Absence Of Taxol Research Articles

Tumor necrosis factor-related apoptosis inducing ligand overexpression and Taxol treatment suppresses the growth of cervical cancer cells in vitro and in vivo.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a member of tumor necrosis factor (TNF) superfamily and functions to promote apoptosis by binding to cell surface death receptor (DR)4 and DR5. Cancer cells are more sensitive than normal cells to TRAIL-induced apoptosis, and TRAIL-based therapeutic strategies have shown promise for the treatment of cancer. The present study investigated whether enforced overexpression of TRAIL in cervical cancer cells promoted cell death in the presence or absence of Taxol, an important first-line cancer chemotherapeutic drug. Hela human cervical cancer cells were transfected with a TRAIL expression plasmid, and the effects of the combination treatment with Taxol on apoptosis was investigated in vitro and in tumor xenografts in vivo. The results indicated that Taxol treatment and TRAIL overexpression enhanced apoptosis compared with either treatment alone. The present data indicate that Taxol may enhance the pro-apoptotic effects of TRAIL overexpression in HeLa cells by increasing cleaved caspase-3 and DR5 expression levels and decreasing Bcl-2 expression levels. Furthermore, the findings suggest a possible novel treatment option for cervical cancer and uncovers a potential mechanism of the enhancing effects of Taxol on TRAIL-induced apoptosis.

Abstract 833: Photolabeling of β-tubulin isotypes by radiolabeled 2-(m-azidobenzoyl)taxol

Abstract There are seven β-tubulin isotypes present in distinct quantities in cells of different origin. Altered expression of β-tubulin isotypes were found in cells resistant to microtubule stabilizing agents (MSAs), such as Taxol, epothilone B, ixabepilone and discodermolide, all of which bind to β-tubulin. For example, the level of βIII-tubulin was very low in the human ovarian cancer cell line, Hey, but increased 8- to 15-fold in two drug resistant daughter cell lines, EpoB8 and Ixab80, respectively. The βV-tubulin level was increased by 2-fold in EpoB8 cells. In contrast, levels of βI- and βIV-tubulin were unchanged and decreased, respectively, in EpoB8. Therefore, it is important to measure the binding affinity of MSAs to different β-tubulin isotypes and study their influence on the drug resistant phenotype. To study the relative binding affinities of MSAs, tubulins from HeLa cells, bovine brain, porcine brain and chicken erythrocytes were specifically photolabeled with the tritium labeled 2-(m-azidobenzoyl)taxol, in the presence and absence of Taxol, Taxotere, epothilone B, ixabepilone and discodermolide. β-tubulin isotype content from these sources was different, particularly for chicken erythrocytes that contain only one β-tubulin isotype, βVI. The inhibitory effects elicited by these MSAs on photolabeling were distinct for β-tubulins from different sources. Taxol had a minimal inhibitory effect on HeLa, bovine and porcine brain β-tubulin, but a strong inhibitory effect on chicken erythrocyte β-tubulin. Discodermolide exhibited opposite effects on photolabeling, compared to those of Taxol. However, Taxotere appeared to have a stronger inhibitory effect than Taxol on photolabeling of β-tubulins from all sources tested. Ixabepilone had the strongest inhibitory effect on HeLa β-tubulin. The inhibition pattern for β-tubulin from porcine brain correlated very well with that from bovine brain. To determine drug binding affinities for the different β-tubulin isotypes, tubulins from different sources were photolabeled and the isotypes resolved by high resolution isoelectrofocusing (IEF). Approximately 20 bands were seen with bovine and porcine brain tubulin, but only 3-5 bands were resolved with chicken erythrocyte tubulin. Many bands represent posttranslationally modified tubulins. IEF patterns for bovine and porcine brain tubulin were very similar. All bands were analyzed by mass spectrometry following CNBr digestion, and the identity and relative quantity of each β-tubulin isotype determined. After measuring the radioactivity associated with each band, it was found that the binding affinity of 2-(m-azidobenzoyl)taxol to βIII-tubulin was the lowest, compared to other β-tubulin isotypes. Citation Format: Chia-Ping H. Yang, Hui Xiao, Susan Band Horwitz. Photolabeling of β-tubulin isotypes by radiolabeled 2-(m-azidobenzoyl)taxol. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 833. doi:10.1158/1538-7445.AM2014-833

Analysis of the Strength of Interfacial Hydrogen Bonds between Tubulin Dimers Using Quantum Theory of Atoms in Molecules

Microtubules are key structural elements that, among numerous biological functions, maintain the cytoskeleton of the cell and have a major role in cell division, which makes them important cancer chemotherapy targets. Understanding the energy balance that brings tubulin dimers, the building blocks of microtubules, together to form a microtubule is especially important for revealing the mechanism of their dynamic instability. Several studies have been conducted to estimate various contributions to the free energy of microtubule formation. However, the hydrogen-bond contribution was not studied before as a separate component. In this work, we use concepts such as the quantum theory of atoms in molecules to estimate the per-residue strength of hydrogen bonds contributing to the overall stability that brings subunits together in pair of tubulin heterodimers, across both the longitudinal and lateral interfaces. Our study shows that hydrogen bonding plays a major role in the stability of tubulin systems. Several residues that are crucial to the binding of vinca alkaloids are shown to be strongly involved in longitudinal microtubule stabilization. This indicates a direct relation between the binding of these agents and the effect on the interfacial hydrogen-bonding network, and explains the mechanism of their action. Lateral contacts showed much higher stability than longitudinal ones (–462 ± 70 vs. –392 ± 59 kJ/mol), which suggests a dramatic lateral stabilization effect of the GTP cap in the β-subunit. The role of the M-loop in lateral stability in absence of taxol was shown to be minor. The B-lattice lateral hydrogen bonds are shown to be comparable in strength to the A-lattice ones (−462 ± 70 vs. –472 ± 46 kJ/mol). These findings establish the importance of hydrogen bonds to the stability of tubulin systems.

The role of cancer stem cells in the acquired resistance to Taxol of human breast cancer cell line MCF-7

Objective To investigate the role of cancer stem cells (CSC) in the acquired resist ance to Taxol of human breast cancer cell line MCF-7.Methods Taxol-resistant MCF-7 (MCF-7/Taxol) was established in vitro by exposure to low concentration (0.005 mg/L) and subsequently the gradually increased dose of Taxol.The proliferation and sensitivity to Taxol of MCF-7/Taxol before and after application of As2O3 were tested using methyl thiazol tetrazolium (MTT) assay.Real-time quantitative polymerase chain reaction (Real-time PCR) was employed to investigate the expression of ATP-binding cassette subfamily G member 2 (ABCG2) and sex determining region Y-box 2 (SOX-2) in wild type MCF-7 and MCF-7/Taxol cells.Results In the presence of Taxol (0.5 mg/L),the growth rate of MCF-7/ Taxol was significantly higher than the wild type MCF-7 cells (P ＜ 0.01).It was also demonstrated that MCF-7/Taxol ceils treated by Taxol developed similar proliferation to wild type MCF-7 cells in the absence of Taxol (P ＞ 0.05).The 50％ inhibitory dose (IC50) of wild type MCF-7 and MCF-7/Taxol cells to Taxol was about 0.05 mg/L and 4.2 mg/L respectively,and the resistance index was about 84.The resistance of MCF-7/Taxol ceils to Taxol was reduced after treatment of As2O3 (0.20 mg/L) with reversed multiples being 3.82 and relative reversed efficiency being 74.7％ respectively.MCF-7/Taxol cells showed elevated expression of ABCG2 and SOX-2 (P ＜0.01) in comparison with wild type MCF-7 cells.After treatment of As2O3,MCF-7/Taxol cells developed decreased levels of ABCG2 and SOX-2 (P ＜ 0.05).Conclusion CSC is a possible mechanism accounting for the acquired resistance of MCF-7/Taxol cells to Taxol. Key words: Breast cancer; Drug resistance; Taxol; Cancer stem cell

Ion specific effects in bundling and depolymerization of taxol-stabilized microtubules

Microtubules (MTs) are nanometer scale hollow cylindrical biological polyelectrolytes. They are assembled from alpha/beta-tubulin dimers, which stack to form protofilaments (PFs) with lateral interactions between PFs resulting in the curved MT. In cells, MTs and their assemblies are critical components in a range of functions from providing tracks for the transport of cargo to forming the spindle structure during mitosis. Previous studies have, shown that while cations with valence equal to or larger than 3+ tend to assemble tight 3D bundles of taxol-stabilized MTs, certain divalent cations induce relatively loose 2D bundles of different symmetry (D. J. Needleman et al., Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 16099). Similarly, divalent cations form 2D bundles of DNA adsorbed on cationic membranes (I. Koltover et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 14046). The bundling behavior for these biological polyelectrolyte systems is qualitatively in agreement with current theory. Here, we present results which show that, unlike the case for DNA adsorbed on cationic membranes, bundling of taxol-stabilized MTs occurs only for certain divalent cations above a critical ion concentration (e.g. Ca2+, Sr2+, Ba2+). Instead, many divalent cations pre-empt the bundling transition and depolymerize taxol-stabilized MTs at a lower counterion concentration. Although previous cryogenic TEM has shown that, in the absence of taxol, Ca2+ depolymerizes MTs assembling in buffers containing GTP (guanosine triphosphate), our finding is surprising given the know stabilizing effects of taxol on GDP (guanosine diphosphate)-MTs. The ion concentration required for MT depolymerization decreases with increasing atomic number for the divalents Mg2+, Mn2+, Co2+, and Zn2+. GdCl3 (3+) is found to be extremely efficient at MT depolymerization requiring ion concentrations of about 1 mM, while oligolysine(2+), is observed not to depolymerize MTs at concentrations as high as 144 mM. The surprising MT depolymerization results are discussed in the context of divalents either disrupting lateral interactions between PFs (which are strengthened for taxol containing beta-tubulin) or interfering with taxol's ability to induce flexibility at the interface between two tubulin dimers in the same PF (which has been recently suggested as a mechanism by which taxol stabilizes MTs post-hydrolysis with the induced flexibility counteracting the kink between GDP-tublin dimers in a PF).

Structural Evidence for Cooperative Microtubule Stabilization by Taxol and the Endogenous Dynamics Regulator MAP4

Microtubules (MTs) composed of αβ-tubulin heterodimers are highly dynamic polymers, whose stability can be regulated by numerous endogenous and exogenous factors. Both the antimitotic drug Taxol and microtubule-associated proteins (MAPs) stabilize this dynamicity by binding to and altering the conformation of MTs. In the current study, amide hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was used to examine the structural and dynamic properties of the MT complex with the microtubule binding domain of MAP4 (MTB-MAP4) in the presence and absence of Taxol. The changes in the HDX levels indicate that MTB-MAP4 may bind to both the outside and the luminal surfaces of the MTs and that Taxol reduces both of these interactions. The MTB-MAP4 binding induces conformational rearrangements of α- and β-tubulin that promote an overall stabilization of MTs. Paradoxically, despite Taxol's negative effects on MAP4 interactions with the MTs, its binding to the MTB-MAP4-MT complex further reduces the overall deuterium incorporation, suggesting that a more stable complex is formed in the presence of the drug.

Tau isoform‐specific modulation of kinesin‐driven microtubule gliding rates and trajectories as determined with tau‐stabilized microtubules

We have utilized tau-assembled and tau-stabilized microtubules (MTs), in the absence of taxol, to investigate the effects of tau isoforms with three and four MT binding repeats upon kinesin-driven MT gliding. MTs were assembled in the presence of either 3-repeat tau (3R tau) or 4-repeat tau (4R tau) at tau:tubulin dimer molar ratios that approximate those found in neurons. MTs assembled with 3R tau glided at 31.1 μm/min versus 25.8 μm/min for 4R tau, a statistically significant 17% difference. Importantly, the gliding rates for either isoform did not change over a fourfold range of tau concentrations. Further, tau-assembled MTs underwent minimal dynamic instability behavior while gliding and moved with linear trajectories. In contrast, MTs assembled with taxol in the absence of tau displayed curved gliding trajectories. Interestingly, addition of 4R tau to taxol-stabilized MTs restored linear gliding, while addition of 3R tau did not. The data are consistent with the ideas that (i) 3R and 4R tau-assembled MTs possess at least some isoform-specific features that impact upon kinesin translocation, (ii) tau-assembled MTs possess different structural features than do taxol-assembled MTs, and (iii) some features of tau-assembled MTs can be masked by prior assembly by taxol. The differences in kinesin-driven gliding between 3R and 4R tau suggest important features of tau function related to the normal shift in tau isoform composition that occurs during neural development as well as in neurodegeneration caused by altered expression ratios of otherwise normal tau isoforms.

Myofibrillogenesis in skeletal muscle cells in the presence of taxol.

We address the controversy of whether mature myofibrils can form in the presence of taxol, a microtubule-stabilizing compound. Previous electron microscopic studies reported the absence of actin filaments and Z-bands in taxol-treated myocytes [Antin et al., 1981: J Cell Biol 90:300-308; Toyoma et al., 1982: Proc Natl Acad Sci USA 79:6556-6560]. Quail skeletal myoblasts were isolated from 10-day-old embryos and grown in the presence or absence of taxol. Taxol inhibited the formation of multinucleated elongated myotubes. Myocytes cultured in the continual presence of taxol progressed from rounded to stellate shapes. Groups of myocytes that were clustered together after the isolation procedure fused in the presence of taxol but did not form elongated myotubes. Actin filaments and actin-binding proteins were detected with several different fluorescent probes in all myofibrils that formed in the presence of taxol. The Z-bands contained both alpha-actinin and titin, and the typical arrays of A-Bands were always associated with actin filaments in the myofibrils. Myofibril formation was followed by fixing cells each day in culture and staining with probes for actin, muscle-specific alpha-actinin, myosin II, nebulin, troponin, tropomyosin, and non-muscle myosin II. Small linear aggregates of alpha-actinin or Z-bodies, premyofibrils, were detected at the edges of the myocytes and in the arms of the taxol-treated cells and were always associated with actin filaments. Non-muscle myosin II was detected at the edges of the taxol-treated cells. Removal of the taxol drug led to the cells assuming a normal compact elongated shape. During the recovery process, additional myofibrils formed at the spreading edges of these elongated and thicker myotubes. Staining of these taxol-recovering cells with specific fluorescent reagents reveals three different classes of actin fibers. These results are consistent with a model of myofibrillogenesis that involves the transition of premyofibrils to mature myofibrils.

Repeat motifs of tau bind to the insides of microtubules in the absence of taxol.

The tau family of microtubule-associated proteins has a microtubule-binding domain which includes three or four conserved sequence repeats. Pelleting assays show that when tubulin and tau are co- assembled into microtubules, the presence of taxol reduces the amount of tau incorporated. In the absence of taxol, strong binding sites for tau are filled by one repeat motif per tubulin dimer; additional tau molecules bind more weakly. We have labelled a repeat motif with nanogold and used three-dimensional electron cryomicroscopy to compare images of microtubules assembled with labelled or unlabelled tau. With kinesin motor domains bound to the microtubule outer surface to distinguish between alpha- and beta-tubulin, we show that the gold label lies on the inner surface close to the taxol binding site on beta-tubulin. Loops within the repeat motifs of tau have sequence similarity to an extended loop which occupies a site in alpha-tubulin equivalent to the taxol-binding pocket in beta-tubulin. We propose that loops in bound tau stabilize microtubules in a similar way to taxol, although with lower affinity so that assembly is reversible.

Benzimidazole binding to Haemonchus contortus tubulin: a question of structure

Response from R.K. PrichardM. Robinson et al., present an interesting structural analysis. The model that was presented in my review was proposed on the evidence for the role of Phe200 and Phe167 in allowing high-affinity benzimidazole (BZM) binding [1xGenetic variability following selection of Haemonchus contortus with anthelmintics. Prichard, R.K. Trends Parasitol. 2001; 17: 445–453Abstract | Full Text | Full Text PDF | PubMed | Scopus (168)See all References][1]. The evidence that the amino acids Ser166, Phe167, Phe200 and Cys201 cluster together, the importance of having a planar BZM structure in order to obtain high-affinity binding, which could allow a staking interaction of the BZM phenyl ring with the two Phe residues at positions 167 and 200, the location of the approximately parallel B5 and B6 strands containing these residues [2xStructure of the αβ tubulin dimer by electron crystallography. Nogales, E et al. Nature. 1998; 391: 199–203Crossref | PubMed | Scopus (1507)See all References][2] and the loss of high-affinity BZM binding when Phe at either position 200 or 167 becomes substituted with a hydroxy substituent (Tyr). However, the model that was proposed does require experimental analysis; therefore, I welcome their discussion. Robinson and colleagues make valuable comments based on measured separation distances of the Phe–Phe inter-rings and the orientation of the side chain of Cys201 in the crystalline structure obtained following taxol stabilization [2xStructure of the αβ tubulin dimer by electron crystallography. Nogales, E et al. Nature. 1998; 391: 199–203Crossref | PubMed | Scopus (1507)See all References][2] and, in the absence of taxol, these restrictions might not apply. I would welcome further work so that we do understand the BZM binding site better, and hope that the model that I have proposed will stimulate further analysis.

In vitro assembly of plant tubulin in the absence of microtubule-stabilizing reagents

The assembly of microtubules is essential for physiological functions of microtubules. Addition of microtubule-stabilizing reagents or microtubule “seeds” is usually necessary for plant tubulin assemblyin vitro, which hinders the investigation of plant microtubule dynamics. In the present note, highly purified plant tubulins have been obtained from lily pollen, a non-microtubule-stabilizing reagent or microtubule “seed” system for plant tubulin assembly has been established and the analysis of plant tubulin assembly performed. Experiment results showed that purified tubulin polymerizedin vitro, and a typical microtubule structure was observed with electron microscopy. The kinetics curve of tubulin assembly exhibited typical “parabola”. The presence of taxol significantly altered the character of plant tubulin assembly, including that abnormal microtubules were assembled and the critical concentration for plant tubulin assembly was decreased exceedingly from 3 mg/mL in the absence of taxol to 0.043 mg/mL in ihe presence of taxol.

Biophysical properties of stable microtubules in neurites revealed by optical techniques.

We tested the stability of microtubules in the neurites of cultured dorsal root ganglion cells by dissolving the cytoplasmic membrane with detergent and exposing them to defined extracellular medium under the microscope. Smooth cytoplasmic filaments visualized after membrane removal were suggested to be microtubules by the preservation of all of the filaments in the presence but not in the absence of taxol. They were further confirmed to be microtubules by immunostaining with anti-tubulin antibody. Significant number of microtubules in the established neurites remained longer than 1 hour after membrane removal. To investigate their stabilization mechanism, we transected the exposed microtubules by laser microbeam irradiation and observed their length changes with video-enhanced microscopy. Microtubule fragments started to shorten on both sides of the transection site, more rapidly from the newly generated plus ends than from the minus ends. The maximal rate as well as the pattern of shortening correlated with the time of transection; microtubules transected later than 30 min after membrane removal shortened at rates less than 20 microm/min and typically with intermittent pauses, while the more labile microtubules included in the earlier transections shortened continuously at higher rates. Microtubules in neurites were thus stabilized by (1) stopping disassembly at local sites including the plus ends, and (2) slowing disassembly along the length. Observations of the course of disassembly also suggested the presence of specialized points along microtubules which is involved in anchoring microtubules to the substratum or transiently stopping disassembly.

Direct visualization and characterization of stable microtubules from the neurites of cultured dorsal root ganglion cells

We tested the stability of microtubules (MTs) in the neurites of cultured dorsal root ganglion cells by dissolving the cytoplasmic membrane with detergent and exposing them to defined extracellular medium under observation with a video-enhanced differential interference contrast (DIC) microscope. Smooth cytoplasmic filaments visualized after membrane removal were suggested to be MTs by the preservation of all of the filaments in the presence but not in the absence of taxol. They were further confirmed to be MTs by specific immunostaining with anti-tubulin antibody. A significant number of MTs in the established neurites of 6-day-old cultures remained longer than 10 min after membrane removal while MTs in the Schwann cell processes or in the distal regions of the growth cone-bearing neurites of 3-day-old cultures disappeared within 2 min. A population of very stable MTs persisting longer than 30 min was also found specifically in the 6-day-old cultures. Association with other structures or bundling seemed to stabilize the MTs to some degree. The most stable MTs, however, were not associated with some structure along the length but were mainly anchored at points, suggesting that specific point attachments may be another important mechanism operating in MT stabilization. The present method is thus capable of directly demonstrating the unusual stability of neuritic MTs, and provides a new system for further investigation on the mechanism of stabilization.

Subadditive interaction of radiation and taxol in vitro

To examine the dependency of Taxol-radiation interactions on the scheduling of the two agents. The human laryngeal squamous cell carcinoma line SCC20 was used for this study. Cells were irradiated as subconfluent cultures using Cs-137 gamma rays at a dose rate of 1.75 Gy/min. Cultures were pretreated with Taxol (7.5 nM for 12 h, S.F. = 0.4) and then irradiated with graded doses followed by either immediate plating or holding for 6 h either in the absence or presence of 7.5 nM Taxol prior to plating for colony-forming ability. Experiments in which cells were irradiated and then exposed to 7.5 nM Taxol for both 12 and 18 h were also performed. Parallel-flow cytometric analyses of cell-cycle distribution of the various treated populations were carried out. The results indicate that pretreatment with Taxol induced a G2 block which was maintained during 6 h postirradiation holding either in the presence or absence of Taxol. No modification of radiosensitivity in the low-dose region was seen for cells treated with Taxol, irradiated, and plated immediately, with the resulting survival being compatible with an additive effect. However, for Taxol-pretreated cells held for 6 h postirradiation, either in the absence or presence of Taxol, the resulting survival reproducibly demonstrated a marked less than additive effect. This was particularly prominent for cells held in the presence of Taxol. Subsequent experiments in which Taxol was added to cells immediately postirradiation again demonstrated a less than additive effect of the two modalities. The results of this study are consistent with a dual mechanism of action involving Taxol-induced radiation resistance, possibly as a consequence of postirradiation holding in G2, and radiation-induced Taxol resistance through an as-yet-undefined mechanism.

Purification and characterization of the high molecule weight microtubule associated proteins from neonatal rat brain.

The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highest levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide beta-II (422-434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the beta-II tubulin fragment, but it showed interaction with the Affigel-conjugated beta-I (431-444) tubulin peptide. The different MAPs components were characterized by western blots using specific monoclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

The 72-kDa microtubule-associated protein from porcine brain.

A microtubule-associated protein (MAP) with a molecular mass of 72-kDa that was purified from porcine brain by using its property of heat stability in a low pH buffer was characterized. Low-angle rotary shadowing revealed that the 72-kDa protein was a rodlike protein approximately 55-75 nm long. The 72-kDa protein bound to microtubules polymerized from phosphocellulose column-purified tubulin (PC-tubulin) with taxol and promoted the polymerization of PC-tubulin in the absence of taxol. Microtubules polymerized by the 72-kDa protein showed a tendency to form bundles of several microtubules. Quick-freeze, deep-etch electron microscopy revealed that the 72-kDa protein formed short crossbridges between microtubules. We performed peptide mapping to analyze the relationship of the 72-kDa protein to other heat-stable MAPs, and the results showed some resemblance of the 72-kDa protein to MAP2. Cross-reactivity with a monoclonal anti-MAP2 antibody further suggested that the 72-kDa protein and MAP2 are immunologically related. To study the relationship between the 72-kDa protein and MAP2C, a smaller molecular form of MAP2 identified in juvenile rat brain, we prepared the 72-kDa protein from rat brain by the same method as that used for porcine brain. The fact that the 72-kDa protein from juvenile rat brain was also stained with our monoclonal anti-MAP2 antibody also suggested that the 72-kDa protein is an MAP2C homologue of the porcine brain.

The acute response of Schwann cells to taxol after nerve crush.

The effect of taxol, an antimitotic drug which stabilizes microtubules and promotes their assembly, was studied with regard to Schwann cells over a 4-week period following a crush injury to rat sciatic nerve. A single intraneural injection of taxol in dimethyl sulfoxide (DMSO) was given immediately after the crush into the site of injury in one sciatic nerve and was compared with the other side which was crushed but injected with DMSO only. Sampled sites were taken proximal and distal to the lesion, as well as from the lesion itself, and studied by light and electron microscopy. The Schwann cell response was most marked during the degenerative phase immediately following the crush. At this time, there was a decrease of all cytoplasmic structures except microtubules and smooth endoplasmic reticulum. At the site of the crush lesion in taxol-treated nerves, Schwann cells possessed accumulations of myelin debris and lipid droplets. Mitotic Schwann cells were also engorged with myelin breakdown products. Multinucleated Schwann cells, believed to be the result of abnormal mitotic activity, were also apparent and were filled with large numbers of cytoplasmic microtubules. The latter were sometimes regularly arranged around phagocytosed or intracytoplasmic debris. Some recovery from the crush injury was noted with time, although the number of Schwann cells was much lower than would have been anticipated in the absence of taxol, in that long stretches of naked axon bundles were common and microtubule-related abnormalities persisted up to 4 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Limited chymotryptic digestion of bovine adrenal 190,000-Mr microtubule-associated protein and preparation of a 27,000-Mr fragment which stimulates microtubule assembly.

A heat stable microtubule-associated protein of Mr 190,000 (190-kDa MAP) has been purified from bovine adrenal cortex (Murofushi, H., Kotani, S., Aizawa, H., Hisanaga, S., Hirokawa, N., and Sakai, H. (1986) J. Cell Biol. 103, 1911-1919). Limited chymotryptic digestion of 190-kDa MAP produced a fragment of Mr 27,000 (27-kDa fragment), which bound to microtubules reconstituted in the presence of taxol. This fragment was purified with the aid of cosedimentation with microtubules. The purified 27-kDa fragment showed an ability to stimulate tubulin polymerization in the absence of taxol. Electron microscopic observation of microtubules reconstituted from purified 27-kDa fragment and tubulin revealed that the microtubules were in the form of thick bundles and that lateral projections which can be seen in microtubules reconstituted from intact 190-kDa MAP and tubulin were not observed. These results indicate that 27-kDa fragment includes or is a part of microtubule-binding domain of 190-kDa MAP and that this fragment is active in stimulating microtubule assembly. Amino acid analysis revealed that the 27-kDa fragment was rich in lysine, proline, and alanine, the sum of these three being about 45% of the total amino acids and that the contents of methionine, tyrosine, phenylalanine, and histidine were very low. These data suggest that the microtubule binding domain of the 190-kDa MAP comprises an unique structure.

Involvement of the carboxyl-terminal domain of tubulin in the regulation of its assembly.

Limited proteolysis of phosphocellulose-purified tubulin with subtilisin resulted in cleavage of both alpha and beta tubulin subunits, with the formation of two major fragments (S alpha, and S beta, 48 kDa) and a small peptide (4 kDa) containing the carboxyl-terminal region of tubulin. Interestingly, tubulin cleaved under the present conditions showed an increased ability to assemble into large polymers in the absence of MAPs and under conditions that do not promote assembly of undigested tubulin--i.e., low magnesium concentrations and the absence of taxol and polyalcohols. The critical concentrations for the subtilisin-cleaved tubulin assembly was similar to that of MAPs-promoted tubulin assembly. Assembly product from subtilisin-cleaved tubulin consisted mainly of protofilament bundles, hooked polymer, and open tubules, structures showing equatorial and longitudinal spacings of 50 and 40 A, respectively. The existence of junctions between polymer walls indicates that the carboxyl-terminal removal facilitates polymer-polymer interactions. These results, together with previous studies on the involvement of the carboxyl-terminal domain of tubulin in its interaction with MAP-2, suggest a regulatory role for this domain in tubulin assembly. Thus, in general terms the tubulin molecule can be analyzed as a protein containing two essential domains with functional significance, one domain playing a major role in self-association and the other (the carboxyl-terminal moiety) playing a regulatory role in modulating the interactions responsible for self-association.

Taxol-requiring mutant of Chinese hamster ovary cells with impaired mitotic spindle assembly.

In the accompanying paper (Cabral, F., 1982, J. Cell. Biol., 97:22-29) we described the isolation and properties of taxol-requiring mutants of Chinese hamster ovary cells. We now show that at least one of these mutants, Tax-18, has an impaired ability to form a spindle apparatus. Immunofluorescence studies using antibodies to tubulin demonstrate that, when incubated in the absence of taxol, Tax-18 forms only a rudimentary spindle with few and shortened microtubules associated with the spindle poles. Furthermore, midbodies were not observed, consistent with an absence of cytokinesis. Essentially normal spindles and midbodies are seen in the presence of taxol. Electron microscopic examination indicates that centrioles and kinetochores are morphologically normal in the mutant strain. Pole-to-kinetochore microtubules were seen but interpolar microtubules were not. Taxol-deprived mutant cells stained with anti-centrosome serum show an elevated centriole content, indicating that the defect in Tax-18 does not affect centriole replication or prevent progression through the cell cycle. Although Tax-18 cells do not form a complete spindle in the absence of taxol, cytoplasmic microtubule assembly occurs in association with microtubule-organizing centers, and microtubules with apparently normal morphology exist throughout the cytoplasm. Observation of chromosome movement indicates that the defect in these cells occurs after prometaphase. These studies demonstrate that the formation of spindle microtubules requires cellular conditions that are different from those required for cytoplasmic microtubule formation. They further show that a normal spindle may be necessary for cytokinesis but not for progress of the cells through the cell cycle.