The effects of microtubule and microfilament disrupting agents on cytoskeletal arrays and wall deposition in developing cotton fibers

Summary The effects of various cytoskeletal disrupting agents (cholchicine, oryzalin, trifluralin, taxol, cytochalasins B and D) on microtubules, microfilaments and wall microfibril deposition were monitored in developing cotton fibers, using immunocytochemical and fluorescence techniques. Treatment with 10^–4 M colchicine, 10^–6 M trifluralin or 10^–6 M oryzalin resulted in a reduction in the number of microtubules, however, the drug-stable microtubules still appear to influence wall deposition. Treatment with 10^–5 M taxol increased the numbers of microtubules present within 15 minutes of application. New microtubules were aligned parallel to the existing ones, however, some evidence of random arrays was observed. Microtubules stabilized with taxol appeared to function in wall organization but do not undergo normal re-orientations during development. Microtubule disrupting agent had no detectable affect on the microfilament population. Exposure to either 4×10^–5 M cytochalasin B or 2×10^–6M cytochalasin D resulted in a disruption of microfilaments and a re-organization of microtubule arrays. Treatment with either cytochalasin caused a premature shift in the orientation of microtubules in young fibers, whereas in older fibers the microtubule arrays became randomly organized. These observations indicate that microtubule populations during interphase are heterogeneous, differing at least in their susceptibility to disruption by depolymerizing agents. Changes in microtubule orientation (induced by cytochalasin) indicate that microfilaments may be involved in regulating microtubule orientation during development.     

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.     

Modifications of microfilaments and microtubules induced by two hepatic tumor promoters,phenobarbital and biliverdin in non-transformed and transformed hepatic cell lines

Microfilaments and microtubules are components of the cytoskeleton which could be implicated in neoplastic transformation. We studied the effect of two hepatic tumor promoters, phenobarbital (PB) and biliverdin (BV), on microfilaments and microtubules of non-transformed (Cl₃) and transformed (FV) hepatic epithelial cells. Cl₃ non-transformed cells cultured in the presence of 1 × 10^–6M BV for 48 h showed a loss of F-actin, fragmentation of actin and the appearance of star-like structures in the cytoplasm, as well as loosening of the peripheral bundle of actin, and some ruffling of cell membranes. In Cl₃ cells exposed to 0.2 × 10^–3M PB a similar disappearance of F-actin staining and a very prominent ruffling of cell membrane were observed. BV and PB also produced in these cells modifications of microtubules characterized by a disappearance of centrosome staining in numerous cells, a condensed ring of tubulin around the nucleus and a depolymerized aspect of the microtubular network. All these modifications of microfilaments and microtubules closely resembled those observed in FV transformed cells in the absence of any treatment (Solvent DMSO only). We did not observe an effect of BV and PB on FV cells.The present data demonstrate that the cytoskeleton of non-transformed epithelial liver cells is sensitive to the action of liver tumor promoters suggesting that it might play a role as to yet be defined in the promotion mechanism.Abbreviations PB phenobarbital - BV biliverdin - TPA 12-0-tetradecanoyl-phorbol 13 acetate - GGT gamma-glutamyl-transpeptidase - DMSO dimethylsulfoxyde     

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     

INHIBITION OF PHAGOCYTOSIS AND PLASMA MEMBRANE MOBILITY OF THE CULTIVATED MACROPHAGE BY CYTOCHALASIN B : Role of Subplasmalemmal Microfilaments

Functional and morphologic effects of cytochalasin B on the cultivated macrophage were examined to determine the basis for plasma membrane movements of the type required for endocytosis and/or spreading on a substratum. Inhibition of phagocytosis and changes in cell shape by cytochalasin B exhibited nearly identical dose-response curves requiring 2–5 x 10^-6 M and 1–2 x 10^-5 M cytochalasin B to inhibit these functions by 50% and 100%, respectively. In contrast, hexose transport was ten times more sensitive to the drug requiring 2–3 x 10^-7 M cytochalasin B to achieve 50% inhibition of 2-deoxyglucose uptake. Inhibition of phagocytosis and changes in cell shape could not be explained solely by drug effects on hexose transport. Analysis of serial thin sections showed that cytochalasin B doses inhibitory for hexose transport had no effect on distribution or organization of either of the two subplasmalemmal microfilament types. However, cytochalasin B concentrations (2.0 x 10^-5 M) that inhibited phagocytosis and altered cell shape disorganized and/or disrupted oriented bundles of 40–50-Å subplasmalemmal microfilaments, but had no effect on the microfilamentous network. Comparative dose-response studies showing positive correlations among cytochalasin B effects on phagocytosis, changes in cell shape, and alterations in oriented subplasmalemmal microfilament bundles provide additional support for the hypothesis that microfilamentous structures play a role in translocation of plasma membrane required for endocytosis and cell motility.     

Effects of cytochalasin B on water, Na+ and Cl? exchanges in the gill of seawater adapted mulletMugil capito

Summary Electron microscopy study shows that cytochalasin treatment of the mullet damages the microfilaments system in the apex of gill ionocytes: the microfilaments are reduced in number and shortened. Cytochalasin causes a reduction of transgill potential difference and an increase of the Na⁺ and Cl^– blood concentration, of the diffusional water permeability of the gill, of the Na⁺ branchial influx and of Cl^– efflux. The increase of the Na⁺ influx may result in a reduction of the Na⁺ net excretion flux compared to the control. The increased permeability in cytochalasin treated fish facilitates the Cl^– entry probably leading to a reduction of the net Cl^– excretion. The partial inhibition of the K⁺ dependent components of Na⁺ and Cl^– effluxes also contributes to the reduction of Na⁺ and Cl^– excretion. The role of microfilaments in the mechanisms of ionic excretion by the gill is discussed.     

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.     

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.     

Regeneration of plants from primary leaves of cowpea

Plants were regenerated from the in vitro cultured explants of primary leaves of cowpea (Vigna unguiculata L. Walp). Primary leaves, including the intact petiole, were excised from three-day-old seedlings and cultured on Gamborg's B5 basal medium containing 8×10^–7 M 2,4,5-trichlorophenoxyacetic acid, 1×10^–2 M L-glutamine and 1×10^–4 M adenine sulfate. Callus formed at the petiole end. Prolific shoot regeneration occurred when this callus was transferred to B5 basal medium containing 5×10^–6 M 6-benzyl-aminopurine (BAP). Regenerated shoots rooted in growth-regulator-free B5 basal medium and were established in soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA 1-napthalene acetic acid - 2,4,5-T 2,4,5-trichloro-phenoxyacetic acid     

The effect of N-terminal substitutions on the biological activity of MSH fragments

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of α-melanotropin, [Glp⁵]α-MSH(5–10), [Glp⁵, -Phe⁷]α-MSH(5–10), [Sar⁵, -Phe⁷]α-MSH(5–10), [Nle⁴, -Phe⁷]α-MSH(4–10), [N-carbamoyl]α-MSH(5–10), and formyl and acetyl derivatives of α-MSH(5–10), [Gly⁵]α-MSH(5–10) and [Gly⁵, -Phe⁷]α-MSH(5–10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.     

Ecdysteroids in nemerteans: Presence and physiological role

Ecdysteroids were detected in the phylum Nemertea and their physiological role was studied. Radioimmunoassay (RIA) measurements showed ecdysteroid concentrations ranging from 1–47 pg/mg wet weight in several nemertean species from the orders Palaeonemertea, Heteronemertea, and Hoplonemertea. High-performance liquid chromatographic (HPLC) analysis of Paranemertes peregrina displayed peaks of RIA activity with retention times similar to those of authentic ecdysone and 20-hydroxyecdysone standards. Fluctuating ecdysteroid titers were observed in the various life stages of Carcinonemertes errans with the highest concentrations (47 pg/mg wet weight) found in gravid females. RIA of HPLC fractions of Carcinonemertes errans eggs indicated the presence of ecdysteroids (105 pg/mg wet weight). Alterations in the growth of juvenile, male, or female C. errans were not observed when the worms were exposed to 10^–7. 10^–6, or 10^–5 M 20-hydroxyecdysone. However, the eggs of C. errans appeared to be stimulated by 20-hydroxyecdysone. Shorter hatching times were observed in the egg strings exposed to hormone (10^–7 to 10^–5 M) compared to sea water and cholesterol (10^–11 and 10^–9 M) controls. Possible physiological roles and the evolutionary significance of ecdysteroids in nemerteans are discussed.     

Cytoplasmic streaming inChara rhizoids

Summary In-vivo videomicroscopy ofChara rhizoids under 10^–4g demonstrated that gravity affected the velocities of cytoplasmic streaming. Both, the acropetal and basipetal streaming velocities increased on the change to microgravity. The endogenous difference in the velocities of the oppositely directed cytoplasmic streams was maintained under microgravity, yet the difference was diminished as the basipetal streaming velocity increased more than the acropetal streaming velocity. Direction and structure of microfilaments labeled by rhodamine-phalloidin had not changed after 6 min of microgravity.Abbreviations g gravitational acceleration - Nizemi slow rotating centrifuge microscope - Texus technological experiments under reduced gravity     

Heterochromatin dispersion and blebbing of the nuclear membrane induced by melanophore stimulating hormone

Stimulation (in vivo and in vitro) of dermal melanophores of the leaf frog, Agalychnis dacnicolor, by melanophore stimulating hormone (MSH) elicits two responses in addition to the dispersion of melanosomes: (1) dispersion of heterochromatin; and (2) blebbing of the outer membrane of the nuclear envelope. The latter leads to the formation of dilated rough endoplasmic reticulum with the involvement of 80–100 Å microfilaments. N⁶,O²-dibutyryl adenosine 3′,5′-monophosphate (db-cAMP) elicits a similar response. Actinomycin D prevents both heterochromatin dispersion and membrane blebbing while cytochalasin B (CB) prevents only the latter.     

Glomerular binding of angiotensin II in the rainbow trout,Salmo gairdneri

Summary Isolated glomeruli of the rainbow trout have been exposed in vitro to¹²⁵I-angiotensin II (0.88 × 10^–9 M) and binding sites located by light-microscopic autoradiography. These studies provide evidence of specific binding of angiotensin II by glomeruli. Binding was significantly inhibited by excess (10^–5 M) unlabelled angiotensin II, but a high degree of non-specific binding also occurred. The mammalian competitive antagonist, saralasin (3 × 10^–7 M) did not influence¹²⁵I-angiotensin II binding to fish glomeruli. Intense binding of¹²⁵I-angiotensin II was noted at the vascular pole of some glomeruli.     

No direct linkage between proton pumping and photosynthate unloading from seed coats of Phaseolus vulgaris L.

The energization of the active sucrose release from bean seed-coat halves was investigated. For this purpose, seed coat tissues adjacent to the apoplastic space were exposed to a variety of treatments and proton and photosynthate release were measured. Fusicoccin (10^–5 moll^–1) stimulated proton pump activities. Orthovanadate (2×10^–4 moll^–1) and abscisic acid (10^–5 moll^–1) diminished the proton extrusion evoked by fusicoccin. Fusicoccin inhibited sucrose release, whereas orthovanadate and abscisic acid stimulated it. Addition of 100 mmoll^–1 K⁺ had a promotory effect on photosynthate unloading, fading away with time. This extra unloading was linearly related to an enhanced proton loss. It was concluded that the photosynthate unloading apparently is not a proton/sucrose antiport and that a pump-leak system for photosynthate release is unlikely. A tentative model for photosynthate/proton symport not directly linked to proton pumping is presented as the mechanism of unloading.Abbreviations ABA abscisic acid - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DTE diethioerythritol - FC fusicoccin - MES 2-(N-morpholino) ethanesulfonic acid monohydrate - NEM n-ethylmaleimide - PCMBS p-chloromercuriphenylsulfonic acid - TRIS 2-amino-2-(hydroxymethyl) propane-1,3 diol - VAN sodium orthovanadate     

F-actin network may regulate a Cl− channel in renal proximal tubule cells

A variety of mechanisms have been proposed for the regulation of ion channel molecules. As integral membrane proteins, ion channels may interact with the cytoskeleton. Regulation of channels by the actin network may therefore be important. In the present study we used cytochalasin D and exogenous actin to test this possibility. The Cl^– channel of the apical membrane of renal proximal epithelium was detected in its active state after prolonged depolarization. Within 6 sec after its addition, cytochalasin D (0.05 g/ml) significantly decreased the number of open channels and mean open probability (NPo) of the Cl^– channel. Colchicine (1 mm), which affects microtubules, did not influence channel activation. Cytochalasin D is known to not only disrupt the F-actin network but to inhibit polymerization of F-actin as well. The latter effect is also produced by DNaseI. Cytochalasin D, but not DNaseI, inactivated Cl^– channels in cell-free membrane patches, suggesting that cytochalasin D inactivated the channel by disrupting the actin network. Cytochalasin D appeared to specifically affect the channel, as opposed to membrane permeability, since only the activated whole-cell Cl^– currents were altered by cytochalasin D. Addition of actin polymer, but not actin monomer, reactivated the cytochalasin-D-depressed channel. Thus, repair of the disrupted F-actin network with actin polymer apparently restored the activity and number of open Cl^– channels. We therefore conclude that the F-actin network interacts with and possibly regulates the Cl^– channel of renal proximal tubule epithelia.We would like to thank T. Tamatsukuri for technical support. This study was presented to the American Society of Nephrology, Baltimore, 1991.     

The kinetics of reactions around the cytochrome bf complex studied in an isolated system

The kinetics of oxidation and reduction of P700, plastocyanin, cytochrome f and cytochrome b-563 were studied in a reconstituted system consisting of Photosystem I particles, cytochrome bf complex and plastocyanin, all derived from pea leaf chloroplasts. Decyl plastoquinol was the reductant of the bf complex. Turnovers of the system were initiated by laser flashes. The reaction between oxidised P700 and plastocyanin was non-homogeneous in that a second-order rate coefficient of c. 5×10^–7 M^–1 s^–1 applied to 80% of the P700⁺ and c. 0.7×10⁷ M^–1 s^–1 to the remainder. In the presence of bf complex, but without quinol, the electron transfer between cytochrome f and oxidised plastocyanin could be described by a second-order rate coefficient of c. 4×10⁷ M^–1 s^–1 (forward), and c. 1.6×10⁷ M^–1 s^–1 (reverse). The equilibrium coefficient was thus 2.5. Unexpectedly, there was little reduction of cytochrome f ⁺ or plastocyanin⁺ by electrons from the Rieske centre. With added quinol, reduction of cytochrome b-563 occurred. Concomitantly, electrons appeared in the oxidised species. It was inferred that either the Rieske centre was not involved in the high-potential chain of electron transfer events, or that, only in the presence of quinol, electrons were quickly passed from the Rieske centre to cytochrome f ⁺. Additionally, the presence of quinol altered the equilibrium coefficient for the cyt f/PC interaction from 2.5 to c. 5. The reaction between quinol and the bf complex was describable by a second-order rate coefficient of about 3×10⁶ M^–1 s^–1. The pattern of the redox reactions around the bf complex could be simulated in detail with a Q-cycle model as previously found for chloroplasts.Abbreviations AQS anthraquinone sulphonate - cyt cytochrome - cyt b-563(H) high-potential cyt b-563 - cyt b-563(L) low potential cyt b-563 - FeS(R) the Rieske protein of the cyt bf complex, containing an Fe₂S₂ centre - PC plastocyanin - PS photosystem - P700 reaction centre in PS I     

Transfer of plasmids and chromosomal genes amongst subspecies ofBacillus thuringiensis

Summary The plasmids pBC16 and pC194 fromBacilus thuringiensis subsp.israelensis strains A084-16-194 were transferred to 25 subspecies ofB. thuringiensis by a conjugation-like process using broth mating technique. The frequencies of transfer varied considerably between different mating pairs, ranging from 1.1×10^–9 to 9.8×10^–5. Additionally, chromosomal transfer could also be demonstrated in tenB. thuringiensis subspecies with very low frequencies (4.3×10^–9 to 3.7×10^–7). The intersubspecies matings within a group of eight subspecies strains gave higher frequencies of transfer than the matings between the subspecies. Furthermore, the results indicated that the capability to transfer plasmids among these various subspecies did not depend on the presence of large plasmid.     

Development of the structural components of the brush border in absorptive cells of the chick intestine

Summary The spatial and temporal relationships between cytoplasmic filaments and the morphogenesis of the intestinal brush border were examined by transmission electron microscopy of normally developing tissue and of tissue exposed to a variety of experimental conditions in organ culture. Distinct stages in the development of the brush border were identified: (1) Irregular projections of the apical plasma membrane that contain a network of microfilaments are converted to uniform projections filled with a core bundle of straight microfilaments (7–11d of incubation). (2) Rootlets form by an elongation or aggregation of filaments (11–15d). (3) The terminal web forms first as a network of short filaments just below the apical plasma membrane, then secondarily stratifies into two layers (19d of incubation to 3d posthatching). (4) Core filaments elongate as microvilli achieve their maturity (21d of incubation to 5d posthatching). Microvillus formation was not perturbed by culturing 9d tissue in high concentrations of Ca⁺⁺ or Mg⁺⁺, either with or without the ionophore, A23187. Rootlet formation was stimulated by high Mg⁺⁺, with or without A23187, and, for reasons unknown, by ethanol. Terminal web formation was not stimulated by Mg⁺⁺ or Ca⁺⁺, but the integrity of the terminal web was lost when 21d embryonic tissue was cultured with EGTA or cytochalasin B. After stratification, the terminal web could not be disrupted by EGTA, but instead was aggregated to the center of the apical end of the cell.     

Morphological evidence for the existence of a more complex cytoskeleton in Amoeba proteus

Summary Various stabilization and extraction procedures were tested to demonstrate the ultrastructural organization of the cytoskeleton in normal, locomoting Amoeba proteus. Most reliable results were obtained after careful fixation in glutaraldehyde/lysine followed by prolonged extraction in a polyethylene glycol/Triton X-100 solution. Before dehydration in a graded series of ethanol and critical-point drying, the amoebae were split by the sandwich-technique, i.e., by mechanical cleavage of cells mounted between two poly-L-lysine-coated glass slides. Platinum-carbon replicas as well as thin sections prepared from such cell fragments revealed a cytoskeleton composed of at least four different types of filaments: (1) 5–7-nm filaments organized as a more or less ordered cortical network at the internal face of the plasma membrane and probably representing F-actin; (2) 10–12-nm filaments running separately or slightly aggregated through the cytoplasm and probably representing intermediate filaments; (3) 24–26-nm filaments forming a loose network and probably representing microtubules; and (4) 2–4-nm filaments as connecting elements between the other cytoskeleton constituents. Whereas microfilaments are responsible for protoplasmic streaming and other motile phenomena, the function of intermediate filaments and cytoplasmic microtubules in amoebae is still obscure.