Studies on the posterior silk gland of the silkworm Bombyx mori. VI. Distribution of microtubules in the posterior silk gland cells

There are two microtubule systems in the posterior silk gland cells. One is a radial microtubule system in which the microtubules run radially from the basal to the apical cytoplasm and in which fibroin globules (secretory granules of fibroin) and mitochondria are arranged along these microtubules, thus composing a "canal system" which is assumed to be responsible for the intracellular transport of fibroin globules. The other is a circular microtubule system in the apical cytoplasm which is composed of bundles of microtubules and microfilaments running in a circular arrangement around the glandular lumen at an interval of approximately 4 mum at the end of the fifth instar. This system is presumably concerned with secretion and/or intraluminal transport of fibroin.     

Cytoskeletal organization following cannabinoid treatment in undifferentiated and differentiated PC12 cells.

Confocal microscopy in association with three-dimensional reconstruction was used to examine the changes in the microtubules and microfilaments following cannabinoid treatment of PC12 cells. Microtubules and microfilaments were disrupted in a dose-dependent manner following treatment with 10-30 microM delta 9-tetrahydrocannabinol (THC). A disruption of microtubules and microfilaments was observed following treatment with 30 microM cannabidiol and cannabinol. The amount of microtubules and microfilaments was reduced in a dose-dependent manner following treatment with 10 and 20 microM THC. Cannabidiol and cannabinol reduced the amount of microtubules and microfilaments; however, the reduction was less than that observed with THC treatment. Following the addition of nerve growth factor, differentiated PC12 cells were generally more sensitive to cannabinoid treatments than undifferentiated cells. The possible mechanisms that may account for the changes in microtubules and microfilaments following cannabinoid treatment are discussed.     

Interaction of captan with mammalian microtubules

Using turbidometry, electron microscopy and immunofluorescent microscopy experiments we studied the effect of captan, a widely used pesticide on mammalian microtubules and microfilaments. Turbidometry at 350 nm showed a dose-dependent inhibition of tubulin assembly incubated with captan. The pesticide, given at equimolar concentration with tubulin (30 microM), caused the total inhibition of microtubule formation, while at lower concentrations (5-20 microM) the inhibition of tubulin polymerization was less extensive. At the same concentration range (5-30 microM), captan also promoted the disassembly of performed microtubules. The results of the in vitro effects of captan with microtubules were confirmed in parallel by electron microscopic studies. In vivo, captan caused also depolymerization of microtubules in cultured mouse fibroblasts as shown by indirect immunofluorescent staining of tubulin. The extent of microtubules disassembly was concentration- and time-dependent. While incubation of the cells with 10 microM captan for 3 h disturbs totally the microtubular structures, incubation with 5 microM captan needs 12 h for the same effect. Recovery of microtubules was observed, when preincubated cells were extensively washed. No interaction of this drug with equimolar concentration of G- or F-actin could be observed in vitro, as shown by polymerization experiments. In line with this, the fluorescent actin pattern in mouse fibroblasts incubated with 10 mM captan for up to 12 h did not seem to be altered. From these results it is concluded that captan interacts in equimolar concentrations with tubulin affecting the assembly and disassembly of microtubules in vitro and in cultures of mammalian cells.     

Bundling of microtubules by synapsin 1. Characterization of bundling and interaction of distinct sites in synapsin 1 head and tail domains with different sites in tubulin.

Synapsin 1 is a nerve terminal phosphoprotein whose role seems to encompass the linking of small synaptic vesicles to the cytoskeleton. Synapsin 1 can join small synaptic vesicles to neuronal spectrin, microfilaments and microtubules; it can also bundle microtubules and microfilaments. In this paper, the mode of interaction between synapsin 1 and microtubules has been investigated. Bundling is shown to be highly cooperative: the apparent Hill coefficient is 3.06 +/- 0.3, and bundling is half-maximal at 0.63 +/- 0.02 microM. Bundling occurs either when whole synapsin 1 preparations (containing monomers and oligomers) or when monomeric synapsin 1 is added to microtubules. However, it is not clear that synapsin 1 remains monomeric in the presence of microtubules. Synapsin 1-microtubule mixtures contain two types of filament. One type is characterised by microtubules often with synapsin 1 bound to their surface. The other type is composed of filaments of diameter 15 +/- 5 nm. This filament type is granular and made up in part of 14-nm-diameter particles. These dimensions are consistent with their being made up of polymerised synapsin 1. It is possible that microtubules induce the polymerisation of synapsin 1. Synapsin 1 had independent tubulin binding sites in the N-terminal head domain and in the C-terminal tail domain. Whole synapsin 1 can interact with tubulin after it has been digested to remove the tubulin C terminus (des-C-terminal tubulin). The interaction of des-C-terminal tubulin with synapsin 1 appears to be via the head domain, since 125I-des-C-terminal tubulin only shows specific binding to the head domain on gel blots. By contrast intact tubulin binds to both head and tail domains. Binding to the tail domain can be inhibited by a synthetic peptide representing the microtubule-associated protein 2 (MAP2) binding site of class II beta tubulin. These results suggest a model for microtubule bundling by synapsin 1 in which independent sites in the head and tail domains of synapsin 1 cross-link microtubules by interactions with two distinct sites in tubulin.     

Evidence for active interactions between microfilaments and microtubules in myxomycete flagellates

We have previously observed the apparent displacement of microfilaments over microtubules in the backbone structure of permeabilized flagellates of Physarum polycephalum upon addition of ATP (Uyeda, T. Q. P., and M. Furuya. 1987. Protoplasma. 140:190-192). We now report that disrupting the microtubular cytoskeleton by treatment with 0.2 mM Ca2+ for 3-30 s inhibits the movement of the microfilaments induced by subsequent treatment with 1 mM Mg-ATP and 10 mM EGTA. Stabilization of microtubules by pretreatment with 50 microM taxol retarded both the disintegrative effect of Ca2+ on the microtubules and the inhibitory effect of Ca2+ on the subsequent, ATP-induced movement of the microfilaments. These results suggest that the movement of the microfilaments depends on the integrity of the microtubular cytoskeleton. EM observation showed that the backbone structure in control permeabilized flagellates consists of two arrays of microtubules closely aligned with bundles of microfilaments of uniform polarity. The microtubular arrays after ATP treatment were no longer associated with microfilaments, yet their alignment was not affected by the ATP treatment. These results imply that the ATP treatment induces reciprocal sliding between the microfilaments and the microtubules, rather than between the microfilaments themselves or between the microtubules themselves. While sliding was best stimulated by ATP, the movement was partially induced by GTP or ATP gamma S, but not by ADP or adenylyl-imidodiphosphate (AMP-PNP). AMP-PNP added in excess to ATP, 50 microM vanadate, or 2 mM erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) inhibited the sliding. Thus, the pharmacological characteristics of this motility were partly similar to, although not the same as, those of the known microtubule-dependent motilities.     

Cytoplasmic dynein-like ATPase cross-links microtubules in an ATP- sensitive manner

We have prepared dynein-like ATPase from the eggs of the sea urchin Strongylocentrotus purpuratus using differential centrifugation and column chromatography. This ATPase preparation is inhibited by vanadate and erythro-9-(3-[2-hydroxynonyl]) adenine (EHNA) at concentrations similar to those that inhibit reactivated flagellar beating and spindle elongation in lysed cell models. Using microtubule affinity and ATP- induced release, we can purify this ATPase activity to a composition on SDS PAGE of four peptides ranging in molecular weight from 180,000- 300,000. When viewed in darkfield optics, this affinity-purified ATPase caused extensive parallel bundling of microtubule-associated protein- free microtubules. These bundles were dispersed by 1 mM ATP but not by ATP gamma S or AMP-5'-adenylimidodiphosphate. The reformation of microtubule bundles after dispersal by ATP required ATP hydrolysis; bundles did not reform in the presence of 10 microM vanadate. Negative stain electron microscopy of these bundled microtubules revealed that they are arranged in parallel networks with extensive close lateral association.     

In vivo regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracellular diffusion of ADP

Relative diffusivities of ADP and creatine in cardiomyocytes were studied. The isolated rat cardiomyocytes were lysed with saponin (40 micrograms/ml) to perforate or completely disrupt sarcolemma that was evidenced by leakage of 80-100% lactate dehydrogenase. In these cardiomyocytes mitochondria were used as 'enzymatic probes' to determine the average local concentration of substrates exerting acceptor control of respiration--ADP or creatine (the latter activates respiration via mitochondrial creatine kinase reaction)--when their concentrations in the surrounding medium were changed. The kinetic parameters for ADP and creatine in control of respiration of saponin-treated cardiomyocytes were compared with those determined in isolated mitochondria and skinned cardiac fibers. The apparent Km for creatine (at 0.2 mM ATP) was very close and in a range of 6.0-6.9 mM in all systems studied, showing the absence of diffusion difficulties for this substrate. On the contrary, the apparent Km for ADP increased from 18 +/- 1 microM for isolated mitochondria to 250 +/- 59 microM for cardiomyocytes with the lysed sarcolemma and to 264 +/- 57 microM for skinned fibers. This elevation of Km was not eliminated by inhibition of myokinase with diadenosine pentaphosphate. When 25 mM creatine was present, the apparent Km for ADP decreased to 36 +/- 6 microM. These data are taken to indicate specific restrictions of diffusion of ADP most probably due to its interaction with intermediate binding sites in cardiomyocytes. The important role of phosphocreatine-creatine kinase system of energy transport is to overcome the restrictions in regulation of energy fluxes due to decreased diffusivity of ADP.     

Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: role of cyclin A-associated protein kinase

Isolated centrosomes nucleate microtubules when incubated in pure tubulin solutions well below the critical concentration for spontaneous polymer assembly (approximately 15 microM instead of 60 microM). Treatment with urea (2-3 M) does not severely damage the centriole cylinders but inactivates their ability to nucleate microtubules even at high tubulin concentrations. Here we show that centrosomes inactivated by urea are functionally complemented in frog egg extracts. Centrosomes can then be reisolated on sucrose gradients and assayed in different concentrations of pure tubulin to quantify their nucleating activity. We show that the material that complements centrosomes is stored in a soluble form in the egg. Each frog egg contains enough material to complement greater than 6,000 urea-inactivated centrosomes. The material is heat inactivated above 56 degrees C. One can use this in vitro system to study how the microtubule nucleating activity of centrosomes is regulated. Native centrosomes require approximately 15 microM tubulin to begin nucleating microtubules, whereas centrosomes complemented in interphase extracts begin nucleating microtubules around 7-8 microM tubulin. Therefore, the critical tubulin concentrations for polymer assembly off native centrosomes is higher than that observed for the centrosomes first denatured and then complemented in egg extracts. In vivo, the microtubule nucleating activity of centrosomes seems to be regulated by phosphorylation at the onset of mitosis (Centonze, V. E., and G. G. Borisy. 1990. J. Cell Sci. 95:405-411). Since cyclins are major regulators of mitosis, we tested the effect of adding bacterially produced cyclins to interphase egg extracts. Both cyclin A and B activate an H1 kinase in the extracts. Cyclin A-associated kinase causes an increase in the microtubule nucleating activity of centrosomes complemented in the extract but cyclin B does not. The critical tubulin concentration for polymer assembly off centrosomes complemented in cyclin A-treated extracts is similar to that observed for centrosomes complemented in interphase extracts. However, centrosomes complemented in cyclin A treated extracts nucleate much more microtubules at high tubulin concentration. We define this as the "capacity" of centrosomes to nucleate microtubules. It seems that the microtubule nucleating activity of centrosomes can be defined by two distinct parameters: (a) the critical tubulin concentration at which they begin to nucleate microtubules and (b) their capacity to nucleate microtubules at high tubulin concentrations, the latter being modulated by phosphorylation.     

Hyposmolar stimulation of secretion of thyrotropin, prolactin, and luteinizing hormone does not require extracellular calcium and is not inhibited by colchicine, cytochalasin B, ouabain, or tetrodotoxin

Hyposmolar stimulation of thyroid-stimulating hormone, prolactin, and luteinizing hormone secretion by dispersed perifused rat pituitary cells was not depressed by removal of Ca2+ from the perifusion medium or by 0.1 mM colchicine, 20 microM cytochalasin B, 0.1 mM ouabain, or 3 microM tetrodotoxin. The secretory response induced by medium hyposmolarity or by thyrotropin-releasing hormone was not appreciably different at 23, 37, or 43 degrees C, but was markedly reduced or abolished when the experiments were performed at 1 degree C. These data indicate that microtubules or microfilaments, transport of extracellular Ca2+ into the cytoplasm, and plasmalemma ion transport mechanisms sensitive to ouabain or tetrodotoxin are not essential components of the mechanism by which extracellular hyposmolarity induces secretion.     

Microtubule initiation at kinetochores and centrosomes in lysed mitotic cells. Inhibition of site-specific nucleation by tubulin antibody

A lysed cell system was developed to determine whether tubulin antibody can block the nucleation of exogenous tubulin at kinetochores and centrosomes. Mitotic PtK2 cells were pretreated with colcemid to remove all endogenous microtubules and were lysed with Triton X-100 in PIPES-EGTA-Mg++ buffer. This procedure left centrosomes, chromosomes, and kinetochores intact as determined by electron microscopy of thin-sectioned cells. Exposure of the lysed cells to phorphocellulose-purified tubulin dimers at 37 degrees C in the presence of 1 mM GTP resulted in site-specific nucleation of microtubules at centrosomes and kinetochores. Treatment of the lysed cell preparations with tubulin antibody before subsequent exposure to the exogenous tubulin resulted in almost complete blockage of microtubule nucleation, especially at kinetochores. Pretreatment of the lysed cell preparations with control antibody or buffer without antibody had no effect on the ability of centrosomes and kinetochores to initiate microtubule assembly. The implications of these results with respect to the molecular composition of centrosomes and kinetochores are discussed.     

Localization and organization of actin in melanophores

Melanophores of the angelfish, Pterophyllum scalare, were studied in an attempt to demonstrate the existence of actin in these cells although microfilaments had previously not been found. By use of a variety of procedures, including immunofluorescence microscopy of intact and detergent-extracted cells, transmission electron microscopy, high voltage electron microscopy of whole-mount preparations, and labeling with heavy meromyosin-subfragment 1, the presence of a loose cortical mesh of actin filaments is demonstrated. In addition, a more parallel array of filaments is detected in microspike- and microvillus-like surface projections. There seem to be no changes in the arrangement of these filaments as a function of the state of pigment distribution. No actin filaments could be found in association with pigment granules or microtubules in more central cell portions. For reasons presently unknown, the preservation of the cortical filament network in lysed cell preparations depends strongly on the presence of an intact microtubular system. The involvement of this subplasmalemmal actin filament network in pigment granule transport remains unclear.     

Monomer-polymer equilibria in the axon: direct measurement of tubulin and actin as polymer and monomer in axoplasm

The monomer-polymer equilibria for tubulin and actin were analyzed for the cytoskeleton of the squid giant axon. Two methods were evaluated for measuring the concentrations of monomer, soluble (equilibrium) polymer, and stable polymer in extruded axoplasm. One method, the Kinetic Equilibration Paradigm ( KEP ), employs the basic principles of diffusion to distinguish freely diffusible monomer from proteins that are present in the form of polymer. The other method is pharmacological and employs either taxol or phalloidin to stabilize the microtubules and microfilaments, respectively. The results of the two methods agree and demonstrate that 22-36% of the tubulin and 41-47% of the actin are monomeric. The in vivo concentration of monomeric actin and tubulin were two to three times higher than the concentration required to polymerize these proteins in vitro, suggesting that assembly of these proteins is regulated by additional mechanisms in the axon. A significant fraction of the polymerized actin and tubulin in the axoplasm was stable microtubules and microfilaments, which suggests that the dissociation reaction is blocked at both ends of these polymers. These results are discussed in relationship to the axonal transport of the cytoskeleton and with regard to the ability of axons to change their shape in response to environmental stimuli.     

The effect of carbonyl cyanide m-chlorophenylhydrazone on steroid transport in membrane vesicles of Pseudomonas testosteroni

The uncoupler carbonyl cyanide chlorophenylhydrazone (CCCP) was an effective inhibitor of steroid transport in membrane vesicles of Pseudomonas testosteroni between 10 microM and 1 microM CCCP. At these concentrations the inhibition of steroid transport was not due to an inhibition of the 3 beta and 17 beta-hydroxysteroid dehydrogenase enzyme. CCCP also affected testosterone-dependent oxygen consumption at concentrations up to 100 microM and inhibited respiration at 0.5 and 1 microM. The effect of CCCP on testosterone-dependent oxygen consumption indicated that CCCP was acting as an uncoupler. The concurrent inhibition of testosterone transport and stimulation of testosterone-dependent oxygen consumption at 10-100 microM CCCP supported the conclusion that transport and metabolism were tightly coupled processes. When membrane vesicles were pre-incubated with CCCP for 15 min, CCCP did inhibit transport and the 3 beta and 17 beta-hydroxysteroid dehydrogenase activity. However, both transport and enzyme inhibition could be prevented by the addition of NAD+ to the incubation mixture. This indicated that CCCP exhibits the properties of a sulfhydryl reagent under pre-incubated conditions.     

Two separate 18-amino acid domains of tau promote the polymerization of tubulin

Tau is a heat-stable microtubule-associated protein which promotes tubulin polymerization. The assembly promoting region of tau was localized using synthetic peptides modeled after domains found in both human and mouse tau. The design of these synthetic peptides was based on the triple repeat motif found in mouse tau. The first peptide, Tau-(187-204), and the second peptide, Tau-(218-235), are capable of promoting the polymerization of tubulin into microtubules, at concentrations above 100 microM. Two other peptides tested, TauR and Tau-(250-267), were not able to promote the assembly of tubulin over a range of concentrations up to 800 microM. TauR is a random analog of Tau-(187-204). Although TauR is unable to promote polymerization, it can modify Tau-(187-204)-induced tubulin assembly.     

Role of microtubules in LPS-induced macrophage inflammatory protein-2 production from rat pneumocytes

We have recently demonstrated that primary cultured rat pneumocytes produce macrophage inflammatory protein-2 (MIP-2) in response to lipopolysaccharide (LPS) stimulation. In this study, we found that brefeldin A, by blocking anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus, decreased LPS-induced MIP-2 in the culture medium and increased its storage in cells. This suggests that MIP-2 is secreted via a pathway from the ER to the Golgi apparatus, a process commonly regulated by microtubules. We further found that LPS induced depolymerization of microtubules as early as 1 min after LPS stimulation, and it lasted at least for 4 h. Preventing depolymerization of microtubules with paclitaxel (Taxol; 10 nM to 10 microM) partially inhibited LPS-induced MIP-2 production, whereas the microtubule-depolymerizing agents colchicine (1-10 microM) and nocodazole (1-100 microM) increased LPS-induced MIP-2 protein production without affecting MIP-2 mRNA expression. These results suggest that in pneumocytes, LPS-induced microtubule depolymerization is involved in LPS-induced MIP-2 production and that secretion of MIP-2 from pneumocytes is via the ER-Golgi pathway.     

Microtubule depolymerization promotes particle and chromosome movement in vitro

We have developed a system for studying the motions of cellular objects attached to depolymerizing microtubules in vitro. Radial arrays of microtubules were grown from lysed and extracted Tetrahymena cells attached to a glass coverslip that formed the top of a light microscope perfusion chamber. A preparation of chromosomes, which also contained vesicles, was then perfused into the chamber and allowed to bind to the microtubule array. The concentration of tubulin was then reduced by perfusing buffer that lacked both tubulin and nucleotide triphosphates, and the resulting microtubule depolymerization was observed by light microscopy. A fraction of the bound objects detached in the flow and washed away, while others stabilized the microtubules to which they were bound. Some of the particles and chromosomes, however, moved in toward the Tetrahymena ghost as their associated microtubules shortened. The mean speeds for particles and chromosomes were 26 +/- 20 and 15 +/- 12 microns/min, respectively. These motions occurred when nucleotide triphosphate levels were very low, as a result of either dilution or by the action of apyrase. Furthermore, the motions were unaffected by 100 microM sodium orthovanadate, suggesting that these forces are not the result of ATP hydrolysis by a minus end-directed mechanoenzyme. We conclude that microtubule depolymerization provided the free energy for the motions observed. All the objects that we studied in detail moved against a stream of buffer flowing at approximately 100 microns/s, so that the force being developed was at least 10(-7) dynes. This force is large enough to contribute to some forms of motility in living cells.     

Analysis of cytoskeleton structural changes in the granular cells of the frog bladder during the stimulation of water transport

Structural changes of the cytoskeleton of the frog urinary bladder granular cells were examined during low and high water permeability of the epithelium. A tight connection of the microfilaments and microtubules with vacuolar membranes and a great increase in the number of microtubules during a stimulated water flow was shown using different electron microscopic methods. Two populations of microtubules were discovered, respectively, with different diameter and different rate of stability. It is suggested that the thicker microtubules while interacting with actin microfilaments through associated electron dense globules may fulfil the transport function in the cell.     

The involvement of microfilaments and microtubules in the lateral mobility of lectin binding sites of normal and brachypod mouse limb mesenchyme

It has been demonstrated that differences exist in the lateral mobility of Con A- and WGA-binding sites in the membranes of normal and brachypod mouse limb mesenchymal cells (Hewitt et al., 1978). The work presented here investigates the involvement of microtubules and microfilaments as mediators of binding site mobility in this system. Treatment of cells with colchicine suggests that microtubules are not involved in the mobility of either type of lectin-binding site. Disruption of microfilaments with cytochalasin B prevents the redistribution of Con A-binding sites but not those of WGA. the results were found to be the same for both genotypes. This suggests that the differences which have been found between genotypes are related to some mechanism of restraining the lateral mobility of lectin binding sites other than by attachment to microtubules and microfilaments.     

Platelet activation and microfilament bundling

Human platelets were obtained in the fully resting state by treating discoid populations with 1.5 mM tetracaine and in the activated state by treatment with 2 microM A-23187. After gel filtration or washing, respectively, platelet suspensions were lysed with 1% Triton X-100 at pH 6.8. The precipitates from resting platelets viewed by negative staining appeared predominantly granular with a few very short microfilaments. They contained polypeptides of 250, 100, 45, 38, 36.5, and 35 Kdaltons, and three small polypeptides including one with the mobility of profilin on SDS gels. Precipitates from activated platelets lacked this low molecular weight band and contained a major band at 200 Kdaltons with the mobility of myosin; these precipitates had significant K+, Ca++ ATPase activity absent from the precipitate of resting platelets. As seen in negative staining, precipitates from activated platelets contained microfilaments arranged as nets or bundles. The granular resting precipitates were transformed in vitro into microfilament bundles by washing the precipitates in buffer at higher pH (7.6) in the presence of 5 X 10(-5) M calcium chloride.     

Gllcocorticoid inhibition of amino acid transport in rat hepatoma cells

Dexamethasone rapidly and reversibly inhibits the initial rate of transport of α-aminoisobutyric acid in rat hepatoma cells in tissue culture. Colcemid and cytochalasin B neither inhibit transport nor interfere with its inhibition by dexamethasone, arguing that microtubules and microfilaments are not involved in this hormonal effect. Continuous protein synthesis is required both for the dexamethasone inhibition of transport and for its reversal, although cycloheximide alone inhibits transport in control cells by less than 25%. A model for the dexamethasone inhibition of amino acid transport is presented suggesting that glucocorticoids either block the synthesis or enhance the degradation of a rate-limiting protein in the transport system.