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.     

Mechanisms of regulating tubulin synthesis in cultured mammalian cells.

Colchicine and nocadazole both depolymerize microtubules in cultured fibroblasts and lead to a rapid inhibition of tubulin synthesis. The level of translatable tubulin mRNA is greatly reduced in drug-treated cells as demonstrated by translation in a reticulocyte-derived in vitro protein synthesizing system. A model of tubulin synthesis regulation is proposed in which the elevated level of unpolymerized tubulin in drug-treated cells inhibits the formation of new tubulin mRNA and the preexisting message decays rapidly. In agreement with this model, tubulin message is found to be short-lived and has an approximately 2 hr half-life in cells treated with actinomycin D. Another prediction of the proposed model is that destabilization of microtubules without a concomitant increase in free tubulin will not inhibit tubulin synthesis. Vinblastine also disrupts microtubules but leads to the aggregation of tubulin into large paracrystals with an apparent decrease in the concentration of free tubulin. This drug does not inhibit tubulin production but rather leads to a measurable enhancement of tubulin synthesis.     

Synthesis and Turnover of Embryonic Sea Urchin Ciliary Proteins during Selective Inhibition of Tubulin Synthesis and Assembly

When ciliogenesis first occurs in sea urchin embryos, the major building block proteins, tubulin and dynein, exist in substantial pools, but most 9+2 architectural proteins must be synthesized de novo. Pulse-chase labeling with [³H]leucine demonstrates that these proteins are coordinately up-regulated in response to deciliation so that regeneration ensues and the tubulin and dynein pools are replenished. Protein labeling and incorporation into already-assembled cilia is high, indicating constitutive ciliary gene expression and steady-state turnover. To determine whether either the synthesis of tubulin or the size of its available pool is coupled to the synthesis or turnover of the other 9+2 proteins in some feedback manner, fully-ciliated mid- or late-gastrula stage Strongylocentrotus droebachiensis embryos were pulse labeled in the presence of colchicine or taxol at concentrations that block ciliary growth. As a consequence of tubulin autoregulation mediated by increased free tubulin, no labeling of ciliary tubulin occurred in colchicine-treated embryos. However, most other proteins were labeled and incorporated into steady-state cilia at near-control levels in the presence of colchicine or taxol. With taxol, tubulin was labeled as well. An axoneme-associated 78 kDa cognate of the molecular chaperone HSP70 correlated with length during regeneration; neither colchicine nor taxol influenced the association of this protein in steady-state cilia. These data indicate that 1) ciliary protein synthesis and turnover is independent of tubulin synthesis or tubulin pool size; 2) steady-state incorporation of labeled proteins cannot be due to formation or elongation of cilia; 3) substantial tubulin exchange takes place in fully-motile cilia; and 4) chaperone presence and association in steady-state cilia is independent of background ciliogenesis, tubulin synthesis, and tubulin assembly state.     

The abundance of alpha-tubulin mRNA increases during ciliary regeneration in Tetrahymena, and this occurs independently of the soluble tubulin content

Control mechanisms of tubulin synthesis are analyzed during ciliary regeneration of the ciliate Tetrahymena. Titration of the alpha-tubulin mRNA concentrations during the regeneration period reveal that enhancement of tubulin synthesis is preceded and accompanied by increased concentrations of tubulin mRNA molecules. Stimulation of tubulin synthesis is independent of the pool size of soluble tubulin molecules, as suggested by at least two independent lines of evidence: First, like cells of normal phenotype a temperature sensitive size mutant enhances tubulin synthesis as well as tubulin mRNA concentration during ciliary regeneration, although these large mutant cells have a much higher concentration and amount of soluble tubulin molecules in the cytoplasm. Second, slowly regenerating cells of normal phenotype shift-up their concentration of tubulin mRNA molecules already before a time, when ciliary outgrowth might cause a significant depletion of the pool of soluble tubulin molecules. Thus, neither an induction of tubulin synthesis nor an increase in tubulin mRNA molecules is mediated via changes in the pool size of soluble tubulin molecules.     

Microtubule assembly in cultivated Greene melanoma cells is stimulated by dibutyryl adenosine 3'':5''-cyclic monophosphate or cholera toxin

Both dibutyryl cyclic AMP (DBcAMP) and cholera toxin promote the formation and elongation of processes of cultivated Greene hamster melanoma cells. The formation and maintenance of these processes, which contain many microtubules, are sensitive to colcemid and vinblastine. Tubulin was measured by [3H]colchicine binding and by acrylamide gel electrophoresis. We found that DBcAMP or cholera toxin increases the ratio of polymerized to unpolymerized tubulin but not the total amount of tubulin per cell. The sum of the lengths of microtubules per unit area was significantly greater in cells treated with DBcAMP than in control cells. Our findings support the hypothesis that cyclic AMP promotes the elongation of cell processes by stimulating the assembly of microtubules from existing tubulin.     

Microtubule formation from maternal tubulins during sea urchin embryogenesis: measurement of soluble and insoluble tubulin pools

The mass of tubulin protein in developing embryos of the sea urchin Lytechinus pictus was measured using a radiodilution immunoassay based on densitometric analysis of immunoprecipitated tubulins resolved electrophoretically. The tubulins constitute an average of 360 +/- 35 pg per egg, or 0.66% of the total protein, and there is no significant change in their concentration during embryogenesis. The masses of soluble and polymerized tubulin were measured for extracts prepared under conditions that stabilize microtubules. In eggs, a maximum of 14% of the tubulin is insoluble, and this increases throughout embryogenesis to 67% at pluteus stage (72 hr). The concentration of tubulin in eggs is at least 500 micrograms/ml, well above the critical concentration for tubulin assembly in vitro, yet microtubules have not been observed in eggs. The mass of newly synthesized tubulin, estimated from the mass of tubulin mRNA per embryo, accounts for a small fraction of the total tubulin by the end of gastrulation but for over half of the tubulin by the 72-hr pluteus stage. These observations are consistent with a model in which the declining level of unpolymerized tubulin controls the stability of tubulin mRNa, providing an autogenous regulation of the ontogenetic pattern of tubulin synthesis during sea urchin embryogenesis (Gong and Brandhorst, Development 102: 31-43).     

Autoregulation of tubulin expression is achieved through specific degradation of polysomal tubulin mRNAs

We have utilized protein synthesis inhibitors to investigate the autoregulatory mechanism that uses the concentration of unpolymerized tubulin subunits to specify tubulin mRNA content in animal cells. Puromycin and pactamycin, both of which remove RNAs from polysomes, completely unlink tubulin RNA content from the level of free subunits, whereas pretreatment of cells with cycloheximide, which traps mRNAs onto stalled polyribosomes, enhances the specific degradation of tubulin RNAs in response to increases in the subunit content. Moreover, in the absence of protein synthesis inhibitors, the tubulin RNAs that are lost from cells with elevated free tubulin subunit levels are those that are associated with polyribosomes. Further, beta-tubulin mRNAs encoding a truncated translation product of only 26 amino acids (and that cannot be polyribosomal) are not substrates for autoregulation. We conclude that autoregulation of tubulin synthesis is achieved by specifically altering the stability of tubulin RNAs that are bound to polyribosomes.     

Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine

At metaphase, the amount of tubulin assembled into spindle microtubules is relatively constant; the rate of tubulin association equals the rate of dissociation. To measure the intrinsic rate of dissociation, we microinjected high concentrations of colchicine, or its derivative colcemid, into sea urchin embryos at metaphase to bind the free tubulin, thereby rapidly blocking polymerization. The rate of microtubule disassembly was measured from a calibrated video signal by the change in birefringent retardation (BR). After an initial delay after injection of colchicine or colcemid at final intracellular concentrations of 0.1-3.0 mM, BR decreased rapidly and simultaneously throughout the central spindle and aster. Measured BR in the central half-spindle decreased exponentially to 10% of its initial value within a characteristic period of approximately 20 s; the rate constant, k = 0.11 +/- 0.023 s-1, and the corresponding half-time, t 1/2, of BR decay was approximately 6.5 +/- 1.1 s in this concentration range. Below 0.1 mM colchicine or colcemid, the rate at which BR decreased was concentration dependent. Electron micrographs showed that the rapid decrease in BR corresponded to the disappearance of nonkinetochore microtubules; kinetochore fiber microtubules were differentially stable. As a control, lumicolchicine, which does not bind to tubulin with high affinity, was shown to have no effect on spindle BR at intracellular concentrations of 0.5 mM. If colchicine and colcemid block only polymerization, then the initial rate of tubulin dissociation from nonkinetochore spindle microtubules is in the range of 180-992 dimers per second. This range of rates is based on k = 11% of the initial polymer per second and an estimate from electron micrographs that the average length of a half-spindle microtubule is 1- 5.5 micron. Much slower rates of tubulin association are predicted from the characteristics of end-dependent microtubule assembly measured previously in vitro when the association rate constant is corrected for the lower rate of tubulin diffusion in the embryo cytoplasm. Various possibilities for this discrepancy are discussed.     

Rapid Degradation of Newly Synthesized Tubulin in Lithium-Treated Sensory Neurons

When cultured chick sensory neurons were labeled with [35S]methionine for 1 h or longer in the presence of 5-25 mM LiCl, we found a dose-dependent reduction in the level of radiolabeled tubulin, to one third of control levels, with no noticeable effect on other proteins. The magnitude of this response was identical after a 1-h or 72-h preincubation in 25 mM LiCl and returned to control values within 1 h after removal of LiCl. Short (5-min) pulse-chase experiments revealed that tubulin synthesis was not affected by Li+, but that newly synthesized tubulin was rapidly degraded, such that 50% of the labeled beta-tubulin was lost within 5 min. There was no enhanced degradation of tubulin present before exposure to Li+. Addition of LiCl at various times before and after a 10-min pulse suggested that tubulin becomes completely refractory to Li(+)-induced degradation within 10 min after translation. Although Li+ treatment resulted in a decrease in the fraction of extant tubulin present in the unassembled form, the Li(+)-induced degradation of nascent tubulin is not a consequence of shifts in assembly state, because colcemid or taxol treatment did not lead to rapid degradation of newly synthesized tubulin, and neither drug altered the response to Li+. We suggest that Li+ interferes with the correct folding of tubulin polypeptides, exposing sites, normally hidden, to the action of a protease(s).     

Primary cilia cycle in PtK1 cells: effects of colcemid and taxol on cilia formation and resorption

The effects of colcemid (0.16-1.0 microM) and taxol (10 microM) on the primary cilia cycle in PtK1 cells were studied by antitubulin immunofluorescence microscopy and by high-voltage electron microscopy of serial 0.25-micron sections. Although these drugs induce a fully characteristic rearrangement (taxol) or disassembly (colcemid) of cytoplasmic microtubules, neither affects the structure of primary cilia formed prior to the treatment or the resorption of primary cilia during the initial stages of mitosis. Cells arrested in mitosis by taxol or colcemid remain in mitosis for 5-7 h at 37 degrees C and then form 4N "micronucleated" restitution nuclei. Formation of primary cilia in these micronucleated cells is blocked by colcemid in a concentration-dependent fashion: normal cilia with expanded (ie, bulbed) distal ends form at the lower (0.16-0.25 microM) concentrations, while both cilia formation and centriole replication are inhibited at the higher (greater than or equal to 1.0 microM) concentrations. However, even in the presence of 1.0 microM colcemid, existing centrioles acquire the appendages characteristically associated with ciliating centrioles and attach to the dorsal cell surface. Continuous treatment with colcemid thus produces a population of cells enriched for the early stages of primary cilia formation. Micronucleated cells formed from a continuous taxol treatment contain two normal centriole pairs, and one or both parenting centrioles possess a primary cilium. Taxol, which has been reported to stabilize microtubules in vitro, does not inhibit the cell-cycle-dependent assembly and disassembly of axonemal microtubules in vivo.     

Modulation of tubulin mRNA levels by interferon in human lymphoblastoid cells.

Blot hybridization with labeled tubulin cDNA showed that treatment of Ramos cells, a human cell line of lymphoblastoid origin, with either alpha or beta interferon (IFN) induced a marked increase in the amount of tubulin mRNA sequences. The level of tubulin mRNA sequences increased rapidly after exposure of cells to IFN-alpha and reached a maximum after 1 h of treatment, which was four times the control level. Treatment with IFN-beta induced a maximal increase after 4 h; the amount of tubulin mRNA sequences was seven times higher than the control level. The mRNA extracted from IFN-treated and nontreated cells was translated in vitro in a reticulocyte lysate cell-free system containing [35S]methionine. Electrophoretic analysis of the labeled cell-free products showed an increase in the amount of translatable tubulin mRNA that parallels the time course of induction of tubulin mRNA sequences. Two-dimensional gel electrophoresis of the labeled protein products directed by mRNA indicates that IFN caused a more pronounced increase in the level of alpha-tubulin than beta-tubulin mRNA. Treatment with colchicine, which disrupts the cell microtubules, caused a marked decrease in the tubulin mRNA content. Concomitant treatment of the cells with colchicine and IFN abolished the interferon-dependent induction of tubulin mRNA.     

Inhibitory effect of taxol, a microtubule stabilizing agent, on induction of DNA synthesis is dependent upon cell lines and growth factors.

Growth-arrested rat fibroblasts, 3Y1, and human diploid fibroblasts, TIG-1, were induced to synthesize DNA by stimulation with various agents such as fetal bovine serum (FBS), epidermal growth factor (EGF), colcemid, or colchicine. Taxol, a microtubule-stabilizing agent, blocked the induction of DNA synthesis after stimulation with colcemid or colchicine in both cell lines. Taxol inhibited the induction of DNA synthesis after stimulation with FBS or EGF in TIG-1, but did not in 3Y1. 12-O-tetradecanoylphorbol-13-acetate (TPA) induced DNA synthesis in TIG-1, which was reduced only partly by taxol. Taxol stabilized or polymerized microtubules in both cell lines. These results indicate that the inhibitory effect of taxol on the induction of DNA synthesis varied among cell lines and among growth factors, and suggest that signal transduction processes may be differentiated by taxol sensitivity. In TIG-1 cells, when taxol was added within 6 h, about halfway into the initiation of DNA synthesis after the addition of FBS or EGF, the inhibition of DNA synthesis still occurred. Taxol did not inhibit the induction of c-fos and c-myc genes by FBS or EGF stimulation. Colchicine itself did not induce these genes in TIG-1. Thus, taxol appeared to inhibit the induction of DNA synthesis not by blockage in the early transduction process of the growth signal from the cell surface to nuclei but by blockage in processes operating in the mid- or late-prereplicative phase.     

Taxol inhibits stimulation of cell DNA synthesis by human cytomegalovirus

The microtubule (MT)-stabilizing drug, taxol, inhibited human cytomegalovirus (CMV)-initiated cell DNA synthesis by up to 100% in serum-arrested mouse embryo (ME) fibroblasts that were abortively infected by CMV. Taxol concentrations known to increase MT polymerization and to stabilize existing MTs (10 to 20 micrograms/ml) blocked CMV-stimulated cell DNA synthesis, while taxol concentrations of 2.5 micrograms/ml, or less, did not. Taxol maximally inhibited CMV initiation of cell DNA synthesis when added 3 h after virus infection and inhibited this initiation by greater than 50% when added up to 12 h after CMV infection. Control experiments suggest that taxol specifically inhibited CMV-stimulated cell DNA synthesis. Pretreatment of CMV stock with taxol did not reduce the stimulatory effect of CMV on cell DNA synthesis and taxol had no detectable effect on CMV-specific early protein synthesis. Moreover, taxol did not appear to alter thymidine pool sizes, affect cell viability, or compromise the DNA synthetic machinery in CMV-infected cells. Since taxol increases tubulin polymerization and inhibits MT disassembly, these results suggest that dynamic changes in MTs or in the pool of free tubulin subunits are necessary for CMV to stimulate cell entry into a proliferative cycle.     

Anion-induced increases in the affinity of colcemid binding to tubulin

Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37 degrees C the association rate constant for colcemid binding is 1.88 X 10(6) M-1 h-1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high-affinity site (Ka = 0.7 X 10(5) M-1 at 37 degrees C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low-affinity site (Kb = 1.2 X 10(4) M-1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high-affinity site) and the reaction is entropy driven (163 J K-1 mol-1). Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower-affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower-affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine. We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B-ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher-affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.