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1.
P L Konen S J Currier A V Rutherford M M Gottesman I Pastan M C Willingham 《The journal of histochemistry and cytochemistry》1989,37(7):1141-1145
Double-label fluorescence microscopy was used to demonstrate the efflux activity of the multidrug transporter in single cultured cells. NIH3T3 cells expressing a transfected MDR1 gene (NIH3T3-MDR) were treated with vinblastine or daunomycin. The accumulation of vinblastine was monitored by examining the morphology of tubulin in cells, using immunofluorescence. Overnight treatment of drug-sensitive cells caused disassembly of microtubules and formation of paracrystals; the absence of vinblastine effects was evident by the presence of intact microtubules. Daunomycin accumulation was detected in nuclei using the inherent fluorescence of the drug with rhodamine epifluorescence microscopy. Drug efflux in multidrug-resistant cells was inhibited with verapamil. When multidrug-resistant cells were treated overnight in vinblastine, an effect of 0.5 microM vinblastine on microtubules was seen only in the presence of verapamil. Similarly, when cells were treated with daunomycin, this drug accumulated in nuclei only when verapamil was present. When cells incubated with vinblastine and verapamil were washed free of drugs, they did not accumulate daunomycin in a subsequent incubation, indicating that the multidrug transporter was still active; this occurred even though the morphologic effects of vinblastine persisted. Cells incubated with vinblastine alone showed an immediate inhibition of efflux activity when verapamil was subsequently added with daunomycin. These results show that the efflux activity of the multidrug transporter can be rapidly manipulated by agents such as verapamil, despite a prior history of drug treatment, and that the effects of inhibition of the transporter are rapidly reversible. 相似文献
2.
Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins. 总被引:37,自引:0,他引:37
In this paper we describe a procedure for detecting proteins associated with cytoplasmic microtubules in vivo. Detergent-extracted cytoskeletons of NIL8 hamster cells are prepared under conditions which preserve the microtubules. The cytoskeletons are then extracted in the presence of calcium, which depolymerizes the microtubules and quantitatively extracted cytoskeletons are prepared from cells that have been incubated with colchicine. The cytoskeletons from these cells contain no microtubules or tubulin. Electrophoretic analysis of the calcium extracts of the colchicine-treated and untreated cells reveals several radioactively labeled polypeptides. There is, however, no apparent quantitative or qualitative difference between the two extracts other than the tubulin polypeptides. Each of the extracts is mixed with an excess of unlabeled calf brain microtubule protein and carried through cycles of temperature-dependent microtubule assembly. Distinct species from each extract co-assemble at a constant ratio, but only one polypeptide is uniquely derived from cells containing intact microtubules. The molecular weight of this polypeptide is similar to that proposed for the tau species detected in brain microtubule preparations. 相似文献
3.
Hideki Takanari Joji Morita Hidetaka Yamanaka Kenichiro Yada Asahiko Takahashi Kosaku Izutsu 《Biology of the cell / under the auspices of the European Cell Biology Organization》1994,82(1):51-57
Summary— The pH-related change in morphology of vinblastine (VLB)-induced paracrystals formed in Chinese hamster ovary (CHO) cells was examined immunohistochemically in order to determine both the mechanism of tubulin crystallization and the influence of acidic pHs on cytoskeletal microtubules. Lowering the extracellular pH (pHe) rapidly reduced the intracellular pH (pHi) in CHO cells. Lowering the pHi to near the neutral range significantly accelerated the growth of VLB-induced paracrystals, compared to that of paracrystals formed at a physiological pHe. However, further cytoplasmic acidification caused by the addition of sodium azide into the culture medium induced the disappearance of typical paracrystals and the appearance of a highly organized meshwork of tubulin appearing as short, thick filaments at the light microscopic level. Treatments using different concentrations of VLB at different pHe's showed that low pHi's (6.7 and 6.3) suppressed paracrystal-formation at lower concentrations of VLB (5×10?6 M and 10?5 M). At higher concentrations of VLB (5×10?5 M and 10?4 M), only short filaments were formed at pHi 6. 3. Electron microscopy revealed that the filaments had a ladder-like structure probably consisting of a stacked series of fused rings. This indicates that paracrystals may be modified by extremely low pH. These results show that paracrystals are unstable in living cells and that their formation is regulated by environmental pH. 相似文献
4.
Effects of anti-microbule drugs on tubulin polymerizationin vitro were investigated using purified mung bean (Vigna radiata) tubuli. Colchicine induced the formation of macrotubules at the relatively low concentration of 10 μM. and the appearance
of corkscrew-like filaments from the ends of the macrotubules at concentrations of more than 100 μM. Vinblastine substantially
inhibited polymerization at 1 μM and caused the formation of paracrystals at concentrations greater than 10 μM. Oryzalin inhibited
polymerization at 1 μM partially and at 10 μM completely. Paracrystal formation was also induced by cremart at 10 μM, but
these paracrystals appeared to be more rigid than those induced by vinblastine. Amiprophos methyl (APM), with a chemical configuration
similar to cremart, substantially inhibited polymerization at 1 μM, but the formation of paracrystals was weak. Griseofulvin
at 10 μMalso inhibited the polymerization of tubulin while at higher concentrations aggregates of helices were formed. Inhibition
of polymerization by phenylcarbamate herbicides was more effective than that caused by benzimidazoylcarbamate fungicides.
The effects of drugs onin vitro preformed (MTs) were also investigated. Colchicine and vinblastine showed identical effects to those on the polymerization
process. Griseofulvin, cremart and APM induced only macrotubule formation while the other drugs tested had no major effects 相似文献
5.
In vivo and in vitro studies on the role of HMW-MAPs in taxol-induced microtubule bundling 总被引:3,自引:0,他引:3
The involvement of high molecular weight microtubule-associated proteins (HMW-MAPs) in the process of taxol-induced microtubule bundling has been studied using immunofluorescence and electron microscopy. Immunofluorescence microscopy shows that HMW-MAPs are released from microtubules in granulosa cells which have been extracted in a Triton X-100 microtubule-stabilizing buffer (T-MTSB), unless the cells are pretreated with taxol. 1.0 microM taxol treatment for 48 h results in microtubule bundle formation and the retention of HMW-MAPs in these cells upon extraction with T-MTSB. Electron microscopy demonstrates that microtubules in control cytoskeletons are devoid of surface structures whereas the microtubules in taxol-treated cytoskeletons are decorated by globular particles of a mean diameter of 19.5 nm. The assembly of 3 X cycled whole microtubule protein (tubulin plus associated proteins) in vitro in the presence of 1.0 microM taxol, results in the formation of closely packed microtubules decorated with irregularly spaced globular particles, similar in size to those observed in cytoskeletons of taxol-treated granulosa cells. Microtubules assembled in vitro in the absence of taxol display prominent filamentous extensions from the microtubule surface and center-to-center spacings greater than that observed for microtubules assembled in the presence of taxol. Brain microtubule protein was purified into 6 s and HMW-MAP-enriched fractions, and the effects of taxol on the assembly and morphology of these fractions, separately or in combination, were examined. Microtubules assembled from 6 s tubulin alone or 6 s tubulin plus taxol (without HMW-MAPs) were short, free structures whereas those formed in the presence of taxol from 6 s tubulin and a HMW-MAP-enriched fraction were extensively crosslinked into aggregates. These data suggest that taxol induces microtubule bundling by stabilizing the association of HMW-MAPs with the microtubule surface which promotes lateral aggregation. 相似文献
6.
Colchicine-binding protein of the liver. Its characterization and relation to microtubules 总被引:6,自引:2,他引:4
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《The Journal of cell biology》1975,66(3):609-620
Colchicine-binding activity of mouse liver high-speed supernate has been investigated. It has been found to be time and temperature dependent. Two binding activities with different affinities for colchicine seem to be present in this high-speed supernate, of which only the high-affinity binding site (half maximal binding at 5 x 10(-6) M colchicine) can be attributed to microtubular protein by comparison with purified tubulin. Vinblastine interacted with this binding activity by precipitating it when used at high concentrations (2 x 10(- 3) M), and by stabilizing it at low concentrations (10(-5) M). Lumicolchicine was found not to compete with colchicine. The colchicine-binding activity was purified from liver and compared with that of microtubular protein from brain. The specific binding activity of the resulting preparation, its electrophoretic behavior, and the electron microscope appearance of the paracrystals obtained upon its precipitation with vinblastine permitted its identification as microtubular protein (tubulin). Electrophoretic analysis of the proteins from liver supernate that were precipitated by vinblastine indicated that this drug was not specific for liver tubulin. Preincubation of liver supernate with 5 mM EGTA resulted in a time- dependent decrease of colchicine-binding activity, which was partly reversed by the addition of Ca++. However, an in vitro formation of microtubules upon lowering the Ca++ concentration could not be detected. Finally, a method was developed enabling that portion of microtubular protein which was present as free tubulin to be measured and to be compared with the total amount of this protein in the tissue. This procedure permitted demonstration of the fact that, under normal conditions, only about 40% of the tubulin of the liver was assemled as microtubules. It is suggested that, in the liver, rapid polymerization and depolymerization of microtubules occur and may be an important facet of the functional role of the microtubular system. 相似文献
7.
M Hayashi 《Journal of biochemistry》1979,85(3):691-698
Brain microtubules are found to disperse rods of skeletal muscle myosin and become decorated with amorphous aggregates of myosin. Then microtubules are partially depolymerized by myosin. Myosin shows high Mg2+-GTPase activity which is not influenced by microtubules, and induces the partial depolymerization of microtubules by exhaustion of GTP in the solution. H-meromyosin depolymerizes microtubules like myosin does. H-meromyosin is, however, contaminated with a trace amount of trypsin, which irreversibly depolymerizes microtubules. 相似文献
8.
H Takanari T Yosida J Morita K Izutsu T Ito 《Biology of the cell / under the auspices of the European Cell Biology Organization》1990,70(1-2):83-90
The processes of tubulin paracrystal induction in Chinese hamster ovary cells treated with a Vinca alkaloid, ie, vinblastine or vincristine, and treated simultaneously with one of the Vinca alkaloids and colcemid or colchicine were followed by four different microscopic techniques, in particular by tubulin-immunofluorescence. Vinca alkaloid alone, in lower concentrations, induced basically tactoid or needle-shaped (N-shaped) paracrystals. However, the formation of crystalloid was greatly enhanced by increasing the concentration of Vinca alkaloid. Square or barrel-shaped (S-shaped) and hexagonal paracrystals were also commonly induced by simultaneous treatment with a Vinca alkaloid and colcemid or colchicine. Large rectangular paracrystals often displayed fibrillar or lamellar fine structures which ran perpendicular to the long axis but tended to cleave into fragments by spontaneous splitting. Electron micrographs revealed the fine structure of crystalloids to be aggregates of numerous filaments. The growth of paracrystals, particularly N-shaped crystals, was markedly inhibited when cells were exposed to drug(s) at a low temperature (4 degrees C). We confirmed that both N- and S-shaped paracrystals dissociated rapidly after the culture medium was replaced with fresh, drug-free medium. Glutaraldehyde-fixed paracrystals treated with RNase solution were stained with acridine orange, showing a weak orange color. Possible factors involved in the assembly and disassembly of tubulin paracrystals are discussed. 相似文献
9.
Summary Vinblastine sulfate was administered to adult rats by intravenous injections. Kidney cortex was fixed after 1, 2, or 5 hours of treatment and studied by routine transmission electron microscopy.In control animals, cells of distal convoluted tubules possessed numerous microtubules with an average diameter of 280 Å. In treated animals, the microtubules of these cells were reduced in number, and paracrystalline inclusions characteristic of vinblastine treatment were common. Macrotubules (570 Å average diameter) were also present and often were seen close to, or in apparent continuity with, paracrystals. Since the work of others indicates that vinblastine-induced paracrystals contain microtubular protein (tubulin), observation of continuities between paracrystals and macrotubules is interpreted as evidence that macrotubules are also composed of tubulin and that macrotubules may become incorporated into paracrystals.Unlike the ordinary microtubules of cells of the distal tubules, vinblastine-induced macrotubules exhibited cross-striations in longitudinal view and subunit structure in cross section.Macrotubules and paracrystals were also observed in cells of the proximal convoluted tubule, mesangium, glomerular endothelium, parietal epithelium of Bowman's capsule, and visceral epithelium of Bowman's capsule. Continuities between macrotubules and paracrystals, although relatively common in occurrence in distal tubule cells, were only rarely seen in the other kinds of cells examined.
Acknowledgements. The authors gratefully acknowledge the technical help of Mrs. Dawn Bockus, Miss Judy Groombridge, Mrs. Jeri Hunter, Mrs. Jolan Pinter, Miss Franque Remington, Miss Mary Stewart, Miss Louise Young, Mr. Reginald Pickering, and Mr. W. J. Masten. This research was supported by N.I.H. grants AM 16 236, GM 00 100, and HE 03 174, by Institutional Cancer Grant IN-26L from the American Cancer Society, and by the Graduate School Research Fund of the University of Washington. 相似文献
10.
In vitro and in vivo effects of vincristine on the Golgi complex of leukaemic lymphoblasts were studied. The cells incubated in vitro for 4 hours with vincristine of 1.25 x 10(-5) M concentration lacked microtubules, but regularly contained paracrystals and parallel arrays of macrotubules associated with ribosomes. The Golgi complex in control lymphoblasts was represented by 1-3 dictyosomes (stacks of cisternae) grouped in one area. After exposure to vincristine the dictyosomes lay at a considerable distance from each other. In many of them the cisternae were shorter than in controls and distended or transformed into large vacuoles. In cells incubated in vitro with lower concentrations of vincristine (1.25 x 10(-6) and 1.25 x 10(-7) M) and in cells obtained after the second therapeutic dose of vincristine (in the course of normal clinical treatment) neither changes in the Golgi complex nor formation of paracrystals and macrotubules were observed. 相似文献
11.
Kinetic analysis of tubulin exchange at microtubule ends at low vinblastine concentrations 总被引:10,自引:0,他引:10
We have investigated the effects of vinblastine at micromolar concentrations and below on the dynamics of tubulin exchange at the ends of microtubule-associated-protein-rich bovine brain microtubules. The predominant behavior of these microtubules at polymer-mass steady state under the conditions examined was tubulin flux, i.e., net addition of tubulin at one end of each microtubule, operationally defined as the assembly or A end, and balanced net loss at the opposite (disassembly or D) end. No dynamic instability behavior could be detected by video-enhanced dark-field microscopy. Addition of vinblastine to the microtubules at polymer-mass steady state resulted in an initial concentration-dependent depolymerization predominantly at the A ends, until a new steady-state plateau at an elevated critical concentration was established. Microtubules ultimately attained the same stable polymer-mass plateau when vinblastine was added prior to initiation of polymerization as when the drug was added to already polymerized microtubules. Vinblastine inhibited tubulin exchange at the ends of the microtubules at polymer-mass steady state, as determined by using microtubules differentially radiolabeled at their opposite ends. Inhibition of tubulin exchange occurred at concentrations of vinblastine that had very little effect on polymer mass. Both the initial burst of incorporation that occurs in control microtubule suspensions following a pulse of labeled GTP and the relatively slower linear incorporation of label that follows the initial burst were inhibited in a concentration-dependent manner by vinblastine. Both processes were inhibited to the same extent at all vinblastine concentrations examined. If the initial burst of label incorporation represents a low degree of dynamic instability (very short excursions of growth and shortening of the microtubules at one or both ends), then vinblastine inhibits both dynamic instability and flux to similar extents. The ability of vinblastine to inhibit tubulin exchange at microtubule ends in the micromolar concentration range appeared to be mediated by the reversible binding of vinblastine to tubulin binding sites exposed at the polymer ends. Determination by dilution analysis of the effects of vinblastine on the apparent dissociation rate constants for tubulin loss at opposite microtubule ends indicated that a principal effect of vinblastine is to decrease the dissociation rate constant at A ends (i.e., it produces a kinetic cap at A ends), whereas it has no effect on the D-end dissociation rate constant. 相似文献
12.
Immunoelectron microscopic localization of the 210,000-mol wt
microtubule-associated protein in cultured cells of primates 总被引:7,自引:7,他引:0
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《The Journal of cell biology》1981,91(2):438-445
Results from ultrastructural immunocytochemistry on glutaraldehyde- fixed cells confirmed and extended findings previously obtained with immunofluorescence. A microtubule-associated protein (MAP) of 210,000 molecular weight was shown to be specifically associated with all cytoplasmic and mitotic microtubules along their entire length in primate cells. Specific labeling with the anti-MAP antibody could not be detected on any other subcellular structures, notably the centrosomes, kinetochores, microfilaments, and intermediate filaments. Treatment with the microtubule-disrupting drug, nocodazole, induced diffusion of the MAP throughout the cytoplasm. During repolymerization of microtubules following disassembly by nocodazole, the association of the MAP with the microtubules was intermediate and complete. When cells were treated with vinblastine, the tubulin paracrystals formed were heavily stained by the antibody. Neither sodium azide nor taxol affected the association of the MAP with microtubules. 相似文献
13.
A L Kovács J Kovács 《Virchows Archiv. B, Cell pathology including molecular pathology》1980,32(2):97-104
Spontaneous autophagocytosis was observed in mouse seminal vesicle cells during incubation for 2 h in vitro. The number of autophagic vacuoles formed was greatest at 37 degrees C and decreased when the temperature was lowered. At 22 degrees C it reached the near-zero value characteristic of non-incubated control cells. Incubation of the cells at 37 degrees C in the presence of 0.1 mg/ml vinblastine sulfate resulted in a marked increase in the number of autophagic vacuoles, but the drug was ineffective at 22 degrees C. Puromycin (10(-3) M) exerted no influence on spontaneous autophagocytosis, but cycloheximide in concentrations from 10(-7) M to 10(-3) M inhibited both spontaneous and vinblastine-induced autophagocytosis. The formation of tubulin paracrystals in vinblastine treated cells was not prevented either by low (22 degrees C) temperature or in the presence of cycloheximide. 相似文献
14.
G Akner K G Sundqvist M Denis A C Wikstr?m J A Gustafsson 《European journal of cell biology》1990,53(2):390-401
The cellular distribution of the glucocorticoid receptor (GR) in relation to various intracellular and plasma membrane structures in human fibroblasts was studied using indirect immunofluorescence techniques with monoclonal and polyclonal antibodies. During interphase, GR was located predominantly in the cytoplasm, showing a similar pattern as tubulin. In mitotic cells, GR and tubulin were localized in mitotic spindles and in telophase midbodies. Colchicine and vinblastine induced a similar redistribution of GR and tubulin to the cell periphery. This redistribution was reversible for colchicine but not for vinblastine. Vinblastine also induced paracrystals containing GR and tubulin. These results support the hypothesis that GR interacts in vivo with cytoplasmic microtubules. 相似文献
15.
A monoclonal antibody to neuronal microtubule-associated protein MAP-2 was produced. Immunoblotting of lysates of cultured cells revealed that the antibody, called MA-01, bound to a protein of Mr 210 kDa. Double immunofluorescence microscopy showed that the antibody stained microtubules. No fibrillar structures were observed in cells treated with Colcemid, but the antibody stained vinblastine paracrystals. In cytochalasin B-treated Leydig cells, MA-01 antibody stained star-like structures that codistributed with actin patches and with a star-like arrangement of vimentin. These observations indicate that the protein immunologically related to MAP-2 in Leydig cells could be involved in the interaction of microtubules with intermediate filaments or microfilaments. 相似文献
16.
The formation of axons induced by dibutyryl-adenosine 3′,5′-cyclic monophosphate (db-cAMP) in neuroblastoma cells was inhibited by concanavalin A (ConA) and vinblastine. These compounds also caused the retraction of existing axons. After removal of ConA or vinblastine, addition of db-cAMP again resulted in axon formation. The cytotoxicity of ConA and vinblastine for neuroblastoma cells was reduced when cell multiplication was inhibited by db-cAMP. Linearly growing normal fibroblasts were also more sensitive to the cytotoxic effect of ConA than confluent non-multiplying fibroblasts. The effects of ConA and vinblastine were additive both in their effects on axon formation and cytotoxicity. Wheat germ agglutinin (WGA) and lumicolchicine did not affect axon formation or reduce cell viability. It is suggested that ConA bound to the cell surface can interfere with the assembly of cytoplasmic microtubules involved in axon formation and cell division. 相似文献
17.
At low concentrations, vinblastine binds rapidly and reversibly to a very limited number of high affinity sites on steady-state bovine brain microtubules (mean Kd, 1.9 × 10?6m; 16.8 ± 4.3 vinblastine binding sites per microtubule) which appear to be located at one or both ends of the microtubules. At high concentrations, vinblastine binds to a high binding capacity class of sites of undetermined affinity, located on helical strands of protofilaments which form at the ends of depolymerizing microtubules, and/or along the surface of the microtubules. Substoichiometric inhibition of microtubule assembly, which occurs at low vinblastine concentrations, appears to be due to the binding of vinblastine to the high affinity class of sites. Fifty per cent inhibition of tubulin addition to the net assembly ends of steady-state microtubules occurred at 1.38 × 10?7m-drug, and at this concentration, 1.16 ± 0.27 molecules of vinblastine were bound to the high affinity class of sites. Vinblastine appeared to bind directly to the microtubule ends, and our results indicate that vinblastine inhibits the assembly of steady-state bovine brain microtubules by binding rapidly and with high affinity to one or two molecules of tubulin at the net assembly ends. Splaying and peeling of protofilaments at microtubule ends and the active depolymerization of microtubules occurred only at vinblastine concentrations greater than 1 × 10?6 to 2 × 10?6m. This action of vinblastine is associated with and may be due to the binding of vinblastine to the high capacity class of sites. Both actions of vinblastine may be due to the binding of vinblastine to the same binding sites on the tubulin molecule, with the sites exhibiting either a high or low affinity depending upon the location in the microtubule. 相似文献
18.
Electron microscopic investigations on the foraminifer Allogromia laticollaris showed that after treatment with 10(-3) M vinblastine tubulin paracrystals can be demonstrated in intermitotic nuclei. As these paracrystals are either membrane coated or lie free in the karyoplasm, and as in the perinuclear cytoplasm, membrane coated paracrystals can be demonstrated as well, it is assumed that the cytoplasmic tubulin which is composing the intranuclear division spindle can transverse the intact nuclear envelope via vesicle transport. 相似文献
19.
Antitubulin immunofluorescent staining was used to examine the relationship among crystal formation, mitotic arrest, and recovery potential in vinblastine-treated Chinese hamster cells. Although vinblastine caused a mitotic block at concentrations as low as 5 x 10(-9) M, it induced tubulin crystal formation only at concentrations higher than 10(-6) M. At these higher concentrations, cells took 48-72 h to recover after return to normal medium. This extended period of time was apparently needed for breakdown of the crystals and regeneration of normal cytoplasmic microtubules. At concentrations less than 10(-6) M, although the mitotic block was still effective, no crystals were present. Possibly because of this lack of crystal formation, the cells recovered rapidly, generating cytoplasmic microtubules within 30 min, and beginning to undergo mitosis within 60 min. These findings tend to support biochemical evidence that tubulin binds to vinblastine at two types of binding site: a high affinity, low capacity site, responsible for tubulin disaggregation; and a low affinity site, responsible for protofilament splaying. 相似文献
20.
Tritium-labeled viblastine, specific activity 107 Ci/mol, was prepared by acetylation of desacetylvinblastine with [3H]acetic anhydride, and has been employed in a study of vinblastine binding to tubulin. There are two high affinity vinblastine-binding sites per mole of embryonic chick brain tubulin (KA = 3-5 X 10(5) l./mol). Binding to these sites was rapid, and relatively independent of temperature between 37 and 0degreeC. Vincristin sulfate and desacetylvinblastine sulfate, two other active vinca alkaloid derivatives, competitively inhibited the binding of vinblastine. The inhibition constant for vincristine was 1.7 X 10(-5) M; and for desacetylvinblastine, 2 X 10(-5) M. The vinblastine binding activity of tubulin decayed upon aging, but this property was not studied in detail. Vinblastine did not depolymerize stable sea urchin sperm tail outer doublet microtubules, nor did it bind to these microtubules. However, tubulin solubilized from the B subfiber of the outer doublet microtubules possessed the two high affinity binding sites (KA = 1-3 X 105 l./mol). These data suggest that vinblastine destroys microtubules in cells primarily by inhibition of microtubule polymerization, and does not directly destroy preformed microtubules. 相似文献