Vegetative Cell Cycle Research Articles

G1-Phase Arrest Is Not a Prerequisite for Encystment inPhysarum

Amoebae of the MyxomycetePhysarum polycephalumform resistant, walled cysts when the food bacteria in a culture have been consumed. No G1 phase has been detected in the vegetative amoebal cell cycle, most of which comprises the G2 phase. Mature cysts are also in G2, but it has been reported that a G1 phase of roughly 24 h, followed by an S phase, is obligatory prior to encystment. We used flow cytometry to determine the distribution of DNA contents in amoebal cultures at intervals during vegetative growth and encystment. In all cultures, the cells were predominantly in G2 phase, and the percentage of cells with G1 DNA content remained very low. We conclude that an extended G1 phase of 24 h did not occur in our cultures and cannot be a prerequisite for encystment.

Relevance of histone H1 kinase activity to the G2/M transition during the cell cycle ofDictyostelium discoideum.

The implication of histone H1 kinase activity for the G2/M transition during the cell cycle was investigated usingDictyostelium discoideum Ax-2. Histone H1 kinase with its activity was purified from cell extracts by the use of p13(suc1) affinity gel. In the vegetative cell cycle, the activity of histone H1 kinase including Cdc2 kinase was found using synchronized Ax-2 cells to be highest just before the entry into mitosis. The activity also was markedly enhanced just prior to the M phase from which developing cells (possibly prespore cells) reinitiate their cell cycle at the mound-tipped aggregate stage. These results strongly suggest the importance of Cdc2 kinase activity in the G2 to M phase transition during the cell cycle, as the case for other eukaryotic cells.

Timing of life cycle morphogenesis in synchronous samples of Sterkiella histriomuscorum I. The vegetative cell cycle

Summary We describe standardized growth conditions and protocols for manipulating the life cycle of a common stichotrich ciliate, Sterkiella histriomuscorum (Oxytrichidae), previously isolated as Oxytricha fallax . High density cultures were obtained with short cell cycles and with predictable kinetics of growth or differentiation. By manipulating the culture conditions we could obtain mass cultures undergoing conjugation, excystment, encystment or cannibalism. Under poor growth conditions, cells down-size by rescaling the cytostome and macronuclear DNA content, as do other Oxytrichidae. Using synchronous samples obtained from cultures in each of the life cycle phases, we describe in detail the timing of morphogenesis stages in the vegetative cell cycle, in encysting cells and in excysting cells. The timing of the nuclear and cortical events are correlated to obtain a cell cycle diagram. This establishes a firm base for study and manipulation of the life cycle differentiation and cell cycle regulation of a stichotrich ciliate at the genetic and physiological level.

Alteration of the Cell Surface during the Vegetative Cell Cycle of the Unicellular Green Alga Chlamydomonas reinhardtii

Alterations of the cell surface during the vegetative cell cycle of the unicellular green alga Chlamydomonas reinhardtii were investigated using polyclonal antibodies against the purified and subsequently deglycosylated insoluble cell wall component and against a 100 kDa polypeptide of the deglycosylated, chaotrope-soluble wall fraction, respectively. Both antibodies recognized epitopes within the non-glycosylated domains of a ‘150 kDa’ chaotrope-soluble glycoprotein (=GP3B) localized in the outer layers of the C. reinhardtii cell wall. Immunofluorescence studies indicated that both antibodies reacted with the surface of ‘late’ sporangia (harvested 1 h before liberation of the zoospores), but not with the cell surfaces of released zoospores, growing cells and young sporangia, respectively. After pretreatment with aqueous LiCl, however, the cell surfaces of zoospores, growing cells and young sporangia became accessible to these particular antibodies. Highly purified preparations of the insoluble wall fraction revealed strong immunofluorescence with both antibodies but not with the corresponding preimmune sera. Based on these data, we concluded that the antigenic sites of the insoluble glycoprotein framework of the C. reinhardtii wall are masked by LiCl-soluble glycoproteins in single cell stages and young sporangia, but not or to a lesser extent in the case of the mother walls of ‘late’ sporangia. The conclusion was supported by findings that (I) the multilayered structure of the mother-cell wall was disturbed in ‘late’, but not in young sporangia and that (II) the amounts of chaotropesoluble cell wall glycoproteins present in the LiCl-extracts from intact sporangia decreased during ripening of the sporangia.

The SLT2(MPK1) MAP kinase is activated during periods of polarized cell growth in yeast.

The SLT2(MPK1) mitogen-activated protein kinase signal transduction pa thway has been implicated in several biological processes in Saccharomyces cerevisiae, including the regulation of cytoskeletal and cell wall structure, polarized cell growth, and response to nutrient availability, hypo-osmotic shock and heat shock. We examined the conditions under which the SLT2 pathway is activated. We found that the SLT2 kinase is tyrosine phosphorylated and activated during periods in which yeast cells are undergoing polarized cell growth, namely during bud formation of vegetative cell division and during projection formation upon treatment with mating pheromone. BCK1(SLK1), a MEK kinase, is required for SLT2 activation in both of these situations. Upstream of BCK1(SLK1), we found that the STE20 kinase was required for SLT2 activation by mating pheromone, but was unnecessary for its activation during the vegetative cell cycle. Finally, SLT2 activation during vegetative growth was partially dependent on CDC28 in that the stimulation of SLT2 tyrosine phosphorylation was significantly reduced directly after a temperature shift in cdc28 ts mutants. Our data are consistent with a role for SLT2 in promoting polarized cell growth.

Expression of ubiquitin genes in Chlamydomonas reinhardtii: involvement in stress response and cell cycle

Applying different stresses (heat shock, photoinhibition, and chilling) to the unicellular green alga Chlamydomonas reinhardtii led to a characteristic transcription pattern of mRNAs encoding ubiquitin extension proteins [0.7-kb transcript(s)] as well as polyubiquitin (2.3-kb transcript). Heat shock resulted in an increase in the amount of polyubiquitin mRNA (up to tenfold compared with control cells). Chilling in the light led to a sevenfold increase in the amount of 2.3-kb transcript and to a twofold increase in the amount of 0.7-kb mRNA(s), whereas a less pronounced effect on the level of the polyubiquitin transcript was observed after applying either chilling in darkness or photoinhibition. The latter stress, however, led to a dramatic decrease in mRNAs encoding ubiquitin extension proteins (down to 23% of control cells). Experiments performed with a temperature-sensitive cell-cycle mutant of C. reinhardtii showed that the 2.3-kb polyubiquitin mRNA was no longer transcribed when cells were shifted to a non-permissive temperature, and thus were blocked in their vegetative cell cycle. This leads to the assumption that the expression of the corresponding gene is necessary for completion of the C. reinhardtii vegetative cell cycle.

Purification and Characterization of a Vegetative Lytic Enzyme Responsible for Liberation of Daughter Cells during the Proliferation ofChlamydomonas reinhardtii

A vegetative lytic enzyme (VLE) of Chlamydomonas reinhardtii mediates digestion of the cell walls of mother cells (sporangia) to allow release of daughter cells after miotic cell division in the vegetative cell cycle. This enzyme is secreted into the culture medium concurrently with the appearance of daughter cells in synchronized cultures. Using an assay that monitors digestion of the mother cell wall, we purified VLE by ion-exchange and gel-filtration chromatography from the medium of synchronized cultures. The purified enzyme was a basic glycoprotein with an apparent molecular mass of 120 kDa on gel filtration and 130 kDa on SDS-PAGE. Thus, VLE appeared to behave as a monomer. The enzyme acted specifically on the mother cell wall and was unable to digest the cell walls derived from single vegetative cells. The enzymatic activity was inhibited by PMSF, p-APMSF, TLCK, HgCl2, iodoacetate, EGTA, EDTA and 1, 10-phenanthroline. VLE cleaved several synthetic model peptides on the carboxyl side of a Lys or Arg residue, indicating that it is a protease that acts on protein in the mother cell wall in vivo to release the daughter cells.

The spines of the centric diatomCorethron criophilum: Light microscopy of vegetative cell division

As part of a study of the biology of the marine planktonic centric diatom Corethron criophilum, cells have been followed through the vegetative cell cycle from immediately before cytokinesis until separation of the two daughter cells. After cytoplasmic cleavage, which lasts 30 min, each of the daughter cells becomes shorter and leaves space for the spines to develop on each of the new hypovalves. The relationship of the two cingula of each cell remains static for approximately 12 h while the new valves are developed. Expansion of both of the daughter cells in the pervalvar axis then withdraws the girdles from one another thus revealing the mature spines of the new hypovalves which now spring out to assume their final positions. The function of the cingulum in protecting and releasing the spines in this diatom is discussed.

Isolation of the cell cycle control gene cdc2 from Paramecium tetraurelia

The polymerase chain reaction was used to amplify a cdc2 homologous sequence from Paramecium tetraurelia. The corresponding amino acid sequences exhibits about 50% similarity to the cdc2 proteins of other species. The Paramecium cdc2 encoded protein is 11 amino acids longer than S. pombe p34 cdc2 . There is one amino acid change in the conserved PSTAIRE region. Southern blot analysis indicates that Paramecium has multiple cdc2 genes. Northern blotting results shows that Paramecium cdc2 is much more expressed in actively dividing cells. It is almost undetectable in starved stationary cells. The mRNA level of cdc2 gene does not change during the vegetative cell cycle.

Establishment of cell polarity in yeast.

The establishment of cell polarity is a central feature of morphogenesis in many types of cells (Schnepf 1986; Horvitz and Herskowitz 1992; Rodriguez-Boulan and Nelson 1993; Shapiro 1993; Priess 1994). Polarity establishment involves selection of an axis of polarization followed by the asymmetric organization of cytoskeletal elements, membranous organdies, components of the plasma membrane, and components of the extracellular matrix or cell wall along this axis. In budding yeasts such as Saccharomyces cerevisiae, cell polarization is vividly manifested during the vegetative cell cycle by the appearance and selective growth of the bud, which depends on the highly polarized movement of secretory vesicles carrying new cell-surface material, and perhaps of the Golgi cisternae that generate such vesicles (Preuss et al. 1992), to the bud site and into the growing bud. This movement appears to depend primarily on the actin/myosin system (Bretscher et al. 1994; Welch et al. 1994; Govindan et al. 1995), but other cytoskeletal elements such as the cytoplasmic microtubules and the septin-containing neck filaments also polarize before bud emergence (Byers 1981; Kilmartin and Adams 1984; Ford and Pringle 1991; Kim et al. 1991; Snyder et al. 1991) and appear to play roles in modulating the pattern of cellsurface growth and/or in the segregation of organdies along the mother-bud axis (Adams 1984; Adams and Pringle 1984; Jacobs et al. 1988; Palmer et al. 1992; Li et al. 1993; Muhua et al. 1994). Thus, the central questions about the establishment of polarity in budding yeast cells concern how the axes of polarization (bud sites) are chosen, how this choice is communicated to the cytoskeletal systems, and how these processes are coordinated with other events in the cell cycle. Yeast cells also polarize during another phase of the life cycle: During mating, a cell polarizes its cytoskeleton and cell-surface growth toward its partner of opposite mating type (Tkacz and MacKay 1979; Byers 1981; Ford and Pringle 1986; Hasek et al. 1987; Read et al. 1992; Chenevert 1994), apparently in response to the gradient of secreted mating pheromone (Jackson and Hartwell 1990; Segall 1993; Chenevert 1994; Dorer et al. 1995).

The Dictyostelium cell cycle and its relationship to differentiation

The dictyostelium vegetative cell cycle is characterized by a short mitotic period followed immediately by a short S-phase (less than 30 min) and a long and variable G2 phase. The cell cycle continues during differentiation despite a decrease in cell mass: DNA replication and mitosis occur early in development and also at the tipped aggregate stage. Cells that are in mitosis, S-phase or early G2, when starved differentiate into prestalk cells and cells that are in the middle of G2 differentiate into prespore cells. We postulate that there is a restriction point late in the G2 phase, about 1-2 h before mitosis, where the cells can be arrested either by starvation and the initiation of development, by growing into stationary phase, or by prolonged incubation at low temperature. During development, this block persists to the tipped aggregate stage, where it is specifically released in prespore cells, and these cells then go through one more round of cell division. Genes encoding components of the cell cycle machinery have recently been isolated and attempts to specifically block the cell cycle by reverse genetics to study the effects on differentiation have been initiated.

The Dictyostelium cell cycle and its relationship to differentiation.

The dictyostelium vegetative cell cycle is characterized by a short mitotic period followed immediately by a short S-phase (less than 30 min) and a long and variable G2 phase. The cell cycle continues during differentiation despite a decrease in cell mass: DNA replication and mitosis occur early in development and also at the tipped aggregate stage. Cells that are in mitosis, S-phase or early G2, when starved differentiate into prestalk cells and cells that are in the middle of G2 differentiate into prespore cells. We postulate that there is a restriction point late in the G2 phase, about 1-2 h before mitosis, where the cells can be arrested either by starvation and the initiation of development, by growing into stationary phase, or by prolonged incubation at low temperature. During development, this block persists to the tipped aggregate stage, where it is specifically released in prespore cells, and these cells then go through one more round of cell division. Genes encoding components of the cell cycle machinery have recently been isolated and attempts to specifically block the cell cycle by reverse genetics to study the effects on differentiation have been initiated.

Cell cycle mutation in Paramecium tetraurelia discriminates between sexual and vegetative functions

AbstractThe temperature‐sensitive mutation cc1 blocks a number of cell cycle processes in Paramecium including macronuclear DNA synthesis, oral morphogenesis, and the later stages of micronuclear mitosis. Oral morphogenesis and micronuclear mitosis also occur in the sexual pathway. This study shows that cc1 cells can proceed through conjugation or autogamy under restrictive conditions; neither stomatogenesis nor micronuclear mitosis is blocked. Fertilization and macronuclear determination occur normally, but DNA synthesis in macronuclear anlagen is blocked. Therefore, this mutation discriminates between oral replacement during meiosis and vegetative prefission stomatogenesis, and between mitotic spindle elongation during the pregamic and postzygotic divisions and spindle elongation during the vegetative cell cycle. These results point to a fundamental regulatory difference between morphogenesis in the vegetative and sexual pathways. © 1994 Wiley‐Liss, Inc.

Interspecific mating reactions between Euplotes octocarinatus and Euplotes patella syngen 2

Euplotes octocarinatus and E. patella syngen 2 were tested for their capacity to undergo interspecific mating reactions. Pheromones which the cells of both species secrete to induce cells of other mating types of the same species to prepare for conjugation by changing their cell surface properties were found to act only species-specific. Cells which have changed their surface properties under the influence of their species-specific pheromones, however, are able to form interspecific conjugant pairs. Our cytological investigations show that the micronuclear processes of about 75% of interspecific conjugant pairs become arrested in the course of the premeiotic division. In these cases the co-conjugants separate from each other and return to the vegetative cell cycle. About 25 % of the interspecific pairs enter meiosis, but the nuclear processes in the co-conjugants are not well synchronized and finally become blocked at metaphase of the first meiotic division. The fate of such co-conjugants is always cell death. The results show that sexual incompatibility between the two species is not only due to different pheromones; in addition, it appears to be necessary that the progress of the nuclear events in a cell is stimulated repeatedly by species-specific signals provided by the co-conjugant.

Costimulation-Induced Rounding in Tetrahymena thermophila: Early Cell Shape Transformation Induced by Sexual Cell-to-Cell Collisions between Complementary Mating Types

Mixing of starved cells of complementary mating types of Tetrahymena thermophilia induces shortening of their longitudinal length within 10 min of mixing. This early morphogenetic transformation in preconjugant sexual interaction (costimulation period) was named "costimulation-induced rounding" (CIR). CIR is the earliest morphological change that has ever been found in the costimulation period and differs from "synchronous rounding" in the vegetative cell cycle, because CIR cells are still able to form food vacuoles, while cells in synchronous rounding do not have this ability. When sexual cell-to-cell collisions between two mating types were hampered by unidirectional stirring for 20 min after mixing of the two mating types, both CIR and conjugation were delayed by 20 min. When secreted materials needed for the onset of costimulation were removed by washing the cells with 10 m M Tris-HCl, pH 7.4, before mixing the two mating types, both CIR and conjugation were delayed by about 30 min. CIR-like rounding was not induced by cell-free medium either from the opposite mating type or from mixed costimulated cells. These results indicate that CIR is induced when cells are activated to form conjugating pairs by cell-to-cell collisions between complementary mating types in the presence of secreted molecules.

An Abundant High-Mobility-Group-Like Protein Is Targeted to Micronuclei in a Cell Cycle-Dependent and Developmentally Regulated Fashion in Tetrahymena thermophila

In this report, we have demonstrated for the first time that an abundant high-mobility-group (HMG)-like protein, HMG B, previously thought to be specific to macronuclei in Tetrahymena thermophila, is also present in micronuclei. Biochemical data document the fact that HMG B is extremely labile in micronuclei. Unless extreme precautions are taken during the isolation of nuclei (addition of 1% formaldehyde to the nucleus isolation buffer), HMG B is not detected in micronuclei. Using polyclonal antibodies highly selective for HMG B, immunoblotting and immunofluorescence analyses show that the presence of HMG B in micronuclei is dynamic, correlating well with known periods of micronuclear DNA replication. This is the case not only during the vegetative cell cycle but also during early stages of the sexual cycle, conjugation, when the presence of HMG B in micronuclei is also closely correlated with meiotic DNA recombination and repair. Since micronuclei are transcriptionally inactive during vegetative growth, our data lend support to the idea that HMG B does not function exclusively in the establishment of transcriptionally competent chromatin. However, micronuclei are transcriptionally active during early stages of conjugation. Evidence that HMG B is strongly synthesized and deposited into micronuclei during this stage is presented. Therefore, it is tempting to suggest that HMG B may play an important role in remodeling micronuclear chromatin into an "active," more open configuration. We favor a model wherein HMG B, like other abundant, low-specificity HMG box-containing proteins, functions to wrap DNA, presumably modulating higher-order chromatin structure for a broad range of biological processes, including transcription and replication.

An abundant high-mobility-group-like protein is targeted to micronuclei in a cell cycle-dependent and developmentally regulated fashion in Tetrahymena thermophila.

In this report, we have demonstrated for the first time that an abundant high-mobility-group (HMG)-like protein, HMG B, previously thought to be specific to macronuclei in Tetrahymena thermophila, is also present in micronuclei. Biochemical data document the fact that HMG B is extremely labile in micronuclei. Unless extreme precautions are taken during the isolation of nuclei (addition of 1% formaldehyde to the nucleus isolation buffer), HMG B is not detected in micronuclei. Using polyclonal antibodies highly selective for HMG B, immunoblotting and immunofluorescence analyses show that the presence of HMG B in micronuclei is dynamic, correlating well with known periods of micronuclear DNA replication. This is the case not only during the vegetative cell cycle but also during early stages of the sexual cycle, conjugation, when the presence of HMG B in micronuclei is also closely correlated with meiotic DNA recombination and repair. Since micronuclei are transcriptionally inactive during vegetative growth, our data lend support to the idea that HMG B does not function exclusively in the establishment of transcriptionally competent chromatin. However, micronuclei are transcriptionally active during early stages of conjugation. Evidence that HMG B is strongly synthesized and deposited into micronuclei during this stage is presented. Therefore, it is tempting to suggest that HMG B may play an important role in remodeling micronuclear chromatin into an "active," more open configuration. We favor a model wherein HMG B, like other abundant, low-specificity HMG box-containing proteins, functions to wrap DNA, presumably modulating higher-order chromatin structure for a broad range of biological processes, including transcription and replication.

Nucleolus behaviour during the cell cycle of a primitive dinoflagellate eukaryote, Prorocentrum micans Ehr., seen by light microscopy and electron microscopy

ABSTRACT Light-microscopy observation of the dinoflagellate Prorocentrum micans after silver-staining of the argyrophilic proteins of the nucleolar organizing region (Ag-NOR staining) showed the presence of nucleolar material throughout the vegetative cell cycle, and in particular during all the mitotic stages. This contrasts with the case in most higher eukaryotes, in which nucleoli disappear at the end of prophase and are reconstituted in daughter cells during telophase. Electron-microscope (EM) observations after conventional or fast-freeze fixation revealed that during interphase several functional nucleoli with three compartments (NORs, the fibrillogranular and the preribosomal granular compartments) are present in a nucleus in which the envelope is persistent and the chromosomes are always compact. During early prophase, when chromosomes are beginning to split, the nucleoli remain functional, whereas in late prophase they contain only a NOR and the granular component, and the chromosomes are surrounded by many protein masses. In early telophase, the nucleolar material coating the chromosomes migrates along with the chromosomes. Nucleologenesis occurs through the formation of prenucleolar bodies around lateral or telomeric nucleofilaments extruding from the chromosomes. Several chromosomes can contribute to the formation of one nucleolus. The behaviour of these ‘persistent nucleoli’ in a closed-nucleus model such as that of the dinoflagellates is discussed with regard to the higher eukaryotes.

Timing of micronuclear mitosis and its relation to commitment to division inParamecium tetraurelia

AbstractThis study examines the timing of micronuclear mitosis during the vegetative cell cycle and shows that mitosis begins early in the division process and coincides approximately with the earliest stages of oral morphogenesis (about 0.6 in the cell cycle in synchronous cell samples). The cc1 mutation blocks cell cycle progression prior to the point of commitment to division. Although the cc1 mutation blocks macronuclear DNA synthesis under restrictive conditions, it does not block micronuclear DNA synthesis. However, absence of functional cc1 gene product leads to blockage of micronuclear mitosis prior to completion of anaphase. This point coincides with commitment to division and is also the point at which oral morphogenesis is blocked in cc1 cells. The tim‐ings of the transition points for micronuclear mitosis and oral morphogenesis in cc1 cells are closely associated in both synchronous cell samples and in asynchronous cultures. © 1992 Wiley‐Liss, Inc.

Relationship between gametic differentiation and the cell cycle in the green alga Chlamydomonas eugametos

Summary: The relationship between the vegetative cell cycle and sexual competence in the green alga Chlamydomonas eugametos was studied using synchronized cultures. The gametic stage was found to be a regular part of the cell cycle; it extended from the beginning of the cell cycle to about 2 h prior to the commitment point for cell division. All variations in the length of this period caused by external growth conditions were accompanied by corresponding changes in the period of mating competence. The period varied depending on the fluence rate, temperature, and nitrate or CO2 concentration. We conclude that the length of the period in the cell cycle in which cells are mating competent is determined by the length of the precommitment period and that environmental factors do not influence mating competence directly, but rather affect the growth rate of the cells.