Roles of nucleoside triphosphates in microtubule assembly

Depolymerization of microtubules in the ATP-reassembly buffer permitted the preparation of GDPETNGTP. Incubation of this tubulin fraction at 35 degrees with ATP induced the phosphorylation of E-site GDP into GTP, which was then dephosphorylated during microtubule assembly. Incubation of GDPETNGTP with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40] also induced polymerization. Depolymerization of microtubules in the GTP-reassembly buffer yielded GTPETNGTP, which was capable of polymerizing into microtubules even in the absence of free GTP. In the presence of 4 M glycerol, GDPETNGTP assembled into microtubules with no change in the bound nucleotides.     

Glucocorticoid receptor inhibits microtubule assembly in vitro

The effect of glucocorticoid hormones, purified glucocorticoid receptor (GR) and purified heat shock protein M(r) 90,000 (hsp90) on microtubule (MT) assembly in vitro was tested by a spectrophotometric MT assembly assay and electron microscopy. GR significantly prolonged the nucleation phase, slowed down the assembly rate and reduced the maximal amplitude of MT assembly compared with control. The effects were partially reversed by the addition of glucocorticoid hormone. GR associated with MTs. These results indicate that GR affects MT assembly in vitro, which may be a functional correlate to the structural association of GR with MTs. This implies that factors affecting GR may affect MT assembly in vivo.     

Inability to detect Chlamyodomonas microtubule assembly in vitro: possible implications to the in vivo regulation of microtubule assembly

It has been demonstrated that the in vitro assembly of microtubules from Chlamydomonas preparations does not occur under a wide range of conditions, including those efficacious for mammalian brain tubulin. This incompetence of Chlamydomonas extracts to form microtubules is independent of the tubulin concentration, the presence of added nucleotides or an added seed, temperature, or the concentration of divalent cation. However, an amorphous aggregate was observed under certain conditions, who composition was mainly tubulin. The in vitro reassembly of microtubules in gerbil brain extracts is inhibited by Chlamydomonas preparations. Fractionation of the Chlamydomonas extracts by column chromatography suggests that the inhibitory component is Chlamydomonas tubulin itself. The mechanism of this inhibition is unknown, but reassembly experiments indicate that the 2 types of tubulins cannot copolymerize. We suggest that the Chlamydomonas tubulin, derived from a cytoplasmic pool, requires to be activated prior to its in vivo polymerization into microtubules.     

Synthesis of guanosine 5'-triphosphate analogues and their effect on microtubule assembly

More than 20 base-modified analogues of guanosine 5'-triphosphate including 2'-deoxyguanosine derivatives were synthesized and examined their effect on tubulin polymerization into microtubules (Mts). Among those, 2,6-diamino-8-oxopurineriboside 5'-triphosphate (1d), 2,6-diamino-2'-deoxypurineriboside 5'-tri phosphate (1b) and 8-bromoguanosine 5'-tri phosphate (1g) were shown to have remarkable effect to promote microtubule assembly.     

Promotion of microtubule assembly by oligocations: cooperativity between charged groups

The rate and, to a lesser degree, the extent of microtubule assembly from rat brain tubulin is enhanced by oligocations such as polyamines, melittin, polybasic drugs, oligolysines, and oligoarginines. The effect is cooperative for degrees of polymerization up to seven for oligolysines and up to five for oligoarginines and is interpreted as an interaction with up to seven closely spaced anionic charges. Microtubules so formed appear to be normal by electron microscopy, and by salt, colchicine, and cold sensitivities. Lysyl residues in excess of seven (or five for arginine) in larger oligomers interact nearly noncooperatively. Separation of lysyl charges by intercalation of alanyl residues reduced assembly promoting potency for hexalysines. The cooperative portion of the response is most likely associated with the highly acidic extreme C termini of tubulin because their removal with limited subtilisin treatment markedly reduces oligolysine potency. However, some cooperative interactions with oligocations can also occur with more widely spaced anionic charges elsewhere in tubulin. The potential role of oligocations in the intracellular regulation of microtubule assembly is discussed.     

A new model for microtubule-associated protein (MAP)-induced microtubule assembly. The Pro-rich region of MAP4 promotes nucleation of microtubule assembly in vitro

The microtubule-binding domains of microtubule-associated protein (MAP) 2, tau, and MAP4 are divided into three distinctive regions: the Pro-rich region, the AP sequence region and the tail region (Aizawa, H., Emori, Y., Murofushi, H., Kawasaki, H., Sakai., H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855). Electron microscopic observation showed that the taxol-stabilized microtubules alone and those mixed with the A4T fragment (containing the AP sequence region and the tail region) had a long, wavy appearance, while those mixed with the PA4T fragment (containing the Pro-rich region, the AP sequence region, and the tail region) or the PA4 fragment (containing the Pro-rich region and the AP sequence region) were shorter and straighter. Stoichiometries of the binding between the fragments and the tubulin dimers were approximately between 1 and 2, suggesting that not all of the AP sequences in the AP sequence region bound to tubulin. Binding affinity of the PA4T fragment is only four times higher than that of the A4T fragment, while the microtubule nucleating activity of the PA4T fragment is far greater. Based on these results, we propose that the nucleation of microtubule assembly is promoted by the bridging activity of the Pro-rich region in the MAPs.     

A comparison of microtubule assembly in brain extracts from young and old rats

The effect of repeated doses of indomethacin on mean peak velocity (MPV) and time-averaged mean velocity in the middle cerebral artery was assessed in 10 ventilated neonates with a patent ductus arteriosus using colour/duplex Doppler technique prior to, and 10, 30, and 120 min after the first and the third dose. Velocities were significantly reduced up to 120 min after the first dose. The third dose resulted in a significant reduction in MPV at 10 and 30 min following treatment. This reduction was half of that observed after the first dose. Systemic blood pressure (BP) and heart rate did not change significantly after each separate dose. However, by the third dose, mean and diastolic BP were significantly increased from pretreatment levels. The attenuated response of cerebral blood flow (CBF) velocities to the third dose of indomethacin compared with the first dose is probably related to altered haemodynamics. Indomethacin should be used cautiously in infants with other conditions which are known to decrease CBF such as hypotension, hypocarbia and polycythaemia.     

Effects of glutathione-oxidizing agents on microtubule assembly and microtubule-dependent surface properties of human neutrophils

In human peripheral blood polymorphonuclear leukocytes and lymphocytes, GSH-oxidizing agents promote the movement of surface-bound concanavalin A (Con A) into caps and inhibit the assembly of microtubules (MT) that is normally induced by Con A binding. Con A capping and inhibition of MT assembly occur when GSH levels in cell suspensions are decreased by 30-70%, and return to GSH to control levels is accompanied by the appearance of cytoplasmic MT and by inhibition of the capping response with Con A. Oxidation of GSH markedly stimulates the hexose monophosphate shunt, and regeneration of GSH occurs rapidly. The data indicate that MT cannot be assembled or maintained in the face of decreased GSH levels. Thus, GSH homeostasis becomes critical during physiological events such as phagocytosis which simultaneously induce the assembly of MT and the production of agents like H2O2 that can oxidize GSH.     

Localization of critical histidyl residues required for vinblastine-induced tubulin polymerization and for microtubule assembly

Vinblastine-induced tubulin polymerization is electrostatically regulated and shows pH dependence with a pI approximately 7.0 suggesting the involvement of histidyl residues. Modification of histidyl residues of tubulin with diethylpyrocarbonate (DEPC) at a mole ratio of 0.74 (DEPC/total His residues) for 3 min at 25 degreesC completely inhibited vinblastine-induced polymerization with little effect on microtubule assembly. Under these conditions DEPC reacts only with histidyl residues. For complete inhibition two histidyl residues have to be modified. Demodification of the carboxyethyl histidyl derivatives by hydroxylamine led to nearly complete recovery of polymerization competence. Labeling with [14C]DEPC localized both of these histidyl residues on beta-tubulin at beta227 and beta264. Similarly, tubulin modification with DEPC for longer times (8 min) resulted in complete inhibition of microtubule assembly, at which time approximately 4 histidyl residues had been modified. This inhibition by DEPC was also reversed by hydroxylamine. The third histidyl residue was found on alpha-tubulin at alpha88. Thus, two charged histidyl residues are obligatorily involved in vinblastine-induced polymerization, whereas a different histidyl residue on a different tubulin monomer is involved in microtubule assembly.     

Cloning and assembly of PCR products using modified primers and DNA repair enzymes

We present a method for the creation of ligatable 3' overhangs by the incorporation of a modified base, uracil, at a specific position in the PCR primer and subsequent treatment with the DNA-modifying enzyme uracil DNA glycosylase and then either T4 endonuclease V or human apurinic/apyrimidinic endonuclease 1. In this study, we describe the cloning of a fragment specifying the chloramphenicol-resistance gene into a SacI vector site. To further test this method, three segments of the lacZ gene were amplified by PCR, and after treatment with the DNA-modifying enzymes, the properly oriented segments were ligated into a SacI-cleaved plasmid. Using the methods described, we were able to assemble PCR products into appropriate structures.     

Inhibition of microtubule assembly in tumor cells by 3-bromoacetylamino benzoylurea, a new cancericidal compound

We have synthesized a new compound, 3-bromoacetylamino benzoylurea (3-BAABU), which showed strong cancericidal activity by inducing irreversible mitotic arrest and subsequently apoptosis in human T cell leukemic cells (CEM), human biphenotypic leukemic cells (SP), a human prostate cancer cell line (PC-3), murine melanoma cells (B-16), and murine lymphoma/leukemia cells (EL4) in vitro with an ID50 in the range of 0.013-0.07 microg/ml (0.04-0.22 microM). Treatment of tumor cells for 12-24 h with 3-BAABU resulted in mitotic arrest at prometaphase/metaphase/anaphase, with separation and dispersion of chromosomes and with the absence of mitotic spindle apparatus in cytoplasm. Treatment with 3-BAABU had no cytotoxic and mitotic blocking effect in normal human lymphocytes, proliferating fibroblast cells (3T3), or proliferating myocardial cells (MOT). Cell cycle analyses showed that most treated leukemic cells accumulated at M phase 12 h after treatment. By the end of 48 h of treatment, the cells underwent apoptosis with DNA fragmentation. 3-BAABU inhibited the assembly of microtubules from tubulin but did not interfere with the disassembly of microtubules. The presence and the position of bromine and urea groups on the benzoic ring are the determining factors for its inhibition of microtubule assembly. Replacing bromine with chlorine yielded much less mitotic blocking activity and increased the ID50 40-fold. Substitution of the urea group with ethyl ester abrogated the activity of blocking mitosis but induced apoptosis. Moving the bromoacetylamino group from the 3-position to the 4-position removed blocking activity for mitosis but induced necrosis. These results suggest that 3-BAABU possesses a unique and functional structure and is a potential agent for cancer chemotherapy.     

Coronin promotes the rapid assembly and cross-linking of actin filaments and may link the actin and microtubule cytoskeletons in yeast

Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts. Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 x 10(-9) M). Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Genetic analyses with a crn1Delta deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability. Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization. Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization. Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments. Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B. These activities, considered with microtubule defects observed in crn1Delta cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast.     

Tubulin polyglycylation in Platyhelminthes: diversity among stable microtubule networks and very late occurrence during spermiogenesis

The distribution of glycylated tubulin has been analyzed in different populations of stable microtubules in a digenean flatworm, Echinostoma caproni (Platyhelminthes). Two cellular types, spermatozoa and ciliated excretory cells, have been analyzed by means of immunofluorescence, immunogold, and immunoblotting techniques using two monoclonal antibodies (mAbs), AXO 49, and TAP 952, specifically directed against differently glycylated isoforms of tubulin. The presence of glycylated tubulin in the two cell types was shown. However, the differential reactivities of TAP 952 and AXO 49 mAbs with the two axoneme types suggest a difference in their glycylation level. In addition, within a single cell, the spermatozoon, cortical microtubules underlying the flagellar membrane, and axonemal microtubules were shown to comprise different tubulin isoforms, the latter ones only being labelled with one of the antiglycylated tubulin mAbs, TAP 952. Similarly, the antiacetylated (6-11B-1) and polyglutamylated (GT335) tubulin mAbs decorated the two types of axonemal microtubules, but not the cortical ones. From these data, a subcellular sorting of posttranslationally modified tubulin isoforms within spermatozoa, on the one hand, and a cellular sorting of glycylated isoforms inside the whole organism, on the other hand, is demonstrated in the flatworm E. caproni. Last, a sequential occurrence of tubulin posttranslational modifications was observed in the course of spermiogenesis. Acetylation appears first, followed shortly by glutamylation; glycylation takes place at the extreme end of spermiogenesis and, specifically, in a proximo-distal process. Thus in agreement with, and extending other studies [Bré et al., 1996], glycylation appears to close the sequence of posttranslational events occurring in axonemal microtubules during spermiogenesis.     

Sulphonate buffers affect the recovery of microtubule-associated proteins MAP1 and MAP2: evidence that MAP1A promotes microtubule assembly

The influence of two commonly used sulphonate buffers, PIPES and MES, on the in vitro assembly of bovine brain microtubule protein was examined. Microtubule assembly was monitored by turbimetry and, after centrifugation, the polymerised protein was analysed by SDS-PAGE and western blotting. Assembly in MES when compared with PIPES resulted in a higher recovery of microtubule proteins at both pH 6.4 and pH 6.9 and in an altered protein composition. The buffer pH affected the total amount of protein polymerised but did not significantly affect the protein composition. At both pH conditions the recovery of HMW-MAPs was markedly increased in MES buffer and this increase was mostly due to an increase in the amount of MAP1.     

The yeast spindle pole body component Spc72p interacts with Stu2p and is required for proper microtubule assembly

We have previously shown that Stu2p is a microtubule-binding protein and a component of the Saccharomyces cerevisiae spindle pole body (SPB). Here we report the identification of Spc72p, a protein that interacts with Stu2p. Stu2p and Spc72p associate in the two-hybrid system and can be coimmunoprecipitated from yeast extracts. Stu2p and Spc72p also interact with themselves, suggesting the possibility of a multimeric Stu2p-Spc72p complex. Spc72p is an essential component of the SPB and is able to associate with a preexisting SPB, indicating that there is a dynamic exchange between soluble and SPB forms of Spc72p. Unlike Stu2p, Spc72p does not bind microtubules in vitro, and was not observed to localize along microtubules in vivo. A temperature-sensitive spc72 mutation causes defects in SPB morphology. In addition, most spc72 mutant cells lack cytoplasmic microtubules; the few cytoplasmic microtubules that are observed are excessively long, and some of these are unattached to the SPB. spc72 cells are able to duplicate and separate their SPBs to form a bipolar spindle, but spindle elongation and chromosome segregation rarely occur. The chromosome segregation block does not arrest the cell cycle; instead, spc72 cells undergo cytokinesis, producing aploid cells and polyploid cells that contain multiple SPBs.     

Observation and quantification of individual microtubule behavior in vivo: microtubule dynamics are cell-type specific

Recent experiments have demonstrated that the behavior of the interphase microtubule array is cell-type specific: microtubules in epithelial cells are less dynamic than microtubules in fibroblasts (Pepper-kok et al., 1990; Wadsworth and McGrail, 1990). To determine which parameters of microtubule dynamic instability behavior are responsible for this difference, we have examined the behavior of individual microtubules in both cell types after injection with rhodamine-labeled tubulin subunits. Individual microtubules in both cell types were observed to grow, shorten, and pause, as expected. The average amount of time microtubules remained within the lamellae of CHO fibroblasts, measured from images acquired at 10-s intervals, was significantly shorter than the average amount of time microtubules remained within lamellae of PtK1 epithelial cells. Further analysis of individual microtubule behavior from images acquired at 2-s intervals reveals that microtubules in PtK1 cells undergo multiple brief episodes of growth and shortening, resulting in little overall change in the microtubule network. In contrast, microtubules in lamellae of CHO fibroblasts are observed to undergo fewer transitions which are of longer average duration, resulting in substantial changes in the microtubule network over time. A small subset of more stable microtubules was also detected in CHO fibroblasts. Quantification of the various parameters of dynamic instability behavior from these sequences demonstrates that the average rates of both growth and shortening are significantly greater for the majority of microtubules in fibroblasts than for microtubules in epithelial cells (19.8 +/- 10.8 microns/min, 32.2 +/- 17.7 microns/min, 11.9 +/- 6.5 microns/min, and 19.7 +/- 8.1 microns/min, respectively). The frequency of catastrophe events (1/interval between catastrophe events) was similar in both cell types, but the frequency of rescue events (1/time spent shrinking) was significantly higher in PtK1 cells. Thus, individual microtubules in PtK1 lamellae undergo frequent excursions of short duration and extent, whereas most microtubules in CHO lamellae undergo more extensive excursions often resulting in the appearance or disappearance of microtubules within the field of view. These observations provide the first direct demonstration of cell-type specific behavior of individual microtubules in living cells, and indicate that these differences can be brought about by modulation of the frequency of rescue. These results directly support the view that microtubule dynamic instability behavior is regulated in a cell-type specific manner.     

MAPs, MARKs and microtubule dynamics

Recent phylogenetic analyses reveal that many eukaryotic nuclear genes whose prokaryotic ancestry can be pinned down are of bacterial origin. Among them are genes whose products function exclusively in cytosolic metabolism. The results are surprising: we had come to believe that the eukaryotic nuclear genome shares a most recent common ancestor with archaeal genomes, thus most of its gene should be 'archaeal' (loosely speaking). Some genes of bacterial origin were expected as the result of transfer from mitochondria, of course, but these were thought to be relatively few, and limited to producing proteins reimported into mitochondria. Here, I suggest that the presence of many bacterial genes with many kinds of functions should not be a surprise. The operation of a gene transfer ratchet would inevitably result in the replacement of nuclear genes of early eukaryotes by genes from the bacteria taken by them as food.