Effect of colchicine on polymerization of tubulin from rats, mice, hamsters and guinea-pigs.

Colchicine-inhibition of polymerization of tulbulin from rats, mice, golden hamsters and guinea-pigs was studied to determine if species differences in tubulin sensitivity to colchicine might parallel species variation in colchicine toxicity. It was found that polymerization of tubulin is nearly equally sensitive to colchicine in all four species.     

Comparison of the in vitro effects of colchicine and its derivative colchiceine on chondrocyte morphology and function

Colchiceine is a colchicine-metabolite which has been reported to inhibit axonal transport although not binding to brain tubulin. In the present study, colchiceine was shown not to depolymerize cytoplasmic microtubules, nor to mimic other effects of colchicine on the ultrastructure of cultured chondrocytes. In addition, the synthesis of proteoglycans was inhibited by colchicine but slightly stimulated by colchiceine. These results support the idea that the disturbances in cultured chondrocytes caused by colchicine are specifically related to a loss of cytoplasmic microtubules.     

Comparison of the in vitro effects of colchicine and its derivative colchiceine on chondrocyte morphology and function

Summary Colchiceine is a colchicine-metabolite which has been reported to inhibit axonal transport although not binding to brain tubulin. In the present study, colchiceine was shown not to depolymerize cytoplasmic microtubules, nor to mimic other effects of colchicine on the ultrastructure of cultured chondrocytes. In addition, the synthesis of proteoglycans was inhibited by colchicine but slightly stimulated by colchiceine. These results support the idea that the disturbances in cultured chondrocytes caused by colchicine are specifically related to a loss of cytoplasmic microtubules.This work was supported by grants from the Swedish Medical Research Council (proj. No. 12X-03355), the King Gustaf V 80th Birthday Fund, the Swedish Society of Medical Sciences, and from the funds of Karolinska Institutet.     

Is tubulin involved in the electrically-induced mechanical activity of the isolated rat sciatic nerve?

Summary The electrically-induced mechanical activity of isolated segments of rat sciatic nerves remains unaffected following incubation with 10^–4 M colchicine, vinblastine or melatonin. Vinblastine depressed tubulin levels in incubated nerves. These results suggest that microtubules are not involved in nerve mechanical activity in vitro.This work was supported by grant No. 6638 from the CONICET (Argentina). It is dedicated to the memory of C.E. Valenti, decreased November 1979.     

A structural view of microtubule dynamics

The essential microtubule property of dynamic instability is based on the binding, hydrolysis and exchange of GTP in each tubulin dimer. The recent high-resolution structures of tubulin and the microtubule have given us the first view at atomic level of properties such as nucleotide exchangeability, the linkage between polymerization and nucleotide hydrolysis. and the origin of microtubule destabilization, as well as the mode of action of antimitotic agents such as taxol.     

Microtubule targeting agents: from biophysics to proteomics

This review explores various aspects of the interaction between microtubule targeting agents and tubulin, including binding site, affinity, and drug resistance. Starting with the basics of tubulin polymerization and microtubule targeting agent binding, we then highlight how the three-dimensional structures of drug–tubulin complexes obtained on stabilized tubulin are seeded by precise biological and biophysical data. New avenues opened by thermodynamics analysis, high throughput screening, and proteomics for the molecular pharmacology of these drugs are presented. The amount of data generated by biophysical, proteomic and cellular techniques shed more light onto the microtubule–tubulin equilibrium and tubulin–drug interaction. Combining these approaches provides new insight into the mechanism of action of known microtubule interacting agents and rapid in-depth characterization of next generation molecules targeting the interaction between microtubules and associated modulators of their dynamics. This will facilitate the design of improved and/or alternative chemotherapies targeting the microtubule cytoskeleton.     

Isoforms of soluble alpha-tubulin in oocytes and brain of the frog (genus Rana): changes during oocyte maturation

Rana oocytes have previously been shown to contain much more soluble tubulin than does the brain, suggesting different assembly and disassembly dynamics of frog oocyte tubulin compared to that in brain. By using centrifugation, SDS-PAGE, two-dimensional gel electrophoresis and Western blots, probed with anti-α-tubulin monoclonal antibodies, polymorphic α-tubulins (isoforms) were compared in brains and follicle-enclosed oocytes of northern (Rana pipiens) and southern (R. berlandieri) frogs. Oocyte tubulin in both species had isoforms with greater ranges of isoelectric point (pI) than those of brain tubulins; in particular, the oocyte tubulin pIs ranged further into the acidic region of the isoelectric-focusing gels than corresponding brain tubulin. This difference may, in part, be responsible for the previously reported assembly differences between oocyte tubulin (undetectable assembly) and brain tubulin (high assembly). Isoforms of α-tubulin with relat ively acidic pI were more abundant in northern frog brain and oocyte soluble extracts than in analogous extracts from southern frogs. Furthermore, additional acidic α-tubulin isoforms were found in progesterone-treated oocytes (i.e., eggs), indicating increased heterogeneity of acidic a-tubulin isoforms during oocyte meiotic maturation. Among northern frog oocyte soluble components fractionated on Superose-6b columns, tubulin complexes with apparent molecular mass of about 1800 kDa were found to contain acidic α-tubulin isoforms while the putative oligomeric tubulins with an apparent molecular mass of about 250 kDa contained an additional relatively basic α-tubulin isoform. The acidic α-tubulin isoforms, therefore, are proposed to be associated with cold-adaptable cells of brain and oocytes, and may also be involved in stabilization of large soluble tubulin complexes in oocytes of the northern frog. Received 1 October 2002; accepted 9 October 2002 RID="*" ID="*"Corresponding author.     

Calmodulin mediates melatonin cytoskeletal effects

In this article, we review the data concerning melatonin interactions with calmodulin. The kinetics of melatonin-calmodulin binding suggest that the hormone modulates cell activity through intracellular binding to the protein at physiological concentration ranges. Melatonin interaction with calmodulin may allow the hormone to modulate rhythmically many cellular functions. Melatonin's effect on tubulin polymerization, and cytoskeletal changes in MDCK and N1E-115 cells cultured with melatonin, suggest that at low concentrations (10^–9 M) cytoskeletal effects are mediated by its antagonism to Ca²⁺-calmodulin. At higher concentrations (10^–5 M), non-specific binding of melatonin to tubulin occurs thus overcoming the specific melatonin antagonism to Ca²⁺-calmodulin. Since the structures of melatonin and calmodulin are phylogenetically well preserved, calmodulin-melatonin interaction probably represents a major mechanism for regulation and synchronization of cell physiology.     

Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin A-4

The African tree Combretum caffrum (Combretacae) has been found to contain a powerful inhibitor of tubulin polymerization (IC50 2-3 microM), the growth of murine lymphocytic leukemia (L 1210 and P 388 with ED50 approximately 0.003 microM and human colon cancer cell lines [(e.g. LoVo (ED50 = 0.005 microgram/ml), HT 29 (ED50 0.02 microgram/ml, Colo 205 (ED50 = 0.07 microgram/ml), DLD-1 (ED50 = 0.005 microgram/ml) and HCT-15 (ED50 = 0.0009 microgram/ml] designated combretastatin A-4 (1c). The structure assigned by spectral techniques was confirmed by synthesis.     

Effects of castration, estradiol and testosterone on tubulin levels of the medial basal hypothalamus and the adenohypophysis of the rat.

Tubulin levels of the medial basal hypothalamus (MBH) were greater in male than in female rats. Orchidectomy brought about a decrease of MBH tubulin concentration, whereas testosterone injection augmented it in the MBH and adenohypophysis. Estradiol administration augmented MBH tubulin and protein concentration.     

Effects of castration,estradiol and testosterone on tubulin levels of the medial basal hypothalamus and the adenohypophysis of the rat

Summary Tubulin levels of the medial basal hypothalamus (MBH) were greater in male than in female rats. Orchidectomy brought about a decrease of MBH tubulin concentration, whereas testosterone injection augmented it in the MBH and adenohypophysis. Estradiol administration augmented MBH tubulin and protein concentration.This study was supported in part by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina (CONICET) (grant No. 6638), and the Programa Latinoamericano de Investigación en Reproducción Humana (PLAMIRH 2.3.1.75 R).     

Tubulin pools in human erythrocytes: altered distribution in hypertensive patients affects Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase activity

The presence of tubulin in human erythrocytes was demonstrated using five different antibodies. Tubulin was distributed among three operationally distinguishable pools: membrane, sedimentable structure and soluble fraction. It is known that in erythrocytes from hypertensive subjects (HS), the Na⁺, K⁺-ATPase (NKA) activity is partially inhibited as compared with erythrocytes from normal subjects (NS). In erythrocytes from HS the membrane tubulin pool is increased by ~150%. NKA was found to be forming a complex with acetylated tubulin that results in inhibition of enzymes. This complex was also increased in erythrocytes from HS. Treatment of erythrocytes from HS with nocodazol caused a decrease of acetylated tubulin in the membrane and stimulation of NKA activity, whereas taxol treatment on erythrocytes from NS had the opposite effect. These results suggest that, in erythrocytes from HS, tubulin was translocated to the membrane, where it associated with NKA with the consequent enzyme inhibition.     

5'-Nucleotidase activity in liver homogenates of rats treated with CCl4, colchicine, cycloheximide, emetine, ethanol, ethionine and 5-fluorotryptophan

5'-Nucleotidase activity an enzyme marker of the plasma membranes, increases in female rat liver homogenates following ethionine administration, while homogenates from males show no changes. Treatment with CCl4, colchicine, cycloheximide, emetine, ethanol and 5-fluorotryptophan does not significantly modify the 5'-nucleotidase activity of liver homogenates of either female or male rats.     

Colchicine inhibition of ADH effect on frog skin permeability

ADH and AMPc enhance both thiourea unidirectional fluxes in frog skin. This effect is completely abolished by colchicine pretreatment. The ADH increase of thiourea discharge with or without colchicine led us to suppose that colchicine does not directly affect ADH action on outer membrane permeability, but exerts its effects on a site which is limiting for the ADH action on transepithelial permeability.     

Melatonin antagonizes colchicine-induced mitotic arrest.

Melatonin, in concentrations up to 10(-3) M, showed no effect on mitosis in cultures of HeLa or KB cells. However, when melatonin at 10(-4) M was preincubated with HeLa cells prior to addition of 10(-7) M colchicine, a reduction in the mitotic index, in comparison to colchicine alone, was observed.     

The role of kinesin, dynein and microtubules in pancreatic secretion

The regulated secretion of pancreatic zymogens depends on a functional cytoskeleton and intracellular vesicle transport. To study the dynamics of tubulin and its motor proteins dynein and kinesin during secretion in pancreatic acinar cells, we infused rats with 0.1 μg/kg/h caerulein. Electron and fluorescence microscopy detected neither dynein nor kinesin at the apical secretory pole, nor on the surface of mature zymogen granules. After 30 min of secretagogue stimulation, kinesin and the Golgi marker protein 58 K were reallocated towards the apical plasma membrane and association of kinesin with tubulin was enhanced. Disruption of acinar cell microtubules had no effect on initial caerulein-induced amylase release but completely blocked secretion during a second stimulus. Our results suggest that mature zymogen granule exocytosis is independent of intact microtubules, kinesin and dynein. However, microtubule-dependent mechanisms seem to be important for the replenishment of secretory vesicles by redistribution of Golgi elements towards the apical cell pole. J. Schnekenburger and I.-A. Weber have contributed equally to this work.     

A basic protein from bovine brain that co-precipitates with tubulin in vitro

A 193 kDa protein consisting of 58 kDa subunits, which has pI values of 8.50 and 8.65, was purified from bovine brain cytosol. It formed heavy precipitates with tubulin, and the molar ratio of tubulin dimer to this protein in the precipitate was 3.2. In contrast to microtubules containing ordinary microtubule-associated proteins, these complexes remained stable against cold and 1 mM CaCl2.     

Colchicine inhibition of ADH effect on frog skin permeability

Summary ADH and AMPc enhance both thiourea unidirectional fluxes in frog skin. This effect is completely abolished by colchicine pretreatment. The ADH increase of thiourea discharge with or without colchicine led us to suppose that colchicine does not directly affect ADH action on outer membrane permeability, but exerts its effects on a site which is limiting for the ADH action on transepithelial permeability.The present work has been supported by C.N.R., Rome.     

Changes in total and polymerized tubulin of the medial basal hypothalamus and adenohypophysis of castrated or hormone-injected rats

Summary Treatment of orchidectomized rats with LH, FSH or prolactin decreased the tubulin content of the medial basal hypothalamus (MBH), whereas FSH or prolactin augmented it in the adenohypophysis (AH). After castration, negative correlations existed between serum LH and total or polymerized MBH tubulin, whereas in the AH positive correlations were found. After estradiol-progesterone treatment of spayed rats a significant correlation was found between serum LH and the percentage of AH tubulin in the polymerized form.Deceased November 24, 1979This study was supported in part by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CON-ICET), Argentina (grant n^o 6638) and a John Simon Guggenheim Memorial Foundation Fellowship to DPC.