-type Irr1p/Scc3p and it is able to interact with chromosomes. The irr1-1/IRR1 diploid exhibits mitotic and meiotic chromosome segregation defects, irregularities in mitotic divisions and is severely affected in meiosis. These defects are gene-dosage dependent, and experiments with synchronous cultures suggest that they may result from the malfunctioning of the spindle assembly checkpoint. The partial structure of Irr1p/Scc3p was predicted and the F658G substitution was found to induce marked changes in the general shape of the predicted protein. Nevertheless, the mutant protein retains its ability to interact with Scc1p, another component of the cohesin complex, as shown by coimmunoprecipitation.

FEMS Microbiol Rev. 2008 Jun 26;
Fu Y, Pastushok L, Xiao W

After exposure to DNA-damaging agents, both prokaryotic and eukaryotic Cells activate stress responses that result in specific alterations in patterns of gene expression. Bacteria such as Escherichia coli possess both lesion-specific responses as well as an SOS response to general DNA damage, and the molecular mechanisms of these responses are well studied. Mechanisms of DNA damage response in lower eukaryotes such as Saccharomyces cerevisiae are apparently different from those in bacteria. It becomes clear that many DNA damage-inducible genes are coregulated by the Cell-cycle checkpoint, a signal transduction cascade that coordinates replication, repair, transcription and Cell-cycle progression. On the other hand, among several well-characterized yeast DNA damage-inducible genes, their effectors and mechanisms of transcriptional regulation are rather different. This review attempts to summarize the current state of knowledge on the molecular mechanisms of DNA damage-induced transcriptional regulation in this model lower eukaryotic microorganism.

Cytometry A. 2008 Jul 8;
Calvert ME, Lannigan JA, Pemberton LF

Budding yeast Saccharoymyces cerevisiae is a powerful model system for analyzing eukaryotic Cell cycle regulation. yeast Cell cycle analysis is typically performed by visual analysis or flow cytometry, and both have limitations in the scope and accuracy of data obtained. This study demonstrates how multispectral imaging flow cytometry (MIFC) provides precise quantitation of Cell cycle distribution and morphological phenotypes of yeast Cells in flow. Cell cycle analysis of wild-type yeast, nap1Delta, and yeast overexpressing NAP1, was performed visually, by flow cytometry and by MIFC. Quantitative morphological analysis employed measurements of Cellular length, thickness, and aspect ratio in an algorithm to calculate a novel feature, bud length. MIFC demonstrated reliable quantification of the yeast Cell cycle compared to morphological and flow cytometric analyses. By employing this technique, we observed both the G2/M delay and elongated buds previously described in the nap1Delta strain. Using MIFC, we demonstrate that overexpression of NAP1 causes elongated buds yet only a minor disruption in the Cell cycle. The different effects of NAP1 expression level on Cell cycle and morphology suggests that these phenotypes are independent. Unlike conventional yeast flow cytometry, MIFC generates complete Cell cycle profiles and concurrently offers multiple parameters for morphological analysis. (c) 2008 International Society for Advancement of Cytometry.

PLoS ONE. 2008; 3(7): e2623
Dinh TN, Nagahisa K, Hirasawa T, Furusawa C, Shimizu H

BACKGROUND: Microorganisms can adapt to perturbations of the surrounding environment to grow. To analyze the adaptation process of the yeast Saccharomyces cerevisiae to a high ethanol concentration, repetitive cultivation was performed with a stepwise increase in the ethanol concentration in the culture medium. METHODOLOGY/PRINCIPAL FINDINGS: First, a laboratory strain of S. cerevisiae was cultivated in medium containing a low ethanol concentration, followed by repetitive cultivations. Then, the strain repeatedly cultivated in the low ethanol concentration was transferred to medium containing a high ethanol concentration and cultivated repeatedly in the same high-ethanol-concentration medium. When subjected to a stepwise increase in ethanol concentration with the repetitive cultivations, the yeast Cells adapted to the high ethanol concentration; the specific growth rate of the adapted yeast strain did not decrease during repetitive cultivation in the medium containing the same ethanol concentration, while that of the non-adapted strain decreased during repetitive cultivation. A comparison of the fatty acid composition of the Cell membrane showed that the contents in oleic acid (C(18:1)) in ethanol-adapted and non-adapted strains were similar, but the content of palmitic acid (C(16:0)) in the ethanol-adapted strains was lower than that in the non-adapted strain in media containing ethanol. Moreover, microscopic observation showed that the mother Cells of the adapted yeast were significantly larger than those of the non-adapted strain. CONCLUSIONS: Our results suggest that activity of Cell growth defined by specific growth rate of the yeast Cells adapted to stepwise increase in ethanol concentration did not decrease during repetitive cultivation in high-ethanol-concentration medium. Moreover, fatty acid content of Cell membrane and the size of ethanol-adapted yeast Cells were changed during adaptation process. Those might be the typical phenotypes of yeast Cells adapted to high ethanol concentration. In addition, the difference in sizes of the mother Cell between the non-adapted and ethanol strains suggests that the Cell size, Cell cycle and adaptation to ethanol are thought to be closely correlated.

Eukaryot Cell. 2008 Jul 7;
Houchens CR, Perreault A, Bachand F, Kelly TJ

The initiation of eukaryotic DNA replication is preceded by the assembly of pre-replication complexes (pre-RCs) at chromosomal origins of DNA replication. Pre-RC assembly requires the essential DNA replication proteins ORC, Cdc6, and Cdt1 to load the MCM DNA helicase onto chromatin. Saccharomyces cerevisiae ScNoc3, an evolutionarily conserved protein originally implicated in 60S ribosomal subunit trafficking, has been proposed as an essential regulator of DNA replication that plays a direct role during pre-RC formation in budding yeast. We have cloned Spnoc3(+), the Schizosaccharomyces pombe homolog of the budding yeast ScNOC3 gene, and functionally characterized the requirement for SpNoc3 protein during ribosome biogenesis, Cell cycle progression, and DNA replication in fission yeast. We showed that fission yeast SpNoc3 is a functional homolog of budding yeast ScNoc3 that is essential for Cell viability and ribosome biogenesis. We also showed that SpNoc3 is required for the normal completion of Cell division in fission yeast. However, in contrast to the proposal that ScNoc3 plays an essential role during DNA replication in budding yeast, we demonstrated that fission yeast Cells do enter and complete S phase in the absence of SpNoc3, suggesting that SpNoc3 is not essential for DNA replication in fission yeast.

Biotechnol Bioeng. 1992 Mar 25; 39(7): 775-80
Nandi R, Bhattacharyya PK, Bhaduri AN, Sengupta S

Formate hydrogenlyase (FHL) activity was induced in a strain of Escherichia coli S13 during anaerobic growth in yeast extract-tryptone medium containing 100 mM formate. The Cells obtained at the optimum growth phase were immobilized in 2.5% (w/v) agar gel when 50-60% of the whole Cell FHL activity was retained. The immobilized FHL system had good storage stability and recycling efficiency. In the lysis of formate, an increase of formate concentration to 1.18M increased QH(2) (initial) value of the immobilized Cell, and subsequently Cells, hydrogen evolution, in general, ceased after 6 to 8 of incubation, resulting in incomplete lysis of formate. Presence of small amount of glucose (28 mM) was more or less quantitatively lysed with concomitant disappearence of glucose from the medium. Synthesis of formate from hydrogen and bicarbonate solution by the immobilized Cells was also characterized. Presence of glucose (10 mM) in 50 mM bicarbonate solution stimulated formate synthesis by immobilized Cells. The pH optimum range, K(m), and specific activity of the immobilized Cells for the lysis of formate were 6.8-7.2 0.4M, and 66 mL/g Cell-h, respectively. The Cells could fix hydrogen to the extent of 24.4% (w/w) of its own wet Cell mass in a 72-h reaction cycle. Potentiality of the immobilized FHL system for biotechnological exploitation was discussed.

Biotechnol Bioeng. 1992 Feb 5; 39(3): 343-50
Huls PG, Nanninga N, van Spronsen EA, Valkenburg JA, Vishcer NO, Woldringh CL

An integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important Cell cycle parameters like the fraction of daughters or budded Cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D-image processing program for rapid and accurate determination of volume distributions of the different sub-populations. The program combines the capacity of image processing and volume calculation by contour-rotation, with the potential of visual evaluation of the Cells. High-contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the Cells to shrink to 35% of their hydrated volume. As an alternative, hydrated Cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid Cell shape were underestimated as compared to volumes derived from 2D-image analysis and contour rotation, because of a deviating Cell shape, especially in the older parent Cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using Cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2-D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded Cells.

Rev Inst Med Trop Sao Paulo. 2008 May-Jun; 50(3): 169-75
Lopera D, Aristizabal BH, Restrepo A, Cano LE, González A

In order to determine the role of lysozyme, an antimicrobial peptide belonging to the innate immune system, against the dimorphic fungus Paracoccidioides brasiliensis, co-cultures of the MH-S murine alveolar macrophages Cell line with P. brasiliensis conidia were done; assays to evaluate the effect of physiological and inflammatory concentrations of lysozyme directly on the fungus life cycle were also undertaken. We observed that TNF-alpha-activated macrophages significantly inhibited the conidia to yeast transition (p = 0.0043) and exerted an important fungicidal effect (p = 0.0044), killing 27% more fungal propagules in comparison with controls. Nonetheless, after adding a selective inhibitor of lysozyme, the fungicidal effect was reverted. When P. brasiliensis propagules were exposed directly to different concentrations of lysozyme, a dual effect was observed. Physiologic concentrations of the enzyme facilitated the conidia-to-yeast transition process (p < 0.05). On the contrary, inflammatory concentrations impaired the normal temperature-dependent fungal transition (p < 0.0001). When yeast Cells were exposed to lysozyme, irrespective of concentration, the multiple-budding ability was badly impaired (p < 0.0001). In addition, ultra-structural changes such as subCellular degradation, fusion of lipid vacuoles, lamellar structures and interruption of the fibrillar layer were observed in lysozyme exposed conidia. These results suggest that lysozyme appears to exert a dual role as part of the anti-P. brasiliensis defense mechanisms.

Cell cycle. 2008 Jul; 7(13): 1907-10
Lu J, Gilbert DM

Although it is tempting to speculate that the transcription-dependent heterochromatin assembly pathway found in fission yeast may operate in higher mammals, transcription of heterochromatin has been difficult to substantiate in mammalian Cells. We recently demonstrated that transcription from the mouse pericentric heterochromatin major (gamma) satellite repeats is under Cell cycle control, being sharply downregulated at the metaphase to anaphase transition and resuming in late G(1)-phase dependent upon passage through the restriction point. The highest rates of transcription were in early S-phase and again in mitosis with different RNA products detected at each of these times.(1) Importantly, differences in the percentage of Cells in G(1)-phase can account for past discrepancies in the detection of major satellite transcripts and suggest that pericentric heterochromatin transcription takes place in all proliferating mammalian Cells. A similar Cell cycle regulation of heterochromatin transcription has now been shown in fission yeast,(2,3) providing further support for a conserved mechanism. However, there are still fundamental differences between these two systems that preclude the identification of a functional or mechanistic link.

PLoS ONE. 2008; 3(7): e2556
Sahin A, Daignan-Fornier B, Sagot I

BACKGROUND: Polarity establishment and maintenance are crucial for morphogenesis and development. In budding yeast, these two intricate processes involve the superposition of regulatory loops between polarity landmarks, RHO GTPases, actin-mediated vesicles transport and endocytosis. Deciphering the chronology and the significance of each molecular step of polarized growth is therefore very challenging. PRINCIPAL FINDINGS: We have taken advantage of the fact that yeast quiescent Cells display actin bodies, a non polarized actin structure, to evaluate the role of F-actin in bud emergence. Here we show that upon exit from quiescence, actin cables are not required for the first steps of polarized growth. We further show that polarized growth can occur in the absence of actin patch-mediated endocytosis. We finally establish, using latrunculin-A, that the first steps of polarized growth do not require any F-actin containing structures. Yet, these structures are required for the formation of a bona fide daughter Cell and Cell cycle completion. We propose that upon exit from quiescence in the absence of F-actin, secretory vesicles randomly reach the plasma membrane but preferentially dock and fuse where polarity cues are localized, this being sufficient to trigger polarized growth.

Curr Biol. 2008 Jul 8; 18(13): 1001-5
Geil C, Schwab M, Seufert W

Cdc14 phosphatase is an important regulator of mitosis in budding yeast [1]. Cdc14 antagonizes cyclin-dependent kinases and promotes multiple postmetaphase events, including segregation of the ribosomal RNA gene array (rDNA) and the nucleolus assembled around this gene cluster [2-6]. During most of the Cell cycle, Cdc14 is anchored to the nucleolus and kept inactive by binding to Net1 (also known as Cfi1) [7-9]. Cdc14 and Net1 are part of a larger nucleolar-protein network, which also contains the Net1-related protein Tof2 [10-12]. Tof2 contributes to the transcriptional silencing of rDNA regions [12], but the precise Cellular and molecular functions of Tof2 remain unclear. Here, we report that, like Net1, Tof2 can bind to Cdc14 directly. Unlike Net1, however, Tof2 did not inhibit Cdc14 but supported Cdc14 phosphatase activity and in vivo function. Deletion of TOF2 delayed rDNA segregation with little effect on mitotic exit, impaired relocalization of condensin to the nucleolus in anaphase, and caused rDNA-dependent synthetic lethality when a cdc14 mutation was present. Thus, Tof2 collaborates with Cdc14 specifically in rDNA segregation, presumably by targeting Cdc14 phosphatase activity to the nucleolus during anaphase to support resolution and compaction of this repetitive and highly transcribed DNA locus.