MEK kinase 1 (MEKK1) transduces c-Jun NH2-terminal kinase activation in response to changes in the microtubule cytoskeleton

Cell shape change and the restructuring of the cytoskeleton are important regulatory responses that influence the growth, differentiation, and commitment to apoptosis of different cell types. MEK kinase 1 (MEKK1) activates the c-Jun NH2-terminal kinase (JNK) pathway in response to exposure of cells to microtubule toxins, including taxol. MEKK1 expression is elevated 3-fold in mitosis and microtubule toxin-treated cells accumulated at G2/M of the cell cycle. Targeted disruption of MEKK1 expression in embryonic stem cells resulted in the loss of JNK activation and increased apoptosis in response to taxol. Targeted disruption of the MEK kinase 2 gene had no effect on activation of the JNK pathway in response to microtubule toxins demonstrating a specific role of MEKK1 in this response. Cytochalasin D-mediated disruption of actin fibers activates JNK and stimulates apoptosis similarly in MEKK1(-/-) and wild type cells. The results show that MEKK1 is required for JNK activation in response to microtubule but not actin fiber toxins in embryonic stem cells. MEKK1 activation can protect cells from apoptosis in response to change in the integrity of the microtubule cytoskeleton.     

Reversible inhibition of microtubules and cell growth by the bicyclic colchicine analogue MTC

2-methoxy-5-(2,3,4-trimethoxyphenyl) 2,4,6-cycloheptatrien-1-one (MTC) is a synthetic colchicine analogue, lacking the B ring of the alkaloid (Fitzgerald: Biochem. Pharmacol. 25:1381-1387, 1976). MTC has been shown to bind reversibly to the colchicine binding site of tubulin and to inhibit microtubule assembly in vitro (Andreu et al: Biochemistry 23:1742-1752, 1984; Bane et al: J. Biol. Chem. 259:7391-7398, 1984). Its action on different cultured cell lines (PtK2, Pk15, and SV-3T3) has now been studied. 0.2 X 10(-6) M MTC stopped Pk15 and SV-3T3 cell growth, inducing an accumulation of mitoses in a few hours. Removal of MTC from the culture medium rapidly restored normal mitotic index and growth rates. Partial depolymerization of the cytoplasmic microtubules of PtK2 cells was observed at concentrations ranging from 2 to 5 X 10(-7) M. Maximal microtubule network depolymerization was obtained after 4 h of treatment with 2 to 5 X 10(-6) M MTC or at a higher MTC concentration (2 X 10(-5) M) for less than 2 h. Removal of 2 X 10(-5) M MTC (the highest MTC concentration used) from the culture medium resulted in almost complete microtubule polymerization after 10 min of drug recovery and a normal microtubule network in 20-30 min. MTC constitutes an antimitotic drug directed to the colchicine site. It is water-soluble, shows a fast and reversible action, and may therefore be employed as a convenient tool to study cellular microtubule-dependent functions.     

Normal and Ha-ras-1 oncogene transformed Buffalo rat liver (BRL) cells show differential resistance to cytoskeletal protein inhibitors.

In the present study, using immunofluorescence microscopy, we have demonstrated that normal and Ha-ras-1 transformed Buffalo rat liver (BRL) cells which were exposed to cytoskeletal protein inhibitors, showed a differential resistance of their microfilament and microtubule networks. One hour exposure of normal BRL cells to 10(-5) M cytochalasin B provoked a clear and already total breakdown of actin filaments. However, at this concentration of cytochalasin B, the microfilaments of transformed BRLHO6T1-1 cells were not seriously affected; a higher cytochalasin B concentration (> or = 2 x 10(-5) M) was required to induce a significant breakdown of microfilaments in these transformed cells. The two cell lines also demonstrated differential microtubule stability when they were treated with either colchicine or triethyllead. Three hours exposure to 10(-6) M of either antimicrotubule agents was sufficient to disrupt the microtubules of normal BRL cells, without affecting their counterparts in the transformed BRLHO6T1-1 cells. A 10-fold higher drug concentration (10(-5) M) was required to induce microtubular breakdown in the transformed BRL cells. The differential stability of microfilaments and microtubules in normal and transformed BRL cells that was observed could not be attributed to a differential internalization of the agents, as shown by experiments on the uptake of [3H]-cytochalasin B and triethyllead. In addition, the transformed BRLHO6T1-1 cells did not express altered actin and tubulin isoforms, as demonstrated by isoelectric focusing followed by immunoblotting analysis. We conclude that the transformation of BRL cells with the Ha-ras-1 oncogene results in a greater stability of microfilaments and microtubules, leading to a structurally firmer cell shape.(ABSTRACT TRUNCATED AT 250 WORDS)     

The G protein-coupled oestrogen receptor 1 agonist G-1 disrupts endothelial cell microtubule structure in a receptor-independent manner

The G protein-coupled oestrogen receptor GPER1, also known as GPR30, has been implicated in oestrogen signalling, but the physiological importance of GPER1 is not fully understood. The GPER1 agonist G-1 has become an important tool to assess GPER1-mediated cellular effects. Here, we report that this substance, besides acting via GPER1, affects the microtubule network in endothelial cells. Treatment with G-1 (3 μM) for 24 h reduced DNA synthesis by about 60 % in mouse microvascular endothelial bEnd.3 cells. Treatment with 3 μM G-1 prevented outgrowth of primary endothelial cells from mouse aortic explants embedded in Matrigel. Treatment with G-1 (0.3-3 μM) for 24 h disrupted bEnd.3 cell and HUVEC microtubule structure in a concentration-dependent manner as assessed by laser-scanning confocal immunofluorescence microscopy. G-1-induced (3 μM) disruption of microtubule was observed also after acute (3 and 6 h) treatment and in the presence of the protein synthesis inhibitor cycloheximide. Disruption of microtubules by 3 μM G-1 was observed in aortic smooth muscle cells obtained from both GPER1 knockout and wild-type mice, suggesting that G-1 influences microtubules through a mechanism independent of GPER1. G-1 dose dependently (10-50 μM) stimulated microtubule assembly in vitro. On the other hand, microtubules appeared normal in the presence of 10-50 μM G-1 as determined by electron microscopy. We suggest that G-1-promoted endothelial cell anti-proliferation is due in part to alteration of microtubule organization through a mechanism independent of GPER1. This G-1-promoted mechanism may be used to block unwanted endothelial cell proliferation and angiogenesis such as that observed in, e.g. cancer.     

A novel microtubule protein in the marginal band of human blood platelets

A characterization is reported of the major cytoskeletal protein, called IEF (isoelectric focusing)-51K, of marginal band microtubule coils from human blood platelets (Kenney, D. M. and Linck, R. W. (1985) J. Cell Sci. 78, 1-22). IEF-51K is a unique biochemical species which is distinguishable from platelet and mammalian neuronal alpha-tubulin and beta-tubulin by 1) its faster mobility on discontinuous sodium dodecyl sulfate electrophoresis corresponding to an apparent Mr 51,000; 2) its more alkaline relative isoelectric point at pH 5.7 compared with that of alpha- and beta-tubulin at pH 5.3 and 5.5, respectively; 3) lack of coincidence in peptide maps prepared with chymotrypsin or Staphylococcus aureus V8 protease; and 4) lack of immunochemical cross-reactivity of polyclonal anti-IEF-51K with alpha- and beta-tubulin and of monoclonal anti-alpha-tubulin and anti-beta-tubulin with IEF-51K. In contrast to its chemical uniqueness, IEF-51K is tubulin-like in some of its properties. IEF-51K is localized in the marginal band of intact platelets by immunofluorescence; it undergoes cycles of microtubule disassembly/reassembly both in vitro and in vivo. Furthermore, IEF-51K was not extracted from isolated Taxol-stabilized marginal band microtubules by elevated NaCl concentrations (to 0.45 M), conditions that do not disrupt the polymeric structure of alpha- and beta-tubulin. These results indicate that IEF-51K together with alpha-tubulin and beta-tubulin are the major structural polypeptides of platelet marginal band microtubules. The unusual subunit composition of the platelet marginal band microtubule may be related to specialization(s) of microtubule structure and function in the marginal band coil of platelets.     

Ionophore-induced disassembly of blood platelet microtubules: effect of cyclic AMP and indomethacin

Cytoplasmic calcium levels are believed to be important in blood platelet activation. Upon activation, the discrete marginal microtubule band, which maintains the discoid shape of non-activated platelets, becomes disrupted. Present studies demonstrate that the extent of assembly of the marginal microtubule band is related to cytoplasmic calcium levels. The divalent cationophore, A23187, causes platelet aggregation, secretion, and contraction by promoting calcium transport from intraplatelet storage sites into the cytoplasm. A23187 caused disassembly of platelet microtubules. Quantitation of electron micrographs revealed that numbers of microtubules were reduced by approximately 80% after A23187 treatment. Secondly, assembled microtubules in homogenates of platelets, in which microtubules were stabilized prior to homogenization, were decreased in favor of free tubulin in A23187-treated platelets. Thirdly, A23187 increased 14C-colchicine binding by intact platelets; this also indicated a shift in the microtubule subunit equilibrium to favor free, colchicine-binding tubulin subunits. In control experiments, A23187 did not affect the stability of platelet tubulin, the colchicine binding reaction, or the total tubulin content of platelets. Stimulation of colchicine binding depended on A23187 concentration (0.05-0.5 microM) and did not require extracellular calcium. A23187-stimulation of colchicine binding was blocked by dibutyryl cyclic AMP (0.80 mM) and/or 3-isobutyl-1-methylxanthine (50 microM) and by indomethacin (10 microM). Cyclic AMP or indomethacin also interferes with A23187-induced platelet activation, but indomethacin is not likely to completely inhibit the perturbation of intraplatelet calcium gradients by A23187. It is suggested that A23187-induced microtubule disassembly may be an indirect effect of calcium on microtubules.     

Interaction of tubulin and cellular microtubules with the new antitumor drug MDL 27048. A powerful and reversible microtubule inhibitor

We have characterized the binding of trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2- propen- 1-one (MDL 27048) to purified procine brain tubulin, and the inhibition of microtubule assembly by this compound in vitro and using cultured cells. Binding measurements were performed by difference absorption and fluorescence spectroscopy. MDL 27048 binds to one site/tubulin heterodimer with an apparent equilibrium constant Kb = (2.8 +/- 0.8) X 10(6) M-1 (50 mM 2-(N-morpholino)ethanesulfonic acid, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.5 mM MgCl2, 0.1 mM GTP buffer, pH 6.7, at 25 degrees C). Podophyllotoxin displaced the binding of MDL 27048, suggesting an overlap with the colchicine-binding site. Assembly of purified tubulin into microtubules was inhibited by substoichiometric concentrations of MDL 27048, which also induced a slow depolymerization of preassembled microtubules. The cytoplasmic microtubules of PtK2 cells were disrupted in a concentration and time-dependent manner by MDL 27048, as observed by indirect immunofluorescence microscopy. Maximal depolymerization took place with 2 X 10(-6) M MDL 27048 in 3 h. When the inhibitor was washed off from the cells, fast microtubule assembly (approximately 8 min) and complete reorganization of the cytoplasmic microtubule network (15-30 min) were observed. MDL 27048 also induced mitotic arrest in SV40-3T3 cell cultures. Due to all these properties, this anti-tumor drug constitutes a new and potent microtubule inhibitor, characterized by its specificity and reversibility.     

Microtubules and beta cell function: effect of colchicine on microtubules and insulin secretion in vitro by mouse beta cells

A monolayer culture system was developed to study the role of microtubules in insulin secretion. Cultured cells were obtained to study the role of microtubules in insulin secretion. Cultured cells were obtained by enzymatic digestion of pancreases from C57BL-KsJ mice 6-12 wk of age. On day 4 of culture, the medium was changed, control or treatment medium added, and frequent samples were removed for insulin assay. Microtubules and beta cells were identified by indirect immunofluorescence with monospecific antibodies to tubulin and insulin. An extensive microtubule network radiates from the perinuclear region of the beta cell to the plasma membrane. Although alterations in the calcium concentration of the medium did not affect the microtubule pattern, the absence of calcium or glucose in the medium inhibited insulin secretion (P less than 0.001). Optimum insulin release occurred at a calcium concentration of 2.5 mM. Colchicine, in concentrations of 10(-10) M, did not affect the microtubule immunofluorescent pattern, whereas concentrations of 1 and 5 x 10(-7) M decreased the number of microtubules, and microtubules could not be identified in cultures treated with 10(-6) M colchicine for 2 h. After a 2-h preincubation, the prolonged release of insulin at either 2.0 or 4.5 mg/ml of glucose was decreased by 10(-6) M colchicine (P less than 0.02). The immediate release of insulin was similar to that in control plates and occurred in cultures with no identifiable microtubules. Microtubules and insulin secretion were not altered by 10(-6) M lumicolchicine and prolonged insulin secretion recovered 24 h after removal of colchicine. These studies show that the microtubules facilitate sustained secretion of insulin but are not required for the immediate release of the hormone. Alterations in the extracellular calcium concentration which play an essential role in insulin secretion do not alter the microtubule pattern in the beta cell.     

Modification in Cell Shape Unrelated to Cellulose Microfibril Orientation in Growing Thallus Cells of Chaetomorpha moniligera

Cellulose microfibril orientation patterns in thallus cellsof Chaetomorpha moniligera were studied, and the relationshipbetween the microfibril and the peripheral microtubule arrangementsduring cell-shape modification by colchicine was examined. Inthe cuttings from growing thalli, linearly arranged cylindricalcells developed into cask-shaped cells during 4–6 daysof culture at 27?C. In the cylindrical cells, microfibrils formingthe innermost portion of the wall were arranged alternatelyin longitudinal and transverse directions, but peripheral microtubuleswere always arranged only in a longitudinal direction. Thesefeatures were also noted in the cask-shaped cells. Colchicineat 10^–3M and 3?10^–3M accelerated both cell expansionand wall thickening with matrix deposition, but the directionsin which both microfibrils and microtubules were arranged werethe same as those of the cylindrical cells. These results indicatethat (1) the microfibril and microtubule arrangements of Chaetomorphaare not necessarily correlated, (2) changes in cell shape ofChaetomorpha are not necessarily accompanied by changes in thearrangement of cell-wall microfibrils, and (3) colchicine playsa role in the loosening and thickening of cell walls by enhancingmatrix deposition. (Received June 2, 1986; Accepted February 13, 1987)     

Isolated cytoskeletons of human blood platelets: dark-field imaging of coiled and uncoiled microtubules

Detergent extraction of human blood platelets pre-treated with Taxol to stabilize microtubules allows isolation of marginal band (MB) cytoskeletons. We studied MB cytoskeleton structure using dark-field light microscopy and negative stain electron microscopy (EM). Dark-field illumination clearly demonstrated the "hoop" shape of MB cytoskeletons in unfixed suspensions where the microtubule coils had a mean diameter of 2.87 microns (+/- 0.18 micron, SD). Microtubules were uncoiled by brief exposure to trypsin (2 ng/micrograms protein) or by NaCl (154-600 mM) but not by DNase I, which removed approximately 40% of total actin, but had no effect on dark-field images of microtubule coils. As microtubules uncoiled, a single fiber emerged from the hoop and gradually lengthened as the brightness of the hoop diminished; these fibers correspond to the single microtubules seen by EM. Polypeptides of coiled and uncoiled MB cytoskeletons were analyzed by SDS-PAGE. When microtubules became uncoiled, no changes in the major components (alpha- and beta-tubulin, IEF-51K, or actin) were found. However, a number (greater than 10) of minor polypeptides, each less than 5% of total cytoskeletal protein and with an Mr ranging from 80,000- greater than 260,000, were decreased in "uncoiled" MB cytoskeletons. These results implicate one or more of these minor polypeptides in maintenance of hoop integrity. Dark-field light microscopy thus provides an approach toward investigating the mechanism(s) involved in maintaining the microtubule coil of the platelet marginal band.     

Microtubules,protoplasts and plant cell shape

Indirect immunofluorescence has been used to study the function of cytoplasmic microtubules in controlling the shape of elongated carrot cells in culture. Using a purified wall-degrading preparation, the elongated cells are converted to spherical protoplasts and the transverse hoops of bundled microtubules are disorganised but not depolymerised in the process. Since microtubules remain attached to fragments of protoplast membrane adhering to coverslips and are still seen to be organised laterally in bundles, it would appear that re-orientation of the transverse bundles is due to loss of cell wall and not to the cleavage of microtubule bridges. After 24 h treatment in 10^-3 M colchicine, microtubules are depolymerised in elongated cells but, at this time, the cells retain their elongated shape. This suggests that wall which was organised in the presence of transverse microtubule bundles can retain asymmetric shape for short periods in the absence of those tubules. However, after longer periods of time the cells become spherical in colchicine. Neither wall nor tubules therefore exert individual control on continued cellular elongation and so we emphasize the fundamental nature of wall/microtubule interactions in shape control. It is concluded that the observations are best explained by a model in which hooped bundles of microtubules—which are directly or indirectly associated with molecules involved with cellulose biosynthesis at the cell surface—act as an essential template or scaffolding for the orientated deposition of cellulose.     

Relationship between 1-chloro2,4-dinitrobenzene-induced cytoskeletal perturbations and cellular glutathione

Exposure of 3T3 cells to micromolar doses of 1-chloro-2,4-dinitrobenzene, a substrate for glutathione-S-transferase, resulted in a rapid depletion of total cellular glutathione accompanied by disassembly of microtubules as visualized by fluorescence microscopy. However, prolonged incubation resulted in cellular recovery from 1-chloro-2,4-dinitrobenzene insult as evidenced by a steady rise in total cellular glutathione accompanied by microtubule reassembly to their normal organization 5 hours after treatment. To evaluate the role of total cellular glutathione in modulating the 1chloro-2,4-dinitrobenzene-induced cytoskeletal perturbation, we used 1-chloro-2,4-dinitrobenzene and/or buthiomine sulfoximine, an effective irreversible inhibitor of glutathione synthesis, to manipulate cellular glutathione levels. Incubation of 3T3 cells with 2.5 M 1-chloro-2,4-dinitrobenzene and 250 M buthiomine sulfoximine for 5 hours resulted in a complete depletion of total cellular glutathione accompanied by essentially complete loss of microtubules and marked alterations in the density and distribution pattern of microfilaments. Buthionine sulfoximine enhanced markedly the extent and duration of cellular glutathione depletion and the severity of microtubule disruption of 3T3 cells over the level achieved by 1-chloro-2,4-dinitrobenzene treatment alone. Furthermore, buthiomine sulfoximine also prevented the restoration of cellular glutathione content and microtubule reassembly that normally were evident 5 hours after 1-chloro-2,4-dinitrobenzene treatment. Exposure of 3T3 cells to 50 M 2-cyclohexene-l-one, which depletes free glutathione by conjugation, resulted in a comAbbreviation BSO DL-buthiomine-S-R-sulfoximine - CDNB 1-chloro-2,4-dinitrobenzene - CHX 2-cyclohexene-l-one - GSH glutathione - GST glutathione-S-transferase - MAPS microtubule-associated proteins - MF microfilaments - MT microtubules.     

1-(3′,4′,5′-Trimethoxyphenyl)-3-(3″,4″-dimethoxy-2″-hydroxyphenyl)-propane with microtubule-depolymerizing ability induces G2/M phase arrest and apoptosis in HepG2 cells

1-(3′,4′,5′-Trimethoxyphenyl)-3-(3″,4″-dimethoxy-2″-hydroxyphenyl)-propane (DP), a novel synthesized 1,3-diarylpropanes compound, showed growth inhibitory effect on human hepatoma HepG2 cells in a concentration-dependent manner. The growth inhibitory effect of DP on HepG2 cells was associated with microtubule depolymerization, G2/M phase arrest and apoptosis induction. The G2/M phase arrest induced by DP resulted from its microtubule-depolymerizing ability, and DP-treated HepG2 cells finally underwent caspase-dependent apoptosis. DP increased the levels of death receptor 4 (DR4), death receptor 5 (DR5) and pro-apoptotic protein Bax, but decreased the levels of anti-apoptotic protein Bcl-2. Meanwhile, the decrease in the mitochondrial membrane potential (MMP) and the release of cytochrome c from mitochondria were observed in DP-treated HepG2 cells. DP increased the levels of reactive oxygen species (ROS) in HepG2 cells, and antioxidant N-acetylcysteine (NAC) completely blocked DP-induced ROS accumulation and the disruption of the balance between Bax and Bcl-2 proteins, and effectively blocked the decreased MMP and apoptosis, but had no effect on the activation of caspase-8 and the up-regulations of DR4 and DR5 induced by DP. These results suggest that DP induces G2/M phase arrest through interruption of microtubule network followed by the death receptor- and ROS-mediated apoptosis in HepG2 cells.     

Effect of colchicine on rat mast cells

In the mast cell, a well-developed array of microtubules is centered around the centrioles. Complete loss of microtubules is observed when mast cells are treated with 10(-5) M colchicine for 4 h at 37 degrees C. The loss of ultrastructurally evident microtubules is associated with a marked change in the shape of mast cells from spheroids to highly irregular, frequently elongated forms with eccentric nuclei. In colchicine-treated cells the association of nucleus, Golgi apparatus, and centrioles is also lost. Mast cells exposed to 10(-5) M colchicine for 4 h at 37 degrees C retain 80% of their capacity to release histamine when stimulated by polymyxin B. Exocytosis is evident in stimulated cells pretreated with colchicine and lacking identifiable microtubules. When the conditions of exposure of mast cells to colchicine are varied with respect to the concentration of colchicine, the length of exposure, and the temperature of exposure, dissociation between deformation of cell shape and inhibition of histamine secretion is observed. These observations indicate that microtubules are not essential for mast cell histamine release and bring into question the assumption that the inhibitory effect of colchicine on mast cell secretion depends on interference with microtubule integrity.     

Mechanics of microtubule bundles in pillar cells from the inner ear.

The mechanical properties of cross-linked microtubule bundles were measured from outer pillar cells isolated from the mammalian inner ear. Measurements were made using a three-point bending test and were incorporated into a mathematical model designed to distinguish between the stiffness contributions from microtubules and their cross-linking proteins. Outer pillar cells were composed of 1000-3000 parallel bundled microtubules in a square array that was interdigitated and cross-linked with actin filaments. The average midpoint bending stiffness of intact cells was 7 x 10(-4) N/m. After removal of both the actin filaments and cross-links with detergent in the presence of DNase I, the square array was disrupted and the stiffness decreased by a factor of 4, to 1.7 x 10(-4) N/m. The bending modulus for individual microtubules was calculated to be 7 x 10(-23) Nm2, and the Young's modulus for these 15 protofilament microtubules was 2 x 10(9) Pa. The shear modulus between microtubules in intact cells was calculated to be 10(3) Pa. It was concluded that cross-linking proteins provided shear resistance between microtubules, which resulted in a fourfold increase in stiffness. The model can be used to estimate the mechanical properties of cross-linked microtubule bundles in cells from which direct measurements are not available.     

Effect of colcemid on the centrosome and microtubules in dermal melanophores of Xenopus laevis larvae in vivo.

An electron microscopy study showed that in melanophores with dispersed and aggregated pigment the sensitivity of the centrosome and the stability of microtubules were different and depended on the colcemid concentration. The structure of the centrosome didn't change upon exposure to colcemid in dispersed melanophores. In aggregated melanophores, on exposure to 10(-6) M colcemid, the centrosome retained its structure; colcemid at 10(-5)-10(-3) M caused a dramatic collapse of the centrosome. Treatment of aggregated melanophores with colcemid resulted in the complete disassembly of the microtubules; though microtubules in dispersed melanophores appear to be colcemid resistant. Light microscopy studies indicated that in Xenopus melanophores with aggregated or dispersed pigment melanosomes didn't change their location after exposure to 10(-3)-10(-6) M colcemid. Subsequent incubation in colcemid-free medium revealed that the cells retained their ability to translocate melanosomes in response to hormone stimulation. Electron microscopy data revealed the inactivation of the centrosome as MTOC (microtubule-organizing center) in dispersed melanophores with melatonin substituted for MSH in the presence of colcemid. In contrast, with melanocyte-stimulating hormone (MSH) substituted for melatonin, we observed the activation of the centrosome in aggregated cells. We showed that in aggregated melanophores pigment movement proceeded in the complete absence of microtubules, suggesting the involvement of a microtubule-independent component in the hormone-induced melanosome dispersion. However, we observed abnormal aggregation along colcemid-resistent microtubules in dispersed melanophores, suggesting the involvement of not only stable but also labile microtubules in the centripetal movement of melanosomes. The results raise the intriguing questions about the mechanism of the hormone and colcemid action on the centrosome structure and microtubule network in melanophores with dispersed and aggregated pigment.     

Actin cytoskeleton of resting bovine platelets

Actin filaments in resting discoid bovine platelets were examined by fluorescence and electron microscopy. Rhodamine-phalloidin staining patterns showed a characteristic wheel-like structure which consisted of a central small circle connected by several radial spokes to a large peripheral circle. This wheel-like structure was composed of actin filaments forming a characteristic arrowhead structure with heavy meromyosin from muscle. Actin filaments were densely arrayed in parallel with a marginal microtubule band and radiated out from the center to the periphery. Platelets treated with colchicine lost their marginal microtubule band but retained their wheel-like structure and normal discoid form. Cytochalasin B disrupted the wheel-like structure but not the marginal microtubule band or the normal discoid form. After simultaneous treatment with both cytochalasin B and colchicine, platelets lost their discoid shape. These results suggest that actin filaments and microtubules both play important roles in the maintenance of the discoid shape of resting bovine platelets.     

Morphological modifications of cells of Clostridium pasteurianum caused by growth on sulfite.

The morphology of Clostridium pasteurianum cells grown on 10(-2) M SO32- showed significant alteration in cell shape and the absence of the electron translucent reserve polysaccharide (amylopectin) when compared to sulfate-grown cells. At the lower sulfite concentrations (10(-3) and 10(-4)M SO32-) the cells showed the cytoplasmic changes noted above but the cell shapes were not modified.     

Rigidity of microtubules is increased by stabilizing agents

Microtubules are rigid polymers that contribute to the static mechanical properties of cells. Because microtubules are dynamic structures whose polymerization is regulated during changes in cell shape, we have asked whether the mechanical properties of microtubules might also be modulated. We measured the flexural rigidity, or bending stiffness, of individual microtubules under a number of different conditions that affect the stability of microtubules against depolymerization. The flexural rigidity of microtubules polymerized with the slowly hydrolyzable nucleotide analogue guanylyl-(alpha, beta)- methylene-diphosphonate was 62 +/- 9 x 10(-24) Nm2 (weighted mean +/- SEM); that of microtubules stabilized with tau protein was 34 +/- 3 x 10(-24) Nm2; and that of microtubules stabilized with the antimitotic drug taxol was 32 +/- 2 x 10(-24) Nm2. For comparison, microtubules that were capped to prevent depolymerization, but were not otherwise stabilized, had a flexural rigidity of 26 +/- 2 x 10(-24) Nm2. Decreasing the temperature from 37 degrees C to approximately 25 degrees C, a condition that makes microtubules less stable, decreased the stiffness of taxol-stabilized microtubules by one-third. We thus find that the more stable a microtubule, the higher its flexural rigidity. This raises the possibility that microtubule rigidity may be regulated in vivo. In addition, the high rigidity of an unstabilized, GDP-containing microtubule suggests that a large amount of energy could be stored as mechanical strain energy in the protein lattice for subsequent force generation during microtubule depolymerization.