Average cell size is a factor reflecting the interaction of CHO cells during their proliferation

Changes in cell area during CHO cell cultivation have been studied with time-lapse microscopy. Capture was started the day after cell plating. It was found that the size of daughter cells after mitosis remained less than the size of the mother cell for a long time (up to 6 h). Nevertheless, the average cell area of the whole population was constant during the observation period (up to 18 h). We assume that this phenomenon is a result of interaction between dividing and nondividing cells. The experimental data we obtained confirm this conclusion.     

Dynamics of L-929 cell spreading after mitosis

Changes in the cell shape of L-929 (NCTC, clone 929) during the cell cycle were analyzed with time-lapse microscopy. It was found that the cells pass through three spreading stages. The maximal cell spreading was observed during the first 1.5 h after mitosis. In this period, the cell area increases in correspondence with the sigmoid dependence and enlarges by approximately 3.0–3.5 times. After a short plateau, the cell area begins to increase, also correspondence with the sigmoid dependence. This period is longer (up to 6 h after the beginning of cell division), with an additional 1.5-fold increase in the cell size. Later, cell area enlargement continues linearly up to the beginning of the next mitosis.     

Dynamics of spreading of cells of L-929 line after the mitosis

Using time-lapse microscopy, the changes in L-929 cells shape were analyzed during a cell cycle. During this time the cells were established to pass through three spreading stages. The highest rate of the cell spreading was observed during the first 1.5 h of mitosis. In this period, the cell area increases approximately 3-3.5 times following sigmoid dependence. After a short plateau the augmentation of the cell area starts also as a sigmoid dependence. This period is longer (up to 6 h after the beginning of cell division) with an additional 1.5-fold augmentation of the cells size. Next, the augmentation of the cells area goes linearly up to the beginning of the following mitosis. After the mother L-929 cell division, the daughter cells remained to be bridged together in the fission furrow site almost in 100% cases. The structure known as an intercellular bridge is related to a late telophase. In this connected state the L-cells are spreading and migrating up to 2.13 +/- 0.06 h where upon they are separated. Transition of the daughter cells from a round shape to the spread one occurring with the simultaneous maintenance of the intercellular bridge during a strictly determined time allows us to consider this phenomenon as independent and not relating to mitosis. We suggest naming this junction between the daughter cells as the "posttelophase intercellular bridge".     

Topography of the insertion of LamB protein into the outer membrane of Escherichia coli wild-type and lac-lamB cells

The appearance of newly induced LamB protein at the cell surface of Escherichia coli was followed topographically by immuno-electron microscopy. LamB protein was induced in E. coli wild-type or lac-lamB cells for a short period of time (4 to 6 min), such that the overall level of LamB protein in induced cells was at least twofold higher than that in uninduced cells. Antibodies bound to LamB protein exposed at the cell surface were labeled with a protein A-gold probe, and the probe distribution in briefly induced cells was compared to that in uninduced cells. Analysis of large numbers of cells showed that newly inserted LamB protein appeared homogeneously over the entire cell surface, both in wild-type cells and in lac-lamB cells. A peak of insertion which was observed at the division site of the cell was also observed in the absence of induction and in control experiments in which a nonspecific probe was used. It is concluded therefore that insertion of LamB protein into the cell envelope of E. coli occurs at multiple sites over the entire cell surface. The average amount of LamB protein which appeared at the cell surface after induction was determined for various cell size classes. It was found that cells of various size classes all synthesized LamB protein after induction, indicating that synthesis of the protein was not restricted to cells in a particular stage of the cell cycle. However, the rate of LamB synthesis was found to vary during the cell cycle: this rate was constant regardless of cell size in nondividing cells, whereas it increased in dividing cells. It is concluded that the accumulation of newly induced LamB protein follows a linear pattern.     

Modeling the impact of single-cell stochasticity and size control on the population growth rate in asymmetrically dividing cells

Microbial populations show striking diversity in cell growth morphology and lifecycle; however, our understanding of how these factors influence the growth rate of cell populations remains limited. We use theory and simulations to predict the impact of asymmetric cell division, cell size regulation and single-cell stochasticity on the population growth rate. Our model predicts that coarse-grained noise in the single-cell growth rate λ decreases the population growth rate, as previously seen for symmetrically dividing cells. However, for a given noise in λ we find that dividing asymmetrically can enhance the population growth rate for cells with strong size control (between a “sizer” and an “adder”). To reconcile this finding with the abundance of symmetrically dividing organisms in nature, we propose that additional constraints on cell growth and division must be present which are not included in our model, and we explore the effects of selected extensions thereof. Further, we find that within our model, epigenetically inherited generation times may arise due to size control in asymmetrically dividing cells, providing a possible explanation for recent experimental observations in budding yeast. Taken together, our findings provide insight into the complex effects generated by non-canonical growth morphologies.     

Simple method for a cell count of the colonial Cyanobacterium, Microcystis sp

The cell counting of colonial Microcystis spp. is a rather difficult and error-prone proposition, as this genus forms irregularly-shaped and irregularly-sized colonies, which are packed with cells. Thus, in order to facilitate a cell count, four methods of dividing the colonies into single cells were compared, including vortexing, sonication, TiO2 treatment, and boiling. As a result, the boiling method was determined to generate the greatest number of single cells from a colony, and all colonies were found to have divided completely after only 6 min of treatment. Furthermore, no significant cell destruction, which might alter the actual cell density, was detected in conjunction with the boiling method (P = 0.158). In order to compute the cell number more simply, the relationship between the colony size and the cell number was determined, via the boiling method. The colony volume, rather than the area or diameter was correlated more closely with the cell number (r2 = 0.727), thereby suggesting that the cell numbers of colonial Microcystis sp. can also be estimated effectively from their volumes.     

Cellular parameters of the shoot apical meristem in Arabidopsis.

The shoot apical meristem (SAM) is a small group of dividing cells that generate all of the aerial parts of the plant. With the goal of providing a framework for the analysis of Arabidopsis meristems at the cellular level, we performed a detailed morphometric study of actively growing inflorescence apices of the Landsberg erecta and Wassilewskija ecotypes. For this purpose, cell size, spatial distribution of mitotic cells, and the mitotic index were determined in a series of optical sections made with a confocal laser scanning microscope. The results allowed us to identify zones within the inflorescence SAM with different cell proliferation rates. In particular, we were able to define a central area that was four to six cells wide and had a low mitotic index. We used this technique to compare the meristem of the wild type with the enlarged meristems of two mutants, clavata3-1 (clv3-1) and mgoun2 (mgo2). One of the proposed functions of the CLV genes is to limit cell division rates in the center of the meristem. Our data allowed us to reject this hypothesis, because the mitotic index was reduced in the inflorescence meristem of the clv3-1 mutant. We also observed a large zone of slowly dividing cells in meristems of clv3-1 seedlings. This zone was not detectable in the wild type. These results suggest that the central area is increased in size in the mutant meristem, which is in line with the hypothesis that the CLV3 gene is necessary for the transition of cells from the central to the peripheral zone. Genetic and microscopic analyses suggest that mgo2 is impaired in the production of primordia, and we previously proposed that the increased size of the mgo2 meristem could be due to an accumulation of cells at the periphery. Our morphometric analysis showed that mgo2 meristems, in contrast to those of clv3-1, have an enlarged periphery with high cell proliferation rates. This confirms that clv3-1 and mgo2 lead to meristem overgrowth by affecting different aspects of meristem function.     

The effect of CaCl2 and verapamil on the binding of cisplatin to cultures of normal and transformed human fibroblasts

Normal and transformed human fibroblasts were treated for either 1 sec or 1 h with the antitumor drug cis-dichlorodiamine platinum (cisplatin). The dose response of drug binding and cell survival was determined for cells treated with the drug in the presence or absence of 3.0 mM CaCl2. The levels of drug initially bound to both cell types was similar and was not affected by the presence of Ca2+. The dividing non-transformed cells were most sensitive to killing by short treatment with cisplatin compared to the transformed cells or the confluent non-transformed cultures. After 1 h of cisplatin treatment, the levels of drug bound to the cells were significantly less than that recovered after the shorter treatment. This time-dependent loss of cisplatin was inhibited both by CaCl2 and by the calcium channel blocking agent, verapamil. The higher levels of cisplatin bound after 1 h in the presence of these agents, however, did not in all cases result in decreased survival; the effects were dependent on cell type and on whether the cells were dividing or confluent. Analysis of cisplatin binding to cell cultures indicated that initially the cisplatin was weakly attached to the pericellular and substratum attached material but that with time, the drug bound to this material decreased. This time-dependent removal from the extracellular matrix was much less in the transformed cell cultures and was inhibited by calcium. We propose that the major site of interaction of cisplatin with these cells is in the extracellular matrix and with time the cultures alter their extracellular matrix to decrease this binding. This removal process appears to involve calcium or calcium transport since CaCl2 and verapamil both block these changes.     

Effect of Protistan Grazing on the Frequency of Dividing Cells in Bacterioplankton Assemblages

Grazing by phagotrophic flagellates and ciliates is a major source of mortality for bacterioplankton in both marine and freshwater systems. Recent studies have demonstrated a positive relationship between clearance rate and prey size for bacterivorous protists. We tested the idea that, by selectively grazing the larger (more actively growing or dividing) cells in a bacterial assemblage, protists control bacterial standing stock abundances by directly cropping bacterial production. Samples of estuarine water were passed through 0.8-μm-pore-size filters (bacteria only) or 20-μm-mesh screens (bacteria and bacterivorous protists) and placed in dialysis tubing suspended in 7 liters of unfiltered water. Changes in total bacterial biovolume per milliliter (bacterial biomass), frequency of dividing cells (FDC), and average per cell biovolume were followed over a period of 24 h. In three experiments, the FDC increased more rapidly and attained higher values in water passed through 0.8-μm-pore-size filters (average, 5.1 to 8.9%; maximum, 15.5%) compared with FDC values in water passed through 20-μm-mesh screens (average, 2.7 to 5.3%; maximum, 6.7%). Increases in bacterial biomass per milliliter lagged behind increases in FDC by about 4 to 6 h. Grazed bacterial assemblages were characterized by lower total biomasses and smaller average cell sizes compared with those of cells in nongrazed assemblages. We conclude that bacterivorous protists control bacterial standing stock abundances partly by preferentially removing dividing cells. Selective grazing of the more actively growing cells may also explain, in part, the ability of slow-growing cells to persist in bacterioplankton assemblages.     

Characterization and efficacy of PKH26 as a probe to study the replication history of the human hematopoietic KG1a progenitor cell line

The PKH26 dye can, in principle, be used for the study of asymmetric cell divisions (ASDs). A requirement for the identification of ASDs based on fluorescence intensity is that the PKH26 dye is distributed equally between daughter cells at each division, but this has not been demonstrated at a single-cell level. The efficacy of PKH26 as a probe for the study of ASDs was examined using the human hematopoietic KG1a cell. An automated time-lapse fluorescent microscope system was used to determine changes in cell size and fluorescence intensity during culture, and track cell divisions. The images of daughter cells were analyzed using the Isee software to determine the distribution of PKH26 dye between daughter cells. Ratios of cell size, mean fluorescence intensity, and total fluorescence intensity were calculated by dividing the values for one daughter cell by the value of the other daughter cell. The ratios for cell size, mean intensity, and total intensity were 1.13 +/- 0.12, 1.08 +/- 0.07, and 1.15 +/- 0.14 (mean +/- SD), respectively. Thus, PKH26 is not distributed equally to both daughter cells upon cell division. However, the replication history of individual KG1a cells can be reliably deduced for up to three divisions based solely on the mean and total fluorescence intensity of the PKH26 dye, using PKH26 concentrations below the chemical and phototoxic limits (2 microM).     

Resuscitation of Vibrio vulnificus from the viable but nonculturable state.

Stationary-phase-grown cells of the estuarine bacterium Vibrio vulnificus became nonculturable in nutrient-limited artificial seawater microcosms after 27 days at 5 degrees C. When the nonculturable cells were subjected to temperature upshift by being placed at room temperature, the original bacterial numbers were detectable by plate counts after 3 days, with a corresponding increase in the direct viable counts from 3% to over 80% of the total cell count. No increase in the total cell count was observed during resuscitation, indicating that the plate count increases were not due to growth of a few culturable cells. Chloramphenicol and ampicillin totally inhibited resuscitation of the nonculturable cells when added to samples that had been at room temperature for up to 24 h. After 72 h of resuscitation, the inhibitors had an easily detectable but reduced effect on the resuscitated cells, indicating that protein and peptidoglycan synthesis were still ongoing. Major changes in the morphology of the cells were discovered. Nonculturable cells of V. vulnificus were small cocci (approximately 1.0 micron in diameter). Upon resuscitation, the cells became large rods with a size of mid-log-phase cells (3.0 microns in length). Four days after the cells had become fully resuscitated, the cell size had decreased to approximately 1.5 micron in length and 0.7 micron in width. The cells were able to go through at least two cycles of nonculturability and subsequent resuscitation without changes in the total cell count. This is the first report of resuscitation, without the addition of nutrient, of nonculturable cells, and it is suggested that temperature may be the determining factor in the resuscitation from this survival, or adaptation, state of certain species in estuarine environments.     

Demonstrating the localization of hemoproteins by an electron cytochemical method]

The dynamics of division and interphase cell size in the growing ovule of Pinus silvestris was studied on histological slides in the year of fertilization. A constant extension in size of dividing cells of the endosperm was shown which, in this respect, do not differ much from the interphase ones. Cell division and elongation in this tissue occur simultaneously to be completed only with the transition of cell to differentiation. The size extention of the dividing cell does not influence the frequency of cell division. In the integument and the nucleus, the value of dividing cells is relatively constant. At the transition of these cells to elongation their mitotic activity decreases shaply to stop completely after-wards. Cell division and elongation are divided here in time.     

Conformation of cytoskeletal elements during the division of infected Lupinus albus L. nodule cells

Lupin nodule cells maintain their ability to divide for several cycles after being infected by endosymbiotic rhizobia. The conformation of the cytoskeletal elements of nodule cells was studied by fluorescence labelling, immunocytochemistry, and laser confocal and transmission electron microscopy. The dividing infected cells showed the normal microtubule and actin patterns of dividing plant cells. The clustered symbiosomes were tethered to the spindle-pole regions and moved to the cell poles during spindle elongation. In metaphase, anaphase, and early telophase, the symbiosomes were found at opposite cell poles where they did not interfere with the spindle filaments or phragmoplast. This symbiosome positioning was comparable with that of the organelles (which ensures organelle inheritance during plant cell mitosis). Tubulin microtubules and actin microfilaments appeared to be in contact with the symbiosomes. The possible presence of actin molecular motor myosin in nodules was analysed using a monoclonal antibody against the myosin light chain. The antigen was detected in protein extracts of nodule and root cytosol as bands of approximately 20 kDa (the size expected). In the nodules, an additional polypeptide of 65 kDa was found. Immunogold techniques revealed the antigen to be localized over thin microfilaments linked to the cell wall, as well as over the thicker microfilament bundles and surrounding the symbiosomes. The pattern of cytoskeleton rearrangement in dividing infected cells, along with the presence of myosin antigen, suggests that the positioning of symbiosomes in lupin nodule cells might depend on the same mechanisms used to partition genuine plant cell organelles during mitosis.     

Plasmolysis during the division cycle of Escherichia coli

Cells of Escherichia coli were plasmolyzed with sucrose. They were classified according to length by way of electron micrographs taken from samples prepared by agar filtration. The percentage of plasmolyzed cells increased about two- and threefold between mean cell sizes of newborn and separating cells. However, dividing cells were less frequently plasmolyzed than nondividing cells of the same length class. Analysis of cell halves (prospective daughters) in dividing cells showed that they behaved as independent cellular units with respect to plasmolysis. The results indicate that compressibility of the protoplast (given a certain plasmolysis space) is inversely related to cell size. That a dividing cell does not react as one osmotic compartment to osmotic stress may suggest that cell size-dependent strength of the cell membrane-cell wall association, rather than variation in turgor, plays a role during the cell division cycle.     

The organization of the mitotic apparatus poles in etoposide-treated CHO-K1 cells

In this study, we have examined the organization of the mitotic spindle poles in CHO-K1 cells dividing after treatment with the etoposide (1 h, 25 microM). We studied at various periods after the treatment: 1) the distribution of gamma-tubulin in mitotic cells by immunofluorescent staining; 2) the level of posttranslational modification of a-tubulin in the spindle microtubules by immunoelectron microscopy; 3) the ultrastructure of the mitotic apparatus poles by standard electron microscopy. In 48 h after the addition of the agent we identified considerable changes in the ultrastructure of poles in etoposide-treated CHO-K1 cells with bipolar and multipolar spindles. The number of centrioles increased. The centrioles were unevenly distributed among the poles, and some centrioles were not explicitly involved in the organization of mitotic spindle, furthermore they can differ in the number of outgrowing microtubules. Most centrioles were without fibrillar halo. In 48 h after the addition of etoposide, electron microscopy of cells after immunoperoxidase staining with antibodies to acetylated and tyrosinated alpha-tubulin has shown that different poles of a multipolar spindle within the same cell are stained differently for tyr-tubulin but not for acet-tubulin. Immunofluorescence staining for gamma-tubulin also points to different organization of poles in the same spindle. Our findings provide the first evidence that the pattern of immunostaning and the ultrastructure of mitotic apparatus poles differ in the cells dividing at various periods after etoposide treatment.     

High efficiency lentiviral gene delivery in non-dividing cells by deoxynucleoside treatment

Background

Gene therapy has recently been advanced by the development of HIV‐based vectors that are able to transduce some non‐dividing cells. The manipulation of most non‐dividing cells remains, however, scarcely efficient. One of the biological mechanisms postulated to prevent powerful transduction of quiescent cells by lentiviral vectors is the paucity of deoxynucleotides (dNTPs). In this study, a novel delivery strategy is developed to improve significantly the efficiency of HIV‐based vectors in transducing non‐dividing cells. This approach is based on increasing the intracellular availability of dNTPs by incubating target cells with the dNTP precursors, deoxynucleosides (dNSs).

Methods

Mature human monocyte‐derived macrophages (14–21 days old) were transduced at a low multiplicity of infection (MOI) of HIV vectors carrying a reporter gene. dNSs were added to the medium during transduction (5 mM dNS) and immediately before post‐transduction culture (2.5 mM dNS). Macrophages were harvested 2–7 days after transduction and assayed for transgene expression by cytofluorimetry.

Results

The addition of dNS to the medium significantly enhanced the efficiency of transduction of human macrophages by HIV‐based vectors. The percentage of cells expressing the transgene rose up to 50% in the presence of dNS, increasing the basal transduction levels up to 35‐fold (average=10.8‐fold). Furthermore, treatment with dNTP precursors compensated for the wide inter‐donor variability, allowing the highest enhancement effects in donors with the lowest basal transduction efficiencies.

Conclusions

This is the first demonstration that a single treatment of non‐dividing target cells with exogenous dNS can enhance the efficiency of lentiviral‐mediated transduction of cells, allowing for high efficiency gene transfer. The effects of dNTP precursors compensated for both the poor basal levels and the wide inter‐donor variability, two major limitations for the transduction of non‐dividing cells. Macrophages are a representative model of cells whose permissiveness to gene delivery was increased up to levels suitable for genetic manipulation applications. This simple approach might be transferred to a broader range of quiescent cell types that are scarcely susceptible to lentiviral‐based gene delivery due to low dNTP levels. Copyright © 2002 John Wiley & Sons, Ltd.

     

The influence of cell contact on the division of mouse 8-cell blastomeres

The pattern of division of polarized 8-cell blastomeres with respect to the axis of cell polarity has been compared (i) for cells dividing alone with cells dividing in pairs, and (ii) for early and late dividing cells within a pair. Cell interactions do not seem to influence significantly the overall pattern of division within the population. The only significant difference found was that the second dividing cell in a pair tended to divide in the same way as its earlier dividing companion slightly more frequently than expected. These results suggest that cell interactions immediately prior to and during division do not influence strongly the orientation and position of the division plane. In contrast, interactions between the cells within an intact early 8-cell embryo, which is subsequently disaggregated to singletons or pairs, do influence the type of progeny generated at division to the 16-cell stage, and seem to do so via an effect on the size of the microvillous region generated at the cell apex.     

A new cell surface marker of aging in human diploid fibroblasts

The relationship of cell surface changes to proliferative decline of human diploid fibroblasts was investigated using the concanavalin A-mediated red blood cell adsorption assay. The amount of the red blood cells adsorbed to human diploid fibroblasts via concanavalin A increased continuously from the early phases of cell passage up through cell senescence, while the amount of 3H-concanavalin A binding did not change to a significant extent. The red blood cell adsorption is not a function of cell cycle phase and time spent in culture. Cocultivation of young cells with old cells also did not affect the adsorption capacity of respective cells. Thus, the concanavalin A-mediated red blood cell adsorption can be expected to serve as a new cell surface marker for aging in vitro. Using this marker, it was revealed that transient cell size or 3H-thymidine incorporating capacity di not have a direct relationship with the division age of a cell. Small rapidly dividing cells in old populations resemble large slowly dividing or nondividing cells of the same populations and differ from small rapidly dividing cells in young populations, in terms of cell surface properties.     

Cell cycle studies in chorionic villi

Summary We have studied the cell cycle of cells obtained from chorionic villi in direct and culture preparations by incorporation of the thymidine analogue BrdU to produce latelabelling or sister chromatid differentiation patterns. We have, therefore, been able to estimate the duration of the cell cycle and, more specifically, the length of some of its phases. While results for chorionic villus sample cells in culture resembled those obtained for fibroblasts, data for the spontaneously dividing trophoblastic cells in direct preparations were different. Villi exposed to BrdU immediately after sampling showed a slight delay in the incorporation of the analogue and a lower percentage of labelled cells compared to villi treated after an overnight incubation, probably due to a temporary effect of the sampling technique. Results from semi-direct protocols suggest that cells have a G2 of no more than 4h, and a mid-S phase of 10–16h. The G1 period is very variable. After 48 h incubation with BrdU, only 4% of cells reach their second generation, whereas this percentage increases up to 70% after 72h, indicating that under these experimental conditions most cells have a cell cycle of approximately 36 h. The average number of sister chromatid exchanges was similar in both direct preparations and cultures: 5.2±2.1 SCE per cell.     

The isolation and culture of lily pollen protoplasts

Methods for the enzymatic isolation of lily protoplasts and their successful culture are described. When pre-anthesis binucleate pollen (immature pollen grains) was treated in enzyme solution containing macerozyme and cellulase, up to 80% lost their exine and gave rise to intact protoplasts within 1 h. These pollen protoplasts were uniform in size and densely cytoplasmic with two prominent generative and vegetative nuclei. The isolated pollen protoplasts regenerated a cell wall within 1 day of culture and produced a structure resembling a pollen tube after 10–12 days of culture. During this culture period, dividing generative nuclei or 2 sperm nuclei were observed in many protoplasts with regenerated cell walls.