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.     

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.     

Stimulation of Tubulin-Dependent ATPase Activity in Microtubule Proteins from Porcine Brain by Taxol

Taxol, an antimitotic agent that induces microtubule assembly, stimulated tubulin-dependent Mg2+-ATPase activity of microtubule-associated proteins (MAPs). A concentration-dependent increase in the rate of ATP hydrolysis was observed. Taxol acted through its binding to the tubulin molecule on MAP ATPase, and maximal stimulation, which was found at approximately equal concentrations of taxol and tubulin, reached about 140% of the original level in the absence of taxol. Taxol enhanced ATP hydrolysis by a mixture of MAPs and tubulin, and this continued at a steady linear rate even when the polymerization had approached a plateau. In the presence of taxol, a large portion of ATPase activity and protein was recovered in the pellet after centrifugation at 70,000 g for 60 min at 25 degrees C. Both colchicine and podophyllotoxin inhibited taxol-stimulated ATPase activity via the same mechanism by which they inhibited taxol-induced microtubule polymerization. The stimulation by taxol was not found in the presence of Ca2+ alone but required Mg2+. We conclude that tubulin effectively stimulates Mg2+-ATPase activity of MAPs under conditions that induce tubulin polymerization.     

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.     

Taxol affects both the microtubular arrays of heliozoan axonemes and their microtubule-organizing center

Short and long-term effects of the antitumor drug taxol on the microtubular axonemes and on the microtubule-organizing centroplast of the centrolhelidian Heterophrys marina have been investigated. Short-term treatment reveals that general aspects of cell structure remain substantially unaffected and that additional microtubule (MT) assembly in individual axonemes is accompanied by disassembly in others. However, the interaction between MTs, via cross-bridging associated proteins, is seriously affected as indicated by the numerous softly-bent axopods with disturbed arrays of the normally hexagonal pattern. Stabilization of MTs becomes evident by the reexpansion of axopods during low temperature incubation and also by the rapid inhibition of the saltatory movement of the extrusive organelles. Rapidly reexpanding axonemes of cells incubated at higher temperatures and in high taxol concentrations arise asymmetrically from the microtubule-organizing centrosomal structure (centroplast) and form a single thick and thorn-like axopodium, indicating a certain disarrangement of the centrally located microtubule-organizing center (MTOC), which obviously is severely damaged after long-term treatment. With increasing disorganization of the centroplast's structure, these cells reveal themselves unable to sustain their regular microtubular axonemal cytoskeleton. Paradoxically, polymerization of free microtubules from the tubulin pool does not take place. Instead, paracrystalline arrays of twisted filaments appear within the cytoplasm. It is concluded that heliozoan MTs can only persist if stabilized by additional factors, such as permanent interaction with the intact centroplast, and that even in the presence of taxol, MTs unattached to such an MTOC will be intrinsically unstable.     

Impact of taxol-mediated stabilization of microtubules on nuclear morphology,ploidy levels and cell growth in maize roots

Direct contact of the radiating perinuclear microtubules (MTs) with the nuclear envelope was visualized with an immunogold technique using specific monoclonal tubulin antibody. The possibility that these perinuclear MT arrays are involved in establishing and maintaining nuclear organization during the interphase of cycling cells in maize root meristems was tested using taxol, a MT-stabilizing agent. Taxol not only stabilized all MTs against the action of the MT-disrupters colchicine and oryzalin but also prevented these agents from their usual induction of nuclear enlargement and decondensation of nuclear chromatin. On the contrary, nuclear size decreased and the chromatin became more compact in mitotically cycling cells of the taxol-treated root apices. Moreover, taxol prevented the stimulation, by colchicine and oryzalin, of the onset of the S phase in cells of the quiescent centre and proximal root meristem. Exposure of maize roots to taxol strongly decreased final cell volumes, suggesting that the more condensed nuclear chromatin is less efficient in genome expression and that this accounts for the restriction of cellular growth. All these findings support the hypothesis that MT arrays, radiating from the nuclear surface, are an essential part of an integrated plant ‘cell body’ consisting of nucleus and the MT cytoskeleton, and that they regulate, perhaps via their impact on chromatin condensation and activity, progress through the plant cell cycle.     

Inhibitory factor of microtubule assembly from sea urchin egg cortex. I. Preparation and characterization

A new inhibitory factor of the microtubule (MT) assembly system was isolated from unfertilized sea urchin egg cortex. This factor not only suppressed spontaneous brain MT assembly, but also induced depolymerization of the reconstituted MTs. The factor did not suppress initial MT growth initiated by ciliary outer fiber fragments but the assembled MTs were soon depolymerized with time. The inhibitory activity was heat-stable but sensitive to trypsin or urea. The mode of the inhibition was distinct from the inhibitory effects of RNA on the MT assembly. The inhibitory factor partially purified on DEAE-Sephadex A-50 completely inhibited tubulin polymerization in a factor: tubulin ratio of 0.013.     

The microtubule cytoskeleton does not integrate auxin transport and gravitropism in maize roots

The Cholodny-Went hypothesis of gravitropism suggests that the graviresponse is controlled by the distribution of auxin. However, the mechanism of auxin transport during the graviresponse of roots is still unresolved. To determine whether the microtubule (MT) cytoskeleton is participating in auxin transport, the cytoskeleton was examined and the movement of 3 H-IAA measured in intact and excised taxol, oryzalin, and naphthylphthalamic acid (NPA)-treated roots of Zea mays cv. Merit. Taxol and oryzalin did not inhibit the graviresponse of roots but the auxin transport inhibitor NPA greatly inhibited both auxin transport and graviresponse. NPA had no effect on MT organization in vertical roots, but caused MT reorientation in horizontally placed roots. Regardless of treatment, the organization of MTs in intact roots differed from that in root segments. The MT inhibitors, taxol and oryzalin had opposite effects on the MTs, namely, depolymerization (oryzalin) and stabilization and thickening (taxol), but both treatments caused swelling of the roots. The data indicate that the MT cytoskeleton does not directly interfere with auxin transport or auxin-mediated growth responses in maize roots.     

Preferential Inhibition of Herpes-Group Viruses by Phosphonoacetic Acid: Effect on Virus DNA Synthesis and Virus-Induced DNA Polymerase Activity

In tissue culture phosphonoacetic acid (PAA) specifically inhibited DNA synthesis of human cytomegalovirus (CMV), murine CMV, simian CMV, Epstein-Barr virus, and Herpesvirus saimiri. Fifty to one hundred micrograms per milliliter PAA completely inhibited viral DNA synthesis with no significant damage to host cell DNA synthesis. In vitro DNA polymerization assays showed that 10 μg/ml of PAA specifically inhibited partially purified human CMV-induced DNA polymerase, while little inhibition of host-cell DNA polymerase activity was found. The specific inhibition of herpes-group virus DNA synthesis with little toxicity to host cells suggests that PAA has great potential as an antiherpesvirus therapeutic agent.     

Independence of centriole formation and initiation of DNA synthesis in Chinese hamster ovary cells

The relationship between centriole formation and DNA synthesis was investigated by examining the effect of taxol on the centriole cycle and the initiation of DNA synthesis in synchronized cells. The centriole cycle was monitored by electron microscopy of whole-mount preparations [Kuriyama and Borisy, J. Cell Biol., 1981, 91:814-821]. A short daughter centriole appeared in perpendicular orientation to each parent during late G1 or early S and elongated slowly during S to G2. Addition of 5-20 micrograms/ml taxol to a synchronous population of cells in S phase did not inhibit centriole elongation; rather, elongation was accelerated. In contrast, when taxol was added to M phase or early G1 cells, centriole duplication was completely inhibited. The taxol block was reversible since nucleation and elongation of centrioles resumed as soon as the drug was removed. Cells exposed to taxol progressed through the cell cycle and became blocked in mitosis, as indicated by an increase in the mitotic index, but eventually the mitotic arrest was overcome, resulting in formation of multinucleated cells. A peak in mitotic index was seen in the following generation, indicating that chromosomes duplicated in the presence of taxol. Incorporation of 3H-thymidine followed by autoradiography confirmed that DNA synthesis was initiated in the presence of taxol even though formation of daughter centrioles was inhibited. It seems, therefore, that centriole duplication is not a prerequisite for entry into S phase. Since DNA synthesis has already been demonstrated not to be necessary for centriole duplication, these two events, normally coordinated in time, appear to be independent of each other.     

Studies on the role of microtubules in myofibrillogenesis

Co-localization of microtubule (MT) and muscle myosin (MHC) myofibril immunofluoresoonoe in developing myotubes of chicken skeletal muscle cultures was observed by using double staining of tubulin and MHC indirect immunofluorescence.120-tetradecanoyl-phorbol-12-acetate (TPA) selectively and reversibly blocks myofibrillogenesis and alters the morphology of myotubes in to myosacs where MTs are present in radiating pattern.When the arrested myogenic cells recover and start myofibrillogenesis after released from TPA,prior to the emergence of myofibrils,the pre-ecisting MTs become bipolarly aligned coincidently with the tubular restoration of cell shape.Single nascent myofibrils overlapping with MTs extend into the base of growth tips where MTs go farther to the end of the tips.That MT might act as scaffold in guiding the bipolar elongation of the growing myofibrils was suggested.Taxol and colcemid disturbed MT polymerization and disposition,and interfered with the normal spatial assembly of myofibrils in developing myotubes.     

Inhibition of spindle elongation by taxol.

During anaphase B spindle elongation, interzonal microtubules lengthen to accomplish pole-pole separation, while at the same time remaining highly dynamic [Shelden and Wadsworth, J. Cell Sci. 97:273-281, 1990]. To further examine the role of microtubule polymerization and dynamics during spindle elongation, cells have been treated with taxol, which induces microtubule polymerization and stabilizes microtubules. Taxol was added to PtK1 cells 3 minutes after initial chromatid separation, so that the effect on anaphase B could be observed with minimal disruption to anaphase A movement. In 20 microM taxol, the rate and extent of pole-pole separation, measured from time-lapse video records, are reduced to 4% and 9.5% of controls, respectively. The organization of microtubules in taxol treated cells was examined using tubulin immunofluorescence and confocal fluorescence microscopy. Taxol induces a dramatic reorganization of interzonal microtubules resulting in a narrow gap, which is nearly completely lacking in MTs, across the center of the interzone. Furthermore, microtubules in taxol treated cells are resistant to nocodazole induced microtubule disassembly. Our results reveal that taxol rapidly inhibits anaphase B spindle elongation; inhibition is accompanied by a depletion of interdigitated interzonal microtubules and a reduction in microtubule dynamic behavior.     

Phospholipase C signaling involvement in macrotubule assembly and activation of the mechanism regulating protoplast volume in plasmolyzed root cells of Triticum turgidum

The role of phosphoinositide-specific phospholipase C (PI-PLC) signaling in the macrotubule-dependent protoplast volume regulation in plasmolyzed root cells of Triticum turgidum was investigated. At the onset of hyperosmotic stress, PI-PLC activation was documented. Inhibition of PI-PLC activity by U73122 blocked tubulin macrotubule formation in plasmolyzed cells and their protoplast volume regulatory mechanism. In neomycin-treated plasmolyzed cells, macrotubule formation and protoplast volume regulation were not affected. In these cells the PI-PLC pathway is down-regulated as neomycin sequesters the PI-PLC substrate, 4,5-diphosphate-phosphatidyl inositol (PtdInsP(2)). These phenomena were unaffected by R59022, an inhibitor of phosphatidic acic (PA) production via the PLC pathway. Taxol, a microtubule (MT) stabilizer, inhibited the hyperosmotic activation of PI-PLC, but oryzalin, which disorganized MTs, triggered PI-PLC activity. Taxol prevented macrotubule formation and inhibited the mechanism regulating the volume of the plasmolyzed protoplast. Neomycin partly relieved some of the taxol effects. These data suggest that PtdInspP(2) turnover via PI-PLC assists macrotubule formation and activation of the mechanism regulating the plasmolyzed protoplast volume; and the massive disorganization of MTs that is carried out at the onset of hyperosmotic treatment triggers the activation of this mechanism.     

Axonal tubulin and microtubules: morphologic evidence for stable regions on axonal microtubules

Biochemical studies indicate that axonal tubulin is composed of at least two distinct pools that differ in cold solubility and biochemical composition [Brady et al: J. Cell Biol. 99:1716-1724]. To determine the morphologic correlate of cold-insoluble tubulin, segments of rat optic nerves were exposed to a series of in vitro experimental conditions that affect microtubules (MTs), including cold, podophyllotoxin (PT), triflupromazine (TFP), and taxol, and then examined by electron microscopy. Longitudinal sections of control axons showed MTs oriented parallel to the long axis of the axons. Axons exposed to cold, PT, and TFP showed short segments of MTs in association with cytoskeletal disarray. Morphometric studies were used to distinguish between a simple malorientation of MTs (undulation or zigzags in their course) and the loss of labile segments of MTs, leaving the stable portions behind. The lengths of MT segments were measured in longitudinal sections, and the numbers of MTs were determined in the cross sections. All MT segment-length histograms showed a unimodal distribution. Cold and PT produced a simple shift of the control histogram to the shorter length MTs. In cross sections the numbers of MTs in cold- and PT-exposed axons were significantly decreased, indicating that the presence of short segments of MTs in the longitudinal plane of sections was due to a loss of portions of MTs. Taxol, an agent that promotes MT assembly, reversed the cold effect partially and resulted in increases in both MT segment length and number. These studies indicate that stable MT segments are portions of longer MTs containing both stable and labile regions. Furthermore, these findings are consistent with the hypothesis that cold-insoluble tubulin functions as a transportable MT-organizing complex in the axon.     

Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus

A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4°C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60–68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.     

Calmodulin colocalization with cold-stable and nocodazole-stable microtubules in living PtK1 cells

To investigate the association of calmodulin (CaM) with microtubules (MTs) in the mitotic apparatus (MA), the distributions of both CaM and tubulin were examined in mitotic PtK1 cells in which MT subclasses had been selectively removed or altered by treatment with cold or with the MT inhibitor, nocodazole. A fluorescent CaM conjugate with tetramethylrhodamine isothiocyanate (CaM-TRITC) was microinjected into living cells, and the CaM distribution in the living cell was compared to the distribution of MTs indicated by tubulin immunofluorescence. In cells which had been treated for 2 h at 0 to 4 degrees C or with a low (0.03 micrograms/ml) dose of nocodazole, the only MTs remaining appeared to be kinetochore MTs (kMTs). The distribution of microinjected CaM-TRITC in these cells was indistinguishable from that found in untreated cells and appeared to be colocalized with the kMTs. In cells which were treated with a high (3.0 micrograms/ml) dose of nocodazole, only short MTs remained. When CaM-TRITC was injected into these cells, it formed a somewhat punctate distribution near the chromosomes and, after tubulin immunofluorescence processing, colocalized with what appeared to be remnants of kMTs. We believe that these observations support the hypothesis that CaM exists in the MA in a structural association with kMTs.     

A novel acetylation of β-tubulin by San modulates microtubule polymerization via down-regulating tubulin incorporation

Dynamic instability is a critical property of microtubules (MTs). By regulating the rate of tubulin polymerization and depolymerization, cells organize the MT cytoskeleton to accommodate their specific functions. Among many processes, posttranslational modifications of tubulin are implicated in regulating MT functions. Here we report a novel tubulin acetylation catalyzed by acetyltransferase San at lysine 252 (K252) of β-tubulin. This acetylation, which is also detected in vivo, is added to soluble tubulin heterodimers but not tubulins in MTs. The acetylation-mimicking K252A/Q mutants were incorporated into the MT cytoskeleton in HeLa cells without causing any obvious MT defect. However, after cold-induced catastrophe, MT regrowth is accelerated in San-siRNA cells while the incorporation of acetylation-mimicking mutant tubulins is severely impeded. K252 of β-tubulin localizes at the interface of α-/β-tubulins and interacts with the phosphate group of the α-tubulin-bound GTP. We propose that the acetylation slows down tubulin incorporation into MTs by neutralizing the positive charge on K252 and allowing tubulin heterodimers to adopt a conformation that disfavors tubulin incorporation.     

Detyrosination of alpha tubulin does not stabilize microtubules in vivo [published erratum appears in J Cell Biol 1990 Sep;111(3):1325-6]

The relationship between alpha tubulin detyrosination and microtubule (MT) stability was examined directly in cultured fibroblasts by experimentally converting the predominantly tyrosinated MT array to a detyrosinated (Glu) array and then assaying MT stability. MTs in mouse Swiss 3T3 cells displayed an increase in Glu immunostaining fluorescence approximately 1 h after microinjecting antibodies to the tyrosinating enzyme, tubulin tyrosine ligase. Detyrosination progressed to virtual completion after 12 h and persisted for 30-35 h before tyrosinated subunits within MTs were again detected. The stability of these experimentally detyrosinated MTs was tested by first injecting either biotinylated or Xrhodamine-labeled tubulin and then measuring bulk turnover by hapten-mediated immunocytochemistry or fluorescence recovery after photobleaching, respectively. By both methods, turnover was found to be similarly rapid, possessing a half time of approximately 3 min. As a final test of MT stability, the level of acetylated tubulin staining in antibody-injected cells was compared with that observed in adjacent, uninjected cells and also with the staining observed in cells whose MTs had been stabilized with taxol. Although intense Glu staining was observed in both injected and taxol-treated cells, increased acetylated tubulin staining was observed only in the taxol-stabilized MTs, indicating that the MTs were not stabilized by detyrosination. Together, these results demonstrated clearly that detyrosination does not directly confer stability on MTs. Therefore, the stable MTs observed in these and other cell lines must have arisen by another mechanism, and may have become posttranslationally modified after their stabilization.     

TAXOL REDUCES THE RATE OF CYTOKINESIS IN PtK1CELLS

Mitotic PtK₁cells were treated both during mid-anaphase and at furrow initiation with the potent microtubule (MT) stabilizing agent, taxol, to determine the role of MTs in the rate of cytokinetic events. Rates of cytokinesis (μm/min) were measured by changes in furrow diameter. Incubation of PtK₁cells during mid-anaphase with 5 μg/ml taxol slows the rate of cytokinesis by an average of 43%. Instead of furrow initiation to midbody formation taking an average of 10.7 min (1.6 μm/min), furrowing to midbody formation was completed in an average of 19.0 min (0.9 μm/min), which does not include the 7-min period between taxol application in mid-anaphase and furrow initiation. Application of 5 μg/ml taxol to cells at furrow initiation had a reduced effect on decreasing the rate of cytokinesis and midbody formation; furrowing to midbody formation took an average of 14.6 min (1.2 μm/min). These data suggest that delays in the rate of cytokinesis is dependent on the mitotic stage at which taxol is applied. Ultrastructural analysis shows that taxol treatment of anaphase cells prevents midbody formation during early G₁, yet MT number and organization in the furrowed region is not significantly altered from untreated cells. There is little change in the organization and amount of contractile ring microfilaments, yet filaments are also found parallel to midbody MTs. Our results may be explained by the fact that taxol tends to stabilize MTs which probably affects the rate at which they depolymerize in the terminal phases of cytokinesis. Reduction in depolymerization rates of a stable population of MTs could serve to regulate the rate of cytokinesis.     

Microtubule orientation and dynamics in elongating characean internodal cells following cytosolic acidification, induction of pH bands, or premature growth arrest

Summary Cortical microtubules (MTs) at indifferent zones in immatureNitella internodes were investigated by injection of fluorescently tagged sheep brain tubulin into living cells and by immunofluorescence on fixed material. Nearly identical MT patterns and numbers were detected with the two techniques, indicating that sheep brain tubulin incorporated into all cortical MTs. MTs were aligned transversely to the long axis of the cell and approximately one MT was present every micrometer of longitudinal cell distance. Treatment of internodes with propionic acid to acidify cytosolic pH caused depolymerization of MTs and an increase in the unpolymerized tubulin pool. Transfer of young, vigorously elongating cells to media inducing premature growth cessation resulted in a slight decrease in microtubule numbers but did not significantly alter microtubule orientation patterns or microtubule lifespans. MTs remained transverse for days following growth cessation before finally assuming a more random alignment characteristic of mature, non-growing internodes. No differences in MT numbers, orientation, or dynamics were detected between acid and alkaline bands in internodes incubated in a band-inducing medium. Thus, properties of cortical MT arrays were not closely coupled to growth status or to regional differences in cellular physiology associated with pH banding.Abbrevations BIM band-inducing medium - CCM Chara culture medium - CF carboxyfluorescein - FRAP fluorescence redistribution after photobleaching - MT microtubule