Colchicine and the mitotic spindle of the aquatic phycomycete Allomyces

Summary Exposure of germlings of Allomyces neo-moniliformis to colchicine for 0 to 5 min after zoospore encystment was found to block 30% of germlings derived from flagellated zoospores and 55% of germlings derived from deflagellated zoospores in C-metaphase configurations at the first mitotic division. The zoospore lacks a pool of colchicine binding protein, and protein synthesis is absent during the time when colchicine first becomes effective in inducing C-metaphase. From these observations it is concluded that the microtubule subunit protein of the spindle apparatus of the first mitotic division to a large extent is derived from the depolymerization of the cytoplasmic microtubules of the zoospore. GTP, Mg²⁺, and ATP were observed to be antagonistic to the action of colchicine in vivo. It is suggested that these compounds may compete with colchicine for binding to the subunit protein in vivo. Germlings derived from flagellated zoospores are appreciably less subject to the action of colchicine in the presence of the antagonistic compounds than are germlings derived from deflagellated zoospores. This differential sensitivity to colchicine is interpreted as reflecting a difference in the quantity of microtubule subunit protein present at the time of exposure to colchicine.     

Characterization of a colchicine receptor protein in rat epididymal adipose tissue

Colchicine was found to be taken up by adipose tissue and therein to bind to a soluble macromolecule not sedimented by centrifugation for 2 h at 100 000 × g. A similar binding occurred when soluble extracts of adipose tissue were incubated with colchicine. The binding reaction is temperature dependent and shows a pH optimum between 6.8 and 7.0. Double reciprocal plots of colchicine concentration versus amounts of colchicine bound to protein in the steady state disclosed an apparent Km of 0.250 to 1.5 ωM. The colchicine binding activity of soluble tissue extracts decreased when the extracts were incubated at 37°C. Addition of guanosine triphosphate and Mg²⁺ retarded the loss of colchicine binding activity. The molecular weight of the colchicine complex was estimated to be 115 000 and its sedimentation coefficient 5.8 S. All of these characteristics are remarkably similar to those of the protein tubulin which has been isolated from other tissues. Since it is now well known that tubulin is a protein subunit of cytoplasmic microtubules, it is suggested that the previously reported metabolic effects of colchicine on adipose tissue result from the dissolution of microtubules by colchicine.     

H-colchicine binding : Failure to detect any binding to soluble proteins from various lower organisms

No ³H-colchicine binding could be detected to post-ribosomal supernatants from Schizosaccharomyces pombe, Chlamydomonas reinhardii, Tetrahymena pyriformis, pea root tips, or Zea mays coleoptiles. However, under the same conditions, ³H-colchicine bound to supernatants from mouse brain and unfertilised sea urchin eggs. A protein with similar characteristics to sea urchin and mouse brain tubulins in terms of its molecular weight and elution properties from DEAE-cellulose columns has been detected in supernatants from the yeast S. pombe. Similar results (unpublished) have been obtained for both Zea mays and Tetrahymena. This protein is tentatively identified as tubulin. The lack of binding of ³H-colchicine by supernatants from lower organisms is consistent with the variable sensitivity of nuclear division to colchicine, and with the possible evolution of the colchicine-binding site of tubulin.     

An Improved DEAE-Cellulose Filter Assay for Colchicine Binding Protein (During Mouse Brain Development)

A DEAE-cellulose filter assay for colchicine binding protein (CBP) has been modified to include 1 m -sucrose in the incubation medium (single-point assay). Due to the much greater stability of colchicine binding capacity under the conditions described, multiple time-point assays (time-decay method) were not necessary for accurate determination of CBP in brain samples, as shown by the agreement of results obtained using the two methods to measure CBP in C129F₁ hybrid mouse brains at different stages of development. In this study, the highest concentrations of CBP (millimoles of colchicine bound per milligram of total protein) were observed in the 160,000g supernatant and pellet of newborn-brain homogenate. The CBP concentrations in these two fractions were approximately twice those observed in the earliest stage measured (13.5 days gestation) and in the 11-month-old adult. However, CBP concentration, in terms of millimoles of colchicine bound per gram of brain wet weight, reached a maximum at the end of the 1st week after birth. Between 17.5 days gestation and the end of the 1st postnatal week, more CBP accumulated in the pellet fraction than in the supernatant. Tse C. and Doherty R. A. An improved DEAE-cellulose filter assay for colchicine binding protein (during mouse brain development). J. Neurochem. 35, 767–774 (1980).     

Resistance of Rosa microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin

The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24°C with an apparent inhibition constant (K _i) of 1.4·10^-4 M for rose tubulin and an apparent K _i=8.8·10^-7 M for brain tubulin. The binding of [³H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2–3 h to reach equilibrium at 24°C, and was not affected by vinblastine sulfate. The binding of [³H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7·10² M^-1 and an estimated molar binding stoichiometry (r) of 0.47 at 24°C. The values for brain tubulin were K=2.46·10⁶ M^-1 and r=0.45 at 37°C. The binding of [³H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin.Abbreviations MT microtubule - TC tubulin-colchicine complex     

Characterization of colchicine binding with normal and glycated albumin: In vitro and molecular docking analysis

The transport of more than 90% of the drugs viz. anticoagulants, analgesics, and general anesthetics in the blood takes place by albumin. Hence, albumin is the prime protein needs to be investigated to find out the nature of drug binding. Serum albumin molecules are prone to glycation at elevated blood glucose levels as observed in diabetics. In this piece of work, glycation of bovine serum albumin (BSA) was carried out with glyceraldehyde and characterized by molecular docking and fluorometry techniques. Glycation of BSA showed 25% loss of free amino groups and decreased protein fluorescence (60%) with blue shift of 6 nm. The present study was also designed to evaluate the binding of colchicine (an anti-inflammatory drug) to native and glycated BSA and its ability to displace 8-analino-1-nephthalene sulfonic acid (ANS), from the BSA–ANS complex. Binding of ANS to BSA showed strong binding (K_a = 4.4 μM) with native conformation in comparison to glycated state (K_a = 8.4 μM). On the other hand, colchicine was able to quench the fluorescence of native BSA better than glycated BSA and also showed weaker affinity (K_a = 23 μM) for glycated albumin compared with native state (K_a = 16 μM). Molecular docking study showed that both glyceraldehyde and colchicine bind to common residues located near Sudlow’s site I that explain the lower binding of colchicine in the glycated BSA. Based on our results, we believe that reduced drugs-binding affinity to glycated albumin may lead to drugs accumulation and precipitation in diabetic patients.     

Colchicine binding to an oligomer of tubulin

An oligomeric form of tubulin present in microtubule protein prepared from mammalian brain, the 36S double ring containing tau protein, is reported to bind colchicine. Colchicine binds to each individual 6S tubulin subunit in the 36S ring without apparent effect on quarternary structure. The colchicine-oligomer complex forms by colchicine binding directly to the tubulin ring; alternatively, complexes formed by colchicine with 6S tubulin subunits associate in the presence of tau protein to form the colchicine-oligomer complex.     

Effect of experimental colchicine encephalopathy on brain protein synthesis and tubulin metabolism

Colchicine blocks axoplasmic flow and produces neurofibrillary degeneration. Brain slices from mice injected intracerebrally with colchicine incorporated more [¹⁴C]leucine into protein and had a decreased uptake of [¹⁴C]leucine into the perchloric acid-soluble pool than did their controls. Brain RNA content was decreased and free leucine increased by colchicine-induced encephalopathy. The specific activities of proteins from subcellular fractions of colchicine-injected brain were increased in the nuclear fraction, the 100,000-g supernatant, and its vinblastine-precipitable tubulin. The ratio of the specific activity of the crude mitochondrial fraction to that of the total homogenate was decreased, as would consistent with impaired movement of newly labeled protein into synaptosomes. Colchicine-injected brain extracts contained one or more cytosol fractions that stimulated ribosomal incorporation of [¹⁴C]leucine into protein in a cell-free system. Colchicine-binding-activity measurements indicated loss of soluble and particulate tubulin in colchicine-injected brains; the decrease of soluble tubulin was verified by its selective precipitation with vinblastine. Colchicine encephalopathy did not affect the rate of spontaneous breakdown of in vitro colchicine binding activity. Similarities of colchicine encephalopathy to the neuron's response to axonal damage suggest that colchicine-induced increase in protein synthesis may, in part, reflect a neuronal response to blockage of neuroplasmic transport.     

Tubulin from Mimosa pudica and its involvement in leaf movement

The sensitive plant Mimosa pudica is made insensitive by a brief treatment with colchicine. A high concentration of colchicine binding protein is present in the fresh actively moving leaves of M. pudica. This protein was partially characterized and compared with the animal brain tubulin. This colchicine binding activity is very low in the insensitive variety of Mimosa, namely Mimosa rubricaulis.     

Pollen proteins after two-dimensional gel electrophoresis and pollen morphology of the amphiploids Aegilops kotschyi and Ae. variabilis with Secale cereale

Proteins from pollen of parent forms and amphiploids Aegilops variabilis ×Secale cereale and Ae. kotschyi×S. cereale, obtained by in vitro propagation or colchicine treatment of F₁ hybrids, were subjected to a study by two-dimensional (2-D) electrophoresis. Qualitative and quantitative diversities of protein patterns were revealed for the amphiploid pollen. The majority of peptides found in the parent forms were also present in the patterns of the amphiploid pollen; however, some of the parent-form-peptides were not expressed and proteins characteristic only of the amphiploids appeared. In the 2-D combined protein pattern obtained for the parent forms, amphiploids Ae. variabilis × S. cereale produced pollen with a poorer spectrum of proteins. In amphiploid 408B, obtained from treated the F₁ generation with colchicine, the 2-D pattern revealed the presence of less than 50% of the proteins recorded for the parent forms. Pollen grain morphology was studied under a scanning microscope. The structure and shape of exines differed from those of the parents. In the parent forms the pollen grains had only one pore, while in amphiploid pollen, one, two or three pores were observed. Possible explanations for the differences in the 2-D patterns of amphiploids and their parent forms (impoverishment of the protein spectrum and appearance of new peptides) are (1) somaclonal variation and mutagenic activity of colchicine, (2) suppression of structural genes, (3) activity of regulators and (4) translocations. Pollen grains with two or even three pores could appear as a result of the independent activity of the genes from three amphiploidal genomes. Received: 6 March 2001 / Accepted: 26 June 2001     

Colchicine binding of cell extracts from colchicine-resistant mutants of Chlamydomonas reinhardi

The colchicine-binding activity of a high speed supernatant from fourteen colchicine- and/or vinblastine-resislant mutants of Chlamydomonas reinhardi has been compared to that of wild type. Four of the mutants have reduced binding per unit protein. The low level of binding of one of these mutants is unusually stable. Three other mutants have normal initial binding levels, but show altered kinetics of decay of binding activity. Most of the mutants with altered colchicine-binding activity produce abnormally large cells. Seven other mutants showed only slight or no differences in colchicine binding from wild type.     

Distinct and overlapping binding sites for IKP104 and vinblastine on tubulin

IKP104 is one of a group of tubulin-binding drugs whose interaction with tubulin suggests that it may bind to the protein at or close to the region where vinblastine binds. By itself IKP104 is a potent enhancer of tubulin decay as evidenced by the fact that it induces the exposure of the sulfhydryl groups and hydrophobic areas on tubulin. In this respect, IKP104 differs from vinblastine and other drugs such as phomopsin A, dolastatin 10, rhizoxin, and maytansine which are competitive or noncompetitive inhibitors of vinblastine binding. In contrast, however, in the presence of colchicine, IKP104 behaves differently and strongly stabilizes tubulin, to an extent much greater than does colchicine alone. IKP104 appears to have two classes of binding site on tubulin, differing in affinity; the acceleration of decay appears to be mediated by the low-affinity site (Chaudhuriet al., 1998,J. Protein Chem., in press). We investigated the relationship of the binding of IKP104 and vinblastine. We found that the high-affinity site or sites of IKP104 overlap with or interact with the vinblastine-binding sites, but that the low-affinity site is distinctly different.     

Distinct and overlapping binding sites for IKP104 and vinblastine on tubulin

IKP104 is one of a group of tubulin-binding drugs whose interaction with tubulin suggests that it may bind to the protein at or close to the region where vinblastine binds. By itself IKP104 is a potent enhancer of tubulin decay as evidenced by the fact that it induces the exposure of the sulfhydryl groups and hydrophobic areas on tubulin. In this respect, IKP104 differs from vinblastine and other drugs such as phomopsin A, dolastatin 10, rhizoxin, and maytansine which are competitive or noncompetitive inhibitors of vinblastine binding. In contrast, however, in the presence of colchicine, IKP104 behaves differently and strongly stabilizes tubulin, to an extent much greater than does colchicine alone. IKP104 appears to have two classes of binding site on tubulin, differing in affinity; the acceleration of decay appears to be mediated by the low-affinity site (Chaudhuriet al., 1998,J. Protein Chem., in press). We investigated the relationship of the binding of IKP104 and vinblastine. We found that the high-affinity site or sites of IKP104 overlap with or interact with the vinblastine-binding sites, but that the low-affinity site is distinctly different.     

Binding of colchicine to a soluble fraction of carrot cells grown in suspension culture

The binding of [³H]colchicine to soluble component prepared from carrot (Daucus carota L.) cells in suspension culture was assayed by the diethylaminoethyl(DEAE)-cellulose powder method. The binding activity was very labile and the time course of the binding indicated that the colchicine-bound complex was also unstable. The reaction was enhanced by vinblastine but lumicolchicine had no effect. The optimum temperature for the reaction was 30° C, and the colchicine binding constant was calculated to be 3.5·10⁴ l mol^–1 at 30° C.     

The effect of colchicine on pyrin and pyrin interacting proteins

MEFV which encodes pyrin, cause familial Mediterranean fever (FMF), the most common auto‐inflammatory disease. Pyrin is believed to be a regulator of inflammation, though the nature of this regulatory activity remains to be identified. Prophylactic treatment with colchicine, a microtubule toxin, has had a remarkable effect on disease progression and outcome. It has been thought that, inhibition of microtubule polymerization is the main mechanism of action of colchicine. But, the exact cellular mechanism explaining the efficacy of colchicine in suppressing FMF attacks is still unclear. Given the ability of colchicine treatment to be considered as a differential diagnosis criteria of FMF, we hypothesized that colchicine may have a specific effect on pyrin and pyrin interacting proteins. This study showed that colchicine prevents reticulated fibrils formed by PSTPIP1 filaments and reduces ASC speck rates in transfected cells. We further noted that, colchicine down‐regulates MEFV expression in THP‐1 cells. We also observed that colchicine causes re‐organization of actin cytoskeleton in THP‐1 cells. Pyrin is an actin‐binding protein that specifically localizes with polymerizing actin filaments. Thus, MEFV expression might be affected by re‐organization of actin cytoskeleton. The data presented here reveal an important connection between colchicine and pyrin which might explain the remarkable efficacy of colchicine in preventing FMF attacks. J. Cell. Biochem. 113: 3536–3546, 2012. © 2012 Wiley Periodicals, Inc.     

Isolation, Crystallization, and Investigation of Ribosomal Protein S8 Complexed with Specific Fragments of rRNA of Bacterial or Archaeal Origin

The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restricted by nucleotides 588-602 and 636-651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA fragments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and synthesized in preparative amounts in vitro using T7 RNA-polymerase. Stable S8–RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bacterial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37-nucleotide rRNA fragment from the same organism suitable for X-ray analysis were obtained.     

Tubulin-dependent secretion of S100A6 and cellular signaling pathways activated by S100A6-integrin β1 interaction

S100A6 is a calcium binding protein expressed mainly in fibroblasts and epithelial cells. Interestingly, S100A6 is also present in extracellular fluids. Recently we have shown that S100A6 is secreted by WJMS cells and binds to integrin β1 (Jurewicz et al., 2014). In this work we describe for the first time the mechanism of S100A6 secretion and signaling pathways activated by the S100A6-integrin β1 complex. We show that colchicine suppressed the release of S100A6 into the cell medium, which indicates that the protein might be secreted via a tubulin–dependent pathway. By applying double immunogold labeling and immunofluorescence staining we have shown that S100A6 associates with microtubules in WJMS cells. Furthermore, results obtained from immunoprecipitation and proximity ligation assay (PLA), and from in vitro assays, reveal that S100A6 is able to form complexes with α and β tubulin in these cells, and that the S100A6-tubulin interaction is direct. We have also found that the S100A6 protein, due to binding to integrin β1, activates integrin-linked kinase (ILK), focal adhesion kinase (FAK) and p21-activated kinase (PAK). Our results suggest that binding of S100A6 to integrin β1 affects cell adhesion/proliferation due to activation of ILK and FAK signaling pathways.     

Podophyllotoxin and nocodazole counter the effect of IKP104 on tubulin decay

Tubulin, the subunit protein of microtubules, undergoes a time-dependent loss of functional properties known as decay. We have previously shown that the drug 2-(4-fluorophenyl)-1-(2-chloro-3,5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone (IKP104) accelerates decay, but that in the presence of colchicine, IKP104 becomes a stabilizer of tubulin. To see if this is due to conformational effects specific to colchicine or simply to occupancy at the colchicine site, we examined the effects of nocodazole and podophyllotoxin, two well-known competitive inhibitors of colchicine for binding to tubulin, on IKP104’s acceleration of decay. We found that podophyllotoxin abolished IKP104’s accelerating effect and, like colchicine, turned it into a stabilizer of tubulin. Nocodazole’s effects were similar to those of podophyllotoxin and colchicine, in that it abolished IKP104-induced enhancement of decay; however, in the presence of nocodazole, IKP104 caused little or no stabilization of tubulin. Since colchicine, nocodazole, and podophyllotoxin have very different interactions with tubulin, but all inhibit the IKP104-induced enhancement of decay, our findings suggest that this inhibition arises from occupancy of the colchicine site rather than from a direct conformational effect of these two drugs.     

Colchicine-binding protein and the secretion of thyroid hormone

The role of microtubules in the thyrotropin- or adenosine 3'',5'' cyclic monophosphate (cyclic AMP)-stimulated accumulation of cytoplasmic colloid droplets and secretion of iodine from the mouse thyroid gland has been investigated by means of different classes of agents that affect the stability of microtubules. The onset of inhibition of secretion by colchicine, the uptake of colchicine-³H by thyroid lobes, and the binding of colchicine-³H to thyroidal soluble protein are shown to have similar time courses Colloid droplet accumulation is also inhibited and does not readily resume upon removal of colchicine from the medium. This appears to be due to the slow washout of the drug (t _½ ∼ hr). Thyroids contain a soluble colchicine-binding protein that resembles microtubule proteins of other tissues with respect to apparent K_m for colchicine, pH optimum, and stability characteristics Colchicine analogues inhibit iodine secretion and colchicine binding in a parallel manner and as a function of their antimitotic potencies. Microtubule-stabilizing agents such as hexylene glycol and D₂O also inhibit secretion. Thus, inhibition of thyroid secretion by antimitotic agents appears to be mediated by an effect on microtubules. The inhibitory locus of colchicine inhibition occurs after the generation of cyclic AMP, since stimulation of secretion by this nucleotide is blocked by colchicine, whereas thyroid-stimulating hormone—induced accumulation of cyclic AMP is not affected. Thus, the functioning microtubule appears to play a role in the induction of colloid endocytosis.     

Organization of Lrp-binding sites upstream of ilvlH in Salmonella typhimurium

Lrp, a major regulatory protein in Escherichia coli, controls the expression of numerous operons, including ilvlH. Lrp binds to six sites upstream of ilvlH, and Lrp binding is required for ilvlH expression. We show here that an Lrp-like protein is also present in Salmonella typhimurium. This protein can bind both E. coli and S. typhimurium ilvlH DNA, as can E. coli Lrp. Methidiumpropyl-EDTA footprinting studies were performed with purified E. coli Lrp and S. typhimurium ilvlH DNA. Six binding sites were defined, three of them being similar to corresponding sites in E. coli, and three being organized differently. A consensus derived from six S. typhimurium sites is compatible with that derived from a similar analysis of E. coli sequences.