Uncoupling growth from the cell cycle

How does a Drosophila wing grow to the appropriate size and shape? Although a collaboration of cell division with the patterning of cell fates seems obvious, almost nothing is known about how these two processes are coordinated during development. A recent paper¹ finds that blocking cell division uncouples cell growth from the cell division cycle, displaying remarkable flexibility in the ability of the wing primordia to achieve the right proportions with fewer than normal cells. BioEssays 20: 283-286, 1998.© 1998 John Wiley & Sons, Inc.     

The initiation of cell wall synthesis in parasynchronous cultures of Schizosaccharomyces pombe

Summary Schizosaccharomyces pombe has been grown in parasynchronous culture to study the synthesis of cell wall material. After a lag period of 2.5h following inoculation the cells began to grow, as measured by optical density, dry weight and cell size. The cell number remained constant until 4.5h after inoculation when approximately 70% of the population divided synchronously. Immunofluorescence studies of the growing cells have shown that new wall material is inserted at the cell apices from 2.5 h after inoculation; this result is supported by radio-isotope labelling data which indicated that synthesis of new cell wall material also commenced 2.5 h after inoculation. The incorporation experiments also demonstrated an interruption in cell wall synthesis during the cell separation stage. The composition of the cell wall material varied during the growth cycle, with maximum nitrogen levels at inoculation and following cell division. No serological differences could be detected in the cell walls during the growth cycle.     

Division Cycle of Myxococcus xanthus III. Kinetics of Cell Growth and Protein Synthesis

The kinetics of cell growth and protein synthesis during the division cycle of Myxococcus xanthus was determined. The distribution of cell size for both septated and nonseptated bacteria was obtained by direct measurement of the lengths of 8,000 cells. The Collins-Richmond equation was modified to consider bacterial growth in two phases: growth and division. From the derived equation, the growth rate of individual cells was computed as a function of size. Nondividing cells (growth phase) comprised 91% of the population and took up 87% of the time of the division cycle. The absolute and specific growth rates of nondividing cells were observed to increase continually throughout the growth phase; the growth rate of dividing cells could not be determined accurately by this technique because of changes in the geometry of cells between the time of septation and physical separation. The rate of protein synthesis during the division cycle was measured by pulselabeling an exponential-phase culture with radio-active valine or arginine and then preparing the cells for quantitative autoradiography. By measuring the size of individual cells as well as the number of grains, the rate of protein synthesis as a function of cell size was obtained. Nondividing cells showed an increase in both the absolute and specific rates of protein synthesis throughout the growth phase; the specific rate of protein synthesis for dividing cells was low when compared to growthphase cells. Cell growth and protein synthesis are compared to the previously reported kinetics of deoxyribonucleic acid and ribonucleic acid synthesis during the division cycle.     

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.     

Controls over the timing of DNA replication during the cell cycle of fission yeast

The controls acting over the timing of DNA replication (S) during the cell cycle have been investigated in the fission yeast Schizosaccharomyces pombe. The cell size at which DNA replication takes place has been determined in a number of experimental situations such as growth of nitrogen-starved cells, spore germination and synchronous culture of wee mutant and wild-type strains. It is shown that in wee mutant strains and in wild type grown under conditions in which the cells are small, DNA replication takes place in cells of the same size. This suggests that there is a minimum cell size beneath which the cell cannot initiate DNA replication and it is this control which determines the timing of S during the cell cycle of the wee mutant. Fast growing wild-type cells are too large for this size control to be expressed. In these cells the timing of S may be controlled by the completion of the previous nuclear division coupled with a requirement for a minimum period in G1. Thus in S. pombe there are two different controls over the timing of S, either of which can be operative depending upon the size of the cell at cell division. It is suggested that these two controls may form a useful conceptual framework for considering the timing control over S in mammalian cells.     

Control and maintenance of mammalian cell size

Background  

Conlon and Raff propose that mammalian cells grow linearly during the division cycle. According to Conlon and Raff, cells growing linearly do not need a size checkpoint to maintain a constant distribution of cell sizes. If there is no cell-size-control system, then exponential growth is not allowed, as exponential growth, according to Conlon and Raff, would require a cell-size-control system.     

SYNCHRONOUS GROWTH OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE): A REVIEW OF OPTIMAL CONDITIONS1

Chlamydomonas reinhardtii Dangeard was synchronized at optimal growth conditions under a 12:4 LD regime at 35 C and 20,000 lx with serial dilution to a standard starting cell density of (1.4 ± 0.2) × 10⁶ cells/ml. Synchronous growth and division were characterized by measuring cell number, cell volume and size distribution, dry weight, protein, carbon, nitrogen, chlorophyll, carotenoids, nucleic acids, nuclear and cytoplasmic division during the vegetative life cycle. The main properties of the present system are: Exponential growth with high productivity, high degrees of synchrony and reproducibility during repeated life cycles. The degree of synchrony of this light-dark synchronization system was evaluated and compared with those described in the literature using probit analysis of the time course of DNA synthesis, nuclear and cytoplasmic division and sporulation (increase in cell number). The results showed that the degree of synchrony is highest for cells grown under optimal conditions.     

DIEL VARIATIONS IN OPTICAL PROPERTIES OF MICROMONAS PUSILLA (PRASINOPHYCEAE)1

Micromonas pusilla (Butcher) Manton et Parke, a marine prasinophyte, was used to investigate how cell growth and division affect optical properties of phytoplankton over the light:dark cycle. Measurements were made of cell size and concentration, attenuation and absorption coefficients, flow cytometric forward and side light scattering and chl fluorescence, and chl and carbon content. The refractive index was derived from observations and Mie scattering theory. Diel variations occurred, with cells increasing in size, light scattering, and carbon content during daytime photosynthesis and decreasing during nighttime division. Cells averaged 1.6 μm in diameter and exhibited phased division, with 1.3 divisions per day. Scattering changes resulted primarily from changes in cell size and not refractive index; absorption changes were consistent with a negligible package effect. Measurements over the diel cycle suggest that in M. pusilla carbon‐specific attenuation varies with cell size, and this relationship appears to extend to other phytoplankton species. Because M. pusilla is one of the smallest eukaryotic phytoplankton and belongs to a common marine genus, these results will be useful for interpreting in situ light scattering variation. The relationship between forward light scattering (FLS) and volume over the diel cycle for M. pusilla was similar to that determined for a variety of phytoplankton species over a large size range. We propose a method to estimate cellular carbon content directly from FLS, which will improve our estimates of the contribution of different phytoplankton groups to productivity and total carbon content in the oceans.     

Cell elongation and division probability during the Escherichia coli growth cycle.

A new method is presented for determining the growth rate and the probability of cell division (separation) during the cell cycle, using size distributions of cell populations grown under steady-state conditions. The method utilizes the cell life-length distribution, i.e., the probability that a cell will have any specific size during its life history. This method was used to analyze cell length distributions of six cultures of Escherichia coli, for which doubling times varied from 19 to 125 min. The results for each culture are in good agreement with a single model of growth and division kinetics: exponential elongation of cells during growth phase of the cycle, and normal distributions of length at birth and at division. The average value of the coefficient of variation was 13.5% for all strains and growth rates. These results, based upon 5,955 observations, support and extend earlier proposals that growth and division patterns of E. coli are similar at all growth rates and, in addition, identify the general growth pattern of these cells to be exponential.     

GROWTH AND CELL CYCLE OF TWO CLOSELY RELATED RED TIDE-FORMING DINOFLAGELLATES: GYMNODINIUM NAGASAKIENSE AND G. CF. NAGASAKIENSE1

Small-sized vegetative cells were found to co-occur with normal-sized cells in populations of the European bloom-forming dinoflagellate Gymnodinium cf. nagasakiense Takayama et Adachi, currently known as Gyrodinium aureolum Hulburt, but not in populations of the closely related Japanese species Gymnodiniumn agasakiense. We examined how cell size differentiation may influence growth and cell cycle progression under a 12:12-h light: dark cycle in the European taxon, as compared to the Japanese one. Cell number and volume and chlorophyll red fluorescence in both species varied widely during the photocycle. These variations generally appeared to be related lo the division period, which occurred at night, as indicated by the variations of the fraction of binucleated cells (mitotic index) as well as the distribution of cellular DNA content. “Small” cells of G. cf. nagasakiense divided mainly during the first part of the dark period, although a second minor peak of dividing cells could occur shortly before light onset. In contrast, “large” cells displayed a sharp division peak that occurred 9 h after the beginning of the dark period. The lower degree of synchrony of “small” cells could be a consequence of their faster growth. Alternatively, these data may suggest that cell division is lightly controlled by an endogenous clock in “large” cells and much more loosely controlled in “small” cells. Cells of the Japanese species, which were morphologically similar to “large” cells of the European taxon, displayed an intermediate growth pattern between the two cell types of G. cf. nagasakiense, with a division period that extended to most of the dark period.     

The Arabidopsis SMO2, a homologue of yeast TRM112, modulates progression of cell division during organ growth

Cell proliferation is integrated into developmental progression in multicellular organisms, including plants, and the regulation of cell division is of pivotal importance for plant growth and development. Here, we report the identification of an Arabidopsis SMALL ORGAN 2 (SMO2) gene that functions in regulation of the progression of cell division during organ growth. The smo2 knockout mutant displays reduced size of aerial organs and shortened roots, due to the decreased number of cells in these organs. Further analyses reveal that disruption of SMO2 does not alter the developmental timing but reduces the rate of cell production during leaf and root growth. Moreover, smo2 plants exhibit a constitutive activation of cell cycle‐related genes and over‐accumulation of cells expressing CYCB1;1:β‐glucuronidase (CYCB1;1:GUS) during organogenesis, suggesting that smo2 has a defect in G₂–M phase progression in the cell cycle. SMO2 encodes a functional homologue of yeast TRM112, a plurifunctional component involved in a few cellular events, including tRNA and protein methylation. In addition, the mutation of SMO2 does not appear to affect endoreduplication in Arabidopsis leaf cells. Taken together we postulate that Arabidopsis SMO2 is a conserved yeast TRM112 homologue and SMO2‐mediated cellular events are required for proper progression of cell division in plant growth and development.     

Cell elongation and septation are two mutually exclusive processes in Escherichia coli

Bacterial rod morphogenesis was studied in synchronously growing cells of Escherichia coli C600 during the reshaping process that follows the removal of mecillinam, a β-lactam antibiotic that specifically inhibits lateral wall formation of gram-negative rods and causes transition to coccal shape. Removal of mecillinam after 30 min of action did not affect the timing of subsequent cell division, but removal after 50 min delayed resumption of cell division for approximately one generation time. In order to study the interplay between lateral wall elongation and septum formation in determining and maintaining the bacterial rod shape, we evaluated the effect of re-adding mecillinam or of adding aztreonam (a specific inhibitor of septum formation) at various stages of the reshaping process. We conclude that mecillinam was active only during the reshaping process, while aztreonam was active only later when the cells were close to dividing again. These results provide further evidence for our previous proposal according to which elongation and septation are two alternating and competing events of the cell cycle and are linked to each other to force bacterial rods to grow to a given length. Received: 23 January 1997 / Accepted: 2 May 1997     

A multilevel analysis of fruit growth of two tomato cultivars in response to fruit temperature

Fruit phenotype is a resultant of inherent genetic potential in interaction with impact of environment experienced during crop and fruit growth. The aim of this study was to analyze the genetic and physiological basis for the difference in fruit size between a small (‘Brioso’) and intermediate (‘Cappricia’) sized tomato cultivar exposed to different fruit temperatures. It was hypothesized that fruit heating enhances expression of cell cycle and expansion genes, rates of carbon import, cell division and expansion, and shortens growth duration, whereas increase in cell number intensifies competition for assimilates among cells. Unlike previous studies in which whole‐plant and fruit responses cannot be separated, we investigated the temperature response by varying fruit temperature using climate‐controlled cuvettes, while keeping plant temperature the same. Fruit phenotype was assessed at different levels of aggregation (whole fruit, cell and gene) between anthesis and breaker stage. We showed that: (1) final fruit fresh weight was larger in ‘Cappricia’ owing to more and larger pericarp cells, (2) heated fruits were smaller because their mesocarp cells were smaller than those of control fruits and (3) no significant differences in pericarp carbohydrate concentration were detected between heated and control fruits nor between cultivars at breaker stage. At the gene level, expression of cell division promoters (CDKB2, CycA1 and E2Fe‐like) was higher while that of the inhibitory fw2.2 was lower in ‘Cappricia’. Fruit heating increased expression of fw2.2 and three cell division promoters (CDKB1, CDKB2 and CycA1). Expression of cell expansion genes did not corroborate cell size observations.     

Cytokinesis-Based Constraints on Polarized Cell Growth in Fission Yeast

The rod-shaped fission yeast Schizosaccharomyces pombe, which undergoes cycles of monopolar-to-bipolar tip growth, is an attractive organism for studying cell-cycle regulation of polarity establishment. While previous research has described factors mediating this process from interphase cell tips, we found that division site signaling also impacts the re-establishment of bipolar cell growth in the ensuing cell cycle. Complete loss or targeted disruption of the non-essential cytokinesis protein Fic1 at the division site, but not at interphase cell tips, resulted in many cells failing to grow at new ends created by cell division. This appeared due to faulty disassembly and abnormal persistence of the cell division machinery at new ends of fic1Δ cells. Moreover, additional mutants defective in the final stages of cytokinesis exhibited analogous growth polarity defects, supporting that robust completion of cell division contributes to new end-growth competency. To test this model, we genetically manipulated S. pombe cells to undergo new end take-off immediately after cell division. Intriguingly, such cells elongated constitutively at new ends unless cytokinesis was perturbed. Thus, cell division imposes constraints that partially override positive controls on growth. We posit that such constraints facilitate invasive fungal growth, as cytokinesis mutants displaying bipolar growth defects formed numerous pseudohyphae. Collectively, these data highlight a role for previous cell cycles in defining a cell''s capacity to polarize at specific sites, and they additionally provide insight into how a unicellular yeast can transition into a quasi-multicellular state.     

Translational control of lipogenic enzymes in the cell cycle of synchronous,growing yeast cells

Translational control during cell division determines when cells start a new cell cycle, how fast they complete it, the number of successive divisions, and how cells coordinate proliferation with available nutrients. The translational efficiencies of mRNAs in cells progressing synchronously through the mitotic cell cycle, while preserving the coupling of cell division with cell growth, remain uninvestigated. We now report comprehensive ribosome profiling of a yeast cell size series from the time of cell birth, to identify mRNAs under periodic translational control. The data reveal coordinate translational activation of mRNAs encoding lipogenic enzymes late in the cell cycle including Acc1p, the rate‐limiting enzyme acetyl‐CoA carboxylase. An upstream open reading frame (uORF) confers the translational control of ACC1 and adjusts Acc1p protein levels in different nutrients. The ACC1 uORF is relevant for cell division because its ablation delays cell cycle progression, reduces cell size, and suppresses the replicative longevity of cells lacking the Sch9p protein kinase regulator of ribosome biogenesis. These findings establish an unexpected relationship between lipogenesis and protein synthesis in mitotic cell divisions.     

Growth Pattern of Single Fission Yeast Cells Is Bilinear and Depends on Temperature and DNA Synthesis

Cell growth and division have to be tightly coordinated to keep the cell size constant over generations. Changes in cell size can be easily studied in the fission yeast Schizosaccharomyces pombe because these cells have a cylindrical shape and grow only at the cell ends. However, the growth pattern of single cells is currently unclear. Linear, exponential, and bilinear growth models have been proposed. Here we measured the length of single fission yeast cells with high spatial precision and temporal resolution over the whole cell cycle by using time-lapse confocal microscopy of cells with green fluorescent protein-labeled plasma membrane. We show that the growth profile between cell separation and the subsequent mitosis is bilinear, consisting of two linear segments separated by a rate-change point (RCP). The change in growth rate occurred at the same relative time during the cell cycle and at the same relative extension for different temperatures. The growth rate before the RCP was independent of temperature, whereas the growth rate after the RCP increased with an increase in temperature, leading to clear bilinear growth profiles at higher temperatures. The RCP was not directly related to the initiation of growth at the new end (new end take-off). When DNA synthesis was inhibited by hydroxyurea, the RCP was not detected. This result suggests that completion of DNA synthesis is required for the increase in growth rate. We conclude that the growth of fission yeast cells is not a simple exponential growth, but a complex process with precise rates regulated by the events during the cell cycle.     

Cell cycle analysis of F''lac replication in Escherichia coli B/r.

The timing and control of replication of an F'lac plasmid was investigated in two substrains of Escherichia coli B/r lac/F'lac growing at a variety of rates. The cellular content of covalently closed circular F'lac deoxyribonucleic acid and the cellular mass at the time of F'lac replication both increased as a function of growth rate. The timing of plasmid replication during the division cycle was determined by measuring the inducibility of beta-galactosidase in cells of different ages in exponentially growing cultures. At all growth rates, the rate of induced beta-galactosidase synthesis increased in a step-wise fashion during the division cycle, indicating that the F'lac plasmid replicated at a discrete time in the cycle. At growth rates greater than one doubling per h, the cell age at F'lac replication was indistinguishable from the cell age at chromosomal lac+ replication in an isogenic F- parent. The ratio of plasmids to chromosomal origins decreased from about 0.7 to 0.4 between growth rates of 1.0 to 2.5 doublings per h. These observations are all consistent with replication of F'lac at about the same time in the division cycle as replication of the homologous chromosomal region at these growth rates. This similarity in timing of replication of homologous deoxyribonucleic acid regions was not evident in slower-growing cells.     

The cell cycle of<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: the role of the commitment point

Chlamydomonas reinhardtii cells can double their size several times during the light period before they enter the division phase. To explain the role of the commitment point (defined as the moment in the cell cycle after which cells can complete the cell cycle independently of light) and the moment of initiation of cell division we investigated whether the timing of commitment to cell division and cell division itself are dependent upon cell size or if they are under control of a timer mechanism that measures a period of constant duration. The time point at which cells attain commitment to cell division was dependent on the growth rate and coincided with the moment at which cells have approximately doubled in size. The timing of cell division was temperature-dependent and took place after a period of constant duration from the onset of the light period, irrespective of the light intensity and timing of the commitment point. We concluded that at the commitment point all the prerequisites are checked, which is required for progression through the cell cycle; the commitment point is not the moment at which cell division is initiated but it functions as a checkpoint, which ensures that cells have passed the minimum cell size required for the cell division.     

CELL CYCLE DURATION IN THE MERISTEM OF NEPHROLEPIS BISERRATA STOLONS: THE ROLE OF THE APICAL CELL

The stolons of Nephrolepis biserrata (sw.) Schott are thin axes that grow rapidly (from 2 to 4 mm per day) in the controlled conditions applied. In the cylindro-conical meristem, three histological zones are defined. Cell cycle duration was determined for each zone by autoradiographic methods after incorporation of tritiated thymidine and confirmed by the colchicine-induced metaphase-accumulation technique. The apical cell and its derivatives (Zone 1) are mitotically more active (cell cycle duration: 80 hr) than the cells of the subapical zones (2 and 3), where cell cycle lengths are 142 hr and 95 hr respectively. These data, compared to previous results, give evidence for the main role played by the relative rate of division of the apical cell compared to that of lateral cells in the organization and the shape of the meristem of pteridophytes. Moreover, the apical cell appears to be unique in having a differentiated cytological aspect not usually associated with an intensely proliferating cell.