Effects of four agents that modify microtubules and microfilaments (vinblastine,colchicine, lidocaine,and cytochalasin B) on macrophage-mediated cytotoxicity to tumor cells

Summary The effects of vinblastine, colchicine, lidocaine, and cytochalasin B on tumor cell killing by BCG-activated macrophages were examined. These four drugs were selected for their action on membrane-associated cytoskeletal components, microtubules, and microfilaments. Colchicine and vinblastine, which block microtubular synthesis, inhibit macrophage-mediated tumor-cell cytotoxicity at a concentration of 10^–6 M. Cytochalasin B, which disrupts microfilaments, enhances tumor cell lysis and stasis due to activated macrophages at a concentration of 10^–7 M. Lidocaine, which may induce the disappearance of both microtubules and microfilaments, has the same inhibiting effect as vinblastine at a concentration of 5×10^–7 M. Whereas vinblastine and lidocaine seem to act on the macrophage itself, cytochalasin B exerts its effect predominantly on the tumor cell. These results suggest that microtubules and microfilaments play a role in the destruction of tumor cells by activated macrophages.     

Morphology and Microtubule Organization in Arabidopsis Roots Exposed to Oryzalin or Taxol

In roots of Arabidopsis thaliana, we examined the effects oflow concentrations of microtubule inhibitors on the polarityof growth and on the organization of microtubule arrays. Intact6 d old seedlings were transplanted onto plates containing inhibitors,and sampled 12 h, 24 h and 48 h later. Oryzalin, a compoundthat causes microtubule depolymerization, stimulates the radialexpansion of roots. The amount of radial swelling is linearlyproportional to the logarithm of the oryzalin concentration,from the response threshold, 170 nM, to 1 µM. Cells inthe zone of division were slightly more sensitive to oryzalinthan were cells in the zone of pure elongation. Radial swellingis also stimulated by taxol, a compound that causes microtubulepolymerization. Taxol at 1 µM causes little swelling,but at 10µM causes extensive radial swelling of cellsin the elongation zone, and does not affect cells in the divisionzone. To examine the microtubules in these roots, we used methacrylatesections with immunofluorescence microscopy. At all concentrationsof oryzalin, cortical arrays are disorganized and depleted ofmicrotubules, and the microtubules themselves often appear fragmented.These effects increase in severity with concentration, but areunmistakable at 170 nM. In taxol, cortical arrays appear tobe more intensely stained than those of controls. At 10 µM,many cells in growing regions of the stele have longitudinalmicrotubules, whereas many cells in the cortex appear to havetransversely aligned microtubules. Taxol affects microtubulesin cells of division and elongation zones to the same extent,despite the observed difference in growth. We conclude thatthe precise, spatial pattern of cortical microtubules may notbe primarily responsible for controlling growth anisotropy;and that control over growth anisotropy may differ between dividingand non-dividing cells. (Received December 6, 1993; Accepted June 7, 1994)     

Inhibitors of cell division and protoplasmic streaming fail to cause a detectable effect on intracellular calcium levels in stamen-hair cells of Tradescantia virginiana L.

The herbicides amiprophos-methyl (APM) and oryzalin disrupt mitosis and cytokinesis in plant cells by causing the depolymerization of microtubules. These drugs have also been shown to affect calcium sequestration by mitochondria. Controversy thus exists as to whether microtubule depolymerization occurs as a result of direct interaction between the drug and tubulin, or because of elevated intracellular calcium levels resulting from drug interference with calcium regulation. In order to clarify this issue we have directly measured the effect of these herbicides and other cell-motility-altering drugs on intracellular calcium levels in stamen-hair cells of Tradescantia. The results indicate that low levels (1–3 M) of APM and oryzalin can act within 3–7 min causing disorganization of mitosis. Studies using the calcium indicator indo-1 injected into stamen-hair cells to monitor internal levels of calcium, show that at drug concentrations where inhibitory effects on mitosis and-or cytokinesis are clearly seen, APM, oryzalin, isopropyl-N-phenyl carbamate, caffeine and cytochalasin D produce no change in intracellular calcium levels. Furthermore, except for cytochalasin D, these drugs do not inhibit cytoplasmic streaming, a calcium-sensitive process. We conclude that the mode of action of these drugs on the cytoskeleton is independent of an effect on intracellular calcium.Abbreviations and Symbols APM amiprophos-methyl - [Ca²⁺]_i free intracellular calcium ion concentration - CD cytochalasin D - DMSO dimethylsulfoxide - IPC isopropyl N-phenylcarbamate - MT(s) microtubule(s) To whom correspondence should be addressedWe thank Dr. L.C. Morejohn, University of Texas, Austin, for encouraging us to perform this study and for his gift of amiprophosmethyl and oryzalin. We also thank our colleagues at the University of Massachusetts for many helpful discussions. This work has been supported by grants from the U.S. Department of Agriculture (88-37261-3727) and the National Science Foundation (DCB-88-01-01750).     

Electric fields affect the orientation of cortical microtubules and cell expansion in pea callus

Summary Cortical microtubules in callus derived fromPisum sativum roots form parallel arrays within cells but are randomly oriented across the tissue. These arrays align perpendicular to the direction of an applied electric field of 6 mV per cell. Application of a field of 6 mV per cell for 4 days resulted in the co-ordinated expansion of cells parallel to the field direction. Cortical microtubule arrays were still aligned perpendicular to the applied field 24 h after removal of the field. The imposition of a field to callus after the removal of cortical microtubules by oryzalin and in the presence of the herbicide resulted in the orientation of recovering microtubules perpendicular to the direction of the field, indicating that microtubules are not directly involved in the detection of the field.Abbreviations EGTA ethylene glycol-bis (-aminoethyl ether) N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule stabilising buffer - PIPES piperazine-N,N-bis(2-ethanesulphonic acid) - oryzalin 3,5-dinitro-N⁴,N⁴ dipropylsulphanil-amide     

The distributional changes and role of microtubules in Nod factor-challenged<Emphasis Type="Italic"> Medicago sativa</Emphasis> root hairs

The normal tip-growing pattern exhibited by root hairs of legumes is disrupted when the hair is exposed to Nod factors generated by compatible bacteria capable of inducing nodule formation. Since microtubules (MTs) play an important role in regulating directionality and stability of apical growth in root hairs [T.N. Bibikova et al. (1999) Plant J 17:657–665], we examined the possibility that Nod factors might affect the MT distribution patterns in root hairs of Medicago sativa L. We observed that Nod factor application caused rapid changes in the pattern of MTs starting as early as 3 min after perfusion. Within 3 to 10 min after Nod factor application, first endoplasmic and then cortical MTs depolymerised, initially at the proximal ends of cells. Twenty minutes after exposure to Nod factors, a transverse band of microtubules was seen behind the tip, while almost all other MTs had depolymerised. By 30 min, very few MTs remained in the root hair and yet by 1 h the MT cytoskeleton re-formed. When Nod factors were applied in the presence of 10 M oryzalin or 5 M taxol, the MTs appeared disintegrated while the morphological effects, such as bulging and branching, became enhanced. Compared to the treatments with oryzalin or taxol alone, the combinatory treatments exhibited higher growth rates. Since microtubule reorganization is one of the earliest measurable events following Nod factor application we conclude that microtubules have an important role in the early phases of the signalling cascade. Microtubule involvement could be direct or a consequence of Nod factor-induced changes in ion levels.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00425-003-1097-1Abbreviations BNM buffered nodulation medium - CLSM confocal laser scanning microscopy - MT microtubule     

Inhibition of glycoconjugate secretion by colchicine and cytochalasin B

Summary The effects have been analyzed of cytochalasin B and colchicine on the secretion of glycoconjugates by human bronchial expiants labeled in vitro with radioactive glucosamine. Both cytochalasin B and colchicine had no effect on baseline ¹⁴C-labeled glycoconjugate release but caused a dose-dependent (10^–7–10^–4 M) inhibition of ¹⁴C-glycoconjugate release and discharge of labeled macromolecules from mucous and serous cells induced by 5 · 10^–5 M methacholine.Quantitative autoradiographic analyses showed that neither cytochalasin B nor colchicine inhibited ³H-threonine or ³H-glucosamine incorporation into mucous and serous cells of the submucosal glands or goblet cells of the airway epithelium. Colchicine (10^–5 M) but not cytochalasin B significantly reduced the rate at which labeled macromolecules were transported through mucous, serous and goblet cells but this effect was not observed until 4 h after the addition of colchicine. Neither cytochalasin B nor colchicine affected the basal rate of labeled-macromolecule discharge from mucous, serous or goblet cells. At a concentration of 10^–5 M, both agents completely inhibited the increase in labeled-macromolecule discharge induced in mucous and serous cells by methacholine.Our results suggest that in the submucosal gland of human airways microtubules and microfilaments may be important in secretagogue-induced but not in baseline cellular glycoconjugate discharge, implying that the mechanisms of the two processes differ significantly. Furthermore, a role for microtubules is suggested in the transport of secretory granules through mucous, serous and goblet cells.Supported by National Institutes of Health Research Grant 5R01HL22444. The authors gratefully acknowledge the technical assistance of Mr. Tudor Williams, Mr. Eduardo Quintanilla and Ms. Maureen Hayes     

Effects of two microtubule-depolymerizing drugs,vincristine and vinblastine,on porphyrin production by primary neural tissue cultures

A porphyrinogenic effect of 10^–5 to 10^–7 M Vincristine (VC) and Vinblastine (VB) was observed on primary neural tissue cultures prepared from chicken embryo brain in the presence of 10^–3 M delta-aminolevulinic acid. This effect is very pronounced in neurocyte cultures, in contrast with the documented neurite elongation defect. The microtubule disassembly by VC and VB changed the quantity of the porphyrins in the cells and medium of glial cell cultures. The correlation was studied between the depolymerization of the microtubules by VC and VB and the porphyrin overproduction of primary neural tissue cultures, the investigation also extending to 10^–7 M taxol. The direct mediation of nucleotide binding proteins of the adenyl-cyclase complex by GTP liberated from beta-tubulin during the disassembly of the microtubules is presumed.     

Rearrangement of cortical microtubules from transverse to oblique or longitudinal in living cells of transgenicArabidopsis thaliana

Summary AGFP-TUA6 (-tubulin 6) gene was transduced in theArabidopsis thaliana genome, and the GFP-TUA6 protein was expressed by 20% of the total -tubulin amount. The expressed GFP-TUA6 protein was incorporated into cortical microtubules (cMTs), so that the cMTs could be visualized under the fluorescence microscope in the living cells. The rearrangement of cMTs was observed at the tangential epidermal cell face of the hypocotyl. At the initial stage of light-induced cMT rearrangement from a transverse to an oblique or a longitudinal orientation, randomly oriented short MTs appeared. These MTs rapidly elongated obliquely or longitudinally as the transverse cMTs shortened. Finally, the transverse cMTs were replaced by the newly organized oblique or longitudinal cMTs. Reorganization of the cMTs took 50–70 min. Treatment of seedlings with 5 × 10^–5 M cytochalasin B induced disarrayed cMTs. The involvement of cytochalasin B in the orientation of developing MTs is discussed.     

Cytoskeleton and ion movements during volume regulation in cultured PC12 cells

Summary The present study investigates the role of cytoskeletal elements, microtubules and microfilaments, on ion transport systems activated during volume regulatory processes in PC12 pheochromocytoma cells. Disruption of microtubule network by colchicine (0.1 mm) or vinblastine sulfate (10 m) has no significant effect on PC12 cell hydration or on changes of the intracellular K⁺, Cl^– and Na⁺ content observed in hypo-osmotic conditions. Disruption of microfilament network by cytochalasin B strongly affects volume regulation in a dose-dependent manner. Cytochalasin B leads to a potentiation of the initial cell swelling and the regulatory volume decrease is suppressed. Although, the internal K⁺ and Cl^– level decreases significantly, as demonstrated by measurements of intracellular ion content and ⁸⁶Rb fluxes. Using the patch-clamp technique, we could demonstrate in PC12 cell membranes an ion channel whose gating is affected by application of a negative hydrostatic pressure (mechanical stress) to the membrane patch, by exposure of the cell to hypoosmotic medium (osmotic stress), or by disruption of the microfilament network with cytochalasin B.Water and ion content measurements, as well as ⁸⁶Rb fluxes have been carried out in the Laboratory of Animal Physiology from Professor R. Gilles, University of Liège, Belgium. M. Cornet was supported by the F.N.R.S., Belgium.     

Taxol maintains organized microtubule patterns in protoplasts which lead to the resynthesis of organized cell wall microfibrils

Summary We have investigated the effects of microtubule stabilizing conditions upon microtubule patterns in protoplasts and developed a new method for producing protoplasts which have non-random cortical microtubule arrays. Segments of elongating pea epicotyl tissue were treated with the microtubule stabilizing drug taxol for 1 h before enzymatic digestion of the cell walls in the presence of the drug. Anti-tubulin immunofluorescence showed that 40 M taxol preserved regions of ordered microtubules. The microtubules in these regions were arranged in parallel arrays, although the arrays did not always show the transverse orientation seen in the intact tissue. Protoplasts prepared without taxol had microtubules which were random in distribution. Addition of taxol to protoplasts with random microtubule arrangements did not result in organized microtubule arrays. Taxol-treated protoplasts were used to determine whether or not organized microtubule arrays would affect the organization of cell wall microfibrils as new walls were regenerated. We found that protoplasts from taxol-treated tissue which were allowed to regenerate cell walls produced organized arrays of microfibrils whose patterns matched those of the underlying microtubules. Protoplasts from untreated tissue synthesized microfibrils which were disordered. The synthesis of organized microfibrils by protoplasts with ordered microtubules arrays shows that microtubule arrangements in protoplasts influence the arrangement of newly synthesized microfibrils.Abbreviations DIC differential interference contrast - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - IgG immunoglobulin G - PIPES piperazine-N,N-bis[2-ethane-sulfonic acid] - PBS phosphate buffered saline     

Microtubules and microfilaments coordinate to direct a fountain streaming pattern in elongating conifer pollen tube tips

This study investigates how microtubules and microfilaments control organelle motility within the tips of conifer pollen tubes. Organelles in the 30-m-long clear zone at the tip of Picea abies (L.) Karst. (Pinaceae) pollen tubes move in a fountain pattern. Within the center of the tube, organelles move into the tip along clearly defined paths, move randomly at the apex, and then move away from the tip beneath the plasma membrane. This pattern coincides with microtubule and microfilament organization and is the opposite of the reverse fountain seen in angiosperm pollen tubes. Application of latrunculin B, which disrupts microfilaments, completely stops growth and reduces organelle motility to Brownian motion. The clear zone at the tip remains intact but fills with thin tubules of endoplasmic reticulum. Applications of amiprophosmethyl, propyzamide or oryzalin, which all disrupt microtubules, stop growth, alter organelle motility within the tip, and alter the organization of actin microfilaments. Amiprophosmethyl inhibits organelle streaming and collapses the clear zone of vesicles at the extreme tip together with the disruption of microfilaments leading into the tip, leaving the plasma membrane intact. Propyzamide and oryzalin cause the accumulation of membrane tubules or vacuoles in the tip that reverse direction and stream in a reverse fountain. The microtubule disruption caused by propyzamide and oryzalin also reorganizes microfilaments from a fibrillar network into pronounced bundles in the tip cytoplasm. We conclude that microtubules control the positioning of organelles into and within the tip and influence the direction of streaming by mediating microfilament organization.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations APM Amiprophosmethyl - FITC Fluorescein isothiocyanate - LATB Latrunculin B     

Effect of microtubule-disrupting drugs on protein and RNA synthesis in Physarum polycephalum amoebae

The effects of the microtubule-disrupting drugs, colchicine, vinblastine, podophyllotoxin, griseofulvin, and lumicolchicine (10^-5 M), on protein and RNA synthesis were studied in Physarum polycephalum amoebae in culture. All, except lumicolchicine, were found to simultaneously reduce the rate of protein synthesis and stimulate RNA synthesis. These results parallel the effects seen in cells exposed to heat shock. Treatment of the cells with a microfilament-disrupting drug, cytochalasin B (10 g/ml in ethanol), resulted in a reduced rate of protein synthesis after 2 h compared to a similar effect by vinblastine in 5–15 min. A morphological abnormality, microtubule paracystals, were seen associated with centrioles in vinblastine-treated cells in which protein synthesis had been reduced by 50%. Vinblastine and podophyllotoxin were shown to interfere with the recovery of protein synthesis after inhibition by low or elevated temperatures. The possible role of microtubules in regulating the translational response of a cell to an external environmental stimulus is discussed.     

Injury toNitella internodal cells alters microtubule organization but microtubules are not involved in the wound response

Summary The arrangement and relative stability of cortical microtubules during and after wound induction in internodal cells ofNitella flexilis andNitella pseudoflabellata were examined by immunofluorescence and by microinjection of fluorescently tagged tubulin. The formation of cellulosic wall appositions (wound walls), induced by treatment with 5×10^–2MCaCl₂, was identicalin young, growing cells and older non-growing internodes, suggesting that the initial microtubule pattern, which differs in growing and non-growing cells, does not influence wound wall formation. Depolymerization of microtubules with oryzalin did not alter wound wall morphology and microtubules were not detected during wound wall formation. After cessation of wound wall growth, microtubules were once again found in the wound site but these were always randomly oriented, even in young cells where the surrounding microtubules were organized into transverse arrays. Microtubules were similarly randomized in chloroplast-free windows induced by laser irradiation. Analysis of microtubule organization in living cells revealed that the microtubules in wound sites are less stable than the microtubules of adjacent transversely oriented arrays. The results indicate that although wounding can alter the relative stability and spatial organization of cortical microtubules, microtubules are neither involved in vesicle transport nor the construction of cellulosic wound walls.Abbreviations AFW artificial fresh water - BSA bovine serum albumin - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline     

The involvement of F-actin inUromyces cell differentiation: The effects of cytochalasin E and phalloidin

Summary The role of F-actin in cell differentiation ofUromyces appendiculatus (bean rust fungus) germlings was examined by treating differentiating and nondifferentiating germlings with the actin-binding drugs cytochalasin E (CE) and phalloidin. Prolonged exposure of urediospores to 5×10^–3–5 × 10^–5 M CE induced nuclear division in up to 28–45% of the resulting germlings, whereas the rate of mitosis in established germlings exposed to these concentrations of CE was significantly lower (4–11%). Germlings treated with CE shifted from polarized apical growth to spherical expansion, cytoplasmic microfilaments were depolymerized, and nuclear inclusions became enlarged. Differentiating germlings exposed to a 10 minute pulse of 5×10^–6M CE before the initiation of septum formation prevented the establishment of the F-actin septal ring and growth of the crosswall delimiting the appressorium. Although these CE treatments resulted in morphological and nuclear events similar to those occurring during normal appressorium formation, transient microfilament depolymerization was not sufficient to induce differentiation. Phalloidin stabilized cytoplasmic microfilaments, especially posteriorly-located microfilaments, but did not affect differentiation, nor did it significantly inhibit the effects of CE.Abbrevations CE cytochalasin E - DAPI 4,6-diamidino-2-phenylindole - DMSO dimethyl sulfoxide - F-actin filamentous actin     

Actin microfilaments and fibroin secretion in the silkgland cells of Bombyx mori : Effects of cytochalasin B

Bombyx mori posterior silkgland cells exhibit an impressive microfilament apparatus located at the cellular apex. It consists of bundles of packed, long microfilaments of 50–70 Å diameter running along circumferences delimiting the lumen of the gland, perpendicularly to the flow of luminal silk. Microfilaments are closely associated with microtubules of the cytoplasmic ‘radial microtubule system’. Immunolabelling with purified antihuman actin antibodies was used to demonstrate their actin-like nature. Apical microfilaments are sensitive to cytochalasin B (CB) which selectively inhibits the secretion of fibroin. Following the removal of the drug, microfilaments recover their normal morphology and secretion resumes. The possible implication of contraction of microfilaments in the process of secretion is discussed.     

Formation and maintenance of viroplasmic centers in Tipula iridescent virus-infected mosquito cells with deranged cytoskeletons

We have examined the role of cytoskeletal elements with respect to the formation and maintenance of viroplasmic centers (VCs) in Tipula iridescent virus (TIV)-infected mosquito Aedes albopictus (C6/36) cells. Filamentous systems consisting of microtubules and microfilaments were detected by immunofluorescence microscopy. Inoculation of cells with TIV resulted in an alteration of microtubule and microfilament organization whether or not VCs developed. The formation of short arrays of microtubules induced by taxol or the depolymerization of microtubules by colchicine, as observed by immunofluorescence microscopy, had no apparent effect upon the development of VCs as detected by Hoechst staining and electron microscopy. The dissolution of the actin-containing filamentous system by cytochalasin B also had no effect upon development. We conclude from these results that microtubules and microfilaments are not involved in the formation or maintenance of VCs in TIV-infected A. albopictus (C6/36) cells.     

Plasma membrane-cell wall connections: Roles in mitosis and cytokinesis revealed by plasmolysis ofTradescantia virginiana leaf epidermal cells

Summary Tradescantia virginiana leaf epidermal cells were plasmolysed by sequential treatment with 0.8 M and 0.3 M sucrose. Plasmolysis revealed adhesion of the plasma membrane to the cell wall at sites coinciding with cytoskeletal arrays involved in the polarisation of cells undergoing asymmetric divisions — cortical actin patch — and in the establishment and maintenance of the division site —preprophase band of microtubules and filamentous (F) actin. The majority of cells retained adhesions at the actin patch throughout mitosis. However, only approximately 13% of cells formed or retained attachments at the site of the preprophase band. After the breakdown of the nuclear envelope, plasmolysis had a dramatic effect on spindle orientation, cell plate formation, and the plane of cytokinesis. Spindles were rotated at abnormal angles including tilted into the plane of the epidermis. Cell plates formed but were quickly replaced by vacuole-like intercellular compartments containing no Tinopal-stainable cell wall material. This compartment usually opened to the apoplast at one side, and cytokinesis was completed by the furrow extending across the protoplast. This atypical cytokinesis was facilitated by a phragmoplast containing microtubules and F-actin. Progression of the furrow was unaffected by 25 g of cytochalasin B per ml but inhibited by 10 M oryzalin. Phragmoplasts were contorted and misguided and cytokinesis prolonged, indicating severe disruption to the guidance mechanisms controlling phragmoplast expansion. These results are discussed in terms of cytoskeleton-plasma membrane-cell wall connections that could be important to the localisation of plasma membrane molecules defining the cortical division site and hence providing positional information to the cytokinetic apparatus, and/or for providing an anchor for cytoplasmic F-actin necessary to generate tension on the phragmoplast and facilitate its directed, planar expansion.Abbreviations ADZ actin-depleted zone - DIC differential interference contrast - GMC guard mother cell - MT microtubule - PPB preprophase band - SMC subsidiary mother cell Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Tungsten affects the cortical microtubules of Pisum sativum root cells: experiments on tungsten–molybdenum antagonism

Tungsten (W) is increasingly shown to be toxic to various organisms, including plants. Apart from inactivation of molybdo-enzymes, other potential targets of W toxicity in plants, especially at the cellular level, have not yet been revealed. In the present study, the effect of W on the cortical microtubule array of interphase root tip cells was investigated, in combination with the possible antagonism of W for the pathway of molybdenum (Mo). Pisum sativum seedlings were treated with W, Mo or a combination of the two, and cortical microtubules were examined using tubulin immunofluorescnce and TEM. Treatments with anti-microtubule (oryzalin, colchicine and taxol) or anti-actomyosin (cytochalasin D, BDM or ML-7) drugs and W were also performed. W-affected cortical microtubules were low in number, short, not uniformly arranged and were resistant to anti-microtubule drugs. Cells pre-treated with oryzalin or colchicine and then treated with W displayed W-affected microtubules, while cortical microtubules pre-stabilized with taxol were resistant to W. Treatment with Mo and anti-actomyosin drugs prevented W from affecting cortical microtubules. Cortical microtubule recovery after W treatment was faster in Mo solution than in water. The results indicate that cortical microtubules of plant cells are indirectly affected by W, most probably through a mechanism depending on the in vivo antagonism of W for the Mo-binding site of Cnx1 protein.     

Flow orientation of brain microtubules studies by linear dichroism

Flow orientation of bovine brain microtubules has been studies using phase-modulation detected linear dichroism, LD, in a Couette cell with radial light propagation. LD could be sensitively measured in a wide flow gradient interval: 10^-3–10³ s^-1, without any apparent degradation of the microtubule structure. An extremely small flow gradient, 10^-3 s^-1 is sufficient to give significant orientation, and 10 s^-1 rapidly produced a very high degree of orientation. It is also shown that thermal convection effectively orients microtubules in vitro.The apparent linear dichroism is dominated by an anisotropic scattering from the aligned microtubules, superimposed on a weaker absorption dichroism due to intrinsic chromophores. The linear dichroism due to anisotropic turbidity, LD, is found to be an excellent tool for monitoring the formation of microtubules and in contrast to ordinary turbidity measurements, non-specific aggregates contribute to a far less extent.Time resolved LD was used to study the orientational relaxation of microtubules upon stopped shear. The relaxation towards random orientation can be described by a slow, multi-exponential decay. With increasing protein concentration the relaxation becomes slower and above approximately 1 mg/ml a fraction with a semipermanent orientation is formed. Finally, the development of orientation with time upon applying a small, constant gradient has been measured and the results are considered in terms of a model for flow orientation of rigid rods.     

Tentacle contraction and ultrastructure inDiscophrya collini: The response to cations

Summary Discophrya collini is a suctorian protozoan with contractile tentacles containing a microtubule-lined canal and microfilaments. The effects of a range of cations on tentacle contraction and ultrastructure have been determined. Treatment with 80 mM CaCl₂ and 95 mM MgCl₂ causes contraction to 28% and 57% of the control length respectively. Re-extension takes over 4 hours in the culture medium, but CaCl₂-treated tentacles are re-extended after a 5 minutes treatment with 10^–2 M EDTA or 5 × 10^–3 M EGTA. CuCl₂ causes a significant contraction at 10^–5 M (to 77%); LaCl₃ at 10^–4 M (to 65%); ZnCl₂ at 10^–2 M (to 65%), but BaCl₂, CoCl₂, MnCl₂, NiCl₂, and SrCl₂ cause significant changes only at 10^–1 M.The cytoplasm of CaCl₂-treated cells contains two forms of membraneous structures when viewed in TEM; that of MgCl₂-treated cells reveals granular areas of medium electron density. None of these features are seen in control cells. The microtubules of the tentacle canal appear to be intact upon its retraction into the cell with no change occurring in the numbers or relative positions of the microtubules. The tentacle cortex is wrinkled. It is suggested from this and previous work that tentacle contraction may be mediated by a microfilament-based mechanism, and that calcium may be involved.