scholarly journals Genetic Interactions among Regulators of Septin Organization

2004 ◽  
Vol 3 (4) ◽  
pp. 847-854 ◽  
Author(s):  
Amy S. Gladfelter ◽  
Trevin R. Zyla ◽  
Daniel J. Lew
Keyword(s):  
Genetic Interactions ◽  
Septin Ring ◽  
Clear Cut ◽  
Cell Functions ◽  
Bud Emergence ◽  
Mutant Strains ◽  
Signaling Center ◽  
Bud Site ◽  
Isogenic Strains ◽  
Initial Assembly

ABSTRACT Septins form a cortical scaffold at the yeast mother-bud neck that restricts the diffusion of cortical proteins between the mother and bud and serves as a signaling center that is important for governing various cell functions. After cell cycle commitment in late G1, septins are assembled into a narrow ring at the future bud site, which spreads to form a mature septin hourglass immediately after bud emergence. Although several septin regulators have been identified, it is unclear how they cooperate to assemble the septin scaffold. We have examined septin localization in isogenic strains containing single or multiple mutations in eight septin organization genes (CDC42, RGA1, RGA2, BEM3, CLA4, GIN4, NAP1, and ELM1). Our results suggest that these regulators act largely in parallel to promote either the initial assembly of the septin ring (CDC42, RGA1, RGA2, BEM3, and CLA4) or the conversion of the ring to a stable hourglass structure at the neck (GIN4, NAP1, and ELM1). Aberrant septin localization patterns in mutant strains could be divided into apparently discrete categories, but individual strains displayed heterogeneous defects, and there was no clear-cut correspondence between the specific mutations and specific categories of defect. These findings suggest that when they are deprived of their normal regulators, septin scaffolds collapse into a limited repertoire of aberrant states in which the nature of the mutant regulators influences the probability of a given aberrant state.

scholarly journals Sensing a bud in the yeast morphogenesis checkpoint: a role for Elm1

2016 ◽  
Vol 27 (11) ◽  
pp. 1764-1775 ◽  
Author(s):  
Hui Kang ◽  
Denis Tsygankov ◽  
Daniel J. Lew
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Division ◽  
Environmental Stresses ◽  
Bud Formation ◽  
Septin Ring ◽  
Bud Emergence ◽  
Bud Neck ◽  
Morphogenesis Checkpoint ◽  
Bud Site ◽  
Mitotic Inhibitor

Bud formation by Saccharomyces cerevisiae must be coordinated with the nuclear cycle to enable successful proliferation. Many environmental stresses temporarily disrupt bud formation, and in such circumstances, the morphogenesis checkpoint halts nuclear division until bud formation can resume. Bud emergence is essential for degradation of the mitotic inhibitor, Swe1. Swe1 is localized to the septin cytoskeleton at the bud neck by the Swe1-binding protein Hsl7. Neck localization of Swe1 is required for Swe1 degradation. Although septins form a ring at the presumptive bud site before bud emergence, Hsl7 is not recruited to the septins until after bud emergence, suggesting that septins and/or Hsl7 respond to a “bud sensor.” Here we show that recruitment of Hsl7 to the septin ring depends on a combination of two septin-binding kinases: Hsl1 and Elm1. We elucidate which domains of these kinases are needed and show that artificial targeting of those domains suffices to recruit Hsl7 to septin rings even in unbudded cells. Moreover, recruitment of Elm1 is responsive to bud emergence. Our findings suggest that Elm1 plays a key role in sensing bud emergence.

scholarly journals Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p

2002 ◽  
Vol 156 (2) ◽  
pp. 315-326 ◽  
Author(s):  
Amy S. Gladfelter ◽  
Indrani Bose ◽  
Trevin R. Zyla ◽  
Elaine S.G. Bardes ◽  
Daniel J. Lew
Keyword(s):  
Transcriptional Activation ◽  
Gtpase Activating Protein ◽  
Gtp Hydrolysis ◽  
Septin Ring ◽  
Turn On ◽  
Ring Assembly ◽  
Bud Emergence ◽  
Assembly Factor ◽  
Gtpase Cycle ◽  
Budding Yeast Cell Cycle

At the beginning of the budding yeast cell cycle, the GTPase Cdc42p promotes the assembly of a ring of septins at the site of future bud emergence. Here, we present an analysis of cdc42 mutants that display specific defects in septin organization, which identifies an important role for GTP hydrolysis by Cdc42p in the assembly of the septin ring. The mutants show defects in basal or stimulated GTP hydrolysis, and the septin misorganization is suppressed by overexpression of a Cdc42p GTPase-activating protein (GAP). Other mutants known to affect GTP hydrolysis by Cdc42p also caused septin misorganization, as did deletion of Cdc42p GAPs. In performing its roles in actin polarization and transcriptional activation, GTP-Cdc42p is thought to function by activating and/or recruiting effectors to the site of polarization. Excess accumulation of GTP-Cdc42p due to a defect in GTP hydrolysis by the septin-specific alleles might cause unphysiological activation of effectors, interfering with septin assembly. However, the recessive and dose-sensitive genetic behavior of the septin-specific cdc42 mutants is inconsistent with the septin defect stemming from a dominant interference of this type. Instead, we suggest that assembly of the septin ring involves repeated cycles of GTP loading and GTP hydrolysis by Cdc42p. These results suggest that a single GTPase, Cdc42p, can act either as a ras-like GTP-dependent “switch” to turn on effectors or as an EF-Tu–like “assembly factor” using the GTPase cycle to assemble a macromolecular structure.

ISOLATION AND GENETIC ANALYSIS OF MMS-SENSITIVE MUS MUTANTS OF NEUROSPORA

1980 ◽  
Vol 22 (4) ◽  
pp. 535-552 ◽  
Author(s):  
E. Käfer ◽  
E. Perlmutter
Keyword(s):  
Dna Repair ◽  
Linkage Group ◽  
Genetic Analysis ◽  
Double Mutant ◽  
Excision Repair ◽  
Methylmethane Sulfonate ◽  
New Genes ◽  
Mutant Strains ◽  
Error Prone Repair ◽  
Isogenic Strains

With the aim of obtaining mutants that affect DNA repair or recombination, mutants sensitive to methylmethane sulfonate (MMS) have been isolated in the ascomycete Neurospora crassa. Seven of these mutants were backcrossed repeatedly to produce isogenic strains for measurements of relative mutagen sensitivities and for analysis of recombination frequencies. The new mus (mutagen sensitives) were compared to four previously known radiation-sensitive mutants which were shown to be cross-sensitive to MMS. Tests for allelism assigned the mus mutants to five new genes, mus-7 to mus-11, each mapping in a different linkage group. In homozygous crosses all mutants were sterile, except the two alleles of gene mus-10 which occasionally produced some viable ascospores. Complementation tests on MMS-media identified double mutant strains from many intercrosses. Such strains can be used for analysis of interactions between mutant alleles from different genes and of possible epistatic groupings for repair-deficient mutants in Neurospora. Four of these double mutant strains, all containing mus-8 and previously known mutants, were checked for survival on MMS media and their sensitivities were compared to those of their parental single mutant strains. Results indicate that mus-8 may be epistatic to uvs-2 which is deficient in excision repair, but not to mutants like uvs-3 that appear to be deficient in error-prone repair.

scholarly journals The Roles of Bud-Site-Selection Proteins during Haploid Invasive Growth in Yeast

2002 ◽  
Vol 13 (9) ◽  
pp. 2990-3004 ◽  
Author(s):  
Paul J. Cullen ◽  
George F. Sprague
Keyword(s):  
Site Selection ◽  
Growth Response ◽  
Invasive Growth ◽  
Glucose Limitation ◽  
Glucose Depletion ◽  
Distal Pole ◽  
Bud Emergence ◽  
Diploid Cells ◽  
Bud Site ◽  
Site Marker

In haploid strains of Saccharomyces cerevisiae, glucose depletion causes invasive growth, a foraging response that requires a change in budding pattern from axial to unipolar-distal. To begin to address how glucose influences budding pattern in the haploid cell, we examined the roles of bud-site-selection proteins in invasive growth. We found that proteins required for bipolar budding in diploid cells were required for haploid invasive growth. In particular, the Bud8p protein, which marks and directs bud emergence to the distal pole of diploid cells, was localized to the distal pole of haploid cells. In response to glucose limitation, Bud8p was required for the localization of the incipient bud site marker Bud2p to the distal pole. Three of the four known proteins required for axial budding, Bud3p, Bud4p, and Axl2p, were expressed and localized appropriately in glucose-limiting conditions. However, a fourth axial budding determinant, Axl1p, was absent in filamentous cells, and its abundance was controlled by glucose availability and the protein kinase Snf1p. In thebud8 mutant in glucose-limiting conditions, apical growth and bud site selection were uncoupled processes. Finally, we report that diploid cells starved for glucose also initiate the filamentous growth response.

scholarly journals Gic2p May Link Activated Cdc42p to Components Involved in Actin Polarization, Including Bni1p and Bud6p (Aip3p)

2000 ◽  
Vol 20 (17) ◽  
pp. 6244-6258 ◽  
Author(s):  
Malika Jaquenoud ◽  
Matthias Peter
Keyword(s):  
Actin Cytoskeleton ◽  
Dominant Negative ◽  
Single Amino Acid ◽  
Polarized Growth ◽  
Hybrid Analysis ◽  
Actin Organization ◽  
Bud Emergence ◽  
Bud Site ◽  
Two Hybrid

ABSTRACT Gic2p is a Cdc42p effector which functions during cytoskeletal organization at bud emergence and in response to pheromones, but it is not understood how Gic2p interacts with the actin cytoskeleton. Here we show that Gic2p displayed multiple genetic interactions with Bni1p, Bud6p (Aip3p), and Spa2p, suggesting that Gic2p may regulate their function in vivo. In support of this idea, Gic2p cofractionated with Bud6p and Spa2p and interacted with Bud6p by coimmunoprecipitation and two-hybrid analysis. Importantly, localization of Bni1p and Bud6p to the incipient bud site was dependent on active Cdc42p and the Gic proteins but did not require an intact actin cytoskeleton. We identified a conserved domain in Gic2p which was necessary for its polarization function but dispensable for binding to Cdc42p-GTP and its localization to the site of polarization. Expression of a mutant Gic2p harboring a single-amino-acid substitution in this domain (Gic2pW23A) interfered with polarized growth in a dominant-negative manner and prevented recruitment of Bni1p and Bud6p to the incipient bud site. We propose that at bud emergence, Gic2p functions as an adaptor which may link activated Cdc42p to components involved in actin organization and polarized growth, including Bni1p, Spa2p, and Bud6p.

scholarly journals Mapping DNA damage-dependent genetic interactions in yeast via party mating and barcode fusion genetics

2017 ◽  
Author(s):  
J. Javier Díaz-Mejía ◽  
Albi Celaj ◽  
Joseph C. Mellor ◽  
Atina Coté ◽  
Attila Balint ◽  
...  
Keyword(s):  
Double Mutant ◽  
Genetic Interaction ◽  
Culture Conditions ◽  
Genetic Interactions ◽  
Specific Gene ◽  
Functional Relationships ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Standard Culture ◽  
Yeast Genetic

AbstractCondition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double mutant strains, does not scale readily to multi-condition studies. Here we describe Barcode Fusion Genetics to map Genetic Interactions (BFG-GI), by which double mutant strains generated via en masse ‘party’ mating can also be monitored en masse for growth and genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to a decrease in the activation or activity of the checkpoint protein kinase Rad53.

scholarly journals Dissection of Filamentous Growth by Transposon Mutagenesis in Saccharomyces cerevisiae

Genetics ◽  
1997 ◽  
Vol 145 (3) ◽  
pp. 671-684 ◽  
Author(s):  
Hans-Ulrich Mösch ◽  
Gerald R Fink
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signaling Pathway ◽  
Filamentous Growth ◽  
Invasive Growth ◽  
Cell Morphogenesis ◽  
Phenotypic Analysis ◽  
Cellular Processes ◽  
Evolutionarily Conserved ◽  
Mutant Strains ◽  
Bud Site

Diploid Saccharomyces cerevisiae strains starved for nitrogen undergo a developmental transition from growth as single yeast form (YF) cells to a multicellular form consisting of filaments of pseudohyphal (PH) cells. Filamentous growth is regulated by an evolutionarily conserved signaling pathway that includes the small GTP-binding proteins Ras2p and Cdc42p, the protein kinases Ste20p, Ste11p and Ste7p, and the transcription factor Ste12p. Here, we designed a genetic screen for mutant strains defective for filamentous growth (dfg) to identify novel targets of the filamentation signaling pathway, and we thereby identified 16 different genes, CDC39, STE12, TEC1, WH13, NAB1, DBR1, CDC55, SRV2, TPM1, SPA2, BNI1, DFG5, DFG9, DFG10, BUD8 and DFG16, mutations that block filamentous growth. Phenotypic analysis of dfg mutant strains genetically dissects filamentous growth into the cellular processes of signal transduction, bud site selection, cell morphogenesis and invasive growth. Epistasis tests between dfg mutant alleles and dominant activated alleles of the RAS2 and STE11 genes, RAS2Val19 and STE11-4, respectively, identify putative targets for the filamentation signaling pathway. Several of the genes described here have homologues in filamentous fungi, where they also regulate fungal development.

Yeast RHO3 and RHO4 ras superfamily genes are necessary for bud growth, and their defect is suppressed by a high dose of bud formation genes CDC42 and BEM1

1992 ◽  
Vol 12 (12) ◽  
pp. 5690-5699 ◽  
Author(s):  
Y Matsui ◽  
A Toh-E
Keyword(s):  
Cell Polarity ◽  
Inhibitory Effect ◽  
High Dose ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bud Formation ◽  
Stabilizing Agents ◽  
Daughter Cells ◽  
Bud Emergence ◽  
Ras Superfamily ◽  
Bud Site

RHO3 and RHO4 are members of the ras superfamily genes of the yeast Saccharomyces cerevisiae and are related functionally to each other. Experiments using a conditionally expressed allele of RHO4 revealed that depletion of both the RHO3 and RHO4 gene products resulted in lysis of cells with a small bud, which could be prevented by the presence of osmotic stabilizing agents in the medium. rho3 rho4 cells incubated in medium containing an osmotic stabilizing agent were rounded and enlarged and displayed delocalized deposition of chitin and delocalization of actin patches, indicating that these cells lost cell polarity. Nine genes whose overexpression could suppress the defect of the RHO3 function were isolated (SRO genes). Two of them were identical with CDC42 and BEM1, bud site assembly genes involved in the process of bud emergence. A high dose of CDC42 complemented the rho3 defect, whereas overexpression of RHO3 had an inhibitory effect on the growth of mutants defective in the CDC24-CDC42 pathway. These results, along with comparison of cell morphology between rho3 rho4 cells and cdc24 (or cdc42) mutant cells kept under the restrictive conditions, strongly suggest that the functions of RHO3 and RHO4 are required after initiation of bud formation to maintain cell polarity during maturation of daughter cells.

scholarly journals mRNAs Encoding Polarity and Exocytosis Factors Are Cotransported with the Cortical Endoplasmic Reticulum to the Incipient Bud in Saccharomyces cerevisiae

2007 ◽  
Vol 27 (9) ◽  
pp. 3441-3455 ◽  
Author(s):  
Stella Aronov ◽  
Rita Gelin-Licht ◽  
Gadi Zipor ◽  
Liora Haim ◽  
Einat Safran ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Nuclear Division ◽  
Mrna Localization ◽  
Dependent Manner ◽  
Protein Enrichment ◽  
Bud Emergence ◽  
Bud Site ◽  
Cortical Endoplasmic Reticulum ◽  
Asymmetrically Distributed

ABSTRACT Polarized growth in the budding yeast Saccharomyces cerevisiae depends upon the asymmetric localization and enrichment of polarity and secretion factors at the membrane prior to budding. We examined how these factors (i.e., Cdc42, Sec4, and Sro7) reach the bud site and found that their respective mRNAs localize to the tip of the incipient bud prior to nuclear division. Asymmetric mRNA localization depends upon factors that facilitate ASH1 mRNA localization (e.g., the 3′ untranslated region, She proteins 1 to 5, Puf6, actin cytoskeleton, and a physical association with She2). mRNA placement precedes protein enrichment and subsequent bud emergence, implying that mRNA localization contributes to polarization. Correspondingly, mRNAs encoding proteins which are not asymmetrically distributed (i.e., Snc1, Mso1, Tub1, Pex3, and Oxa1) are not polarized. Finally, mutations which affect cortical endoplasmic reticulum (ER) entry and anchoring in the bud (myo4Δ, sec3Δ, and srp101) also affect asymmetric mRNA localization. Bud-localized mRNAs, including ASH1, were found to cofractionate with ER microsomes in a She2- and Sec3-dependent manner; thus, asymmetric mRNA transport and cortical ER inheritance are connected processes in yeast.

scholarly journals Interaction between bud-site selection and polarity-establishment machineries in budding yeast

2013 ◽  
Vol 368 (1629) ◽  
pp. 20130006 ◽  
Author(s):  
Chi-Fang Wu ◽  
Natasha S. Savage ◽  
Daniel J. Lew
Keyword(s):  
Cell Cycle ◽  
Site Selection ◽  
Time Lapse ◽  
Yeast Cells ◽  
Bud Emergence ◽  
Ras Family ◽  
Polarity Establishment ◽  
Diploid Cells ◽  
Bud Site ◽  
Time Lapse Imaging

Saccharomyces cerevisiae yeast cells polarize in order to form a single bud in each cell cycle. Distinct patterns of bud-site selection are observed in haploid and diploid cells. Genetic approaches have identified the molecular machinery responsible for positioning the bud site: during bud formation, specific locations are marked with immobile landmark proteins. In the next cell cycle, landmarks act through the Ras-family GTPase Rsr1 to promote local activation of the conserved Rho-family GTPase, Cdc42. Additional Cdc42 accumulates by positive feedback, creating a concentrated patch of GTP-Cdc42, which polarizes the cytoskeleton to promote bud emergence. Using time-lapse imaging and mathematical modelling, we examined the process of bud-site establishment. Imaging reveals unexpected effects of the bud-site-selection system on the dynamics of polarity establishment, raising new questions about how that system may operate. We found that polarity factors sometimes accumulate at more than one site among the landmark-specified locations, and we suggest that competition between clusters of polarity factors determines the final location of the Cdc42 cluster. Modelling indicated that temporally constant landmark-localized Rsr1 would weaken or block competition, yielding more than one polarity site. Instead, we suggest that polarity factors recruit Rsr1, effectively sequestering it from other locations and thereby terminating landmark activity.

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