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".     

Evaluation of L-929 postmitotic daughter cell spreading during their migration on a substrate

Spreading of postmitotic daughter cells was examined using time-lapse microscopy. The work was performed on unsynchronized cells of a permanent L-929 cell line. The study aimed at formalizing the comparison of the moving cell area and estimating whether the area of migrating cells was changed randomly or nonrandomly. Two new parameters are proposed for comparison of cell morphology: the identity indicator and the synchronism indicator. To calculate these parameters, time-dependent changes of the area in cell pairs were measured. The first indicator shows the degree of coincidence between the absolute area values in the cell pairs, whereas the second indicator shows synchronism in the changes of cell areas and does not depend on their absolute values. The low indicators were a high similarity in the time-dependent changes of the cell area. The indicators were shown to be approximately 1.5-fold lower for the pairs of the postmitotic daughter cells than those for any other pair of the cells. The results point to a nonrandom character of the changes of cultured cell morphology.     

Cultivation of Neisseria gonorrhoeae in an L-929 cell culture

N. gonorrhoeae strain b has been found to be capable of retaining its viability in medium 199 with 10% of inactivated cattle serum added and in monolayer cell culture L-929 in the above medium. The characteristics obtained in the present investigation permit simulating the mixed association of gonococci and chlamydiae in the culture system used in this work.     

Respiration of mengovirus-infected L-929 cells

Iglewski, W. J. (The Pennsylvania State University, University Park), and E. H. Ludwig. Respiration of mengovirus-infected L-929 cells. J. Bacteriol. 92:733-738. 1966.-Polarographic techniques were employed to study the oxidative metabolism of L-929 cells during a one-step mengovirus growth cycle. Virus maturation began 3.5 hr after infection and was complete with 7 hr. Virus maturation was accompanied by a decreased rate of endogenous respiration and an increased rate of oxidation of succinate and alpha-glycerophosphate by L-929 cells. The rate of glucose uptake was the same for mengovirus-infected and control L-929 cells. However, there was a decreased oxidation of glucose to carbon dioxide and a decreased production of lactic acid by L cells infected with mengovirus under aerobic conditions. Mengovirus was produced equally well under aerobic and anaerobic conditions. The implications of the alterations in metabolism with respect to virus synthesis are discussed.     

L-929 cells under hyperosmotic conditions

Changes in cell water content resulting from sorbitol addition to the environment of L-929 cells were evaluated gravimetrically using¹⁴C-labeled polyethylene glycol as a probe of extracellular space. Reductions in cell water were proportional to sorbitol supplements up to 0.6 molal, above which no further measurable decrease occurred. No volume regulation occurred for at least 1 h but the percentage of cell water lost was quickly regained when physiological conditions were restored. The amount of cell water lost because of a given hyperosmotic exposure was found to exceed the loss of cell volume. That discrepancy could be the result of an overestimation of extracellular space and/or an underestimation of cell volume reduction as a result of infolding of the cell surface. Na⁺ and K⁺ were also measured in cells of variable water content and volume: no significant change occurred in the amounts of these ions per cell, but large increases in total cell concentration resulted from hyperosmotic exposure. The sum of Na⁺ and K⁺ concentrations exceeds the total osmotic pressure of the medium indicating that an appreciable fraction of Na⁺ and K⁺ must be bound to fixed charges within the cells. The results are evaluated in the context of intracellular organization.     

Analysis of the cell cycle duration of cells of permanent line L-929

The direct measurement of the cell cycle duration in L-929 cells was performed using time-lapse photography. The cell cycle duration was 15.77 +/- 0.08 h with a standard deviation of 1.54 +/- 0.06 h. The experimental value fit to a normal distribution with a correlation coefficient 0.999. High homogeneity of this parameter and a wide range of variability of the karyotype (58-66 chromosomes) indicate that there is no correlation between these characteristics of L-929 cells. It is also shown that the difference between cell cycle durations of daughter cells tent to zero and fits by an exponent.     

Glycolysis in permeabilized L-929 cells.

Mouse L-929 cells permeabilized with dextran sulphate (DSP cells) carry out glycolysis when supplemented with glucose, ATP and NAD+ in a suitable incubation buffer. Glycolytic rates were linear and generally independent of cell density over the range examined (1 x 10(6)-10 x 10(6) cells/ml). Electron microscopy revealed characteristic changes in DSP cell ultrastructure, notably for nuclei and mitochondria. Some cells lacked plasma membranes, while others appeared intact. In the latter case, estimates of the lesion size in plasma membranes were obtained from volume of distribution studies using 14C-labelled proteins, and infiltration of fluorescein isothiocyanate dextran. The results indicated the presence of lesions large enough to allow globular proteins of about 400 kDa to cross the cell surface. In spite of that, only about 10% of total cell protein exited from DSP cells during a 30 min incubation period. We propose that none of the glycolytic enzymes in DSP cells can exist completely in solution in the 'cytosol', suggesting extensive enzyme organization. The results are interpreted within the broader picture of metabolic organization in animal cells and the nature of the 'cytosol'.     

Transient inhibition of L929 cell mitosis and locomotion by argon ion laser irradiation

Summary Argon ion laser irradiation of L929 cells transiently inhibits both entry into and passage through mitosis without affecting clonogenic survival. Anaphase mitotic figures virtually disappear from irradiated cell monolayers although prophase + metaphase mitotic figures can still be identified. The total number of mitotic figures does not change significantly and time-lapse video recording shows that cells do not enter mitosis following irradiation. This effect is dependent on light dose within the 900–2700 J/cm² range and persists for 10–48 h depending on the initial light exposure. Inhibition of cell locomotion and subsequent recovery were observed to occur over a similar time course. The possible contribution of these phenomena must be considered whenever biological systems are exposed to argon ion laser irradiation.     

Effects of reduced cell volume and water content on glycolysis in L-929 cells

Mouse L-929 cells were subjected to increasing concentrations of sorbitol, which remove cell water and reduce volume osmotically. The rate of lactate production from glucose was significantly higher in osmotically perturbed cells than in controls, both in monolayers and in suspensions. L cells can apparently use sorbitol as a glycolytic substrate; however, studies using other solutes (trehalose and sucrose) and permeabilized cells showed that the major effect of sorbitol on glycolysis in intact cells is mediated through a reduction in cell water content and volume. It is possible to explain some of these results by an increase in the chemical potentials of dissolved components of the glycolytic pathway caused by water loss; however, the relationship between water loss and glycolytic rate increase in not a simple linear one, suggesting that the situation is more complex than would result merely from increased concentrations of pathway components. Whatever the complete explanation might be, these studies show that glycolysis continues in an orderly fashion in cells that have lost about 85% of their original water content, suggesting that the operation of this pathway is not unduly sensitive to events taking place in the bulk aqueous phase.     

The characteristics of glucose consumption by an L-929 cell culture infected with Chlamydia

The effect of the intracellular parasite Chlamydia trachomatis on the host cell energy metabolism has been studied. Glucose consumption by L-929 cell cultures infected or uninfected by C. trachomatis was studied in comparison during a 3-days cultivation. The content of glucose in the cultural medium was determined every 5, 24, 48, 72 hrs according to the developmental cycle of the parasite. It was shown that cell infection by C. trachomatis induced the alteration of energy metabolism via an increase in the glucose consumption rate.     

Anti-steroid action in cultured L-929 mouse fibroblasts

L-929 mouse fibroblast growth is arrested by the glucocorticosteroid dexamethasone (dex), which also decreases adhesiveness to culture plates. Both dex effects were abolished when RU 486, a new synthetic anti-hormonal steroid, was added to the culture medium. Using [3H]RU 486, binding studies revealed that RU 486 bound approximately 25,000 sites/cell of the glucocorticosteroid receptor (GR) with affinity higher than that of dex and translocated GR to the nucleus. However, the distribution of steroid-receptor complexes between cytosol and nuclei was different for the agonist and the antagonist, with more nuclear accumulation in the case of dex. Estradiol increases L-929 cell growth and adhesiveness to culture plates, but not if the anti-estrogen tamoxifen (tam) was added. These observations initially made in serum containing medium, were confirmed in serum-free, chemically defined culture medium (SF). In SF medium, tam (1 microM) provoked death of most L-929 cells after 10 days of culture, leading to the selection of a variant clone LB of tam-resistant cells. Tam binds to the estrogen receptor (ER), but with less affinity than estradiol. ER concentration, estimated by the binding of 4-hydroxytamoxifen (OH-tam) and of estradiol was lower in LB cells than in original tam-sensitive L-929 cells. The concentration of specific anti-estrogen binding sites in the particulate fraction of the cells, measured by OH-tam binding, non competed by estradiol, was also significantly diminished in tam-resistant LB cells.     

The association between prolyl hydroxylase metabolism and cell growth in cultured L-929 fibroblasts

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis, its active form is a tetramer (alpha 2 beta 2). In L-929 fibroblasts in the log phase of culture there is a low level of active enzyme. When the cell culture reaches confluency, prolyl hydroxylase activity in cells increases by a process that requires de novo RNA and protein synthesis. The same result may be achieved by crowding the cells (replating log phase cells at the density of stationary phase cells). In the work reported here we further examined induction of the enzyme. RNA synthesis necessary for enzyme induction is complete 6 h after "crowding" while protein synthesis requires 12 h. Thymidine (0.2-0.5 mM) added to log phase cells will also cause enzyme induction to the level found in "crowded" or resting cells. We also looked at the decay of the enzyme activity after subculture. This occurs rapidly (enzyme half-life is 1-2 h) and is concurrent with the re-entry of resting cells into cell cycle; however, thymidine added at the time of subculture to block DNA synthesis does not prevent the loss of prolyl hydroxylase activity. These results suggest that when cells are not engaged in propagation, they begin to synthesize luxury proteins such as prolyl hydroxylase. However, the loss of prolyl hydroxylase during subculture is probably not a direct consequence of DNA synthesis.     

Continuous production of human erythropoietin by immobilized recombinant L-929 cells

Recombinant L-929 cells transfected with the human erythropoietin (EPO) gene were immobilized in a macroporous cellulosic support and its derivatives in which charged groups or cell attachment factors were introduced. The immobilized cells were cultured in serum-containing and serum-free media. Comparable production of EPO was observed even in the serum-free medium when a support modified by polyethyleneimine was used for immobilization. The cells immobilized on the supports were cultured in fluidized-bed and inner-loop type air-lift bioreactors for continuous production of EPO. A high cell density of more than 2 × 10⁷ cells/cm³-support and high EPO productivity were achieved and maintained for 50 d through the use of the inner-loop type air-lift bioreactor. The productivity was 13.4-fold higher than that of conventional static cultures in petri-dishes.     

Protein factors in extracts of ribosomes isolated from L-929 cells during various phases of the cell cycle

Centrifugal elutriation has been utilized in order to separate cultures of L-929 fibroblasts into subpopulations containing cells at different stages of the cell cycle. The subpopulations were characterized by Coulter counter volume determination, [3H]thymidine label into DNA and flow cytometry. When a population of early G1 cells was returned to roller culture it was shown to progress through the cell cycle in a synchronous manner. Ribosomal factor extracts were prepared from cells at various phases during the cell cycle. The amounts of protein in the extracts varied greatly, being lowest in early G1 phase and showing a peak during S phase. Polyacrylamide gel electrophoresis demonstrated that there were differences in the protein species present in the extracts. Some proteins were present in the same amounts throughout the cell cycle, whereas others appeared to show a form of cyclical behaviour.     

Mouse transcobalamin II: Biosynthesis and uptake by L-929 cells

Mouse fibroblast (L-929) cells, in culture, synthesized and secreted into the growth medium a vitamin B₁₂-binding substance which was identical to mouse transcobalamin II (TC II) as judged by the following criteria: (i) gel filtration on Sephadex G-200, (ii) ion-exchange chromatography on DEAE-cellulose and CM-cellulose, and (iii) the ability to facilitate cellular B₁₂ uptake by L-929 cells. The secretion of mouse fibroblast binder was blocked by cycloheximide and puromycin; and in both cases the cells' ability to secrete this binder was partially restored when the inhibitor was removed. Within 30 h after the cells were exposed to [⁵⁷Co]B₁₂ bound to mouse serum TC II (M_r ~ 38,000) the [⁵⁷Co]B₁₂ was bound to a large molecular weight intracellular binder (M_r ~ 120,000) which was not released into the culture medium. During this same incubation period, the cells released free [⁵⁷Co]B₁₂ and [⁵⁷Co]B₁₂ bound to a protein which had the same elution volume as mouse serum TC II on Sephadex G-200.     

Dynamics of chromosome compaction during mitosis

We have quantitatively studied the space-time dynamics of mitotic chromosome compaction in cultured amphibian cells. After collecting digital phase-contrast images we have done digital image analysis to study spatial correlations in density. We find a characteristic distance at which the strongest correlations occur, which provides a quantitative measure of the size of patches of dense chromatin during interphase and early prophase. Later in mitosis, this length corresponds to the thickness of prophase and metaphase chromosomes. We find that during interphase strong correlations exist at a few-micrometer length; during prophase this correlation length progressively drops as the chromosomes are compacted. Our data are explained by a model based on assembly of chromatin loops onto already fiberlike interphase chromosomes. To test this model we have microinjected cobalt hexamine trichloride into interphase nuclei and have observed the rapid condensation of the interphase chromatin into thick fibers with a spacing similar to the native-state interphase correlation length determined from our image analysis.