Centrosome Behavior under the Action of a Mitochondrial Uncoupler and the Effect of Disruption of Cytoskeleton Elements on the Uncoupler-Induced Alterations

Carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) induced in pig kidney embryo cells a loss of rhodamine 123 staining of mitochondria in 2-3 min. Within 5 min after FCCP inoculation of cells prestained with rhodamine 123, the diffuse staining of the cytoplasm was absent. FCCP did not induce changes in the cytoplasmic microtubule complex, but induced nonrandom (preferentially perpendicular to the substrate surface) orientation of maternal centrioles. Nonrandom orientation of maternal centrioles occurred 10 min after treatment and remained for 2 hr. At 30 min after introduction of the drug, FCCP treatment increased the mean number of pericentriolar satellites on maternal centrioles and the frequency of primary cilia. The percentage of centrioles perpendicular to the substrate induced by FCCP treatment was slightly increased by disruption of microtubules and slightly diminished by disruption of microfilaments. In both cases centrioles were oriented significantly differently from random (P < 0.01). These results suggest that microtubules are neither involved in the signaling pathway from plasma membrane to the centriole, nor do they anchor the centrioles perpendicular to the substrate, as proposed by Albrecht-Buehler and Bushnell (Experimental Cell Research 120, 1979).     

Calcium-independent, pH-regulated effects of S-100 proteins on assembly-disassembly of brain microtubule protein in vitro

At alkaline pH, Ca2+ is no longer required for S-100 proteins to inhibit the assembly and to promote the disassembly of brain microtubules in vitro, though the presence of Ca2+ significantly favors the S-100 effects. These effects are inversely related to the microtubule protein concentration and directly related to the S-100 concentration and the pH. Ca2+-independent, pH-regulated inhibition of assembly of phosphocellulose-purified tubulin by S-100 is also described. The microtubule disassembling effect of S-100 is additive to that of alkali (used to raise the pH), and S-100 further disassembles microtubules after alkalinization. Thus the larger inhibitory effect of S-100 on microtubule assembly at alkaline versus acid pH depends on both a decrease in the assembly rate and an increase in the disassembly rate. Together with previous data on this topic, the present findings indicate that S-100 proteins act on microtubule protein in vitro primarily by binding to tubulin, this event being Ca2+-regulated at a given pH, and pH-regulated at a given free Ca2+ concentration.     

Studies on in vitro polymerization of tubulin from renal medullary extracts.

Previous in vivo studies showed that microtubules are involved in the cellular action of vasopressin. In order to analyze the role of renal medullary microtubules, a system was developed which would allow the study of the assembly of tubulin in renal medulla extracts into microtubules in vitro. The assembly of tubulin into microtubules occurred in renal medullary cytosol (100 000 times g supernatant) under specific conditions which include pre-concentration of cytosol by ultrafiltration, the presence of ethylene glycol bis(2-aminoethyl)ether tetraacetic acid (EGTA) and 4 M glycerol, and warming at 37 degrees C. Formation of microtubules, which sedimented at 100 000 times g, was proved by (a) an increase in the apparent [3H]colchicine-binding activity of depolymerized pellets, (b) appearance of typical microtubules as shown by electron microscopy, and (c) by the increase in the quantity of microtubular protein analyzed by polyacrylamide gel electrophoresis. Vinblastine at a concentrationof 10(-6) M completely blocked formation of microtubules. A slight increase of ionized calcium in the polymerization mixture also prevented microtubule assembly; this inhibitory effect of ionized calcium was present at concentrations as low as 10(-4) M. Blockade of microtubule assembly by the increase in concentration of ionized calcium or by vinblastine may be the basis of known inhibitory effect of these two agents upon the hydroosmotic effect of vasopressin in vivo.     

Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes

Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation.     

Heat stress affects the organization of microtubules and cell division in Nicotiana tabacum cells

To investigate the effects of heat stress on the plant cytoskeleton, the structure of microtubule arrays in N. tabacum suspension cells incubated at 38 or 42°C was analysed. Whilst incubation at 42 °C resulted in the disruption of the majority of cellular microtubules after 30 min, in cells exposed to 38 °C all the microtubule arrays were preserved even after 12 h of incubation, although their organization was altered. The most susceptible were the microtubules of the mitotic spindle and the phragmoplast. Several abnormalities were observed: (i) splitting of the spindle into several parts; (ii) elongation of the spindles; (iii) formation of microtubule asters in mitotic cells, and (iv) elongation of phragmoplast microtubules. Exposure of cells to 38 °C caused a decrease in the mitotic index but an accumulation of telophase cells. The recovery of normal microtubule organization occurred after 12 h. Treatment of the cells subjected to heat stress conditions with an inhibitor of protein synthesis, cycloheximide, did not prevent either the alterations of microtubule organization or accumulation of cells containing phragmoplasts. Therefore, heat shock proteins do not seem to be directly responsible for the microtubule disorganization induced by heat stress.     

Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization

To study the effects of microtubule-associated proteins (MAPs) on in vivo microtubule assembly, cDNAs containing the complete coding sequences of a Drosophila 205-kD heat stable MAP, human MAP 4, and human tau were stably transfected into CHO cells. Constitutive expression of the transfected genes was low in most cases and had no obvious effects on the viability of the transfected cell lines. High levels of expression, as judged by Western blots, immunofluorescence, and Northern blots, could be induced by treating cells with sodium butyrate. High levels of MAPs were maintained for at least 24-48 h after removal of the sodium butyrate. Immunofluorescence analysis indicated that all three MAPs bound to cellular microtubules, but only the transfected tau caused a rearrangement of microtubules into bundles. Despite high levels of expression of these exogenous MAPs and the bundling of microtubules in cells expressing tau, transfected cells had normal levels of assembled and unassembled tubulin. With the exception of the tau-induced bundles, microtubules in transfected cells showed the same sensitivity as control cells to microtubule depolymerization by Colcemid. Further, all three MAPs were ineffective in reversing the taxol-dependent phenotype of a CHO mutant cell line. The absence of a quantitative effect of any of these heterologous proteins on the assembly of tubulin suggests that these MAPs may have different roles in vivo from those inferred previously from in vitro experiments.     

Decoration of microtubules by fluorescently labeled microtubule-associated protein 2 (MAP2) does not interfere with their spatial organization and progress through mitosis in living fibroblasts

Microtubule-associated protein 2 (MAP2) derivatized with iodoacetamidotetramethylrhodamine or with iodoacetamidofluorescein binds to microtubules after injection into living interphase cells [Scherson et al, 1984]. The binding of derivatized MAP2 stabilized microtubules in vitro; it was therefore important to check if the binding of MAP2 in vivo perturbed the dynamics and organization of the microtubule network. We have addressed these questions by studying the effect of the injection of derivatized MAP2 on mitosis in PtK 1 cells and on the recovery of the microtubule network from low temperature incubation in interphase cells. We found that the presence of derivatized MAP2 did not change the duration of any mitotic stage and that the injected cell normally completed mitosis. We subsequently showed that the injected MAP2 bound to the microtubules within 5 minutes after injection and remained bound throughout the course of mitosis. The reorganization of the microtubule network upon cooling and rewarming was studied in the cytoplasm of human foreskin fibroblasts (356 cells). During the recovery, the distribution of the fluorescent MAP2 in living cells was identical with the microtubule pattern visualized by immunofluorescence in lysed and fixed cells. In these experiments, the fluorescent MAP2 bound to microtubules can be considered as a nonperturbing reporter of the microtubule network. This result is discussed in terms of the role of MAPs in the dynamics and organization of microtubules in living cells.     

Microtubule depolymerization and phosphorylation of c-Jun N-terminal kinase-1 and p38 were involved in gambogic acid induced cell cycle arrest and apoptosis in human breast carcinoma MCF-7 cells

Gambogic acid (GA), an ingredient isolated from Garcinia hanburyi, has potent anticancer activity both in vitro and in vivo. In the present study, we examined the effects of GA on intracellular microtubules and reconstituted microtubules in vitro. Immunofluorescence microscopy revealed that 2.5 muM GA caused microtubule cytoskeleton disruption and microtubule depolymerization in human breast carcinoma MCF-7 cells, thereby reducing the amount of polymer form of tubulin and increasing the amount of monomer form of tubulin. We further confirmed that GA could depolymerize microtubule associated protein (MAP)-free microtubules and MAP-rich microtubules in vitro. Thus we suggested that GA-induced G2/M phase cell cycle arrest may be attributed to its depolymerization of microtubules. We also revealed that phosphorylation levels of p38 and c-Jun N-terminal kinase-1 (JNK-1) were increased markedly by GA, resulting in apoptosis of MCF-7 cells. Taken together, our results suggested that GA depolymerized microtubules and elevated the phosphorylation levels of JNK1 and p38, which caused G2/M cell cycle arrest and apoptosis in MCF-7 cells.     

A microtubule-binding protein associated with membranes of the Golgi apparatus

A monoclonal antibody (M3A5), raised against microtubule-associated protein 2 (MAP-2), recognized an antigen associated with the Golgi complex in a variety of non-neuronal tissue culture cells. In double immunofluorescence studies M3A5 staining was very similar to that of specific Golgi markers, even after disruption of the Golgi apparatus organization with monensin or nocodazole. M3A5 recognized one band of Mr approximately 110,000 in immunoblots of culture cell extracts; this protein, designated 110K, was enriched in Golgi stack fractions prepared from rat liver. The 110K protein has been shown to partition into the aqueous phase by Triton X-114 extraction of a Golgi-enriched fraction and was eluted after pH 11.0 carbonate washing. It is therefore likely to be a peripheral membrane protein. Proteinase K treatment of an isolated Golgi stack fraction resulted in complete digestion of the 110K protein, both in the presence and absence of Triton X-100. A the 110K protein is accessible to protease in intact vesicles in vitro, it is presumably located on the cytoplasmic face of the Golgi membrane in vivo. The 110K protein was able to interact specifically with taxol-polymerized microtubules in vitro. These results suggest that the 110K protein may serve to link the Golgi apparatus to the microtubule network and so may belong to a novel class of proteins: the microtubule-binding proteins.     

Mitochondrial behavior during oogenesis in zebrafish: a confocal microscopy analysis

The behavior of mitochondria during early oogenesis remains largely unknown in zebrafish. We used three mitochondrial probes (Mito Tracker Red CMXRos, Mito Tracker Green FM, and JC-1) to stain early zebrafish oocyte mitochondria, and confocal microscopy to analyze mitochondrial aggregation and distribution. By using fluorescence recovery after photobleaching (FRAP), we traced mitochondrial movement. The microtubule assembly inhibitor nocodazole and microfilament inhibitor cytochalasin B (CB) were used to analyze the role of microtubules and microfilaments on mitochondrial movement. By using the dual emission probe, JC-1, and oxidative phosphorylation uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), we determined the distribution of active and inactive (low-active) mitochondria. Green/red fluorescence ratios of different sublocations in different oocyte groups stained by JC-1 were detected in merged (green and red) images. Our results showed that mitochondria exhibited a unique distribution pattern in early zebrafish oocytes. They tended to aggregate into large clusters in early stage I oocytes, but in a threadlike state in latter stage I oocytes. We detected a lower density mitochondrial area and a higher density mitochondrial area on opposite sides of the germinal vesicle. The green/red fluorescence ratios in different sublocations in normal oocytes were about 1:1. This implies that active mitochondria were distributed in all sublocations. FCCP treatment caused significant increases in the ratios. CB and nocodazole treatment caused an increase of the ratios in clusters and mitochondrial cloud, but not in dispersed areas. Mitochondria in different sublocations underwent fast dynamic movement. Inhibition or disruption of microtubules or microfilaments resulted in even faster mitochondrial free movement.     

Specific association of STOP protein with microtubules in vitro and with stable microtubules in mitotic spindles of cultured cells.

STOP (Stable Tubule Only Polypeptide) is a neuronal microtubule associated protein of 145 kd that stabilizes microtubules indefinitely to in vitro disassembly induced by cold temperature, millimolar calcium or by drugs. We have produced monoclonal antibodies against STOP. Using an antibody affinity column, we have produced a homogeneously pure 145 kd protein which has STOP activity as defined by its ability to induce cold stability and resistance to dilution induced disassembly in microtubules in vitro. Western blot analysis, using a specific monoclonal antibody, demonstrates that STOP recycles quantitatively with microtubules through three assembly cycles in vitro. Immunofluorescence analysis demonstrates that STOP is specifically associated with microtubules of mitotic spindles in neuronal cells. Further, and most interestingly, STOP at physiological temperature appears to be preferentially distributed on the distinct microtubule subpopulations that display cold stability; kinetochore-to-pole microtubules and telophase midbody microtubules. The observed distribution suggests that STOP induces the observed cold stability of these microtubule subpopulations in vivo.     

Association of tyrosine phosphatase epsilon with microtubules inhibits phosphatase activity and is regulated by the epidermal growth factor receptor

Protein tyrosine phosphatases (PTPs) are key mediators that link physiological cues with reversible changes in protein structure and function; nevertheless, significant details concerning their regulation in vivo remain unknown. We demonstrate that PTPepsilon associates with microtubules in vivo and is inhibited by them in a noncompetitive manner. Microtubule-associated proteins, which interact strongly with microtubules in vivo, significantly increase binding of PTPepsilon to tubulin in vitro and further reduce phosphatase activity. Conversely, disruption of microtubule structures in cells reduces their association with PTPepsilon, alters the subcellular localization of the phosphatase, and increases its specific activity. Activation of the epidermal growth factor receptor (EGFR) increases the PTPepsilon-microtubule association in a manner dependent upon EGFR-induced phosphorylation of PTPepsilon at Y638 and upon microtubule integrity. These events are transient and occur with rapid kinetics similar to EGFR autophosphorylation, suggesting that activation of the EGFR transiently down-regulates PTPepsilon activity near the receptor by promoting the PTPepsilon-microtubule association. Tubulin also inhibits the tyrosine phosphatase PTP1B but not receptor-type PTPmu or the unrelated alkaline phosphatase. The data suggest that reversible association with microtubules is a novel, physiologically regulated mechanism for regulation of tyrosine phosphatase activity in cells.     

The effect of the disruption of the cytoskeletal elements on uncoupler-induced changes in the centrosome

The disruption of microtubules with nocodazole or microfilaments with cytochalasin B did not prevent mother centrioles from nonrandom, preferentially perpendicular orientation with respect to the substrate plane after FCCP treatment. The microtubules affect negatively the reorientation of centrioles, because after their disruption by nocodazole the percentage of centrioles with the perpendicular orientation (the angle is tipped to the substrate plane by over 74 degrees) is seen to increase. The microfilaments have the positive effect, because after their disruption by cytochalasin B the share of centrioles with the perpendicular orientation decreases. Thus, our observations do not support the hypothesis that the long microtubules can provide the perpendicular orientation of centrioles anchoring them in the cytoplasm.     

Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport

In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end- directed movement. The latter was selectively blocked in the rigor- mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo.     

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins.

In this paper we describe a procedure for detecting proteins associated with cytoplasmic microtubules in vivo. Detergent-extracted cytoskeletons of NIL8 hamster cells are prepared under conditions which preserve the microtubules. The cytoskeletons are then extracted in the presence of calcium, which depolymerizes the microtubules and quantitatively extracted cytoskeletons are prepared from cells that have been incubated with colchicine. The cytoskeletons from these cells contain no microtubules or tubulin. Electrophoretic analysis of the calcium extracts of the colchicine-treated and untreated cells reveals several radioactively labeled polypeptides. There is, however, no apparent quantitative or qualitative difference between the two extracts other than the tubulin polypeptides. Each of the extracts is mixed with an excess of unlabeled calf brain microtubule protein and carried through cycles of temperature-dependent microtubule assembly. Distinct species from each extract co-assemble at a constant ratio, but only one polypeptide is uniquely derived from cells containing intact microtubules. The molecular weight of this polypeptide is similar to that proposed for the tau species detected in brain microtubule preparations.     

Is the microtubule disruption-induced alteration of peroxide concentration a factor inhibiting the assembly of ribonucleoprotein stress granules?

It has been examined whether the destruction of cell microtubules affects the increase in the intracellular hydrogen peroxide concentration caused by sodium arsenite, which induces the formation of stress ribonucleoprotein granules. As expected, sodium arsenite caused a 50% increase in hydrogen peroxide concentration in HeLa cells; on the other hand, another stress granule inducer tert-butylhydroquinone did not affect the peroxide concentration. The disruption of microtubules by nocodazole or vinblastine also resulted in some increase in the intracellular peroxide concentration, and the microtubule stabilization by taxol did not affect it. The combined treatment of cells with arsenite and antimicrotubule drugs caused an additive effect, and the peroxide concentration increased twice or more. Thus, the inhibition of stress granule formation after microtubule disruption cannot be explained by a decrease in peroxide concentration as compared with the affect of arsenite.     

33K protein--an inhibitory factor of tubulin polymerization in porcine brain

A factor (33K protein) that modulates tubulin polymerization in vitro has been purified to homogeneity from porcine brain by ammonium sulfate fractionation and Whatman DE52, Toyo-pearl HW65C and Bio-Gel A 0.5 m column chromatographies. The purified fraction was free of nucleic acids and sugars. The activity of the purified 33K protein is pronase E sensitive but apparently heat- and trypsin-resistant though it undergoes tryptic digestion. The 33K protein inhibits polymerization of brain microtubule proteins in a dose-dependent manner and partially depolymerizes preformed microtubules. It also inhibits polymerization of purified starfish tubulin and microtubule elongation involving fragellar outer doublet microtubules and purified porcine brain tubulin. This suggests that the target of the 33K protein is tubulin rather than microtubule-associated proteins. The 33K protein causes incomplete depolymerization of microtubules and a new steady state is quickly attained which is apparently independent of microtubule mass concentration. Divalent cations such as calcium and magnesium do not modulate the inhibitory activity of the 33K protein.     

"Buttonin," a unique button-shaped microtubule-associated protein (75 kD) that decorates spindle microtubule surface hexagonally

A 75-kD protein was purified from sea urchin egg microtubule proteins through gel filtration. It enhanced the polymerization of porcine brain tubulin, but was not heat-stable and did not bind to calmodulin in the presence of calcium as demonstrated by calmodulin affinity column chromatography. Rotary shadowing of the freeze-etched 75-kD protein adsorbed on mica revealed the protein to be a spherical molecule (approximately 9 nm in diameter). Quick-freeze deep-etch electron microscopy revealed that the surface of microtubules polymerized with 75-kD protein was entirely covered with hexagonally packed, round, button-like structures that were quite uniform in shape and size (approximately 9 nm) and similar to the buttons observed on microtubules of mitotic spindles in vivo or microtubules isolated from mitotic spindles. Judging from calibration studies of molecular mass by gel filtration, the 75-kD protein probably exists in a dimeric form (approximately 150 kD) in its native condition. The stoichiometry of tubulin (dimer) versus 75-kD protein (dimer) in the polymerized pellet was 3-3.4:1. Hence, we concluded that the 75-kD protein was a unique microtubule-associated protein that formed the microtubule button in vivo and in vitro. We propose to name this protein "buttonin".     

Enhancement of chemotactic response and microtubule assembly in human leukocytes by ascorbic acid.

The incubation of human leukocytes with ascorbic acid increased chemotaxis of the cells. In addition, ascorbic acid promoted the assembly of intracellular polymorphonuclear leukocyte (PMN) with colchicine blocked the effect of ascorbic acid on promoting microtubule assembly. Not only did ascorbic acid promote the assembly of microtubules in vivo, but it enhanced the assembly of bovine brain tubulin into microtubules in vitro as quantitated by a glass-fiber filtration assay and by promotion of viscosity changes. The enhancement in leukocyte mobility by ascorbate at concentrations achievable in normal tissues correlates with its ability to assemble microtubule organelles.