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Changes in nucleus, nucleolus and cell size accompanying somatic embryogenesis of Theobroma cacao L. I. Relationship between DNA and total protein content and size of nucleus, nucleolus and cell.

There was a linear relation between an increase in DNA content and size of nuclei, nucleoli and cells in callus and proembryos (Theobroma cacao L.). In callus the increase of DNA content was accompanied by proportional increase in nuclear size whereas in proembryos the increase in nuclear size did not match the increasing amount of DNA. The stimulation of embryogenesis by 10(-2) mg/l 2,4-D was associated with increase in nuclear and nucleolar size and with decrease in cell sizes. Inhibition of embryogenesis by 1.0 mg/l 2,4-D+10% coconut water did not change nuclear size, but increased cell size in relation to the control. The process of embryo formation was accompanied by changes in relationship between nuclear, nucleolar and cell size and the total (DNFB-stained) proteins content. In callus as well as in proembryo the increase in total protein content in nucleus was not equivalent to the increasing sizes of nuclei which leads to the decrease in nuclear protein concentration. Similar situation was observed for nucleoli. Differences were found in the concentration of cytoplasmic proteins between the callus and proembryo cells. The stimulation of embryogenesis by low concentration of 2,4-D resulted in decrease in concentration of total proteins in nuclei and nucleoli and the increase in cytoplasm.

Cacao↗

Lhcb Transcription Is Coordinated with Cell Size and Chlorophyll Accumulation (Studies on Fluorescence-Activated, Cell-Sorter-Purified Single Cells from Wild-Type and immutans Arabidopsis thaliana).

To study the mechanisms that integrate pigment and chlorophyll a/b-binding apoprotein biosynthesis during light-harvesting complex II assembly, we have examined [beta]-glucuronidase (GUS) enzyme activities, chlorophyll contents, and cell sizes in fluorescence-activated, cell-sorting-separated single cells from transgenic Arabidopsis thaliana wild-type and immutans variegation mutant plants that express an Lhcb (photosystem II chlorophyll a/b-binding polypeptide gene)/GUS promoter fusion. We found that GUS activities are positively correlated with chlorophyll content and cell size in green cells from the control and immutans plants, indicating that Lhcb gene transcription is coordinated with cell size in this species. Compared with the control plants, however, chlorophyll production is enhanced in the green cells of immutans; this may represent part of a strategy to maximize photosynthesis in the green sectors to compensate for a lack of photosynthesis in the white sectors of the mutant. Lhcb transcription is significantly higher in pure-white cells of the transgenic immutans plants than in pure-white cells from norflurazon-treated, photooxidized A. thaliana leaves. This suggests that immutans partially uncouples Lhcb transcription from its normal dependence on chlorophyll accumulation and chloroplast development. We conclude that immutans may play a role in regulating Lhcb transcription, and may be a key component in the signal transduction pathways that control chloroplast biogenesis.

Journal Article↗

The relationship between electropermeabilization and cell cycle and cell size of Saccharomyces cerevisiae.

Yeast cells (Sacchromyces cerevisiae) in 0.9% NaCl solution containing phloxine B (dye) were treated by an application of a rectangular electric pulse. We input microscopic images of the yeast suspensions after the application into a computer, and measured whether each cell dyes or not, the phase in the cell cycle, and each cell size, using the software we had developed. After those measurements, we discussed the relationship between the yield of electropermeabilization (the ratio of dyed cells to the total cell number) and the phase in the cell cycle, and cell size. From the results, it was found that the yeast cells from S-phase to M-phase (S-M phase) in the cell cycle tend to be more permeated than G1-phase yeast cells, and in both phases the yield decreases with the increase in cell size.

Cell Cycle↗

Proliferative potential of human fibroblasts: an inverse dependence on cell size.

Human foreskin fibroblast-like cells were separated on the basis of DNA content and cell size by fluorescence-activated cell sorting. Subpopulations of "large" or "small" cells with the same (G1) DNA content were clonally expanded and found to contain predominantly nondividing or highly proliferative cells, respectively. From the rate of clonal growth, we deduce that small cells divide faster than large cells. Intermediate-sized cells were found to yield primarily smaller ("attenuated") clones. The clonal data can be incorporated into a previously reported kinetic model of clonal attenuation. This version of the model postulates that small "stem" cells yield larger daughters which have only a limited proliferative potential. We also postulate that a progressive increase in cell size can account for the decreasing concentration of DNA polymerase alpha, which has been reported in older cultures.

Cell Division↗

An automated, direct method for measuring adipocyte cell size.

Current methods for direct determination of fat cell size are optical and non-automated. They are thus tedious, but have the advantage of providing estimates of the variance of a cell size distribution, as well as a measure of the mean cell size. An indirect method based on counting the number of osmium-fixed fat cells derived from a known wet weight of adipose tissue is also available. This method is automated and thus rapid, but does not provide information about the variance of the cell size distribution. In the present paper we describe a direct, automated method of fat cell sizing that provides estimates for both mean cell size and the variance of the cell size distribution. The correlation between our method and in indirect method based on counting osmium-fixed cells was 0.96. Transformation of the volume distribution to diameters indicated that cell diameters appeared to be normally distributed, confirming the observations of others.

Adipose Tissue↗

Flow cytometric analysis of cell-surface antigen expressions on acute myeloid leukemia cell populations according to their cell-size.

Acute myeloid leukemia (AML) cells which expanded from a single leukemic cell show certain degrees of morphological and biological heterogeneity. In the present study, we determined cell-surface antigen expressions (CD13, 33, 34 and 38, and HLA-DR) on AML cells based on their cell-size (large vs small cells) by flow cytometry. We found that the cell-surface antigens were more strongly expressed on the large leukemic cells than the small cells, regardless of FAB subtypes. Furthermore, our preliminary study demonstrated that AML patients who showed a relatively small difference in antigen expression between large and small leukemic cells had longer remission durations and survival periods, compared with those with a more prominent difference in antigen expression. Thus, the heterogeneity of AML cells determined by the combination of cell-surface antigen expressions and cell-size may be associated with clinically important biological behaviors.

Adult↗

In vitro cellular aging in T-lymphocyte cultures: analysis of DNA content and cell size.

Like other normal diploid mammalian cells, human T-lymphocytes display a limited in vitro lifespan. Long-term cultures of normal adult peripheral blood T cells, activated in vitro and passaged in the presence of interleukin-2 undergo 23 +/- 7 cumulative population doublings. Cell cycle analysis revealed that in senescent T cell cultures, restimulation with the original antigen resulted in 16-22% of the cells entering S phase, compared to 60% entering cycle in young cultures. In addition, within 1 week of restimulation, the senescent cultures do not increase in cell number, but 93% of the cells return to the G1/G0 DNA content, a proportion typically seen in quiescent (nonrestimulated) cultures. Coculture of early- and late-passage cells at two different ratios excluded a putative inhibitory factor produced by the old cells and similarly eliminated a possible stimulatory product in early cultures. Flow cytometry measure of forward angle light scatter revealed no difference in cell size between early- and late-passage cells, in contrast to the findings with senescent fibroblasts. Thus, while increasing cell size may contribute to the senescent phenotype of fibroblast cultures, it is not a factor in the senescence of human T-lymphocytes, and it is therefore doubtful that alterations in cell size are fundamental to in vitro cellular aging.

Cell Cycle↗

Kinetic analysis of cell size and DNA content distributions during tumor cell proliferation: Ehrlich ascites tumor study.

In order to study the growth dynamics of proliferating and non-proliferating cells utilizing discrete-time state equations, the cell cycle was divided into a finite number of age compartments. In analysing tumor growth, the kinetic parameters associated with a retardation in the growth rate of tumors were characterized by computer simulation in which the simulated results of the growth curve, the growth fraction, and the mean generation time were adjusted to fit the experimental data. The cell age distibution during the period of growth was obtained and by a linear transformation of the state transition matrices, was employed to specify the cell size and DNA content distributions. In an application of the model, the time-course behavior of cell cycle parameters of Ehrlich ascites tumor is illustrated, and the parameters important for the transition of cells in the proliferating compartment to the non-proliferating compartment are discussed, particularly in relation to the G1-G0 and G2-G0 transitions of non-cycling cells as revealed by the variation of cell size distribution.

Animals↗

Lipolytic response of rat adipocytes to epinephrine: effect of age and cell size.

Isolated fat cells from 3, 12 and 28 month old rats were compared intheir lipolytic response to various doses of L-epinephrine. With increasing age a progressive decline in the response occurred when comparing rats with unmatched mean adipocyte diameters. However, this apparently age-related decrease was no longer evident if the diameters were matched since the 28 month old animals had a lipolytic response less than the 3 month old rats but greater than the 12 month olds. Body weight differences between these ages may account for these observations. The biphasic dose response curve to hormone stimulation in the 3 and 12 month olds was monophasic at 28 months. Within each age group the initial lag in glycerol release decreased and the lipolytic response increased as mean cell size increased. The rate of hormone stimulated glycerol release varied inversely with incubations of 3,700 to 25,000 cells/ml and aging had no effect on this parameter. DNA content per fat cell remained constant with age.

Adipose Tissue↗

Cell size checkpoint control by the retinoblastoma tumor suppressor pathway.

Size control is essential for all proliferating cells, and is thought to be regulated by checkpoints that couple cell size to cell cycle progression. The aberrant cell-size phenotypes caused by mutations in the retinoblastoma (RB) tumor suppressor pathway are consistent with a role in size checkpoint control, but indirect effects on size caused by altered cell cycle kinetics are difficult to rule out. The multiple fission cell cycle of the unicellular alga Chlamydomonas reinhardtii uncouples growth from division, allowing direct assessment of the relationship between size phenotypes and checkpoint function. Mutations in the C. reinhardtii RB homolog encoded by MAT3 cause supernumerous cell divisions and small cells, suggesting a role for MAT3 in size control. We identified suppressors of an mat3 null allele that had recessive mutations in DP1 or dominant mutations in E2F1, loci encoding homologs of a heterodimeric transcription factor that is targeted by RB-related proteins. Significantly, we determined that the dp1 and e2f1 phenotypes were caused by defects in size checkpoint control and were not due to a lengthened cell cycle. Despite their cell division defects, mat3, dp1, and e2f1 mutants showed almost no changes in periodic transcription of genes induced during S phase and mitosis, many of which are conserved targets of the RB pathway. Conversely, we found that regulation of cell size was unaffected when S phase and mitotic transcription were inhibited. Our data provide direct evidence that the RB pathway mediates cell size checkpoint control and suggest that such control is not directly coupled to the magnitude of periodic cell cycle transcription.

Amanitins↗

Cell cycle length, cell size, and proliferation rate in hydra stem cells.

We have analyzed the cell cycle parameters of interstitial cells in Hydra oligactis. Three subpopulations of cells with short, medium, and long cell cycles were identified. Short-cycle cells are stem cells; medium-cycle cells are precursors to nematocyte differentiation; long-cycle cells are precursors to gamete differentiation. We have also determined the effect of different cell densities on the population doubling time, cell cycle length, and cell size of interstitial cells. Our results indicate that decreasing the interstitial cell density from 0.35 to 0.1 interstitial cells/epithelial cell (1) shortens the population doubling time from 4 to 1.8 days, (2) increases the [3H]thymidine labeling index from 0.5 to 0.75 and shifts the nuclear DNA distribution from G2 to S phase cells, and (3) decreases the length of G2 in stem cells from 6 to 3 hr. The shortened cell cycle is correlated with a significant decrease in the size of interstitial stem cells. Coincident with the shortened cell cycle and increased growth rate there is an increase in stem cell self-renewal and a decrease in stem cell differentiation.

Animals↗

Glucose modulation of cell size in yeast.

Saccharomyces cerevisiae cells grown in glucose have larger average size than cells grown in ethanol. Besides, yeast must reach a carbon source-modulated critical cell size in order to enter S phase at Start. This control is of outmost physiological relevance, since it allows us to coordinate cell growth with cell cycle progression and it is responsible for cell size homeostasis. The cell sizer mechanism requires the overcoming of two sequential thresholds, involving Cln3 and Far1, and Clb5,6 and Sic1, respectively. When both thresholds are non-functional, carbon source modulation of cell size at Start is completely abolished. Since inactivation of extracellular glucose sensing through deletion of either the GPR1 or the GPA2 gene causes a marked, but partial, reduction in the ability to modulate cell size and protein content at Start, it is proposed that both extracellular and intracellular glucose signalling is required for properly setting the cell sizer in glucose media.

Cell Size↗

Preparation of cell-size unilamellar liposomes with high captured volume and defined size distribution.

Cell-size unilamellar vesicles were made by removing solvents from microscopic chloroform spherules containing smaller water droplets within. The average diameter of the vesicles in a typical preparation was 9.2 mum, comparable to that of human erythrocytes (7 mum). The standard deviation of the size distribution was 3.0 mum. The unilamellarity and bilayer unit membrane of vesicles were demonstrated by transmission electron microscopy. Materials so far successfully incorporated into vesicles include glucose, sucrose, Arsenazo III and Ponceau S dyes, thymidine triphosphate, methotrexate, agarose, collagen, ferritin, polyadenylic aid, DNA, and whole bacteria. The captured volume per milligram of lipids (up to 144 microliter/mg) was almost an order of magnitude greater than the highest value reported in the literature to date (up to 15.6 microliter/mg) (Szoka, F.C. and Papahadjopoulos, D. (1978) Proc. Natl. Acad, Sci. U.S.A. 75, 4194-4198).

Chloroform↗

Insulin receptor substrate-1, p70S6K, and cell size in transformation and differentiation of hemopoietic cells.

After an initial burst of cell proliferation, the type 1 insulin-like growth factor receptor (IGF-IR) induces granulocytic differentiation of 32D IGF-IR cells, an interleukin-3-dependent murine hemopoietic cell line devoid of insulin receptor substrate-1 (IRS-1). The combined expression of the IGF-IR and IRS-1 (32D IGF-IR/IRS-1 cells) inhibits IGF-I-mediated differentiation, and causes malignant transformation of 32D cells. Because of the role of IRS-1 in changing the fate of 32D IGF-IR cells from differentiation (and subsequent cell death) to malignant transformation, we have looked for differences in IGF-IR signaling between 32D IGF-IR and 32D IGF-IR/IRS-1 cells. In this report, we have focused on p70(S6K), which is activated by the IRS-1 pathway. We find that the ectopic expression of IRS-1 and the inhibition of differentiation correlated with a sustained activation of p70(S6K) and an increase in cell size. Phosphorylation in vivo of threonine 389 and, to a lesser extent, of threonine 421/serine 424 of p70(S6K) seemed to be a requirement for inhibition of differentiation. A role of IRS-1 and p70(S6K) in the alternative between transformation or differentiation of 32D IGF-IR cells was confirmed by findings that inhibition of p70(S6K) activation or IRS-1 signaling, by rapamycin or okadaic acid, induced differentiation of 32D IGF-IR/IRS-1 cells. We have also found that the expression of myeloperoxidase mRNA (a marker of differentiation, which sharply increases in 32D IGF-IR cells), does not increase in 32D IGF-IR/IRS-1 cells, suggesting that the expression of IRS-1 in 32D IGF-IR cells causes the extinction of the differentiation program initiated by the IGF-IR, while leaving intact its proliferation program.

Animals↗

Brood-cell size does not influence the susceptibility of honey bees (Apis mellifera) to infestation by tracheal mites (Acarapis woodi).

Tracheal mites have been associated with the condition in honey bees that devastated colonies in Britain and Ireland in the early 1900s. The first outbreak of this condition, that became known as the 'Isle of Wight' disease, coincided with the period when brood-cell size was increased from about 5.0 mm to about 5.5 mm in width. We undertook an inoculation experiment over a 7-day period to establish if the act of increasing the brood-cell size could have triggered the onset of tracheal mites in honey bees. The standard-sized cells used had a cell width of 5.44 mm and the small-sized cells a width of 5.07 mm. Using callow (newly emerged) bees, from three colonies that had mixed cell sizes, we compared the susceptibility of bees reared in standard-sized cells with that of those raised in small-sized cells. The results indicated similar levels of female mite abundance (0.49 vs. 0.52 mites per bee) and mean fecundity (4.33 vs. 4.22 offspring per female mite), and produced no evidence of any difference in the overall susceptibility between the bees raised in the standard-sized cells versus small-sized brood cells.

Animals↗

Growth during the bacterial cell cycle: analysis of cell size distribution.

Cell volume distributions were determined electronically for steady-state cultures of Escherichia coli, Bacillus megaterium, Bacillus subtilis, and Salmonella typhimurium by use of a Coulter transducer-multichannel analyzer system of good resolution. All of the cell volume distributions had the same general shape, even though cultures were grown at widely different rates. Some results were independent of any particular growth model. Both the variability in the volumes of dividing cells and the fraction of constricted and unseparated doublet cells increased with growth rate. The greater separation to single cells at slow growth rates is in agreement with the general finding that filamentous and hyphal forms are greatly reduced in slowly growing chemostat cultures. The distributions were fitted equally well by simple models which assumed that cell growth was either linear or exponential throughout the entire cell cycle. It is concluded that methods of determining growth rate by analysis of distributions of bacterial volumes do not yet have sufficient resolution to distinguish between a variety of alternative models for growth of bacteria.

Bacillus megaterium↗

Cell size regulation, a mechanism that controls cellular RNA accumulation: consequences on regulation of the ubiquitous transcription factors Oct1 and NF-Y and the liver-enriched transcription factor DBP.

Cell sizes can differ vastly between cell types in individual metazoan organisms. In rat liver, spleen, and thymus, differences in average cell size roughly reflect differences in RNA:DNA ratios. For example, hepatocytes were found to have a cytoplasmic:nuclear volume ratio and an RNA:DNA ratio which were 34- and 21-fold higher, respectively, than those in thymocytes. RNA synthesis per DNA-equivalent in the hepatocytes was 25-fold greater than that in thymocytes, suggesting that differences in overall transcriptional activity, not differences in overall RNA stability, were primarily responsible for determining cellular RNA:DNA ratios. The mechanisms determining the capacity of large cells to synthesize and accumulate more ubiquitous cytoplasmic macromolecules, such as ribosomes, than smaller cells is entitled "cell size regulation." Cell size regulation may have important consequences on the tissue distribution of transcription factors. Thus, in liver, lung, kidney, spleen, and brain, cellular levels of the mRNA encoding the leucine zipper protein DBP correlate closely to cellular RNA:DNA ratios. Our results suggest that DBP mRNA levels, like rRNA levels, are transcriptionally determined. Thus the dbp gene, like the ribosomal genes, may be subject to cell size regulation. As a consequence, nuclei from liver, a tissue with a very large average cell size, accumulated higher levels of DBP protein than nuclei from small-celled tissues, such as spleen or lung. In contrast to DBP, the ubiquitous transcription factors Oct1 and NF-Y escaped cell size control. Nuclei from most tissues contained similar amounts of these factors irrespective of cell size. Likewise, tissues with large or small average cell sizes contained similar levels of the mRNAs encoding Oct1 or NF-Ya, one of the subunits of the heteromeric CCAAT-binding factor NF-Y, per DNA-equivalent. Interestingly, mRNA encoding NF-Yb, another subunit of NF-Y, was subject to cell size regulation. Our results suggest that NF-Yb protein escapes cell size regulation at a posttranslational level.

Animals↗