Tubulin Synthesis Research Articles

Triiodothyronine regulated tubulin biosynthesis in oligodendrocytes during myelinogenesis

l-Triiodothyronine (T(3)) is shown to stimulate the biosynthesis of tubulin in oligodendrocytes isolated from rat brains at different stages of myelinogenesis. The hormone sensitivity appears around day 11, reaches a maximum at day 15 and disappears by day 25 after birth. Dose response studies show that the optimal stimulation of tubulin in the oligodendrocytes from 15 day rat brain occurs with 0.045 nM T(3) in contrast to 4.5 nM T(3) that was previously shown to promote a similar age-dependent induction of tubulin in the astroglial cells from neonatal rat brain. Exposure of the oligodendrocytes to optimal dose of T(3) (0.045 nM) for 2 h elicits a marked and almost selective increase in the level of tubulin. Higher concentrations of T(3) induce additional proteins resulting in a loss of this sensitivity. Studies on the synthesis and turnover of (35)S-labeled tubulin show that the stimulation of tubulin by T(3) is primarily due to enhanced synthesis of the protein. Pulse chase experiments reveal that the half life of tubulin is about 5.5 h and that it remains unaffected by T(3). The crucial role of thyroid hormones and the possible function of tubulin in the two most critical phases of brain maturation viz. synaptogenesis and myelinogenesis is discussed.

A possible regulatory system of microtubule formation among uremic toxins.

Tubulin is an intracellular protein, whose polymerization leads to the formation of microtubules (MT). MT are an essential component of axons of nerve cells, and the rate of axon regeneration depends on the synthesis or polymerization of tubulin 1. Uremic plasma inhibits the growth of nervous fibers in chicken embryo. Moreover, in uremic neuropathy, axonal degeneration is frequently observed 2. Thus, in uremic patients, MT formation from tubulin polymerization may be inhibited. If this effect also occurs in vivo, uremic toxins (UT) might be involved in the pathogenesis of uremic neuropathy.

Autoregulation of tubulin synthesis.

AbstractIn many mammalian cell types, increases in the level of nonpolymerized tubulin cause an inhibition in tubulin synthesis which is accompanied by a decrease in tubulin mRNA levels. To see whether inhibition is caused by nuclear or cytoplasmic events, two groups have recently examined the ability of enucleated cells to autoregulate tubulin synthesis.1,2 These experiments have demonstrated that transcription, processing, and transport of tubulin mRNAs from the nucleus to the cytoplasm are not major sites of autoregulation. Instead, monomeric tubulin must reduce, either directly or indirectly, the translatability of its own message.

Interval between the synthesis and assembly of cytoskeletal proteins in cultured neurons

We have used pulse-chase experiments to study the time interval between the synthesis and assembly of tubulin and neurofilament proteins (NFP) in sympathetic neurons grown in tissue culture. After varying pulse-chase times, cultures were extracted with Triton X-100 such that polymerized tubulin and NFP were insoluble, while unassembled tubulin and NFP were quantitatively solubilized. The partitioning of labeled tubulin and NFP between Triton X-100-soluble and insoluble, or cytoskeletal, fractions was determined with an isoelectric focusing X SDS gel electrophoresis assay. Labeled tubulin and NFP in cultures pulse-labeled for 5-10 min partitions primarily with the soluble fraction. When pulse-labeled cultures were chased for increasing periods of time, relatively more of the total labeled tubulin and NFP partitioned with the cytoskeleton, attaining maximal values after chase times of 60-120 and 15-30 min, respectively. The maximal values for the relative levels of labeled tubulin and NFP in polymer were 70-75 and greater than 90%, respectively. The levels of labeled tubulin and NFP synthesized during a short pulse-label remained constant for at least 2 hr, indicating that selective turnover of soluble tubulin and NFP does not detectably contribute to the changes in solubility properties of these proteins observed in the pulse-chase experiments. These results indicate that newly synthesized tubulin and NFP are rapidly assembled from soluble precursors. The lag between the synthesis and assembly of the 145,000-molecular-weight NFP is not related to its phosphorylation because its initial incorporation into the cytoskeleton occurs prior to its phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochalasin D alters the rate of synthesis of some HEp‐2 cytoskeletal proteins

The most abundant proteins of HEp-2 cells were resolved by two-dimensional gel electrophoresis. The protein spots corresponding to several cytoskeletal proteins (vimentin, alpha-tubulin, beta-tubulin, alpha-actinin, tropomyosins, and cytokeratins) were identified by comigration with protein markers or by immunological techniques. After treatment of HEp-2 cells with 0.2 microM or 2.0 microM cytochalasin D for 20 h, radioautograms of two-dimensional gel patterns of lysates from cells pulse-labeled with [35S]methionine indicated that the drug altered the rate of synthesis of some proteins. The relative rate of synthesis of the identified cytoskeletal proteins was measured. Synthesis of alpha-actinin, the higher-molecular-mass pair of tropomyosins and actin were similarly increased with cytochalasin D treatment, suggesting coordinate induction. Vimentin and tubulin synthesis was depressed. One cytokeratin exhibited an increase in synthesis comparable to actin, another was increased to a lesser extent and one was decreased.

Tubulin synthesis in the regenerating rat superior cervical ganglion: a biphasic response.

Axotomy of a major cranial post ganglionic branch of the superior cervical ganglion of an adult rat resulted in increased radiolabeling of tubulin and microtubule-associated proteins after an in vitro 5-h incubation with [14C]-methionine. The increased incorporation of the radiolabel began at day 1 postoperatively and exhibited a biphasic response, peaking at days 3 and 8.

Thyroidal induction of tubulin in brain development — identification of the target cell

Exposure of organ cultures of newborn rat brains to tri-iodothyronine (T3) followed by cell fractionation as well as direct exposure of prefractionated neuronal (N) and glial (G) cells to the hormone results in an almost selective induction of tubulin in the glial cells. This is established from two independent assays of tubulin, viz. colchicine binding and vinblastin precipitation. In the newborn rat brain, the tubulin content of the G cells is almost 3-fold higher than that of the N cells. Treatment with T3 elicits 40–50% stimulation of tubulin in the G cells within 2 hr without any significant increase in the N cells. Brains from 8- or 50-day-old rats are irresponsive to induction to tubulin by T3. The rate of incorporation of [3H]leucine into total protein is very similar in both N and G cells of newborn rat brain but that into tubulin of G cells is about 3-fold higher than that of N cells. T3 promotes this incorporation by over 30% in the G cells with only a marginal 5% increase in the N cells. The overall results suggest that the glial cells represent the target cells for the T3-induced synthesis of tubulin, the major structural protein of the developing brain.

Retention of autoregulatory control of tubulin synthesis in cytoplasts: demonstration of a cytoplasmic mechanism that regulates the level of tubulin expression.

Virtually all animal cells rapidly and specifically depress synthesis of new alpha- and beta-tubulin polypeptides in response to microtubule inhibitors that increase the pool of depolymerized subunits, or in response to direct elevation of the cellular tubulin subunit content through microinjection of exogenous tubulin subunits. Collectively, these previous findings have documented the presence of an apparent eucaryotic, autoregulatory control mechanism that specifies the level of expression of tubulin in cultured animal cells. Mechanistically, this regulation of tubulin synthesis is achieved through modulation of tubulin mRNA levels. To dissect further the molecular pathway that underlies this autoregulatory phenomenon, we have now investigated whether enucleated cells still retain the requisite regulatory machinery with which to alter tubulin synthetic levels in response to fluctuations in the pool size of unpolymerized tubulin subunits. Using two-dimensional gel electrophoresis to analyze the patterns of new polypeptide synthesis, we have determined that such cytoplasts can indeed respond to drug-induced microtubule depolymerization by specific repression of new beta-tubulin synthesis. Moreover, the response of cytoplasts is, if anything, greater in magnitude than that of whole cells. We conclude that autoregulatory control of beta-tubulin gene expression must derive principally, if not exclusively, from a cytoplasmic control mechanism that modulates beta-tubulin mRNA stability. For alpha-tubulin, although the response of cytoplasts after drug-induced microtubule depolymerization is quantitatively less dramatic than that of whole cells, at least part of the regulatory machinery must also be activated through a cytoplasmic regulatory event.

Autoregulation of tubulin synthesis in hepatocytes and fibroblasts.

Microtubule polymer levels in mouse 3T6 fibroblasts and primary cultures of rat hepatocytes can be manipulated by treatment of cells with long term, low doses of colcemid. Such treatment produces a rather uniform population of cells with microtubules of reduced lengths. Using this system, we demonstrate (a) that the rate of tubulin synthesis is sensitive to small changes (10%) in microtubule polymer mass and (b) that the percent of inhibition of synthesis is proportional to the level of soluble tubulin. Experiments with hepatocytes indicate that not only synthesis but the stability of tubulin protein was also regulated to maintain a specific level of tubulin. Treatment of hepatocytes with colcemid or other microtubule-depolymerizing drugs reduced the half-life of tubulin from 50 to 2 h, whereas taxol, which stabilizes microtubules, increased the half-life. To assess the consequences of altering microtubule polymer mass, we have analyzed the effect of controlled depolymerization of microtubules in rat hepatocytes on the processing of endocytosed ligands and found it sensitive to small changes in microtubule polymer levels.

An analysis of protein synthesis patterns during early embryogenesis of the urodele - Ambystoma mexicanum

Changes in protein synthesis during early Ambystoma mexicanum (axolotl) embryogenesis were monitored using two-dimensional (2-D) polyacrylamide gel electrophoresis. No change in synthesis patterns during progesterone-induced oocyte maturation was detected. In oocytes matured in vivo (unfertilized eggs), however, the synthesis of several oogenetic proteins ceased, only to be resumed later in development. At fertilization, one novel non-oogenetic protein was found. A cleavage-specific protein was also detected. Dramatic changes in protein synthesis patterns were detected at gastrulation in axolotl embryos. About 10% of the proteins synthesized at earlier stages ceased synthesis at gastrulation. Another 10% of the proteins synthesized during gastrulation were novel. A gastrulation-specific protein was also detected. After gastrulation additional novel non-oogenetic proteins were synthesized for most stages examined. A pronounced increase in the number of novel proteins synthesized was observed at the onset of neurulation and during neural fold fusion. Some of those proteins were specific to dorsal or axial structure tissue (AST) and some were specific to ventral or non-axial structure tissue (NAST). Actin and tubulin synthesis was also monitored during axolotl development. While the cytoplasmic gamma- and beta-actins were synthesized at all stages, muscle-specific alpha-actin synthesis began at the head-process stage (stage 23/25).

Autoregulation of tubulin synthesis in enucleated cells.

The effects on tubulin messenger RNA levels and tubulin protein synthesis of treating cells with microtubule-depolymerizing drugs or directly microinjecting cells with tubulin have suggested that non-polymerized tubulin depresses its own synthesis. The precise level of this control is unclear. It has been shown that enucleated cells, termed cytoplasts, retain many properties of the original cell, including maintenance of cell shape, pinocytic activity and locomotion as well as biosynthetic activities such as protein synthesis and replication of cytoplasmic viruses. Furthermore, cytoplasts retain most of the components of the cytoskeleton including the centrioles. If cytoplasmic activities alone are responsible for regulating tubulin biosynthesis, cytoplasts should contain the necessary components. To distinguish between regulation which would occur in the nucleus, that is, alterations in mRNA synthesis or modifications of the mRNA, from alterations in mRNA stability and/or translatability which would take place in the cytoplasm, we examined the autoregulation of tubulin synthesis in enucleated cells. Here, we report that enucleated mouse fibroblasts retain the ability to turn off tubulin protein synthesis in response to microtubule depolymerization, the reduction in tubulin synthesis being accompanied by a corresponding decrease in tubulin mRNA levels. Thus, transcription, processing and transport of tubulin mRNA from the nucleus are not likely to be the loci of regulation. Instead, tubulin must reduce, either directly or indirectly, the translatability of its own mRNA.

Induction of brain tubulin by triidothyronine: dual effect of the hormone on the synthesis and turnover of the protein

The relative effects of triiodothyronine (T 3) on the synthesis and turnover of tubulin in the developing rat and chick brain have been examined. Measurements of rates of turnover of radiolabeled tubulin in the hormone-sensitive tissues show that the half-life of tubulin turnover is 4–6 h in the absence of T 3 and 10–12 h in the presence of the hormone. Analysis of short-term kinetic data on the stimulation of tubulin by T 3 in the chick brain show that the rapid induction is due to a dual effect of the hormone on the metabolism of tubulin —an increase in the rate of synthesis as well as a decline in its rate of turnover.

Reconstruction of appropriate tubulin and actin gene regulation after transient transfection of cloned beta-tubulin and beta-actin genes.

Most animal cells rapidly depress the synthesis of new alpha- and beta-tubulin polypeptides in response to microtubule inhibitors that increase the pool of depolymerized subunits. This apparent autoregulatory control of tubulin synthesis is achieved through the modulation of tubulin mRNA levels. To begin to analyze the molecular mechanism responsible for such regulation, we have introduced exogenous beta-tubulin gene sequences into cultured mouse cells by DEAE-dextran-mediated DNA transfection. We find that the heterologous tubulin genes are expressed and that their RNA transcripts are accurately processed to mature mRNAs. Moreover, after drug-induced microtubule depolymerization, the expression of unintegrated tubulin gene sequences is regulated coordinately with the endogenous mouse alpha- and beta-tubulin RNA transcripts. Such regulation appears to be specific for transfected tubulin genes, since similar down-regulation is not observed in a contransfected beta-actin gene. Curiously, in response to microtubule depolymerization, the amount of RNA transcripts from a transfected beta-actin gene increases twofold, which qualitatively and quantitatively parallels that seen by the RNAs encoded by the endogenous actin genes. Thus, the transient DNA transfection approach may permit the unambiguous elucidation of regulatory sequences involved in establishing the proper level of expression of these two important cytoskeletal gene families.

Reconstruction of appropriate tubulin and actin gene regulation after transient transfection of cloned beta-tubulin and beta-actin genes.

Most animal cells rapidly depress the synthesis of new alpha- and beta-tubulin polypeptides in response to microtubule inhibitors that increase the pool of depolymerized subunits. This apparent autoregulatory control of tubulin synthesis is achieved through the modulation of tubulin mRNA levels. To begin to analyze the molecular mechanism responsible for such regulation, we have introduced exogenous beta-tubulin gene sequences into cultured mouse cells by DEAE-dextran-mediated DNA transfection. We find that the heterologous tubulin genes are expressed and that their RNA transcripts are accurately processed to mature mRNAs. Moreover, after drug-induced microtubule depolymerization, the expression of unintegrated tubulin gene sequences is regulated coordinately with the endogenous mouse alpha- and beta-tubulin RNA transcripts. Such regulation appears to be specific for transfected tubulin genes, since similar down-regulation is not observed in a contransfected beta-actin gene. Curiously, in response to microtubule depolymerization, the amount of RNA transcripts from a transfected beta-actin gene increases twofold, which qualitatively and quantitatively parallels that seen by the RNAs encoded by the endogenous actin genes. Thus, the transient DNA transfection approach may permit the unambiguous elucidation of regulatory sequences involved in establishing the proper level of expression of these two important cytoskeletal gene families.

Actin and tubulin in Tetrahymena.

Tubulin and actin are cytoskeletal proteins known to play a major role in dividing cells. Tetrahymena pyriformis, a ciliated protozoan, was used as a model system for investigating tubulin synthesis during cilia regeneration and during the cell cycle. Until recently the identification of actin in Tetrahymena has been controversial. In this report evidence for the presence of actin in Tetrahymena is reviewed and control of actin gene expression during the cell cycle is discussed.

The effect of heat shock on the cell cycle regulation of tubulin expression in Physarum polycephalum.

In the myxomycete Physarum polycephalum, tubulin synthesis is subject to mitotic cycle control. Virtually all tubulin synthesis is limited to a 2-h period immediately preceding mitosis, and the peak of tubulin protein synthesis is accompanied by a parallel increase in the level of tubulin mRNA. The mechanism by which the accumulation of tubulin mRNA is turned on and off is not clear. To probe the relationship between tubulin regulation and cell cycle controls, we have used heat shocks to delay mitosis and have followed the pattern of tubulin synthesis during these delays. Two peaks of tubulin synthesis are observed after a heat shock. One occurs at a time when synthesis would have occurred without a heat shock, and a second peak immediately precedes the eventual delayed mitosis. These results are clearly due to altered cell cycle regulation. No mitotic activity is detected in delayed plasmodia at the time of the control mitosis, and tubulin behavior is shown to be clearly distinct from that of heat shock proteins. We believe that the tubulin family of proteins is subject to regulation by a thermolabile mitotic control mechanism but that once the cell has been committed to a round of tubulin synthesis the "tubulin clock" runs independently of the heat sensitive system. In delayed plasmodia, the second peak of synthesis may be turned on by a repeat of the commitment event.

Gene amplification-associated cytogenetic aberrations and protein changes in vincristine-resistant Chinese hamster, mouse, and human cells.

We carried out cytogenetic studies of four Chinese hamster, mouse, and human cell lines selected for high levels of resistance (500- to 4,000-fold) to vincristine (VCR) by a multistep selection procedure. All cells examined contained gene amplification-associated metaphase chromosome abnormalities, either homogeneously staining regions (HSRs), abnormally banding regions (ABRs), or double-minute chromosomes (DMs); control actinomycin D- and daunorubicin-resistant hamster lines did not exhibit this type of chromosomal abnormality. VCR-resistant Chinese hamster sublines exhibited both increased synthesis of the protein V19 (Mr 19,000; pl = 5.7) and increased concentrations of V19 polysomal mRNA. When VCR-resistant cells were grown in drug-free medium, level of resistance, synthesis of V19, and amount of V19 mRNA declined in parallel with mean length of the HSR or mean number of DMs per cell. Cross-resistance studies indicate that VCR-resistant cells have increased resistance both to antimitotic agents and to a wide variety of agents unrelated to VCR in chemical structure and/or mechanism of action. Our studies of tubulin synthesis in Chinese hamster cells indicate no overproduction of tubulin or presence of a mutant tubulin species. Comparison with antifolate-resistant Chinese hamster cells known to contain amplified dihydrofolate reductase genes localized to HSRs or ABRs strongly suggests that the HSRs, ABRs, or DMs of the Vinca alkaloid-resistant sublines likewise represent cytological manifestations of specifically amplified genes, possibly encoding V19, involved in development of resistance to VCR.

Tubulin synthesis and auxin-induced root initiation in phaseolus

The auxin-induced formation of roots in the hypocotyls of Phaseolus vulgaris can be prevented by treatment with actinomycin D, colchicine or cytochalasin B if applied within 40 hr of initiation. Shortly after auxin pretreatment, there is an increase in translatable messenger RNA activity. Analysis of the labelled cell-free products indicate, among other changes, a striking increase in a protein co-migrating with tubulin, in the case of RNA isolated from indolebutyric acid (IBA) pretreated hypocotyls. An increase in tubulin content in vivo can also be demonstrated on the basis of SDS-polyacrylamide gel analysis of membrane proteins and functional assays for tubulin polymerization. An increase in the synthesis of tubulin in vivo can also be demonstrated after IBA pretreatment. In addition, the auxin is also able to promote tubulin polymerization when added in vitro. It is suggested that tubulin synthesis and microtubule assembly are early events in auxin-mediated root differentiation.

Abundance of tubulin mRNA on polysomes following deciliation and during the synchronous cell cycle of Tetrahymena.

The protozoan, Tetrahymena pyriformis GL, was used as a model system for studying polysomal mRNA during the cell cycle and during cilia regeneration. Our previous work has shown a substantial induction of tubulin synthesis following deciliation and during G2 of the synchronous cell cycle. In the present study, the abundance of tubulin mRNA on polysomes was examined in order to determine whether more tubulin mRNA was being translated during the periods of peak tubulin synthesis. Polysomes isolated at sequential times following deciliation and during the synchronous cell cycle were translated in a cell-free translation system derived from wheat germ. The abundance of tubulin mRNA on polysomes was inferred from the amount of tubulin translated in vitro. Following deciliation and prior to the peak period of tubulin synthesis, the abundance of tubulin mRNA (at 140 min post-deciliation) increases to 25 times the initial value observed (at 20 min post-deciliation). Since the increase in tubulin mRNA abundance precedes the peak in tubulin synthesis, induction of tubulin synthesis appears to be mRNA-dependent. A similar analysis of tubulin mRNA abundance on polysomes during the synchronous cell cycle revealed a peak of tubulin mRNA prior to each peak of tubulin synthesis. These studies suggest that the periodic fluctuations in the synthesis of tubulin are dependent upon fluctuating levels of tubulin mRNA on polysomes.

Synthesis of putative microtubule-associated proteins by mouse blastocysts during early outgrowth in vitro.

The outgrowth of mouse trophoblast in culture provides a simplified model system analogous in certain ways to blastocyst implantation in vivo. Day-four blastocysts cultured for 3 days in vitro undergo extensive changes in cell shape and motility which are likely to involve the complex cytoskeletal system of the trophoblast cells. To explore the biochemical basis of these changes, one set of cytoskeletal proteins, the microtubule-associated proteins (MAPs), was studied. Day 4 blastocysts were labeled with [35S]methionine and blastocyst outgrowths, after 3 days in culture from the blastocyst stage, were labeled with [3H]methionine. Labeled embryos were disrupted and the soluble supernatants were pooled, and newly synthesized proteins from the two stages were coassembled with taxol-stabilized brain microtubule polymer enriched for MAP-binding sites. Double-labeled coassembly proteins (putative MAPS) were then released from the microtubule polymer by treatment with 0.35 M NaCl and analyzed by one-dimensional polyacrylamide gel electrophoresis. 3H/35S dpm ratios were determined for individual protein bands to compare the relative synthesis rates for day 4 blastocyst and day 3 outgrowth MAPs. In spite of the extensive changes in cell shape and motility associated with blastocyst outgrowth, a common set of putative MAPs characterizes the two stages investigated, including several in the size range of tau factors. No synthesis of high molecular weight MAPs comparable with MAP 1 or MAP 2 from brain was detected. The synthesis rates of individual MAPs relative to each other remain constant over this period and are likely coordinated with total protein and tubulin synthesis.