Isolation and preliminary characterization of the human and mouse homologues of the bacterial cell cycle gene era

Era is an essential GTPase that is required for proper cell cycle progression and cell division in Escherichia coli and is found in nearly all bacteria sequenced to date. To determine whether Era is also present in eukaryotic organisms, we searched the dbEST database and found EST clones coding for proteins that were similar to Era. Full sequencing of these ESTs from human and mouse identified a conserved homologue, ERAL1 (Era-like 1). ERAL1 maps to 17q11.2 in human and is located in the syntenic region of mouse chromosome 11. ERAL1 may be an attractive candidate for a tumor suppressor gene since ERAL1 is located in a chromosomal region where loss of heterozygosity is often associated with various types of cancer.     

The widely conserved Era G-protein contains an RNA-binding domain required for Era function in vivo

Era is a small G-protein widely conserved in eubacteria and eukaryotes. Although essential for bacterial growth and implicated in diverse cellular processes, its actual function remains unclear. Several lines of evidence suggest that Era may be involved in some aspect of RNA biology. The GTPase domain contains features in common with all G-proteins and is required for Era function in vivo. The C-terminal domain (EraCTD) bears scant similarity to proteins outside the Era subfamily. On the basis of sequence comparisons, we argue that the EraCTD is similar to, but distinct from, the KH RNA-binding domain. Although both contain the consensus VIGxxGxxI RNA-binding motif, the protein folds are probably different. We show that bacterial Era binds RNA in vitro and can form higher-order RNA-protein complexes. Mutations in the VIGxxGxxI motif and other conserved residues of the Escherichia coli EraCTD decrease RNA binding in vitro and have corresponding effects on Era function in vivo, including previously described effects on cell division and chromosome partitioning. Importantly, mutations in L-66, located in the predicted switch II region of the E. coli Era GTPase domain, also perturb binding, leading us to propose that the GTPase domain regulates RNA binding in response to unknown cellular cues. The possible biological significance of Era RNA binding is discussed.     

RanGAP is required for post-meiotic mitosis in female gametophyte development in Arabidopsis thaliana

RanGAP is the GTPase-activating protein of the small GTPase Ran and is involved in nucleocytoplasmic transport in yeast and animals via the Ran cycle and in mitotic cell division. Arabidopsis thaliana has two copies of RanGAP, RanGAP1 and RanGAP2. To investigate the function of plant RanGAP, T-DNA insertional mutants were analysed. Arabidopsis plants with a null mutant of either RanGAP1 or RanGAP2 had no observable phenotype. Analysis of segregating progeny showed that double mutants in RanGAP1 and RanGAP2 are female gametophyte defective. Ovule clearing with differential interference contrast optics showed that mutant female gametophytes were arrested at interphase, predominantly after the first mitotic division following meiosis. In contrast, mutant pollen developed and functioned normally. These results show that the two RanGAPs are redundant and indispensable for female gametophyte development in Arabidopsis but dispensable for pollen development. Nuclear division arrest during a mitotic stage suggests a role for plant RanGAP in mitotic cell cycle progression during female gametophyte development.     

A new essential gene of the 'minimal genome' affecting cell division

The complete sequencing of bacterial genomes has offered new opportunities for the identification of essential genes involved in the control and progression of the cell cycle. For this purpose, we have disrupted ten E. coli genes belonging to the so-called 'minimal genome'. One of these genes, yihA, was necessary for normal cell division. The yihA gene possesses characteristic GTPase motifs and its homologues are present in eukaryotes, archaea and most prokaryotes. Depletion of YihA protein led to a severe reduction in growth rate and to extensive filamentation, with a block beyond the stage of nucleoid segregation. Filamentation was correlated with reduced FtsZ levels and could be specifically suppressed by overexpression of ftsQI, ftsA and ftsZ, and to some extent by ftsZ alone. We hypothesize that YihA, like the Era GTPase, may participate in a checkpoint mechanism that ensures a correct coordination of cell cycle events.     

Overview of controls in the Escherichia coli cell cycle

The harmonious growth and cell-to-cell uniformity of steady-state bacterial populations indicate the existence of a well-regulated cell cycle, responding to a set of internal signals. In Escherichia coli, the key events of this cycle are the initiation of DNA replication, nucleoid segregation and the initiation of cell division. The replication initiator is the DnaA protein. In nucleoid segregation, the MukB protein, required for proper partitioning, may be a member of the myosin-kinesin superfamily of mechanoenzymes. In cell division, the FtsZ protein has a tubulin motif, is a GTPase and polymerizes in a ring around midcell during septation; the FtsA protein has an actin-like structure. The nature of the internal signals triggering these events is not known but candidates include cell mass, the superhelical density of the chromosome and the concentration of two regulatory nucleotides, cyclic AMP and ppGpp. The involvement of cytoskeletal-like proteins in key cycle events encourages the notion of a fundamental biological unity in cell cycle regulation in all organisms.     

The fission yeast ran GTPase is required for microtubule integrity

The microtubule cytoskeleton plays a pivotal role in cytoplasmic organization, cell division, and the correct transmission of genetic information. In a screen designed to identify fission yeast genes required for chromosome segregation, we identified a strain that carries a point mutation in the SpRan GTPase. Ran is an evolutionarily conserved eukaryotic GTPase that directly participates in nucleocytoplasmic transport and whose loss affects many biological processes. Recently a transport-independent effect of Ran on spindle formation in vitro was demonstrated, but the in vivo relevance of these findings was unclear. Here, we report the characterization of a Schizosaccharomyces pombe Ran GTPase partial loss of function mutant in which nucleocytoplasmic protein transport is normal, but the microtubule cytoskeleton is defective, resulting in chromosome missegregation and abnormal cell shape. These abnormalities are exacerbated by microtubule destabilizing drugs, by loss of the spindle checkpoint protein Mph1p, and by mutations in the spindle pole body component Cut11p, indicating that SpRan influences microtubule integrity. As the SpRan mutant phenotype can be partially suppressed by the presence of extra Mal3p, we suggest that SpRan plays a role in microtubule stability.     

sodC Is an α-COP-Related Gene Which Is Essential for Establishing and Maintaining Polarized Growth in Aspergillus nidulans

Strains of Aspergillus nidulans carrying the conditional-lethal mutation sodVIC1 (stabilization of disomy) are defective in nuclear division and hyphal extension. The mutation affects both the establishment and maintenance of polar growth, since mutant spores do not germinate at restrictive temperature and preexisting hyphae stop growing upon upshift. The defect is reversible within the first 3-4 h at restrictive temperature but longer periods of incubation are lethal due to cell lysis and morphological abnormalities. There is no evidence for a specific cell cycle lesion, suggesting the existence of a feedback mechanism whereby hyphal extension is coordinated with nuclear partitioning. The sodVIC gene has been cloned from a chromosome VI-specific cosmid library and its product exhibits strong homology to the alpha-COP subunit of the coatomer complex involved in the secretory pathway in yeast and higher organisms. Molecular disruption of the gene is lethal, indicating that SodVIC is essential for growth in A. nidulans.     

Era, an essential Escherichia coli small G-protein, binds to the 30S ribosomal subunit.

Era is an essential G-protein in Escherichia coli identified originally as a homologue protein to Ras (E. coli Ras-like protein). It binds to GTP/GDP and contains a low intrinsic GTPase activity. Its function remains elusive, although it has been suggested that Era is associated with the cytoplasmic membrane, cell division, energy metabolism, and cell-cycle check point. Recently, a cold-sensitive phenotype was found to be suppressed by the overexpression of 16S rRNA methyltransferase, suggesting Era association with the ribosome. Here we demonstrate that Era specifically binds to 16S rRNA and the 30S ribosomal subunit. Both GTP and GDP, but not GMP, inhibit Era binding to ribosomal component. Involvement of Era in protein synthesis is suggested by the fact that Era depletion results in the translation defect both in vitro and in vivo.     

Up-regulation of yggG promotes the survival of Escherichia coli cells containing Era-1 mutant protein

Era is a highly conserved GTPase essential for bacterial growth. Using a digoxigenin-labeled Era protein to screen a phage expression library of Escherichia coli genomic DNA, yggG, a gene that encodes a putative zinc metalloprotease was isolated and characterized. The deduced amino acid sequence of YggG showed high degrees of similarity to some reported heat shock proteins. In this study, the direct interaction between Era and YggG was confirmed, and it was found that the yggG gene, encoding a 25 kDa heat shock protein, was up-regulated at the mRNA level in partially defective Era GTPase mutants (era-1) and in E. coli cells overproducing Era-1. The delta yggG strain displayed the same growth rate as wild-type strain under normal growth conditions and after heat shock. Overexpression of Era-1 in the delta yggG strain resulted in a stronger growth-inhibitory effect than that in the wild-type strain, while coexpression of YggG partially restored the bacterial growth rate. The results indicated that YggG expression is significantly increased in response to stress caused by the Era-1 mutant protein in E. coli, thus promoting the growth of E. coli.     

Autophosphorylation of the Bacterial Tyrosine-Kinase CpsD Connects Capsule Synthesis with the Cell Cycle in Streptococcus pneumoniae

Bacterial capsular polysaccharides (CPS) are produced by a multi-protein membrane complex, in which a particular type of tyrosine-autokinases named BY-kinases, regulate their polymerization and export. However, our understanding of the role of BY-kinases in these processes remains incomplete. In the human pathogen Streptococcus pneumoniae, the BY-kinase CpsD localizes at the division site and participates in the proper assembly of the capsule. In this study, we show that the cytoplasmic C-terminal end of the transmembrane protein CpsC is required for CpsD autophosphorylation and localization at mid-cell. Importantly, we demonstrate that the CpsC/CpsD complex captures the polysaccharide polymerase CpsH at the division site. Together with the finding that capsule is not produced at the division site in cpsD and cpsC mutants, these data show that CPS production occurs exclusively at mid-cell and is tightly dependent on CpsD interaction with CpsC. Next, we have analyzed the impact of CpsD phosphorylation on CPS production. We show that dephosphorylation of CpsD induces defective capsule production at the septum together with aberrant cell elongation and nucleoid defects. We observe that the cell division protein FtsZ assembles and localizes properly although cell constriction is impaired. DAPI staining together with localization of the histone-like protein HlpA further show that chromosome replication and/or segregation is defective suggesting that CpsD autophosphorylation interferes with these processes thus resulting in cell constriction defects and cell elongation. We show that CpsD shares structural homology with ParA-like ATPases and that it interacts with the chromosome partitioning protein ParB. Total internal reflection fluorescence microscopy imaging demonstrates that CpsD phosphorylation modulates the mobility of ParB. These data support a model in which phosphorylation of CpsD acts as a signaling system coordinating CPS synthesis with chromosome segregation to ensure that daughter cells are properly wrapped in CPS.     

Friend cell variants temperature-sensitive for growth

Friend erythroleukemia cells, thermosensitive for growth, have been isolated by a novel selection procedure employing hypoxanthine, aminopterin and bromodeoxyuridine (HAB) with near-visible light. This reagent eliminates both wild-type cells replicating at the non-permissive temperature of 39 °C and cells lacking thymidine kinase activity unable to incorporate bromodeoxyuridine (BUdR), the lethal constituent of HAB. Clones growth arrested at the non-permissive temperature have a temperature-sensitive defect in progression through G1 of the cell cycle. At permissive temperatures these clones have a karyotype similar to that of wild-type cells and are inducible for synthesis of hemoglobin. Clones which have survived the selection by means of an extended generation time are almost tetraploid at permissive temperatures, are larger than wild-type cells and are inducible for hemoglobin synthesis. At 39 °C these cells are defective in accurate mitotic division. This results in a population of cells heterogeneous in size, having chromosome complements ranging from less than the mouse diploid number to approx. 150 chromosomes/ cell. In the latter giant cells, not all nuclei are in mitosis at any one time. Such cells may be defective in cytokinesis.The two distinct classes of ts variant obtained should be useful for

1. 1. the study of whether induction of hemoglobin synthesis is cell-cycle dependent;

2. 2. mapping the chromosomes important in controlling accurate mitotic division.

     

Relationship between cellular autolytic activity, peptidoglycan synthesis, septation, and the cell cycle in synchronized populations of Streptococcus faecium.

Synchronized, slowly growing (TD = 70 to 80 min) cultures were used to study several wall-associated parameters during the cell cycle: rate of peptidoglycan synthesis, septation, and cellular autolytic activity. The rate of peptidoglycan synthesis per cell declined during most of the period of chromosome replication (C), but increased during the latter part of C and into the period between chromosome termination and cell division (D). An increase in cellular septation was correlated with the increased rate of peptidoglycan synthesis. Cellular autolytic capacity increased during the early portion of C, reached a maximum late in C or early in D, and declined during D. Inhibition of DNA synthesis during C prevented the decline in autolytic capacity at the end of the cell cycle, caused a slight reduction in the rate of peptidoglycan synthesis, delayed but did not prevent septation, and prevented the impending cell division by inhibiting cell separation. Inhibition of DNA synthesis during D did not prevent the increase in autolytic capacity during the next C phase, but, once again, prevented the decline at the end of the subsequent cycle. Thus, increased autolytic capacity at the beginning of the cell cycle did not seem to be related to chromosome initiation, whereas decreased autolytic capacity at the end of the cell cycle seemed to be related to chromosome termination. The data presented are consistent with the role of autolytic enzyme activity in the previously proposed model for cell division of S. faecium (G.D. Shockman et al., Ann. N.Y Acad. Sci. 235:161-197, 1974).     

A New Look at Rho GTPases in the Cell Cycle: Their Role in Kinetochore-Microtubule Attachment

Rho GTPases including Rho, Rac and Cdc42 are involved in cell morphogenesis by inducing specific types of actin cytoskeleton and alignment and stabilization of microtubules. Previous studies suggest that they also regulate cell cycle progression; Rho, Rac and Cdc42 regulate the G1-S progression and Rho controls cytokinesis. However, a role of Rho GTPases in nuclear division has not been definitely shown. We have recently found that Cdc42 and its downstream effector mDia3 are involved in bi-orientation and stabilization of spindle microtubules attachment to kinetochores and regulate chromosome alignment and segregation. Here, we discuss how this is coordinated with other events in mitosis, particularly, with the action of Rho in cytokinesis and how attachment of microtubules to kinetochores is achieved and stabilized. We also discuss redundancy of Cdc42 and Cdc42-related GTPase(s) and potential mechanisms of chromosome instability in cancer     

Cell cycle and sporulation in Bacillus subtilis

Recent work on cell division and chromosome orientation and partitioning in Bacillus subtilis has provided insights into cell cycle regulation during growth and development. The cell cycle is an integral part of development and entrance into sporulation is modulated by signals that transmit the status of DNA integrity, chromosome replication and segregation. In addition, B. subtilis modifies cell division and DNA segregation to establish cell-type-specific gene expression during sporulation.     

小麦RAN1在酵母系统中影响微管的完整性和核质运输过程

真核生物的小G蛋白Ran在进化过程中比较保守,它可直接参与细胞周期调控过程,它的缺失突变可以影响很多细胞生理进程.我们已经从小麦(Triticum aestivum L.cv.Jingdong No.1)cDNA文库中克隆到一个新的RanGTPase的同源基因TaRAN1.在此基础上利用裂殖酵母模式系统研究了该基因的功能.研究结果表明,TaRAN1基因超表达可产生缺陷的纺锤体微管,这可能是导致我们以前观察到的异常染色体分离现象的原因.反义TaRAN1基因表达的酵母细胞,微管系统受到破坏.我们推测TaRAN1蛋白在细胞有丝分裂的纺锤体组装和维持微管系统的完整与稳定过程中起着重要作用.透射电镜观察实验结果显示,超表达TaRAN1的酵母细胞具有异常的核膜结构,反义表达TaRAN1的酵母细胞有异常的液泡结构和紊乱的膜结构,由此推测,TaRAN1在整个核质运输事件中可能是必须的.     

Staphylococcus aureus requires at least one FtsK/SpoIIIE protein for correct chromosome segregation

Faithful coordination between bacterial cell division and chromosome segregation in rod‐shaped bacteria, such as Escherichia coli and Bacillus subtilis, is dependent on the DNA translocase activity of FtsK/SpoIIIE proteins, which move DNA away from the division site before cytokinesis is completed. However, the role of these proteins in chromosome partitioning has not been well studied in spherical bacteria. Here, it was shown that the two Staphylococcus aureus FtsK/SpoIIIE homologues, SpoIIIE and FtsK, operate in independent pathways to ensure correct chromosome management during cell division. SpoIIIE forms foci at the centre of the closing septum in at least 50% of the cells that are close to complete septum synthesis. FtsK is a multifunctional septal protein with a C‐terminal DNA translocase domain that is not required for correct chromosome management in the presence of SpoIIIE. However, lack of both SpoIIIE and FtsK causes severe nucleoid segregation and morphological defects, showing that the two proteins have partially redundant roles in S. aureus.     

The bifunctional FtsK protein mediates chromosome partitioning and cell division in Caulobacter

Bacterial chromosome partitioning and cell division are tightly connected cellular processes. We show here that the Caulobacter crescentus FtsK protein localizes to the division plane, where it mediates multiple functions involved in chromosome segregation and cytokinesis. The first 258 amino acids of the N terminus are necessary and sufficient for targeting the protein to the division plane. Furthermore, the FtsK N terminus is required to either assemble or maintain FtsZ rings at the division plane. The FtsK C terminus is essential in Caulobacter and is involved in maintaining accurate chromosome partitioning. In addition, the C-terminal region of FtsK is required for the localization of the topoisomerase IV ParC subunit to the replisome to facilitate chromosomal decatenation prior to cell division. These results suggest that the interdependence between chromosome partitioning and cell division in Caulobacter is mediated, in part, by the FtsK protein.     

PomZ,a ParA‐like protein,regulates Z‐ring formation and cell division in Myxococcus xanthus

Accurate positioning of the division site is essential to generate appropriately sized daughter cells with the correct chromosome number. In bacteria, division generally depends on assembly of the tubulin homologue FtsZ into the Z‐ring at the division site. Here, we show that lack of the ParA‐like protein PomZ in Myxococcus xanthus resulted in division defects with the formation of chromosome‐free minicells and filamentous cells. Lack of PomZ also caused reduced formation of Z‐rings and incorrect positioning of the few Z‐rings formed. PomZ localization is cell cycle regulated, and PomZ accumulates at the division site at midcell after chromosome segregation but prior to FtsZ as well as in the absence of FtsZ. FtsZ displayed cooperative GTP hydrolysis in vitro but did not form detectable filaments in vitro. PomZ interacted with FtsZ in M. xanthus cell extracts. These data show that PomZ is important for Z‐ring formation and is a spatial regulator of Z‐ring formation and cell division. The cell cycle‐dependent localization of PomZ at midcell provides a mechanism for coupling cell cycle progression and Z‐ring formation. Moreover, the data suggest that PomZ is part of a system that recruits FtsZ to midcell, thereby, restricting Z‐ring formation to this position.