Effect of heat shock on growth and division of Stylonychia mytilus

Stylonychia mytilus cells grown at 23 degrees C exhibit an immediate arrest at G1 and S stages in the cell cycle when subjected to a heat shock of 1 h at 35 degrees C. The duration of arrest was seen to be dependent on the stage at which heat shock was given. It varied from 3 to 7 h and was synchronously accompanied by the delay in the completion of cell cycle. G2 and the early dividing stage D1 were found to be even more sensitive to heat shock than G1 and S phases. Cells divide normally when heat shock was given at the late dividing stage D2. However, the G1 stage of progeny cells was prolonged to 30 h from normal 5.5 h. These observations have been compiled from the cytological studies of normal and heat-shocked Stylonychia mytilus cells at different stages of cell cycle.     

Light-sheet microscopy-based 3D single-cell tracking reveals a correlation between cell cycle and the start of endoderm cell internalization in early zebrafish development

Controlling the initiation of cell migration plays a fundamental role in shaping the tissue during embryonic development. During gastrulation in zebrafish, some mesendoderm cells migrate inward to form the endoderm as the innermost germ layer along the yolk syncytial layer. However, how the initiation of inward migration is regulated is poorly understood. In this study, we performed light-sheet microscopy-based 3D single-cell tracking consisting of (a) whole-embryo time-lapse imaging with light-sheet microscopy and (b) three-dimensional single cell tracking in the zebrafish gastrula in which cells are marked with histone H2A-mCherry (nuclei) and the sox17:EGFP transgene (expressed in endoderm cells). We analyzed the correlation between the timing of cell internalization and cell division. Most cells that differentiated into endoderm cells began to internalize during the first half of the cell cycle, where the length of a cell cycle was defined by the period between two successive cell divisions. By contrast, the timing of other internalized cells was not correlated with a certain phase of the cell cycle. These results suggest the possibility that cell differentiation is associated with the relationship between cell cycle progression and the start of internalization. Moreover, the 3D single-cell tracking approach is useful for further investigating how cell migration is integrated with cell proliferation to shape tissues in zebrafish embryos.     

On the constancy of cell division rate in the root meristem

This review examines under what circumstances the rate of cell division among cells of the root meristem is known to vary. First, methods are compared that have been used to quantify cell division rate. These can be grouped as being either cytological, in which the rate of accumulation of cells in a particular phase of the cell cycle is determined based on some form of cytological labeling, or kinematic, in which the rate of cell accumulation is determined from the net movement of cells. Then, evidence is reviewed as to whether cell division rates vary between different tissues or cell types, between different positions in the root, or finally between different environments. The evidence is consistent with cells dividing at a constant rate, and well documented examples where cell division rate changes substantially are rare. The constancy of cell division rate contrasts with the number of dividing cells, which varies extensively, and implies that a major point for cell cycle control is governing the exit from the proliferative state at the basal boundary of the meristem.     

Regulation of cell cycle events in asymmetrically dividing cells: functions required for DNA initiation and chain elongation in Caulobacter crescentus

To study the regulation of cell cycle events after asymmetric cell division in Caulobacter crescentus, we have identified functions that are required for DNA synthesis in the stalked cell produced at division and in the new stalked cell that develops from the swarmer cell 60 min after division. The initiation of DNA synthesis in the two progeny cells is dependent upon at least two common functions. One of these is a requirement for protein synthesis and the other is a gene product identified in a temperature-sensitive cell cycle mutant. DNA chain elongation requires a third common function. The characteristic pattern of DNA synthesis in C. crescentus appears to be controlled in part by the expression of these functions in the two stalked cells at different times after cell division. The age distribution for Caulobacter cells in an exponential population has been calculated (Appendix by Robert Tax) and used to analyze some of the results.     

Increase in cell mass during the division cycle of Escherichia coli B/rA.

Increase in the mean cell mass of undivided cells was determined during the division cycle of Escherichia coli B/rA. Cell buoyant densities during the division cycle were determined after cells from an exponentially growing culture were separated by size. The buoyant densities of these cells were essentially independent of cell age, with a mean value of 1.094 g ml-1. Mean cell volume and buoyant density were also determined during synchronous growth in two different media, which provided doubling times of 40 and 25 min. Cell volume and mass increased linearly at both growth rates, as buoyant density did not vary significantly. The results are consistent with only one of the three major models of cell growth, linear growth, which specifies that the rate of increase in cell mass is constant throughout the division cycle.     

Control of the cell elongation-division cycle by shuttling of PBP1 protein in Bacillus subtilis

The characteristic shape of bacterial cells is mainly determined by the cell wall, the synthesis of which is orchestrated by penicillin-binding proteins (PBPs). Rod-shaped bacteria have two distinct modes of cell wall synthesis, involved in cell elongation and cell division, which are believed to employ different sets of PBPs. A long-held question has been how these different modes of growth are co-ordinated in space and time. We have now identified the cell division protein, EzrA, and a newly discovered protein, GpsB, as key players in the elongation-division cycle of Bacillus subtilis. Mutations in these genes have a synthetic phenotype with defects in both cell division and cell elongation. They also have an unusual bulging phenotype apparently due to a failure in properly completing cell pole maturation. We show that these phenotypes are tightly associated with disturbed localization of the major transglycosylase/transpeptidase of the cell, PBP1. EzrA and GpsB have partially differentiated roles in the localization cycle of PBP1, with EzrA mainly promoting the recruitment of PBP1 to division sites, and GpsB facilitating its removal from the cell pole, after the completion of pole maturation.     

Cell cycle characteristics of thermophilic archaea.

We have performed a cell cycle analysis of organisms from the Archaea domain. Exponentially growing cells of the thermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius were analyzed by flow cytometry, and several unusual cell cycle characteristics were found. The cells initiated chromosome replication shortly after cell division such that the proportion of cells with a single chromosome equivalent was low in the population. The postreplication period was found to be long; i.e., there was a considerable time interval from termination of chromosome replication until cell division. A further unusual feature was that cells in stationary phase contained two genome equivalents, showing that they entered the resting stage during the postreplication period. Also, a reduction in cellular light scatter was observed during entry into stationary phase, which appeared to reflect changes not only in cell size but also in morphology and/or composition. Finally, the in vivo organization of the chromosome DNA appeared to be different from that of eubacteria, as revealed by variation in the relative binding efficiency of different DNA stains.     

The activity of total histone H1 kinases is related to growth and commitment points while the p13suc1-bound kinase activity relates to mitoses in the alga Scenedesmus quadricauda

Under optimal growing conditions, synchronous cultures of the alga Scenedesmus quadricauda underwent three DNA replications and three mitoses during one cell cycle. This resulted in eight daughter cells. By different illumination regimes and temporal addition of cycloheximide, cell divisions resulting in two, four or eight daughter cells per cycle were obtained. The selected cell cycle patterns differed in timing of commitment points, the number of nuclear divisions, and their positioning in the cell cycle. These distinct cell cycle patterns allowed to assess the correlation of histone H1 kinase activity with commitment points and mitoses. The activity of the histone H1 kinases was assayed in cellular protein extracts and after affinity purification using the p13^suc1 protein. The main peaks of kinase activity in the cellular extract were found to correlate with the commitment points. Small histone H1 kinase activity peaks were also found which preceded the nuclear division. Contrary to the histone H1 kinase activity of cellular extracts, the p13^suc1-bound kinase activity preceded the nuclear division, whilst its activity was negligible at the commitment points. Being able to manipulate the timing of commitment points and cell division by manipulating experimental conditions, we could precisely match the commitment points to an as yet unidentified histone H1 kinase activity and mitosis to p13^suc1-bound CDK activity during a particular cell division pattern with overlapping cycles. This provides molecular evidence, that local activation of CDKs regulates distinct events of the cell cycle.     

Effect of cell population density on G2 arrest in Tetrahymena

The ciliated protozoan, Tetrahymena pyriformis strain GL-C, has been used to study the effect of cell population density during starvation on the synchrony obtained after refeeding and on the number of cells arrested in G2 phase of the cell cycle. At high cell densities two peaks of division indices were observed after refeeding while only one was observed at low cell densities. Cell division began earlier in cultures starved at high cell densities. Most importantly, the proportion of cells in G2 was considerably higher in populations starved at high cell densities. When tritiated thymidine was present during the refeeding period, radioautographs of cell samples at different times showed that the first cells to exhibit division furrows contained unlabeled nuclei. The first peak in the division index after refeeding was observed only at higher cell densities and is attributed to the cells arrested in G2. These results suggest that Tetrahymena is an excellent organism to study the concept of resting stages in the cell cycle and their control.     

Varying competence for protein import into chloroplasts during the cell cycle in Chlamydomonas.

By studying the import of radioactively labelled small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) into chloroplasts of the green alga C. reinhardtii cw-15 protein delivery to chloroplasts was found to vary during the cell cycle. Chloroplasts were isolated from highly synchronous cultures at different time points during the cell cycle. When pSS was imported into 'young' chloroplasts isolated early in the light period about three times less pSS was processed to small subunit SS than in 'mature' chloroplasts from the middle of the light period. In 'young' chloroplasts also, less pSS was bound to the envelope surface. During the second half of the light period the import competence of isolated chloroplasts decreased again when based on chlorophyll content or cell volume, but did not change significantly when related to chloroplast number. Measurements of pSS binding to the surface of chloroplasts of different age indicated that the adaptation of protein import competence during the cell cycle is due to a variation of the number of binding sites per chloroplast surface area, rather than to modulation of the binding constant.     

Role of timer and sizer in regulation of Chlamydomonas cell cycle

To estimate the role that time and size had in controlling the Chlamydomonas cell cycle, we used a new on-chip single-cell microcultivation system, which involved the direct observation of single cells captured in microchambers made on a thin glass slide. The dependence of the pattern of energy supply for cells on its cell cycle was examined through a series of different intensities of continuous illumination in a minimal medium, and we found that cell division occurred when cells reached the critical size, which was 2.2 times larger than that of the newly created cells. When illumination stopped before cells reached the critical size, even though growth had stopped, they continued dividing during the delay time, which was shorter when cells were larger. With re-illumination after darkness, cells began to grow again and the timing of cell division was again controlled by the critical size. This indicates that the co-existence of two cell cycle regulation mechanisms and the sizer mechanism had a stronger influence than the timer.     

Embryogenesis in C. elegans after elimination of individual blastomeres or induced alteration of the cell division order

Summary Our earlier studies on embryonic arrest mutants of C. elegans had indicated that early deviations from the normal temporal and spatial pathway of development lead to monstrous terminal phenotypes with little resemblance to a hatched juvenile. To analyze more directly the roles of different parameters for cellular pattern formation, various experiments with a laser microbeam have now been performed and are described in this and the accompanying paper. By ablating early blastomeres we demonstrate here that the establishment of certain cell lineages is not necessary for the generation of a hatching juvenile. However, no replacement of missing cells was observed in these cases, and the resultant animals lacked those structures which are normally produced by the ablated cells. We found that retardation of cell cycle periods in certain cell lineages and thus a change in the normal order of cell divisions is compatible with development to a hatching juvenile. This is also true when, after irradiation of gut precursor cells, their inward migration is considerably delayed. Our results demonstrate that the invariant pattern of early nematode embryogenesis is not a necessary prerequisite for normal development. Studying parameters necessary for gastrulation we found that after irradiation leading to prolonged cell cycle periods the undivided gut founder cell itself rather than its two daughters moves into the center of the embryo. We removed individual early blastomeres and tested whether the typical inward movement of gut precursors still took place. Our results show that the presence of specific neighboring founder cells is not required, indicating that prospective gut cells reduce their cohesive contacts with adjacent blastomeres prior to the onset of gastrulation. Correspondence to: E. Schierenberg     

Commitment to the First Cortical Reorganization During Conjugation in Stylonychia mytilus: An Argument for Homology with Cortical Development During Binary Fission

The asexual nature of the first cortical reorganization of conjugation in Stylonychia was analyzed by comparing the effect of amputation performed at different stages of early conjugation to that performed on vegetative cells at different stages of the cell cycle. Amputation of vegetative cells delineated a point of commitment to binary fission at 0.51–0.57 of the cell cycle. Cells amputated before this point were induced to undergo the regenerative mode of asexual development, but those amputated after this point continued with binary fission. In parallel, during conjugation a similar commitment was made around the time of formation of tight mating-pairs: early conjugants amputated around this time might undergo regeneration, and those operated on after this stage continued with the first cortical reorganization as in typical conjugants. The two mates of a pair might differ in their response to amputation, suggesting that the timing of commitment to the first cortical reorganization is not related to the events of conjugation, but rather is individually determined in the vegetative cycle of the cells before they pair up in mating. These observations provide support for the notion that the first cortical reorganization of conjugants is homologous to the asexual mode of cortical development in dividers, according to the theory of developmental heterochrony in the sexual reproduction of hypotrichs. The timing of commitment to the first cortical reorganization was found to temporally correlate with the entrance of the micronuclei into meiosis. Since the first cortical reorganization can proceed without the micronucleus, this raises the possibility that initiation of micronuclear meiosis is closely coupled with, and may be determined by, the commitment to the first cortical reorganization.     

Uncontrolled septation in a cell division cycle mutant of the fission yeast Schizosaccharomyces pombe.

A temperature-sensitive Schizosaccharomyces pombe mutant, cdc16-116, has been isolated which undergoes uncontrolled septation during its cell division cycle. The mutant accumulates two types of cells after 3 h of growth at the restrictive temperature: (i) type I cells (85% of the population), which complete nuclear division and then form up to five septa between the divided nuclei; and (ii) type II cells (15% of the population), which form an asymmetrically situated septum in the absence of any nuclear division. cdc16-116 is a monogenic recessive mutation unlinked to any previously known cdc gene of S. pombe. It is not affected in a previously reported control by which septation is dependent upon completion of nuclear division. We propose the cdc16-116 is unable to complete septum formation and proceed to cell separation and is also defective in a control which prevents the manufacture of more than one septum in each cell cycle.     

Players between the worlds: multifunctional DNA translocases

DNA translocases play important roles during the bacterial cell cycle and in cell differentiation. Escherichia coli cells contain a multifunctional translocase, FtsK, which is involved in cell division, late steps of chromosome segregation and dimer resolution. In Gram-positive bacteria, the latter two processes are achieved by two translocases, SftA and SpoIIIE. These two translocases operate in a two step fashion, before and after closure of the division septum. DNA translocases have the remarkable ability to translocate DNA in a vectorial manner, orienting themselves according to polar sequences present in bacterial genomes, and perform various additional roles during the cell cycle. DNA translocases genetically interact with Structural Maintenance of Chromosomes (SMC) proteins in a flexible manner in different species, underlining the high versatility of this class of proteins.     

Control of cell division in Paramecium tetraurelia. Effects of abrupt changes in nutrient level on accumulation of macronuclear DNA and cell mass

In the cell cycle of Paramecium there are three points of interaction between cell growth-related processes and the processes of macronuclear DNA replication and cell division: initiation of DNA synthesis, regulation of the rates of growth and DNA accumulation, and initiation of cell division. This study examines the regulation of the latter two processes by analysis of the response of each to abrupt changes in nutrient level brought about either by transferring dividing cells from a steady-state chemostat culture to medium with unlimited food, or by transferring well-fed dividing cells to exhausted medium. The rates of DNA accumulation and cell growth respond quickly to changes in nutrient level. The amounts of these cell components accumulated during the cell cycle following a shift in nutrient level are typical of those occurring during equilibrium growth under post-shift conditions. Commitment to division occurs at a fixed interval prior to fission that is similar in well-fed and nutrient-limited cells. Initiation of cell division in Paramecium is associated with accumulation of a threshold DNA increment, whose level is largely independent of nutritive conditions. The amount of DNA accumulated during the cell cycle varies with nutritional conditions because the rates of growth and DNA accumulation are affected by nutrient level; slowly growing cells accumulated relatively little DNA during the fixed interval between commitment to cell division and fission.     

Cell cycle dependence of transformation expression in mouse cells transformed by a thermosensitive mutant (Ts-121) of polyoma virus.

Change in division capability as a phenotypic expression of cellular transformation was investigated by using one of the temperature-sensitive (ts) mutants of the polyoma virus-transformed cell line, the 121-6-5 cells of BALB/3T3. When contact -inhibited cells were treated with hyaluronidase at 39 degrees C, a single round of cell division was induced after which cell growth was inhibited by cell density. However, if the cells were incubated at 35 degrees C, after the enzyme treatment, density-inhibition block disappeared and the cells entered a second division. This indicates that the release of cells from density-inhibition depends on the low temperature incubation. The ability of cells to complete a second division was examined by shifting the cells from 39 degrees C to 35 degrees C during different phases of the first division cycle after the enzyme-treatment. A 6-hour incubation of S phase cells at 35 degrees C resulted in a second cycle of division, while the 24-hour incubation of G1 cells at 35 degrees C did not induce a second round of division. These results suggest that expression of the transformed phenotype in 121-6-5 cells is clearly dependent upon both the temperature and the phase of the division cycle.     

A protein critical for cell constriction in the Gram‐negative bacterium Caulobacter crescentus localizes at the division site through its peptidoglycan‐binding LysM domains

During division of Gram‐negative bacteria, invagination of the cytoplasmic membrane and inward growth of the peptidoglycan (PG) are followed by the cleavage of connective septal PG to allow cell separation. This PG splitting process requires temporal and spatial regulation of cell wall hydrolases. In Escherichia coli, LytM factors play an important role in PG splitting. Here we identify and characterize a member of this family (DipM) in Caulobacter crescentus. Unlike its E. coli counterparts, DipM is essential for viability under fast‐growth conditions. Under slow‐growth conditions, the ΔdipM mutant displays severe defects in cell division and FtsZ constriction. Consistent with its function in division, DipM colocalizes with the FtsZ ring during the cell cycle. Mutagenesis suggests that the LytM domain of DipM is essential for protein function, despite being non‐canonical. DipM also carries two tandems of the PG‐binding LysM domain that are sufficient for FtsZ ring localization. Localization and fluorescence recovery after photobleaching microscopy experiments suggest that DipM localization is mediated, at least in part, by the ability of the LysM tandems to distinguish septal, multilayered PG from non‐septal, monolayered PG.     

Does protein secretion activity vary during the cell cycle of Escherichia coli?

It is not known whether the activity of the Escherichia coli protein export system changes during the division cycle. To address and answer this question, we took two approaches. First, we pulse-labeled a random culture and size-fractionated the labeled cells in the presence of inhibitors of secretion. Second, we pulse-labeled synchronously growing cells at different phases in the cell cycle. In the latter experiment, we used a new method of synchronization in which a random culture was simply filtered through glass fiber filters. In both cases, the proportions of unprocessed precursor molecules were measured by immunoprecipitation and gel electrophoresis for some representative periplasmic and outer membrane proteins. Within the sensitivity limits of these methods, we could not detect any significant variation in the precursor labeling for cells of different ages. Thus, E. coli cells appear to secrete proteins continuously throughout the division cycle.