Cytoplasmic membrane changes in conjugating Tetrahymena

Summary The formation of stacks of smooth-surfaced cisternae was studied in samples of Tetrahymena pyriformis that were prepared for electron microscopy after starvation for 2 days in buffer, and in samples of organisms of different mating type that were starved for 2 days and then mixed to induce conjugation. The number of stacks of cisternae was greater in starved ciliates than in those from stock cultures, and the size and number of stacks increased further after mixing animals of different mating type. When cells were either starved or mixed in buffer to which the protein synthesis inhibitor cycloheximide had been added, the formation of the membranous stacks was almost completely abolished. Addition of the RNA synthesis inhibitor actinomycin D, however, did not result in a significant decrease in the size and number of the stacks of saccules. Conjugation did not occur in the presence of either cycloheximide or actinomycin. The results suggest that protein synthesis is required for the formation of the stacks, but RNA synthesis is no longer necessary for their formation at this stage. The previous identification of the stacks as a Golgi apparatus and the possible functions of these membranes are considered.Supported by grants from NSF (GB-32285) and the American Cancer Society (E-500).The authors acknowledge the technical assistance of Mrs. Sue Thompson.     

Effect of Actinomycin D and Cycloheximide on Gonadotropin-Induced 17α, 20β-Dihydroxy-4-pregnen-3-one Production by Intact Ovarian Follicles and Granulosa Cells of the Amago Salmon,Oncorhynchus rhodurus*

The effect of actinomycin D and cycloheximide on gonadotropin (partially purified chum salmon gonadotropin, SGA)-induced 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-diOHprog, a maturation-inducing steroid in amago salmon) production was examined in intact ovarian follicles and granulosa cells of postvitellogenic amago salmon, Oncorhynchus rhodurus. Both actinomycin D and cycloheximide blocked gonadotropin-induced 17α, 20β-diOHprog production by intact follicles. In contrast, gonadotropin-induced 17α-hydroxyprogesterone production by intact follicles was not abolished by actinomycin D, but was abolished by cycloheximide, suggesting that postvitellogenic amago salmon ovarian follicles already contain the RNAs necessary for the synthesis of 17α-hydroxyprogesterone. In isolated granulosa cells, chum salmon gonadotropin was able to stimulate 17α, 20β-diOHprog production only when a precursor, 17α-hydroxyprogesterone was provided in the incubation medium, indicating that gonadotropin acts directly on granulosa cells to enhance the activity of 20β-hydroxysteroid dehyrogenase (20β-HSD). Total inhibition of 20β-HSD enhancement in granulosa cells, judged by 17α, 20β-diOHprog production, was achieved when actinomycin D was added between 1 hr before the start of incubation with 17α-hydroxyprogesterone and gonadotropin to 6 hr after. With cycloheximide total inhibition was observed when added in the period of 1 hr before to 9 hr after the start of the incubation. These results suggest that chum salmon gonadotropin acts on granulosa cells to enhance the de novo synthesis of 20β-HSD by a mechanism involving RNA synthesis.     

CELL ARREST IN Gl AND G2 OF THE MITOTIC CYCLE OF VICIA FABA ROOT MERISTEMS

Experiments were performed with cultured excised primary root tips of Vicia faba ‘Longpod’ to determine: (1) the proportion of meristematic cells arrested in Gl and in G2 during carbohydrate starvation, and to determine if the proportion is fixed or can be varied experimentally; (2) the effect of increased starvation on the ability of arrested cells in Gl and G2 to initiate DNA synthesis and mitosis, respectively, when exogenous sucrose was supplied; and (3) whether puromycin, cycloheximide, or actinomycin D prevented the initiation of DNA synthesis and the onset of mitosis. Microspectrophotometry of nuclear DNA and autoradiographic measurements of incorporated ³H-thymidine showed that 72 hr of starvation immediately after excision produced tissue with more than 70 % of the cells arrested in G2 and less than 30 % in Gl. If cultured for three days and then starved for 72 hr, the tissue had nearly equal numbers of cells arrested in Gl and G2. As the duration of starvation increased, the time required to initiate DNA synthesis and to divide when carbohydrate was replenished also increased. Inhibition of protein synthesis by puromycin and cycloheximide prevented the initiation of DNA synthesis and mitosis, but actinomycin D, an inhibitor of RNA synthesis, did not prevent division of cells from G2 nor DNA synthesis by cells from Gl. The experiments demonstrated that the mitotic cycle of Vicia has two major controls, one in Gl and another in G2, and that other factors determine how many cells are affected by either of these cycle controls.     

Influence of Cycloheximide and Actinomycin D on Initiation and Early Development of Adventitious Roots

From leaf cuttings of the bean Phaseolus vulgaris L. adventitious roots form on the petiole. This root formation is stimulated by treatment with auxin. Simultaneous or subsequent application of cycloheximide irreversibly inhibited dedifferentiation, so that root production was completely prevented. The effects of actinomycin D application depended upon the stage of development of the root primordium. Cells in the first stage of dedifferentiation were extremely sensitive. When actinomycin D was applied later than 6 h after cutting, its inhibiting effect gradually diminished. It is concluded that an actinomycin D-sensitive process occurring early in dedifferentiation is crucial for root initiation. A second, less actinomycin D-sensitive process occurring later in dedifferentiation is required for the further development of the root primordium. During the initiation and development of the root primordium protein synthesis is required.     

AGE-DEPENDENT TOXIC PROPERTIES OF ACTINOMYCIN D AND X-RAYS IN CULTURED CHINESE HAMSTER CELLS

A comparative study was made of the toxic properties of actinomycin D and X-rays using synchronized populations of Chinese hamster cells cultured in vitro. X-irradiated cells are most resistant in the latter half of the DNA synthetic period (late S). While cells treated with actinomycin D appear to go through a survival maximum at the same age, they are most resistant after the completion of DNA synthesis; i.e. in G₂ (or G₂-mitosis). In spite of these differences, we found that actinomycin D damage in late S cells interacts with X-ray damage. Thus, a common locus for the site of actions of both agents is suggested which may be in or around the genome of a cell in view of the well-known DNA binding properties of actinomycin D.     

Studies on the amoebo-flagellate transformation inPhysarum polycephalum

Flagellar formation in the true slime mold,Physarum polycephalum, involves a sequence of events during which amoebae are changed into flagellate cells. In the present study a series of inhibitors thought to inhibit RNA and protein synthesis and microtubule assembly were added in an attempt to characterize the metabolic processes associated with this amoebo-flagellate transformation. Proflavin (inhibitor of cellular RNA synthesis), puromycin, cycloheximide and streptomycin (inhibitors of protein synthesis), blocked the transformation; however, actinomycin D (inhibitor of DNA-dependent RNA synthesis) did not block this transformation. On the other hand, 2-mercaptoethanol and dithiothreitol did block flagella formation, but even high concentrations of colchicine failed to have such an effect. Flagellate formation was more strongly inhibited by inhibitors of oxidative phosphorylation than by other respiratory inhibitors; this suggests that oxidative phosphorylation takes part in the energy metabolism of this transformation.     

Microcyst germination in the cellular slime mold, Polysphondylium pallidum: Effects of actinomycin D and cycloheximide on macromolecular synthesis and enzyme accumulation

Germination of microcysts of Polysphondylium pallidum is characterized by an immediate rapid increase in incorporation of [³H]leucine into protein which is cycloheximide-sensitive but unaffected by actinomycin D. Significant RNA synthesis, as measured by [³H]uridine incorporation, does not begin until approx. 2 h after the onset of germination. The increase in [³H]uridine incorporation is prevented by actinomycin D. Germination and the increase in alkaline phosphatase and β-glucosidase enzyme activities are prevented by cycloheximide but unaffected by actinomycin D. The data strongly imply the presence of stable RNA in dormant microcysts and indicate a requirement for a discrete period of protein synthesis for germination of microcysts of P. pallidum.     

DNA SYNTHESIS AND MITOSIS IN MERISTEMS: REQUIREMENTS FOR RNA AND PROTEIN SYNTHESIS

Following provision of sucrose to starved, stationary phase pea root meristems, G₁ and G₂ cells enter DNA synthesis and mitosis, respectively. Puromycin (450 μg/ml) and cycloheximide (5 μg/ml) completely prevent this initiation of progression through the cell cycle. Actinomycin D (10 μg/ml) has no effect on the initial entry of G₁ and G₂ cells into S and mitosis, although later entry is prevented. The resistance of the cells to actinomycin D is lost slowly with time in medium without sucrose, suggesting that an RNA required for the resumption of proliferative activity is being gradually lost. The effects of the inhibitors on transitional and proliferative phase meristem cells indicate that such dividing cells do indeed have sufficient of the requisite RNA for 8-12 hr progression through the cycle, but that protein synthesis is required continuously. It is suggested that this RNA is the one lost slowly during starvation, allowing starved cells to reinitiate progression through the cycle in the presence of actinomycin D.     

Prostaglandin production by methylcholanthrene-transformed mouse BALB/3T3. Requirement for protein synthesis

Stimulation of prostaglandin synthesis in transformed mouse fibroblasts by serum, thrombin, and bradykinin was blocked by actinomycin D and cycloheximide. These RNA and protein synthesis inhibitors did not affect prostaglandin synthetase in vitro or in vivo; nor did they affect the acylation of arachidonic acid into phospholipids. Serum-stimulated release of arachidonic acid and prostaglandins from [3H]arachidonic acid-labeled cells also was inhibited by actinomycin D and cycloheximide. RNA and protein synthesis appear to be required for expression of phospholipase activity; a prerequisite for prostaglandin synthesis by these cells.     

Reformation of the nuclear envelope in melanophores during recovery from a hormone plus puromycin treatment

Previous experiments have shown that treatment of the melanophores of Pachymedusa (Agalychnis) dacnicolor with Melanophore-Stimulating Hormone (MSH) + puromycin causes the nuclear envelope to breakdown leading to the formation of discontinuous vesicles and the hyperdispersion of chromatin. We show here that these cells recover, reform their nuclear envelopes, and recondense their chromatin, both in the presence and in the absence of actinomycin D (actD). After recovery, these cells respond to MSH by melanosome dispersion. From these results, the following conclusions or observations are drawn:

1. 1, Reformation of nuclear envelope does not require that the chromatin be condensed into chromosomes as in mitosis.

2. 2, The new nuclear envelope is derived primarily from reutilization of the membrane vesicles produced during nuclear envelope breakdown, somewhat similar to mitosis. There may also be contributions from other membranous organelles.

3. 3, The hyperdispersed chromatin appears not to be subject to extensive attack by endogenous nucleases as the recovered cells are of good ultrastructure and can respond tropically to MSH.

4. 4, The presence of actD appears not to prevent the conversion of the hyperdispersed chromatin into the normal pattern.

     

Tumour promoter-mediated reversible inhibition of cell-cell communication (electrical coupling) : Relationship with phorbol ester binding and de novo macromolecule synthesis

A tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), reversibly inhibits the onset and maintenance of cell-cell communication measured by electrophysiological method. We have now studied the mechanism by which TPA inhibits communication of human cells (FL) in culture. Using [³H]phorbol-12,13-dibutyrate ([³H]PDBu), we found a class of specific, high-affinity, saturable binding sites in intact FL cells; they have a dissociation constant of 15.4 nM, and at saturation about 3 × 10⁵ PDBu molecules were bound to each cell. The binding of [³H]PDBu to FL cells was inhibited by TPA, phorbol-12-13-didecanoate and mezerein, whereas phorbol and 4α-phorbol-12-13-didecanoate had no effect. There is a close correlation between the ability of the former compounds to inhibit [³H]PDBu binding and their capacity to inhibit cell-cell communication. When FL cells are dispersed with EDTA and plated onto a culture dish, they start to couple electrically within 2 h; such cell coupling was not affected by the presence of cycloheximide or actinomycin D. TPA inhibits the formation of electrical cell coupling as well as its maintenance, even in the presence of cycloheximide; the recovery of cell-cell communication after the removal of TPA was not significantly affected by the addition of cycloheximide or actinomycin D. Taken together, these results suggest that TPA-mediated reversible inhibition of intercellular communication is mediated by specific binding of TPA to cellular receptors and that macromolecular synthesis is not necessary.     

Cadmium and zinc flux in wild-type and cadmium-resistant CHO cells

Cellular flux of cadmium-109 and zinc-65 is characterized in cultured Chinese hamster ovary cells. The transport of cadmium is primarily unidirectional and, following uptake, cadmium is strongly retained. Zinc transport is bidirectional and intracellular zinc continuously leaches out into the medium. Nonradioactive cadmium or zinc enhances the efflux of⁶⁵Zn from prelabeled cells. Transport of these metals into wild-type cells is not affected by azide, ouabain, cycloheximide, or actinomycin D. A cadmium-resistant mutant was isolated that exhibited altered sensitivities to certain inhibitors of macromolecular synthesis as well as quantitative differences in metal transport and accumulation. Although the mutant accumulates less cadmium than the wild-type cell, that which is retained is bound much more tightly. In addition, this lower rate of cadmium uptake is significantly decreased by either cycloheximide or actinomycin D. This suggests that thede novo synthesis of a protein or proteins is required for much of the net cadmium retention by the cadmium-resistant cells.     

Platelet-derived growth factor-modulated translatable mRNAs.

The treatment of density-arrested BALB/c 3T3 cells with electrophoretically homogeneous or highly purified preparations of the platelet-derived growth factor (PDGF) stimulated the rapid and selective accumulation of several species of abundant mRNA identified by cell-free translation. These translatable mRNAs appeared long before entry into the S phase. Less PDGF was required for selective mRNA accumulation than for PDGF-modulated DNA synthesis. The translatable mRNAs also accumulated after addition of the epidermal growth factor but not after addition of insulin or platelet-poor plasma. Their selective accumulation was blocked by addition of actinomycin D. Three classes of PDGF-modulated mRNAs were defined. An early (primary) RNA appeared within 30 to 60 min of PDGF addition; its accumulation was not blocked by cycloheximide. Another early mRNA also appeared within 60 min, but treatment with both PDGF and cycloheximide was required for optimal accumulation. A third class, secondary RNAs, began to accumulate later at 90 to 120 min; the appearance of this class was inhibited by cycloheximide. One- and two-dimensional gel electrophoresis of translation products demonstrated that a spontaneously transformed BALB/c 3T3 (ST2-3T3) cell line, which does not require PDGF or epidermal growth factor for growth, constitutively accumulated the secondary growth factor-regulated mRNAs. The accumulation of these translatable mRNAs may be required for PDGF-modulated DNA synthesis.     

Regulation of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in adrenocortical cell cultures by adrenocorticotropin.

Δ⁵-3β-Hydroxysteroid dehydrogenase-isomerase activity was found to decay in primary cultures of normal rat adrenocortical cells maintained in the absence of adrenocorticotropin for more than 7 days. Physiological concentrations of adrenocorticotropin induced the enzyme complex with a lag period of about 4 hours. Studies with actinomycin D and cycloheximide suggested that both RNA and protein synthesis are required for the induction of steroid dehydrogenase-isomerase activity.     

Commitment to differentiation in Friend cells and initiation of globin mRNA synthesis occurs during the G1 phase of the cell cycle

We have measured the kinetics of specific globin mRNA and Friend virus (FV) RNA synthesis by hybridization to immobilized cDNA after induction of differentiation of two erythroleukemia cell lines (F4N, B8) by butyrate and Me₂SO. The induction with butyrate in these cell lines occurs very rapidly (16–24 h). Cell cycle analysis was made of the populations throughout induction by flow cytofluorometry. The kinetics of commitment of cell populations to terminal differentiation by butyrate was determined by removal of inducer at various times and scoring of benzidine staining cells (hemoglobin producing). In addition, the cell cycle dependence of commitment was determined by flow sorting out of G1 and S+G2 cells various times after addition of inducer and scoring benzidine-stained colonies after growth in methylcellulose. Cells exposed to inducer were also sorted by cell cycle phase using an elutriator rotor. The amount of globin mRNA synthesis in the different cell populations was then determined.

1. 1. It was found that an 8–12 h period in butyrate was required before (a) globin specific mRNA was synthesized; and (b) commitment to differentiation occurred. The time course of globin mRNA synthesis was positively correlated with G1 arrest, as has been also found by others.

2. 2. The increase of FV RNA synthesis was not found during G1 arrest. It occurred early and before commitment.

3. 3. Commitment of cells to irreversible differentiation upon butyrate induction occurs only during the G1 phase of the cell cycle.

4. 4. Globin mRNA synthesis occurs first only in G1 cells.

5. 5. Globin mRNA is synthesized later in all phases of the cell cycle.

These data suggest that (a) commitment to differentiation and globin mRNA accumulation are coupled; and (b) that both events occur only in G 1 cells after a pre-commitment phase of about 12 h.