The 5' end of the pea ferredoxin-1 mRNA mediates rapid and reversible light-directed changes in translation in tobacco

Ferredoxin-1 (Fed-1) mRNA contains an internal light response element (iLRE) that destabilizes mRNA when light-grown plants are placed in darkness. mRNAs containing this element dissociate from polyribosomes in the leaves of transgenic tobacco (Nicotiana tabacum) plants transferred to the dark for 2 d. Here, we report in vivo labeling experiments with a chloramphenicol acetyl transferase mRNA fused to the Fed-1 iLRE. Our data indicate that the Fed-1 iLRE mediates a rapid decline in translational efficiency and that iLRE-containing mRNAs dissociate from polyribosomes within 20 min after plants are transferred to darkness. Both events occur before the decline in mRNA abundance, and polyribosome association is rapidly reversible if plants are re-illuminated. These observations support a model in which Fed-1 mRNA in illuminated leaves is stabilized by its association with polyribosomes, and/or by translation. In darkness a large portion of the mRNA dissociates from polyribosomes and is subsequently degraded. We also show that a significant portion of total tobacco leaf mRNA is shifted from polyribosomal to non-polyribosomal fractions after 20 min in the dark, indicating that translation of other mRNAs is also rapidly down-regulated in response to darkness. This class includes some, but not all, cytoplasmic mRNAs encoding proteins involved in photosynthesis.     

Mechanism of colchicine-induced steroidogenesis in rat adrenocortical cells

Conflicting data for the effects of colchicine on cholesterol transport and steroidogenesis raise the question of the role of microtubules in cholesterol transport from the lipid droplet to mitochondria in steroidogenic cells. In this study, using corticosterone radioimmunoassay and immunofluorescence microscopy, we re-evaluated the effects of colchicine on hormone production and morphological changes of lipid droplets' and studied the signaling pathway involved in colchicine-induced steroidogenesis. Colchicine stimulated steroid production in a dose- and time-dependent manner. The structural integrity of both the microtubules and the lipid droplet capsule was destroyed by colchicine treatment. Disruption of the lipid droplet capsule occurred later than microtubule depolymerization. After cessation of colchicine treatment and a 3 h recovery in fresh medium, capsular protein relocated to the droplet surface before the cytoplasmic microtubule network was re-established. beta-lumicolchicine, an inactive analogue of colchicine, disrupted the capsule and increased hormone production without affecting microtubular structure. Thus, microtubule depolymerization is not required for the increase in steroid production and capsular disruption. To explore the signaling pathway involved in colchicine-induced steroidogenesis, we measured intracellular cAMP levels. Unlike ACTH, colchicine did not increase cAMP levels, suggesting that the cAMP-PKA system is not involved. Colchicine and ACTH had additive effects on corticosterone production, whereas colchicine and PMA did not, implying that part of the PKC signaling mechanism may be involved in colchicine-induced steroidogenesis. Cycloheximide, a protein synthesis inhibitor, completely inhibited colchicine-induced steroidogenesis and capsular disruption. These results demonstrate that the steroid production and lipid droplet capsule detachment induced by colchicine are both protein neosynthesis-dependent and microtubule-independent.     

Distribution of histone messenger RNA among free and membrane-associated polyribosomes of a mouse myeloma cell line

The distribution of histone messenger RNA among free polyribosomes and two classes of membrane-associated polyribosomes was studied in an immunoglobulin-secreting mouse myeloma cell line. The two classes of membrane polyribosomes were distinguished on the basis of their sensitivity to dissociation from membranes upon exposure to high concentrations of salt or during repeated centrifugation through sucrose. Histone messenger RNA is specifically excluded from that class of polyribosomes most tightly associated with cellular membranes.     

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular pH on translocation of protein kinase C isozymes in rat pinealocytes

In rat pinealocytes, cytoplasmic alkalization causes protein kinase C (PKC) translocation, but the isozyme involved is not known. In this study, we investigated the effect of cytoplasmic alkalization on membrane-associated PKCalpha, delta, epsilon, and zeta, four isozymes present in the rat pineal gland. Treatment with NH(4)Cl, which had no effect on PKCzeta, caused a sustained increase in membrane-associated PKCalpha, delta, and epsilon that lasted for at least 60 min. The effect of NH(4)Cl on PKCalpha, delta, and epsilon was reduced by sodium propionate, an agent that counteracts the effect of NH(4)Cl on intracellular pH. Both sodium propionate and 5-(N,N-hexamethylene)amiloride (HMA), two treatments that abolished the effect of norepinephrine on cytoplasmic alkalization, also reduced norepinephrine-mediated increases in membrane-associated PKCalpha, delta, and epsilon. In contrast, these two treatments did not have an effect on the increase in membrane-associated PKC isozymes caused by 4beta-phorbol 12-myristate 13-acetate (PMA), an active phorbol ester, even though HMA was effective in abolishing PMA-mediated increases in intracellular pH. These results, apart from demonstrating that cytoplasmic alkalization by itself can cause translocation of PKCalpha, delta, and epsilon in rat pinealocytes, also indicate that the norepinephrine-stimulated cytoplasmic alkalization plays an important role in transducing signals from the adrenergic receptor to selective PKC isozymes. However, PKC translocation stimulated directly by PMA does not appear to be sensitive to changes in intracellular pH.     

Localization of Polyribosomes Containing Alkaline Phosphatase Nascent Polypeptides on Membranes of Escherichia coli

A procedure has been developed for extracting membranes from bacterial cells under conditions that keep a large fraction of bacterial polyribosomes intact. Freeze-thawing spheroplasts in the presence of deoxyribonuclease, followed by differential centrifugation, permits a separation of free and membrane-associated polyribosomes. The latter fraction contains as much as 40% of cell ribosomal ribonucleic acid (RNA) and 55% of cell messenger RNA (mRNA). Nascent polypeptides were divided almost equally between the two fractions, but 70 to 80% of alkaline phosphatase nascent chains, detected both chemically and immunologically, were derived from polyribosomes associated with the bacterial membrane. Analysis of the fractions for mRNA specific for the lac and trp operons by RNA-deoxyribonucleic acid hydridization showed somewhat larger amounts on membrane than on free polyribosomes, but enrichment for nascent alkaline phosphatase (a secreted protein) on membranes was consistently greater, suggesting that polyribosomes making secreted proteins are more tightly bound to membranes. Electron micrographs of the membrane preparations show relatively intact membranes with clusters of polyribosomes on their inner surfaces.     

Activation of membrane-associated procaspase-3 is regulated by Bcl-2

The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 zymogen was still associated with heavy membranes, its spontaneous activation was blocked. However, Bcl-2 expression had little effect on the levels of cytoplasmic caspase activity in unstimulated cells. Furthermore, the membrane-associated caspase-3 differed from cytosolic caspase-3 in its responsiveness to activation by exogenous cytochrome c. Our results demonstrate that intracellular membranes can generate active caspase-3 by a Bcl-2-inhibitable mechanism, and that control of caspase activation in membranes is distinct from that observed in the cytoplasm. These data suggest that Bcl-2 may control cytoplasmic events in part by blocking the activation of membrane-associated procaspases.     

Differences in sequence content of nuclear and cytoplasmic polyribosomal RNA from adenovirus-infected cells.

RNA molecules from nuclear and cytoplasmic polyribosomes of adenovirus-infected HeLa cells were compared by hybridization to analyse the sequence content. Nuclear polyribosomes were released by exposure of intact detergent-washed nuclei to poly(U) and purified. Cytoplasmic polyribosomes were also purified from the same cells. To show that nuclear polyribosomes contain ribosomes linked by mRNA, polyribosomes were labelled with methionine and uridine in the presence of actinomycin D in adenovirus-infected cells. Purified nuclear polyribosomes were treated with EDTA under conditions which dissociate polyribosomes into ribosomes and subunits with a simultaneous release of mRNA, and sedimented. The treatment dissociated these polyribosomes, releasing the mRNA from them. Radiolabelled total RNA from each polyribosome population was fractionated in sucrose gradients into several pools or hybridized to intact adenovirus DNA to select virus-specific RNA. Sucrose-gradient-fractionated pool-3 RNA (about 28S) and virus-specific RNA were then hybridized to fragments of adenovirus DNA cleaved by restriction endonucleases SmaI, HindIII and EcoRI by the Southern-blot technique and by filter hybridization. The results showed that nuclear RNA contained sequences, from about 0 to 18 map units, which were essentially absent from cytoplasmic RNA. Furthermore, the amount of virus-specific RNA for a particular sequence was also different in the two populations.     

Evidence that microtubule depolymerization early in the cell cycle is sufficient to initiate DNA synthesis

Microtubule disrupting drugs initiated DNA synthesis in serum-free cultures of nonproliferating fibroblast-like cells. The addition of colchicine to chick, mouse and human fibroblasts in serum-free medium stimulated thymidine incorporation at least twofold, with a half-maximal concentration of 1 X 10(-7) M. This stimulation represented up to 75% of the maximal stimulation by thrombin and was paralleled by an increase in the percentage of labeled nuclei. Other microtubule disrupting drugs showed similar stimulation, whereas lumicolchicine had no effect. Indirect immunofluorescent staining of tubulin showed a correlation between microtubule depolymerization and initiation of DNA synthesis by these drugs. A 2 hr treatment with 10(-6) M colchicine caused complete disruption of the microtubular network and stimulated thymidine incorporation (measured 28 hr later) to an even greater extent than continuous colchicine exposure. A similar 2 hr exposure to 10(-6) M colcemid also stimulated thymidine incorporation and led to a 50% increase in cell number. Taxol, a drug which stabilizes cytoplasmic microtubules, blocks initiation of DNA synthesis by colchicine, indicating that microtubule depolymerization is necessary for this initiation. To determine if microtubule depolymerization is involved in stimulation of DNA synthesis by other growth factors, highly purified human thrombin was added to cells with or without colchicine. In no case did colchicine plus thrombin increase DNA synthesis above that of the maximal stimulation by thrombin alone. Furthermore, pretreatment of cultures with taxol (5 micrograms/ml) inhibited approximately 30% of the stimulation of thymidine incorporation by thrombin. Together, these studies demonstrate that microtubule depolymerization is sufficient to initiate both DNA synthesis and events leading to cell division and suggest that microtubule depolymerization may be a required step in initiation of cell proliferation by growth factors such as highly purified human thrombin.     

Temperature-sensitive Yeast Mutant Defective in Ribonucleic Acid Production

A single, recessive mutation in a nuclear gene confers a temperature-sensitive growth response in a mutant of Saccharomyces cerevisiae, ts(-) 136. The mutant grows normally at 23 C, but exhibits a rapid and preferential inhibition of ribonucleic acid (RNA) accumulation after a shift to 36 C, demonstrating a defect in stable RNA production. Cultures of the mutant which were shifted from 23 to 36 C display the following phenomena which indicate that messenger RNA (mRNA), as well as stable RNA production, is defective. The entrance of pulse-labeled RNA into cytoplasmic polyribosomes is even more strongly inhibited than is net RNA accumulation. The rate of protein synthesis, at first unaffected, decreases slowly; this decrease is paralleled by the decay of polyribosomes to monoribosomes with a half-time of 23 min. The polyribosomes which remain after a 30-min preincubation of the mutant at 36 C are active in polypeptide synthesis in vivo, whereas the monoribosomes which accumulate are not. Furthermore, ribosomes isolated from a culture of the mutant preincubated for 1 hr at 36 C are inactive in polypeptide synthesis in vitro, but can be restored to full activity by the addition of polyuridylic acid as mRNA. We conclude that mutant ts(-) 136 is defective either in the synthesis of all types of cytoplasmic RNA, or in the transport of newly synthesized RNA from the nucleus to the cytoplasm, and that the mRNA of a eucaryotic organism (yeast) is metabolically unstable, having a half-life of approximately 23 min at 36 C.     

Shedding of Phosphatidylserine from Developing Erythroid Cells Involves Microtubule Depolymerization and Affects Membrane Lipid Composition

Phosphatidylserine (PS), which is normally localized in the cytoplasmic leaflet of the membrane, flip-flops to the external leaflet during aging of, or trauma to, cells. A fraction of this PS undergoes shedding into the extracellular milieu. PS externalization and shedding change during maturation of erythroid cells and affect the functioning, senescence and elimination of mature RBCs. Several lines of evidence suggest dependence of PS shedding on intracellular Ca concentration as well as on interaction between plasma membrane phospholipids and microtubules (MTs), the key components of the cytoskeleton. We investigated the effect of Ca flux and MT assembly on the distribution of PS across, and shedding from, the membranes of erythroid precursors. Cultured human and murine erythroid precursors were treated with the Ca ionophore A23187, the MT assembly enhancer paclitaxel (Taxol) or the inhibitor colchicine. PS externalization and shedding were measured by flow cytometry and the cholesterol/phospholipids in RBC membranes and supernatants, by ¹H-NMR. We found that treatment with Taxol or colchicine resulted in a marked increase in PS externalization, while shedding was increased by colchicine but inhibited by Taxol. These results indicate that PS externalization is mediated by Ca flux, and PS shedding by both Ca flux and MT assembly. The cholesterol/phospholipid ratio in the membrane is modified by PS shedding; we now show that it was increased by colchicine and A23187, while taxol had no effect. In summary, the results indicate that the Ca flux and MT depolymerization of erythroid precursors mediate their PS externalization and shedding, which in turn changes their membrane composition.     

Membrane-associated ribosomes in producing and nonproducing mouse myeloma cells

Mouse myeloma cell lines synthesize large amounts of immunoglobulin on their membrane-associated polyribosomes. Variants which no longer synthesize immunoglobulins have been studied and shown to have the same number of tightly bound membrane-associated polyribosomes as the parental cell lines. These polyribosomes are still active in the synthesis of nonimmunoglobulin proteins.     

The interaction between newly-formed mRNA and ribosomal particles during retarded translation]

The formation of polyribosomes in mouse liver cells at the reduced-rate translation was studied by treatment with cycloheximide (CHI) and aurintricarboxylic (ATA) acid. An increase of polypeptide synthesis time by 1.7-2.7 times (0.5 mg CHI per 25 g of weight or 15 mg ATA per 25 g) leads to a delay of the entrance of newly formed cytoplasmic D-RNA into polyribosomes. These results are in agreement with the model of polyribosome formation from ribonucleoprotein precursors containing cytoplasmic D-RNA. On the other hand, in the presence of a CHI dose (5 mg/25 g) causing a dramatic (240-fold) increase of polypeptide synthesis time, the kinetics of entrance of newly formed D-RNA into polyribosomes does not differ from the normal one, and amount of the incorporated mRNA is even somewhat higher than under normal conditions. It is suggested that in this situation ribosomes are moving along the newly formed mRNA, and their movement is not accompanied by the synthesis of completed polypeptide chain.     

Association of mitochondria with intermediate filaments and of polyribosomes with cytoplasmic actin

Summary Anti-mitochondrial autoantibody and fluorescent derivatives of insulin stain phase-dense mitochondria in acetone-fixed monolayers of fibroblasts. Double fluorochrome studies show mitochondria in close topographic association with intermediate filaments. In cells treated with vinblastine or colchicine, mitochondria are relocated in sites closely associated with coils of perinuclear intermediate filaments. In contrast, autoantibody to polyribosomes stains granules aligned in the long axis of well spread embryonic cells, in the direction of actin-containing fibrils, an arrangement that is lost in cells pretreated with the actin filament disrupting drug cytochalasin B. In more mature fibroblasts, antiribosomal antibody reacts with phase-dense rough endoplasmic reticulum and this staining pattern is not affected by cytochalasin B. The observations suggest that mitochondria are associated with intermediate filaments and that free polyribosomes, but not polyribosomes attached to rough endoplasmic reticulum, are associated with cytoplasmic actin.Supported by a grant from the Anti-Cancer Council of Victoria. We thank Mrs. I. Burns for technical assistance and Dr. H.A. Ward and staff for preparation of fluorescent conjugates     

Transport of messenger RNA into different classes of membrane-associated polyribosomes in Ehrlich-ascites-tumor cells.

The membrane-bound polyribosomes in Ehrlich ascites tumor cells can be separated into a loosely bound and a tightly bound fraction by means of a high salt treatment. Both membrane fractions as well as the free polyribosomes in the supernatant synthesize about the same set of proteins, suggesting a close relationship between these polyribosome fractions in the Ehrlich cell. Relatively high concentrations of cycloheximide do not prevent newly synthesized poly(A)-containing mRNA from entering the tightly bound polyribosome fraction. Nor had treatment of the cells with puromycin in the presence of cycloheximide, which released about 70% of the nascent chains, any significant effect on the entrance of newly synthesized mRNA into tightly bound polyribosomes. These results suggest that in ehrlich ascites tumor cells nascent polypeptide chains are not involved in the binding of polyribosomes to membranes.     

Free and membrane-bound polyribosomes in BHK cells infected with Sindbis virus.

The data presented in the paper demonstrate that in BHK cells infected with Sindbis virus virtually all the 42S mRNA not in nucleocapsid is associated with free polyribosomes, whereas the 26S mRNA is distributed between free and membrane-bound polyribosomes. We suggest that the 26S RNA polyribosomes are bound to the membranes through the nascent chains of the B1 protein and that a large percentage of 26S RNA polyribosomes free in the cytoplasm may be due to the small amount of rough endoplasmic reticulum in BHK cells. In addition, we found that intracellular nucleocapsid is in the nonmembrane fraction of the cytoplasm of infected cells.     

Kinetics of synthesis of cytoplasmic messenger-like RNA not associated with ribosomes in HeLa cells

The turn-over of cytoplasmic messenger-like RNA not associated with polyribosomes as well as that of polyribosomal mRNA was investigated by labelling with [3H]uridine in conditions of arrested ribosomal RNA and mitochondrial RNA synthesis. The synthesis of ribosomal RNA was inhibited with toyokamycin and that of mitochondrial RNA with ethidium bromide. In both accumulation kinetics and actinomycin-D-chase experiments, cytoplasmic messenger-like ribonucleoprotein particles and polyribosomes were fractionated by buoyant density centrifugation in CsCl gradients. The half-life of free m1RNA was found to be of 1--2 h whereas the bulk of polyribosomal mRNA was stable over the time period considered (up to 8 h) but with a minor short-lived component. Purification of RNA from polyribosomes labelled under the same conditions and fractionation of it into polyadenylated and non-polyadenylated fractions showed that this short-lived minor component of half-life less than 1 h is non-polyadenylated.     

Resonance Raman spectra of heme a derivatives. Evidence for the reaction of peripheral formyl group with HCN and NaHSO3.

A separate and distinct population of polyribosomes exists in the detergent-washed nuclei of adenovirus-infected HeLa cells. These polyribosomes, released by exposure to polynucleotides such as high molecular weight nuclear RNA or poly(U), do not appear to be cytoplasmic contaminants. Nuclear polyribosomes have a considerably lower buoyant density compared to cytoplasmic ones. Nuclear polyribosomes, in a cell-free system of protein synthesis, are six- to eight-fold less active compared to cytoplasmic ones and are insensitive to aurin tricarboxylic acid. They do not complement cytoplasmic polyribosomes in protein synthesis in the cell-free system. Finally, the number of proteins synthesized by nuclear polyribosomes is higher compared with that synthesized by the cytoplasmic ones. Only the virus-specific proteins, including P-VII, are synthesized by cytoplasmic polyribosomes. Nuclear polyribosomes, on the other hand, synthesize virusspecific proteins, including P-VII and VII, and a number of additional proteins not synthesized by the cytoplasmic ones.