scholarly journals cdc12p, a Protein Required for Cytokinesis in Fission Yeast, Is a Component of the Cell Division Ring and Interacts with Profilin

1997 ◽  
Vol 137 (1) ◽  
pp. 169-182 ◽  
Author(s):  
Fred Chang ◽  
David Drubin ◽  
Paul Nurse
Keyword(s):  
Cell Division ◽  
Fission Yeast ◽  
Division Ring ◽  
Genetic Interaction ◽  
Essential Gene ◽  
Single Point ◽  
Actin Ring ◽  
Ring Assembly ◽  
Mutant Cells

As in many other eukaryotic cells, cell division in fission yeast depends on the assembly of an actin ring that circumscribes the middle of the cell. Schizosaccharomyces pombe cdc12 is an essential gene necessary for actin ring assembly and septum formation. Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes. Using indirect immunofluorescence, we show that cdc12p is located in the cell division ring and not in other actin structures. When overexpressed, cdc12p is located at a medial spot in interphase that anticipates the future ring site. cdc12p localization is altered in actin ring mutants. cdc8 (tropomyosin homologue), cdc3 (profilin homologue), and cdc15 mutants exhibit no specific cdc12p staining during mitosis. cdc4 mutant cells exhibit a medial cortical cdc12p spot in place of a ring. mid1 mutant cells generally exhibit a cdc12p spot with a single cdc12p strand extending in a random direction. Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested. Finally, we found that cdc12 and cdc3 mutants show a syntheticlethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

scholarly journals Analysis of MinC Reveals Two Independent Domains Involved in Interaction with MinD and FtsZ

2000 ◽  
Vol 182 (14) ◽  
pp. 3965-3971 ◽  
Author(s):  
Zonglin Hu ◽  
Joe Lutkenhaus
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Functional Domains ◽  
Wild Type ◽  
Terminal Domain ◽  
Ring Assembly ◽  
Z Ring ◽  
Ftsz Assembly

ABSTRACT In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by themin system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts minfunction, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.

scholarly journals Roles of a Fimbrin and an α-Actinin-like Protein in Fission Yeast Cell Polarization and Cytokinesis

2001 ◽  
Vol 12 (4) ◽  
pp. 1061-1077 ◽  
Author(s):  
Jian-Qiu Wu ◽  
Jürg Bähler ◽  
John R. Pringle
Keyword(s):  
Fission Yeast ◽  
Protein Localization ◽  
Normal Growth ◽  
Growth Conditions ◽  
Cell Polarization ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Ring Assembly ◽  
Or Organization

Eukaryotic cells contain many actin-interacting proteins, including the α-actinins and the fimbrins, both of which have actin cross-linking activity in vitro. We report here the identification and characterization of both an α-actinin-like protein (Ain1p) and a fimbrin (Fim1p) in the fission yeast Schizosaccharomyces pombe. Ain1p localizes to the actomyosin-containing medial ring in an F-actin–dependent manner, and the Ain1p ring contracts during cytokinesis. ain1 deletion cells have no obvious defects under normal growth conditions but display severe cytokinesis defects, associated with defects in medial-ring and septum formation, under certain stress conditions. Overexpression of Ain1p also causes cytokinesis defects, and the ain1 deletion shows synthetic effects with other mutations known to affect medial-ring positioning and/or organization. Fim1p localizes both to the cortical actin patches and to the medial ring in an F-actin–dependent manner, and several lines of evidence suggest that Fim1p is involved in polarization of the actin cytoskeleton. Although a fim1deletion strain has no detectable defect in cytokinesis, overexpression of Fim1p causes a lethal cytokinesis defect associated with a failure to form the medial ring and concentrate actin patches at the cell middle. Moreover, an ain1 fim1 double mutant has a synthetical-lethal defect in medial-ring assembly and cell division. Thus, Ain1p and Fim1p appear to have an overlapping and essential function in fission yeast cytokinesis. In addition, protein-localization and mutant-phenotype data suggest that Fim1p, but not Ain1p, plays important roles in mating and in spore formation.

scholarly journals Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast

2010 ◽  
Vol 21 (6) ◽  
pp. 989-1000 ◽  
Author(s):  
Benjamin C. Stark ◽  
Thomas E. Sladewski ◽  
Luther W. Pollard ◽  
Matthew Lord
Keyword(s):  
Motor Activity ◽  
Atpase Activity ◽  
Fission Yeast ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Bound State ◽  
Contractile Ring ◽  
Ring Assembly

Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.

scholarly journals β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

2002 ◽  
Vol 13 (8) ◽  
pp. 2919-2932 ◽  
Author(s):  
Mohan L. Gupta ◽  
Claudia J. Bode ◽  
Douglas A. Thrower ◽  
Chad G. Pearson ◽  
Kathy A. Suprenant ◽  
...  
Keyword(s):  
Essential Gene ◽  
Dynamic Properties ◽  
Microtubule Dynamics ◽  
Cytoplasmic Microtubule ◽  
Bud Emergence ◽  
Spindle Elongation ◽  
Mutant Cells ◽  
Β Tubulin

Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast β-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules. The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells. The single microtubule effectively located the bud site before bud emergence. Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis. Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants. The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth. The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function.

scholarly journals Mutations in RNA Polymerase II and Elongation Factor SII Severely Reduce mRNA Levels in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (10) ◽  
pp. 5771-5779 ◽  
Author(s):  
J. Cale Lennon ◽  
Megan Wind ◽  
Laura Saunders ◽  
M. Benjamin Hock ◽  
Daniel Reines
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Mutant Cells ◽  
Rna Levels

ABSTRACT Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro. The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood. Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2. An SII gene disruption or therpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates. Cells with both mutations had reduced levels of total poly(A)+ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity. In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe. Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not. This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied.

scholarly journals MinD-like ATPase FlhG effects location and number of bacterial flagella during C-ring assembly

2015 ◽  
Vol 112 (10) ◽  
pp. 3092-3097 ◽  
Author(s):  
Jan S. Schuhmacher ◽  
Florian Rossmann ◽  
Felix Dempwolff ◽  
Carina Knauer ◽  
Florian Altegoer ◽  
...  
Keyword(s):  
Cell Division ◽  
Structural Diversity ◽  
Basal Bodies ◽  
Independent Manner ◽  
Bacterial Flagella ◽  
Division Site ◽  
Ring Assembly ◽  
Species Specific ◽  
Regular Patterns

The number and location of flagella, bacterial organelles of locomotion, are species specific and appear in regular patterns that represent one of the earliest taxonomic criteria in microbiology. However, the mechanisms that reproducibly establish these patterns during each round of cell division are poorly understood. FlhG (previously YlxH) is a major determinant for a variety of flagellation patterns. Here, we show that FlhG is a structural homolog of the ATPase MinD, which serves in cell-division site determination. Like MinD, FlhG forms homodimers that are dependent on ATP and lipids. It interacts with a complex of the flagellar C-ring proteins FliM and FliY (also FliN) in the Gram-positive, peritrichous-flagellated Bacillus subtilis and the Gram-negative, polar-flagellated Shewanella putrefaciens. FlhG interacts with FliM/FliY in a nucleotide-independent manner and activates FliM/FliY to assemble with the C-ring protein FliG in vitro. FlhG-driven assembly of the FliM/FliY/FliG complex is strongly enhanced by ATP and lipids. The protein shows a highly dynamic subcellular distribution between cytoplasm and flagellar basal bodies, suggesting that FlhG effects flagellar location and number during assembly of the C-ring. We describe the molecular evolution of a MinD-like ATPase into a flagellation pattern effector and suggest that the underappreciated structural diversity of the C-ring proteins might contribute to the formation of different flagellation patterns.

scholarly journals Contribution of the Ade Resistance-Nodulation-Cell Division-Type Efflux Pumps to Fitness and Pathogenesis of Acinetobacter baumannii

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Eun-Jeong Yoon ◽  
Viviane Balloy ◽  
Laurence Fiette ◽  
Michel Chignard ◽  
Patrice Courvalin ◽  
...  
Keyword(s):  
Cell Division ◽  
Acinetobacter Baumannii ◽  
Pulmonary Infection ◽  
Single Point ◽  
Resistance Mechanism ◽  
Infection Model ◽  
Efflux System ◽  
Biological Cost

ABSTRACT Overexpression of chromosomal resistance-nodulation-cell division (RND)-type efflux systems with broad substrate specificity contributes to multidrug resistance (MDR) in Acinetobacter baumannii . We have shown that modulation of expression of the structural genes for the efflux systems AdeABC and AdeIJK confers MDR and results in numerous alterations of membrane-associated cellular functions, in particular biofilm formation. However, the contribution of these RND pumps to cell fitness and virulence has not yet been studied. The biological cost of an antibiotic resistance mechanism is a key parameter in determining its stability and dissemination. From an entirely sequenced susceptible clinical isolate, we have generated a set of isogenic derivatives having single point mutations resulting in overexpression of each efflux system or with every pump deleted by allelic replacement. We found that overproduction of the pumps results in a significant decrease in fitness of the bacterial host when measured by competition experiments in vitro . Fitness and virulence were also evaluated in vivo both in systemic and pulmonary infection models in immunocompetent mice. A diminished competitiveness of the AdeABC-overexpressing mutant was observed only after intraperitoneal inoculation, but not after intranasal inoculation, the latter mimicking the most frequent type of human A. baumannii infection. However, in mice infected intranasally, this mutant was more virulent and stimulated an enhanced neutrophil activation in the lungs. Altogether, these data account for the observation that adeABC overexpression is common in MDR A. baumannii frequently found in ventilator-associated pneumonia. IMPORTANCE Overproduction of the RND AdeABC efflux system is observed with a high incidence in multidrug-resistant Acinetobacter baumannii and results in increased resistance to several antibiotics of choice for the treatment of infections caused by this nosocomial pathogen. It was therefore important to study the biological cost of the overexpression of the adeABC structural operon which is normally tightly regulated. Fitness diminution of an overexpressing mutant detected in vitro and in vivo in a model that mimics sepsis was not observed in a pulmonary infection model in which the mutant was more virulent. This points out that increased virulence can occur independently from prolonged persistence in the infected organ and can account for the elevated incidence of this resistance mechanism in clinical isolates. The study also indicates that transposon libraries will identify only virulence genes that are expressed under physiological conditions but not those that are tightly regulated.

scholarly journals The mitosis-to-interphase transition is coordinated by cross talk between the SIN and MOR pathways in Schizosaccharomyces pombe

2010 ◽  
Vol 190 (5) ◽  
pp. 793-805 ◽  
Author(s):  
Samriddha Ray ◽  
Kazunori Kume ◽  
Sneha Gupta ◽  
Wanzhong Ge ◽  
Mohan Balasubramanian ◽  
...  
Keyword(s):  
Cell Division ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Signaling Pathway ◽  
Cross Talk ◽  
Cytoskeletal Remodeling ◽  
Ring Assembly

The mechanisms that regulate cytoskeletal remodeling during the transition between mitosis and interphase are poorly understood. In fission yeast the MOR pathway promotes actin polarization to cell tips in interphase, whereas the SIN signaling pathway drives actomyosin ring assembly and cytokinesis. We show that the SIN inhibits MOR signaling in mitosis by interfering with Nak1 kinase-mediated activation of the most downstream MOR component, the NDR family kinase Orb6. Inactivation of the MOR may be a key function of the SIN because attenuation of MOR signaling rescued the cytokinetic defects of SIN mutants and allowed weak SIN signaling to trigger ectopic cytokinesis. Furthermore, failure to inhibit the MOR is toxic when the cell division apparatus is compromised. Together, our results reveal a mutually antagonistic relationship between the SIN and MOR pathways, which is important for completion of cytokinesis and coordination of cytoskeletal remodeling at the mitosis-to-interphase transition.

scholarly journals In vitro reactivation of spindle elongation in fission yeast nuc2 mutant cells.

1990 ◽  
Vol 110 (2) ◽  
pp. 417-425 ◽  
Author(s):  
H Masuda ◽  
T Hirano ◽  
M Yanagida ◽  
W Z Cande
Keyword(s):  
Fission Yeast ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Sensitive Mutant ◽  
Vitro Assay ◽  
Chromosome Separation ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Mutant Cells

To investigate the mechanisms of spindle elongation and chromosome separation in the fission yeast Schizosaccharomyces pombe, we have developed an in vitro assay using a temperature-sensitive mutant strain, nuc2. At the restrictive temperature, nuc2 cells are arrested at a metaphase-like stage with short spindles and condensed chromosomes. After permeabilization of spheroplasts of the arrested cells, spindle elongation was reactivated by addition of ATP and neurotubulin both at the restrictive and the permissive temperatures, but chromosome separation was not. This suggests that the nuc2 cells are impaired in function at a stage before sister chromatid disjunction. Spindle elongation required both ATP and exogenous tubulin and was inhibited by adenylyl imidodiphosphate (AMPPNP) or vanadate. The ends of yeast half-spindle microtubules pulse-labeled with biotinylated tubulin moved past each other during spindle elongation and a gap formed between the original half-spindles. These results suggest that the primary mechanochemical event responsible for spindle elongation is the sliding apart of antiparallel microtubules of the two half-spindles.

scholarly journals Dephosphorylation of Iqg1 by Cdc14 regulates cytokinesis in budding yeast

2015 ◽  
Vol 26 (16) ◽  
pp. 2913-2926 ◽  
Author(s):  
Daniel P. Miller ◽  
Hana Hall ◽  
Ryan Chaparian ◽  
Madison Mara ◽  
Alison Mueller ◽  
...  
Keyword(s):  
Ring Formation ◽  
Cyclin Dependent Kinase ◽  
Filamentous Actin ◽  
Actin Ring ◽  
Ring Assembly ◽  
Cdk Phosphorylation ◽  
Necessary And Sufficient ◽  
Type Ii Myosin ◽  
Actin Rings

Cytokinesis separates cells by contraction of a ring composed of filamentous actin (F-actin) and type II myosin. Iqg1, an IQGAP family member, is an essential protein in Saccharomyces cerevisiae required for assembly and contraction of the actomyosin ring. Localization of F-actin to the ring occurs only after anaphase and is mediated by the calponin homology domain (CHD) of Iqg1, but the regulatory mechanisms that temporally restrict actin ring assembly are not well defined. We tested the hypothesis that dephosphorylation of four perfect cyclin-dependent kinase (Cdk) sites flanking the CHD promotes actin ring formation, using site-specific alanine mutants. Cells expressing the nonphosphorylatable iqg1-4A allele formed actin rings before anaphase and exhibited defects in myosin contraction and cytokinesis. The Cdc14 phosphatase is required for normal cytokinesis and acts on specific Cdk phosphorylation sites. Overexpression of Cdc14 resulted in premature actin ring assembly, whereas inhibition of Cdc14 function prevented actin ring formation. Cdc14 associated with Iqg1, dependent on several CHD-flanking Cdk sites, and efficiently dephosphorylated these sites in vitro. Of importance, the iqg1-4A mutant rescued the inability of cdc14-1 cells to form actin rings. Our data support a model in which dephosphorylation of Cdk sites around the Iqg1 CHD by Cdc14 is both necessary and sufficient to promote actin ring formation. Temporal control of actin ring assembly by Cdk and Cdc14 may help to ensure that cytokinesis onset occurs after nuclear division is complete.

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