Tubulin Pool Research Articles

Assembly and disassembly of axonal microtubules of the toad Xenopus laevis under the effect of temperature.

In toads Xenopus laevis living at 11 degrees (winter), the microtubular density of 4-microns myelinated axons of lumbosacral nerves was assessed with the electron microscope. In controls, the density was 11.2 microtubules/microns2. In nerves incubated at 0 degrees, microtubules decreased following a simple exponential curve with a half time of 4.7 min (k = 0.149 min-1); residual microtubules were 4.5%. After rewarming, the full complement of microtubules reappeared within 60 min. In steady state, the microtubular density exhibited a linear relationship with temperature (range: 0-22 degrees; slope 0.94 microtubules/microns 2 per degree; r, 0.96). After heating the nerve by 11 degrees above the physiological temperature, microtubules increased by 83%, whereby the pool of unpolymerized tubulin was at least 2.7 mg/ml of axoplasm. A seasonal variation of the microtubular density was observed which accorded with the environmental temperature. The macroscopic kinetics of microtubule disassembly in the axoplasm is similar to that reported for purified tubulin but that of assembly is slower. Microtubules of peripheral axons of Xenopus are cold-labile and vary during the annual cycle.

Cytoplasmic microtubular dynamics and chromatin organization during mammalian oogenesis and oocyte maturation

A chronological series of coordinated alterations in oocyte chromosome and microtubule disposition occur during oogenesis and oocyte maturation in the mammal. Timely transitions in meiotic spindle and cytoplasmic microtubules, due to modifications in both the assembly competence of the tubulin pool and nucleation capacity of centrosomes, underscore key nuclear events during the progressive stages of meiosis I and II. The regulation of these transitional states during meiosis is discussed with respect to hormonal influences imparted to the oocyte within the follicular microenvironment, and the possible ways in which environmental perturbations may result in defective chromosomal partitioning during meiosis.

Particulate Tubulin in Interphase and Metaphase Extracts of Oocytes of Spisula.

In 1972 Weisenberg reported that surf clam oocytes contained a particulate and sedimentable pool of tubulin that could be isolated in buffers containing hexylene glycol. This "interphase particulate tubulin" (IPT) copurified with 10-20 {mu}m granular spheres, which were identified as the "tubulin-containing structures" (TCS). Approximately one TCS per oocyte was isolated and the TCS disappeared after nuclear envelope breakdown. Weisenberg postulated that the TCS was comprised of a stored form of tubulin or a microtubule-assembly intermediate. To characterize this intriguing form of stored tubulin, IPT was isolated in hexylene glycol-containing buffers as described by Weisenberg (1972, J. Cell Biol. 54, 266-278) and the amount of sedimentable tubulin was quantitated by immunoblotting during the first meiotic cell cycle. Approximately 10% of the total tubulin in Spisula oocytes sediments at g forces that are too small to pellet tubulin dimers or even single microtubules. Granular spheres, approximately 15 {mu}m in diameter, are present in the sedimentable tubulin fractions. During the first cell cycle, the granular spheres disappear while the sedimentable tubulin levels gradually decrease. Although the disappearance of the spheres corresponds with the loss of sedimentable tubulin, the spheres do not contain tubulin. An initial centrifugation of the oocyte homogenates at 650g leaves most of the tubulin in the supernatant and the granular spheres in the pellet. The tubulin-containing fractions are composed of membranes and an amorphous unidentified material associated with short microtubules. Sedimentable tubulin is not detected in homogenates prepared at 0{deg}C or in the absence of hexylene glycol, conditions that favor microtubule disassembly. It is likely that sedimentable tubulin is composed of hexylene glycol-induced polymers and not unique particulate structures that sequester tubulin. Finally, the granular spheres that contaminate the tubulin preparations are identified as nucleoli. They are morphologically identical to the nucleoli of the intact oocyte and they fluoresce brightly when stained with the Hoechst DNA dye 22358.

Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/105 cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.

Alterations to the cytoskeleton of erythrocytes infected with frog erythrocytic virus: a fluorescence and electron microscopic study.

Erythrocytes of bullfrogs (Rana catesbeiana) infected with frog erythrocytic virus are spheroid and their nucleus is displaced. In contrast, uninfected cells are ellipsoid and have a centralized nucleus. Fluorescent staining revealed that these changes are correlated with alterations to components of the erythrocyte cytoskeleton. Uninfected erythrocytes contained a broad, continuous marginal band of microtubules, which appeared thinner and interrupted in infected cells. The described disruption of microtubules was associated with an inability to polymerize the tubulin pool with the addition of 12 microM taxol. The arrangement of submembranous microfilaments in uninfected erythrocytes was not significantly altered in infected cells. Vimentin filaments were distributed throughout the cytoplasm and around the nucleus of uninfected cells, and concentrated at the cell and nuclear peripheries. Cytoplasmic pockets that did not contain vimentin filaments were associated with the viral assembly site(s) in infected cells. These data suggest that the distortion of viral-infected erythrocytes could be due, in part, to an irreversible depolymerization of microtubules of the marginal band and a reorganization of the vimentin filament network.

Thrombin, epidermal growth factor, and phorbol myristate acetate stimulate tubulin polymerization in quiescent cells: a potential link to mitogenesis.

Previous studies suggest that alterations in the microtubule (MT)-tubulin equilibrium during G0/G1 affect mitogenesis. To determine the effect of growth factors on the MT-tubulin equilibrium, we developed a radioactive monoclonal antibody binding assay (Ball et al.: J. Cell. Biol. 103:1033-1041, 1986). With this assay, 3H-Ab 1-1.1 binding to cytoskeletons in confluent populations of cultured cells is proportional to the number of tubulin subunits polymerized into MTs. We now show that purified alpha-thrombin increases 3H-Ab 1-1.1 binding to cytoskeletons of serum-arrested mouse embryo (ME) fibroblasts from 1.5- to 3-fold. This stimulation is dose-dependent and correlates with concentrations of thrombin required for initiation of DNA synthesis. Other mitogenic factors, epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA), also stimulate MT polymerization. Addition of colchicine (0.3 microM) eight hours after growth factor addition, blocks stimulation of 3H-thymidine incorporation by thrombin, EGF, or PMA, suggesting that tubulin polymerization or subsequent events triggered by MT polymerization are required for cells to enter a proliferative cycle. Consistent with models for autoregulation of tubulin synthesis, thrombin, EGF, and PMA all increase tubulin synthesis 9 to 15 hr after growth factor addition, raising the possibility that the decrease in free tubulin and subsequent stimulation of tubulin synthesis is linked to progression of cells into a proliferative cycle. Colchicine addition to these cells also stimulates DNA synthesis, but colchicine-stimulated cells enter S phase 6 to 8 hr later than those stimulated by growth factors. This delayed stimulation may be related to the time required for degradation of tubulin-colchicine complexes below a critical level. These data suggest that regulation of cell proliferation may be linked to increased MT polymerization and the resulting decrease in free tubulin pools.

Influence of suckling on tubulin-dependent GTPase activity in the anterior pituitary lobe of the lactating rat

A GTPase assay was employed to determine the relative proportions of the enzymatic activity in soluble and polymerized tubulin pools in the anterior pituitary lobe of the lactating rat. The GTPase activity in the tubulin fractions was estimated in 25-50 micrograms protein using [gamma-32P]GTP. The liberation of inorganic phosphate (Pi) was proportional to the protein concentration with either of the tubulin fractions. The enzymatic activity appeared to reach equilibrium by 1 min. Antitubulin antibodies inhibited the enzymatic activity in a concentration-dependent manner in both the tubulin fractions; at a final dilution of 1:2000 the antibody maximally inhibited the enzyme activity in both the tubulin fractions by 39-44%. After establishing the optimal conditions for the GTPase assay, the effect of suckling on pituitary GTPase activity was studied. Soluble and polymerized tubulin fractions were prepared from anterior pituitaries obtained from lactating rats killed after suckling for 30, 60, and 90 min; GTPase activity was assayed in both the tubulin fractions in the absence of antitubulin antibody. Compared to the nonsuckled control, suckling for 60 and 90 min stimulated the enzymatic activity in the soluble tubulin fraction by 80% and 44%, respectively (P less than 0.05). The enzymatic activity in the polymerized tubulin fraction increased by 30% at 60 min and decreased by about 20% at 90 min (P less than 0.05). The suckling-stimulated GTPase activity in the two pituitary fractions cannot be attributed to tubulin alone since there are other proteins also capable of hydrolyzing GTP. Therefore, GTPase activity was assayed in the pituitary tubulin fractions in the presence of antitubulin antibody (1:2000 dilution); tubulin-GTPase activity is the difference between the activity assayed in the absence of the antibody and that which was determined in the presence of the antibody. In the soluble tubulin fraction, tubulin-GTPase activity increased by 166% at 30 min suckling (P less than 0.05), decreased by 40% at 60 min (P less than 0.05), and again increased by 148% at 90 min (P less than 0.05). In the polymerized tubulin fraction, the enzyme activity decreased by 82% at 30 min (P less than 0.05), increased by 742% at 60 min (P less than 0.05), and again decreased by 95% at 90 min (P less than 0.05). Thus, an inverse relationship between tubulin-GTPase activities in the two pituitary fractions was observed and provides further evidence in support of our hypothesis that microtubules are recruited to transport PRL granules from the Golgi apparatus to the plasma membrane.

Influence of suckling on tubulin-dependent GTPase activity in the anterior pituitary lobe of the lactating rat.

A GTPase assay was employed to determine the relative proportions of the enzymatic activity in soluble and polymerized tubulin pools in the anterior pituitary lobe of the lactating rat. The GTPase activity in the tubulin fractions was estimated in 25-50 micrograms protein using [gamma-32P]GTP. The liberation of inorganic phosphate (Pi) was proportional to the protein concentration with either of the tubulin fractions. The enzymatic activity appeared to reach equilibrium by 1 min. Antitubulin antibodies inhibited the enzymatic activity in a concentration-dependent manner in both the tubulin fractions; at a final dilution of 1:2000 the antibody maximally inhibited the enzyme activity in both the tubulin fractions by 39-44%. After establishing the optimal conditions for the GTPase assay, the effect of suckling on pituitary GTPase activity was studied. Soluble and polymerized tubulin fractions were prepared from anterior pituitaries obtained from lactating rats killed after suckling for 30, 60, and 90 min; GTPase activity was assayed in both the tubulin fractions in the absence of antitubulin antibody. Compared to the nonsuckled control, suckling for 60 and 90 min stimulated the enzymatic activity in the soluble tubulin fraction by 80% and 44%, respectively (P less than 0.05). The enzymatic activity in the polymerized tubulin fraction increased by 30% at 60 min and decreased by about 20% at 90 min (P less than 0.05). The suckling-stimulated GTPase activity in the two pituitary fractions cannot be attributed to tubulin alone since there are other proteins also capable of hydrolyzing GTP. Therefore, GTPase activity was assayed in the pituitary tubulin fractions in the presence of antitubulin antibody (1:2000 dilution); tubulin-GTPase activity is the difference between the activity assayed in the absence of the antibody and that which was determined in the presence of the antibody. In the soluble tubulin fraction, tubulin-GTPase activity increased by 166% at 30 min suckling (P less than 0.05), decreased by 40% at 60 min (P less than 0.05), and again increased by 148% at 90 min (P less than 0.05). In the polymerized tubulin fraction, the enzyme activity decreased by 82% at 30 min (P less than 0.05), increased by 742% at 60 min (P less than 0.05), and again decreased by 95% at 90 min (P less than 0.05). Thus, an inverse relationship between tubulin-GTPase activities in the two pituitary fractions was observed and provides further evidence in support of our hypothesis that microtubules are recruited to transport PRL granules from the Golgi apparatus to the plasma membrane.

Direct evidence for the occurrence of simple microtubule organizing centers (MTOCs) and for end-to-end joining of microtubules with modified ends in the reticulopodia of Allogromia laticollaris (foraminifera)

The partial disassembly of filopodial microtubules into helical filaments, respectively the reassembly from paracrystals of exactly registered tubulin helices was recognized as a special adaptation to the highly dynamic reticulopodial movements in the foraminifer Allogromia laticollaris [4, 16]. Electron microscopic investigations of partially extracted or completely lysed filopodial strands revealed some additional adaptations to the local tubulin demands within the widely spread, rapidly changing reticulopodial networks. As probable foci for the initiation of additional microtubules from pools of tubulin during the netto growth phase, small (40 × 60 nm) cube-shaped, electron dense bodies were discovered which could act as mobile MTOCs at very distant sites from the metabolically active cell body within the reticulopodial system. The microtubule ends, proximally as well as distally to the filopodial origins, are characterized by a closed appearance because of a terminal cap which is continuous with the microtubule wall, whereas some other microtubules appear as the usual open cylinders. It is suggested that the closed ends, which occur either freely or decorated by the cube-shaped, electron dense bodies, may play a role in the de novo initiation of microtubules as well as in the transformation to the helical state or vice versa. Points of discontinuity within single longitudinally cut microtubules can be observed directly by electron microscopy and correspond to end-to-end contacts of closed MT-terminations. These junctions probably represent initial contact points of an annealing process between adjacent shorter MT-segments. Microtubule annealing occurs rapidly in vitro [14, 15] and could explain the elongation rates observed during filopodial outgrowth.

Evidence for microtubule capture by filopodial actin filaments in growth cones

We have studied, by immunofluorescence, the organization of microtubules in growth cones in dissociated neuronal cell cultures. Growth cones have a large pool of soluble tubulin and in conventionally fixed cultures the entire growth cone, including the filopodia, is labelled with tubulin antibodies, thus obscuring the microtubules. In contrast, in cultures fixed with fixatives containing detergent, the soluble pool of tubulin is removed allowing clear views of the microtubules. On entering the growth cone from the neurite, microtubules splay out and may extend as far as the base of filopodia. In most growth cones, one or more individual microtubules extend into filopodia, aligning with the parallel bundle of microfilaments. This observation suggests one way growth cone motility and neurite advance may be coupled.

Control of microtubule assembly in growth cones

Growth cones are transient structures present at the tips of growing axons and dendrites (neurites). They are crucial to neuronal development because of their pathfinding ability and their role in synaptogenesis (reviewed by Dodd and Jessell, 1988). In the last few years it has become apparent that growth cones are also involved in the assembly of the cytoskeleton of the elongating neurite (reviewed by Gordon-Weeks, 1989, 1991). We are particularly interested in the assembly of microtubules in the growth cone and its importance for neurite advance. The microtubules in the neurite are bundled into fascicles, presumably by the cross-linking action of microtubule-associated proteins (MAPs), and on entering the growth cone they splay out like the ribs of a fan with their 'plus' ends, the ends at which assembly takes place preferentially, oriented distally. Within the growth cone there is a large pool of assembly competent tubulin that provides subunits for microtubule elongation. Several observations point to the existence of precise mechanisms controlling assembly of this soluble tubulin pool. If the control mechanisms are disrupted the ability of the neurite to advance is severely compromised. We have examined the possibility that MAPs are important components in this control mechanism. Many of the known MAPs are present within growth cones, including MAP 1B, MAP 2 and tau. Experiments with neuronal cultures and growth cones isolated as a subcellular fraction from developing rat brain point particularly toward the phosphorylated form of MAP 1B as an essential component in the concerted assembly of microtubules at the growth cone and in particular in the bundling of microtubules in the neurite.

Quantitation of cellular tubulin in microtubules and tubulin pools by a competitive ELISA

A comprehensive method is described for isolating microtubules from cultured mammalian cells and quantitating the tubulin content of both the microtubules and total cellular tubulin pools with a competitive enzyme-linked immunosorbent assay (ELISA). The microtubule isolation procedure involves detergent lysis of cells in a microtubule stabilizing buffer, high speed centrifugation to collect the cytoskeletons, and subsequent solubilization of tubulin from microtubule-containing pellets. The competitive immunoassay involves preincubating an anti-tubulin monoclonal antibody with an unknown quantity of tubulin in cell extracts or solubilized microtubules to quantitatively reduce the antibody available to bind to a tubulin-coated microtiter plate. Binding of remaining antibody to the microtiter plate is measured spectrophotometrically using an alkaline phosphatase-conjugated secondary antibody. Quantitation is accomplished by comparison with a known quantity of bovine brain tubulin.

Microtubules are stabilized in confluent epithelial cells but not in fibroblasts.

Rhodamine-tagged tubulin was microinjected into epithelial cells (MDCK) and fibroblasts (Vero) to characterize the dynamic properties of labeled microtubules in sparse and confluent cells. Fringe pattern fluorescence photobleaching revealed two components with distinct dynamic properties. About one-third of the injected tubulin diffused rapidly in the cytoplasm with a diffusion coefficient of 1.3-1.6 x 10(-8) cm2/s. This pool of soluble cytoplasmic tubulin was increased to greater than 80% when cells were treated with nocodazole, or reduced to approximately 20% upon treatment of cells with taxol. Fluorescence recovery of the remaining two-thirds of labeled tubulin occurred with an average half-time (t1/2) of 9-11 min. This pool corresponds to labeled tubulin associated with microtubules, since it was sensitive to treatment of cells with nocodazole and since taxol increased its average t1/2 to greater than 22 min. Movement of photobleached microtubules in the cytoplasm with rates of several micrometers per minute was shown using very small interfringe distances. A significant change in the dynamic properties of microtubules occurred when MDCK cells reached confluency. On a cell average, microtubule half-life was increased about twofold to approximately 16 min. In fact, two populations of cells were detected with respect to their microtubule turnover rates, one with a t1/2 of approximately 9 min and one with a t1/2 of greater than 25 min. Correspondingly, the rate of incorporation of microinjected tubulin into interphase microtubules was reduced about twofold in confluent MDCK cells. In contrast to the MDCK cells, no difference in microtubule dynamics was observed in sparse and confluent populations of Vero fibroblasts, where the average microtubule half-life was approximately 10 min. Thus, microtubules are significantly stabilized in epithelial but not fibroblastic cells grown to confluency.

Effect of Ovine Prolactin on Tubulin Function in the Anterior Pituitary Lobe of the Lactating Rat*

Ovine PRL (oPRL) was employed to investigate the role of anterior pituitary lobe microtubules in suckling-induced PRL secretion in the lactating rat. Groups of primiparous rats on days 12-14 postpartum were isolated from their pups for 4-5 h, then suckled for 10 min, and killed, and the anterior pituitary lobes were dissected out. Each pituitary lobe was processed to obtain the two tubulin pools, viz. soluble and polymerized tubulin fractions. After 10 min of suckling the pituitary soluble tubulin levels were reduced by about 25% (P less than 0.05), and polymerized tubulin levels increased by about 40% (P less than 0.05). When 3 mg oPRL were injected 4 h before suckling, the suckling-induced rise in the polymerized tubulin levels in the anterior pituitary lobe as well as plasma PRL levels were significantly inhibited (P less than 0.05). In the suckled rats injected with oPRL a 25% reduction in the total tubulin levels (soluble and polymerized) was observed. In a second experiment, each anterior pituitary lobe obtained from groups of rats suckled for 10 min was processed to obtain the total tubulin fraction. Suckling for 10 min stimulated the in vitro assembly of total tubulin fraction into microtubules by about 150% (P less than 0.05); 3 mg oPRL injected 4 h before suckling inhibited the suckling-induced rise in tubulin assembly (P less than 0.05). In a third experiment, suckling for 10 min stimulated GTPase activity in the total tubulin fraction by about 60% (P less than 0.05). Administration of oPRL 4 h before suckling caused about an 80% increase in GTPase activity. At a 1:2000 dilution, antitubulin antibodies maximally inhibited GTPase activity by about 40%, suggesting that a significant proportion of the enzyme activity can be due to tubulin present in the pituitary. These results suggest that PRL secretion is coupled to pituitary microtubules and that in addition to tubulin, other GTP-binding proteins might be involved in PRL secretion.

Role of dopamine in anterior pituitary tubulin pools in the lactating rat

In lactating rats, suckling stimulated the in-vitro tubulin assembly of cold-labile pituitary tubulin fraction by about 70%. Domperidone and alpha methyl-p-tyrosine stimulated the pituitary tubulin assembly by 90% and 325%, respectively. In another experiment, pituitaries from a group of lactating rats (no reference to suckling) were processed to obtain cold-labile and cold-stable tubulin fractions; total tubulin fraction was obtained from another group. Tubulin assembly in the total and cold-stable tubulin fractions was 17-20 fold higher compared to cold-labile fraction. Since the cold-stable and total tubulin fractions were extracted with a Ca(2+)-containing buffer, these results suggest that dopamine-induced changes in cytosolic Ca2+ levels modulate pituitary microtubules.

Two Drosophila beta tubulin isoforms are not functionally equivalent.

We have tested the functional capacity of different beta tubulin isoforms in vivo by expressing beta 3-tubulin either in place of or in addition to beta 2-tubulin in the male germ line of Drosophila melanogaster. The testes-specific isoform, beta 2, is conserved relative to major metazoan beta tubulins, while the developmentally regulated isoform, beta 3, is considerably divergent in sequence. beta 3-tubulin is normally expressed in discrete subsets of cells at specific times during development, but is not expressed in the male germ line. beta 2-Tubulin is normally expressed only in the postmitotic germ cells of the testis, and is required for all microtubule-based functions in these cells. The normal functions of beta 2-tubulin include assembly of meiotic spindles, axonemes, and at least two classes of cytoplasmic microtubules, including those associated with the differentiating mitochondrial derivatives. A hybrid gene was constructed in which 5' sequences from the beta 2 gene were joined to protein coding and 3' sequences of the beta 3 gene. Drosophila transformed with the hybrid gene express beta 3-tubulin in the postmitotic male germ cells. When expressed in the absence of the normal testis isoform, beta 3-tubulin supports assembly of one class of functional cytoplasmic microtubules. In such males the microtubules associated with the membranes of the mitochondrial derivatives are assembled and normal mitochondrial derivative elongation occurs, but axoneme assembly and other microtubule-mediated processes, including meiosis and nuclear shaping, do not occur. These data show that beta 3 tubulin can support only a subset of the multiple functions normally performed by beta 2, and also suggest that the microtubules associated with the mitochondrial derivatives mediate their elongation. When beta 3 is coexpressed in the male germ line with beta 2, at any level, spindles and all classes of cytoplasmic microtubules are assembled and function normally. However, when beta 3-tubulin exceeds 20% of the total testis beta tubulin pool, it acts in a dominant way to disrupt normal axoneme assembly. In the axonemes assembled in such males, the doublet tubules acquire some of the morphological characteristics of the singlet microtubules of the central pair and accessory tubules. These data therefore unambiguously demonstrate that the Drosophila beta tubulin isoforms beta 2 and beta 3 are not equivalent in intrinsic functional capacity, and furthermore show that assembly of the doublet tubules of the axoneme imposes different constraints on beta tubulin function than does assembly of singlet microtubules.

Effect of polyanions and polycations on detyrosination of tubulin and microtubules at steady state

Microtubule protein preparations purified from rat brain were used to study the effect of polycations and polyanions on the release of the COOH-terminal tyrosine of the α-chain of tubulin catalyzed by tubulin carboxypeptidase. (1) Most of the polycations and polyanions tested, independently of the ionogenic group, inhibited the reaction in a concentration-dependent fashion. Under steady-state conditions, detyrosination of the microtubule pool was inhibited to the same degree as occurred with the non-assembled tubulin pool, except in the case of chondroitin sulphate. This compound inhibited detyrosination of the non-assembled tubulin pool, but not that of microtubules. (2) Heparin, the most potent inhibitor tested, produced the dissociation of the carboxypeptidase from microtubules. Many, but not all, of the other microtubule-associated polypeptides were also dissociated by heparin. (3) Polylysine counteracted the inhibitory and dissociating effects of heparin. (4) Heparin protected tubulin carboxypeptidase against inactivation. Our results and previous reports describing, in nervous tissue, the presence of proteoglycans, RNA and basic proteins that inhibit detyrosination, suggest that tubulin carboxypeptidase might be physiologically modulated by electrically charged macromolecules.

How is the flagellar length of mature sperm determined?: II. Comparison of tubulin synthesis in spermatids between newt and Xenopus in vitro

In order to elucidate mechanisms that control flagellar length of mature sperm, we studied in synchronous cell suspension cultures flagellar growth, tubulin pool, and tubulin synthesis in round spermatids of Xenopus laevis and the newt Cynops pyrrhogaster. The average final length of flagella in Xenopus round spermatids was 35 μm, almost the same length as that in mature sperm, whereas in the newt round spermatids, the length was 210 μm, almost half that of mature sperm. Kinetics of flagellar growth showed that the rate and period of flagellar growth in the newt spermatids were two to threefold those in Xenopus spermatids. The tubulin pool size in newt spermatids was estimated to be about 10-fold greater than that in Xenopus spermatids. But even if all of the pool was used for flagellar growth, it could support only about a seventh to a tenth of the flagellar length in mature sperm in either species. Thus, the possibility that the tubulin pool primarily determines flagellar length was excluded. Since the tubulin pool size did not change throughout the culture period, the possibility that the termination of flagellar growth is due to the exhaustion of the tubulin pool was also excluded. Tubulin synthesis declined over the culture period but continued in newt spermatids longer than in Xenopus spermatids. The period of flagellar elongation almost coincided with the period of tubulin synthesis. The amount of rRNA did not decrease, excluding the possibility that the decline of tubulin synthesis was due to cytoplasmic shedding which might result in the loss of ribosomes. Tubulin synthesis and the amount of rRNA in newt spermatids was more than threefold greater than that in Xenopus spermatids, which may explain the difference in growth rates of their flagella.

Light Regulation of β-Tubulin Gene Expression during Internode Development in Soybean (Glycine max [L.] Merr.)

The relationship between tubulin gene expression and cell elongation was explored in developing internodes of Glycine max (L.) Merr., using light as a variable to alter the rate of elongation. First internodes of etiolated seedlings elongated two to three times more rapidly than did those of seedlings growing under a 12 hour diurnal light/dark cycle. Furthermore, light slowed or completely halted internode elongation in the etiolated seedlings, depending upon the age of the seedlings at the time of the light treatment. Steady state levels of beta-tubulin mRNA were determined in Northern blots and by solution hybridization of poly(A)(+)RNA with a probe derived from the coding region of a previously characterized soybean beta-tubulin gene. (MJ Guiltinan, DP Ma, RF Barker, MM Bustos, RJ Cyr, R Yadegari, DE Fosket [1987] Plant Mol Biol 10: 171-184). Internodes of light-grown seedlings exhibited levels of beta-tubulin mRNA that differed by a factor of three, and varied concomitantly with the elongation rate. Illumination of 10-day-old etiolated seedlings not only stopped first internode elongation, but also brought about a 80% decrease in the steady state level of beta-tubulin mRNA over the course of the subsequent 12 hours. This strong down regulation of beta-tubulin mRNA occurred without significant changes in the size of the soluble tubulin pool and it was accompanied by a marked increase in chlorophyll a/b binding protein mRNA.

Direct visualisation of the soluble pool of tubulin in the neuronal growth cone: immunofluorescence studies following taxol polymerisation

Previously we have shown that the predominant form of soluble α-tubulin in the growth cone is C-terminally tyrosinated (Gordon-Weeks and Lang. Dev. Brain Res., 42 (1988) 156–160). Here we show that when growth cones are incubated in taxol, this soluble pool of α-tubulin is polymerised onto the ends of the neurite microtubules that enter the proximal part of the growth cone indicating that it is assembly-competent.