Microtubule-associated distribution of specific granules and secretion of atrial natriuretic factor in primary cultures of rat cardiomyocytes

A close spatial relationship between specific granules containing atrial natriuretic factor (ANF) and microtubules was demonstrated in primary cultures of neonatal rat cardiac myocytes. For the detection of specific granules and microtubules, the myocytes were double immunolabelled with antibodies against -ANF and -tubulin and examined by conventional fluorescence or laser scanning confocal microscopy. In addition, the ultrastructural distribution of specific granules was demonstrated by electron microscopy. In the atrial myocytes, ANF was stored in numerous specific granules that were mainly localized in the perinuclear sarcoplasm. In the ventricular myocytes, however, a minority of the cells (10%) exhibited limited ANF immunoreactivity after 4 days in culture. Microtubules were present throughout the sarcoplasm of the myocytes. They were most densely packed in the perinuclear regions. Depolymerization of the microtubules with nocodazole was followed by dispersal of ANF immunostaining both in the atrial myocytes and in the ventricular myocytes exhibiting ANF immunoreactivity. When the microtubules were allowed to recover, the perinuclear distribution of specific granules, as seen in non-treated myocytes, reappeared. Measurements of secreted immunoreactive ANF by radioimmunoassay revealed that the secretion of ANF from atrial myocytes into the medium was significantly reduced following nocodazole treatment, whereas a similar decrease in secretion from ventricular myocytes was not observed. These findings indicate that ANF-containing specific granules are closely associated with microtubules within the myocytes. It is suggested that secretion of ANF from the atrial myocytes, in contrast to the ventricular myocytes, is microtubule-dependent.     

Microtubule regulation of cell surface receptor topography during granulosa cell differentiation

Abstract. A possible role for cytoplasmic microtubules in modulating lectin binding site topography has been examined during the hormone-directed differentiation of rat ovarian granulosa cells in vitro. Indirect immunofluorescence staining with anti-tubulin antibodies indicates that undifferentiated cultured granulosa cells contain a network of microtubules which radiate from the cell center to the cell periphery. Cultures induced to differentiate by a three day treatment with 1 μg/ml prolactin exhibit a marginal distribution of microtubules and a centrally-located primary cilium. Prolactin enhances the incidence of granulosa cells containing a primary colium from 9% in undifferentiated cultures to 53% in hormone-treated cultures. The pattern of lectin binding site redistribution induced by Concanavalin A (Con A) is also modified by prolactin treatment. In contrast to undifferentiated cells, which randomly endocytose fluorescein Con A, granulosa cells exposed to prolactin respond to fluorescein Con A by forming central surface caps to a greater extent (75%) than undifferentiated controls (25%). Double label fluorescence microscopy and transmission electron microscopy on Con A labeled cells show that caps form at central cell surface sites which contain the primary cilium. Disruption of cytoplasmic microtubules by colchicine, in undifferentiated granulosa cells, results in the formation of cell surface caps upon Con A addition. These data suggest that cytoplasmic microtubules modulate the topography of lectin bindings sites which is subject to hormonal control during the in vitro differentiation of ovarian granulosa cells.     

Microtubule regulation of cell surface receptor topography during granulosa cell differentiation

A possible role for cytoplasmic microtubules in modulating lectin binding site topography has been examined during the hormone-directed differentiation of rat ovarian granulosa cells in vitro. Indirect immunofluorescence staining with anti-tubulin antibodies indicates that undifferentiated cultured granulosa cells contain a network of microtubules which radiate from the cell center to the cell periphery. Cultures induced to differentiate by a three day treatment with 1 microgram/ml prolactin exhibit a marginal distribution of microtubules and a centrally-located primary cilium. Prolactin enhances the incidence of granulosa cells containing a primary cilium from 9% in undifferentiated cultures to 53% in hormone-treated cultures. The pattern of lectin binding site redistribution induced by Concanavalin A (Con A) is also modified by prolactin treatment. In contrast to undifferentiated cells, which randomly endocytose fluorescein Con A, granulosa cells exposed to prolactin respond to fluorescein Con A by forming central surface caps to a greater extent (75%) than undifferentiated controls (25%). Double label fluorescence microscopy and transmission electron microscopy on Con A labeled cells show that caps form at central cell surface sites which contain the primary cilium. Disruption of cytoplasmic microtubules by colchicine, in undifferentiated granulosa cells, results in the formation of cell surface caps upon Con A addition. These data suggest that cytoplasmic microtubules modulate the topography of lectin bindings sites which is subject to hormonal control during the in vitro differentiation of ovarian granulosa cells.     

Capping of Con A receptors and actin distribution are influenced by disruption of microtubules in Dictyostelium discoideum

Capping of Concanavalin A (Con A) receptors can be inhibited in Dictyostelium by treatment of amoebae with the microtubular drug tubulozole. In cells that were incubated with Con A or with fluorescent Con A conjugate the capping process was completed in 30 min as could be demonstrated by fluorescence microscopy and Con A peroxidase labeling. In the presence of 10(-5) M tubulozole redistribution of the receptors did not proceed beyond a stage that can be characterized as patching. The effect of the drug on microtubule integrity was checked by electron microscopy and immunofluorescence of tubulin. Treatment resulted in shortening of the peripheral parts of the microtubules, in agreement with results described by other authors. Electron microscopy confirmed that the Con A receptor complexes remained on the plasma membrane and were not internalized. The distribution of F-actin in Con A-treated cells showed a pattern closely resembling that of Con A. Cells that were also treated with tubulozole remained spherical and did not resume significant directional movement until tubulozole was removed from the medium. It is concluded that microtubules are involved in the rearrangement of the microfilament network in moving cells.     

Inhibition of modulation of thymus-leukemia antigens by concanavalin A.

When incubated at 37 °C in medium containing antibodies specific for thymus-leukemia (TL) antigens, viable cells bearing these antigens become resistant to the cytolytic effects of guinea pig complement, a process termed antigenic modulation. Antibody-induced membrane redistribution of the TL antigens, detected by indirect immunofluorescence, occurs with a similar pace. When high concentrations of concanavalin A (Con A) were included with antibodies in the incubation medium, TL antigenic modulation as well as antigen patching and capping were markedly inhibited, similar to effects of Con A on membrane immunoglobulin redistribution with murine spleen cells. Colchicine antagonized the inhibition by Con A suggesting the involvement of microtubules. In parallel experiments high concentrations of Con A failed to alter the quantity of TL antigen expression or its rate of change with time during incubation in cognate antisera. These results support the hypotheses that (a) generalized alterations in membrane receptor mobility may be induced by ligand binding to the cell membrane, and (b) under certain conditions stable interactions occur between normally independent cell surface antigens.     

Association between endocrine pancreatic secretory granules and in- vitro-assembled microtubules is dependent upon microtubule-associated proteins

By use of dark-field light microscopy, secretory granules isolated from the anglerfish endocrine pancreas were observed to attach to and release from microtubules assembled in vitro from brain homogenates. Secretory granules only bound to microtubules assembled in the presence of microtubule-associated proteins (MAPs) and not to microtubules assembled from purified tubulin. The addition of a MAP fraction to purified tubulin restored secretory granule binding. The secretory granules were released from MAP-containing microtubules by the addition of Mg-ATP but not by other nucleotides. The number of secretory granules bound to MAP-containing microtubules was increased in the presence of cyclic AMP. In addition to the associations of secretory granules with microtubules, MAP-containing microtubules also associated with each other. These laterally associated microtubules were dispersed by the addition of Mg-ATP. Electron micrographs confirmed that the associations between MAP-containing microtubules and secretory granules as well as the associations of microtubules with one another were mediated by the high molecular weight MAPs known to project from the surface of in-vitro-assembled microtubules.     

Microtubules and cyclic amp in human leukocytes: on the order of things

We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.     

Endothelin-B receptor mediates effect of intravitreal endothelin on mitochondria-associated anterograde axonal transport

HnRNP A2 is an RNA trafficking protein that binds to a specific cis -acting RNA trafficking element (A2RE) in myelin basic protein RNA and other transported RNAs. A2RE/hnRNPA2 determinants mediate several different steps in RNA trafficking including granule assembly, transport to the plus ends of microtubules and translational activation. A yeast two hybrid screen designed to identify proteins that interact with hnRNP A2 selected a clone corresponding to the carboxyl terminal portion of TOG (tumor overexpressed gene), a microtubule-associated protein that regulates microtubule dynamics. Co-immunostaining of oligodendrocytes with antibody to hnRNPA2 and TOG revealed extensive colocalization of TOG with hnRNP A2 granules in the dendrites. A small population of hnRNP A2 granules lacked TOG and some regions of TOG staining lacked hnRNP A2. In oligodendrocytes injected with fluorescent A2RE RNA and stained for TOG, granules containing fluorescent RNA colocalized with TOG. Co-injection of anti-TOG antibody with fluorescent A2RE RNA decreased colocalization with TOG and increased transport of the injected RNA. These observations suggest that molecular interaction between hnRNP A2 and TOG serves to anchor A2RE mRNAs/hnRNPA2 granules at plus ends of microtubules.
Acknowledgements:   Supported by NIH NS19943 (EB) and NS15190 (JHC), and NMSS RG2843 (EB).     

Facilitation of adenylate cyclase stimulation in macrophages by lectins.

Preincubation of guinea pig peritoneal macrophages with concanavalin A (Con A) markedly enhanced the accumulation of 3′,5′-cyclic-adenosine monophosphate (cAMP) in response to the adenylate cyclase (AC) stimulators prostaglandin E₁ (PGE₁) and isoproterenol (IP). Basal cAMP levels were not altered. Maximal enhancement of cAMP accumulation was induced by preincubation with 50–100 μg/ml Con A for 10 min at 37 °C. Con A-induced facilitation of macrophage responsiveness was prevented by α-methyl-d-mannoside (αMM). No facilitation was induced by the divalent derivative, succinyl-Con A or by Con A immobilized on Sepharose beads. Con A-induced facilitation developed normally in macrophages treated with the microfilament blocking agent, cytochalasin B. The responsiveness of macrophages to PGE₁ and IP was also augmented by phytohemagglutinin (PHA) but wheat germ agglutinin (WGA), soy bean agglutinin (SBA), pokeweed mitogen (PWM), and Lotus tetragonolobus lectin (LL) showed no enhancing effect. The effect of Con A on cAMP levels was the result of augmented cAMP synthesis and not of reduced degradation or a block in cAMP egress from the cells. Lectin-induced facilitation of AC stimulation could be mediated via one of the following mechanisms: (i) induction of receptor clustering; (ii) causing a conformational change in the receptors; (iii) inhibition of negative cooperativity; (iv) causing an increase in membrane fluidity; (v) disruption of microtubules by acting as a Ca²⁺ ionophore; or (vi) inactivation of a sugar-containing inhibitor of AC.     

Electron-microscopic and immunohistochemical study of the reorganization of the microtubule system in granular cells of frog urinary bladder after water transport induction]

Structural changes in organization of the microtubule system in granular cells of frog urinary bladder after water transport induction by vasopressin were studied by methods of electron microscopy and immunocytochemistry. It is shown that in steady-state conditions microtubules form a wide network equally distributed in the whole cytoplasm of granular cells. After vasopressin action, the amount of microtubules increases in the apical region of the cytoplasm. A predominant orientation of microtubules, perpendicular to the apical membrane direction, appears. A structural association of microtubules with specific granules and large vacuoles was observed. A supposition is advanced about association of the described microtubule system reorganization with the activation of vectorial intracellular transport occurring after transepithelial water transport induction.     

Effect of pentoxifylline on developmental changes in neutrophil cell surface mobility and membrane fluidity

Polymorphonuclear leukocytes (PMNs) from human neonates respond less efficiently to chemotactic factor stimulation than do PMNs from adults. The biologic mechanisms underlying this developmental process are poorly understood. In previous studies, we have found that pentoxifylline, an agent report to enhance membrane deformability, increased the chemotactic response of neonatal PMNs. In the present studies, we have examined the effect of pentoxifylline on cell surface mobility and membrane fluidity by assessing fluorescent concanavalin A (Con A) capping and fluorescent polarization (FP). Baseline Con A capping was lower in the PMNs of neonates when compared to PMNs from adult controls. Colchicine, which increases capping by disrupting microtubules, exaggerated the differences between the adult and neonatal PMNs. Following exposure of neonatal PMNs to pentoxifylline, colchicine enhanced Con A capping to levels equivalent to those of colchicine-treated PMNs from adults. Employing a fluorescence polarization (FP) assay, we found the fluid state of the membrane of PMNs from neonates was significantly less than that of adult controls. Pentoxifylline alone significantly increased the fluidity of the cell membranes of neonatal PMNs while decreasing elevated basal levels of F-actin in the cell. These data suggest an intrinsic cytoskeletal difference in the PMNs of neonates that may be responsive to pharmacologic manipulation.     

Poster Sessions CP05: Intracellular Transport and Vesicle Trafficking. MBP mRNA trafficking factor HNRNP A2 interacts with microtubules binding protein TOG

HnRNP A2 is an RNA trafficking protein that binds to a specific cis‐acting RNA trafficking element (A2RE) in myelin basic protein RNA and other transported RNAs. A2RE/hnRNPA2 determinants mediate several different steps in RNA trafficking including granule assembly, transport to the plus ends of microtubules and translational activation. A yeast two hybrid screen designed to identify proteins that interact with hnRNP A2 selected a clone corresponding to the carboxyl terminal portion of TOG (tumor overexpressed gene), a microtubule‐associated protein that regulates microtubule dynamics. Co‐immunostaining of oligodendrocytes with antibody to hnRNPA2 and TOG revealed extensive colocalization of TOG with hnRNP A2 granules in the dendrites. A small population of hnRNP A2 granules lacked TOG and some regions of TOG staining lacked hnRNP A2. In oligodendrocytes injected with fluorescent A2RE RNA and stained for TOG, granules containing fluorescent RNA colocalized with TOG. Co‐injection of anti‐TOG antibody with fluorescent A2RE RNA decreased colocalization with TOG and increased transport of the injected RNA. These observations suggest that molecular interaction between hnRNP A2 and TOG serves to anchor A2RE mRNAs/hnRNPA2 granules at plus ends of microtubules. Acknowledgements: Supported by NIH NS19943 (EB) and NS15190 (JHC), and NMSS RG2843 (EB).     

Concanavalin A affects beta-tubulin mRNA expression during neuritic processes of mouse neuroblastoma N18TG2 cells in a different manner from colchicine

Addition of concanavalin A (Con A) to mouse neuroblastoma N18TG2 cells cultured with dibutyryl-cAMP which can stimulate neurite outgrowth, stopped the neuritic processes effectively. The extended neurites showed a gradual retraction for at least 8 hrs after addition of Con A, while addition of colchicine caused rapid retraction of the neurites. Immunocytochemistry showed that the addition of Con A did not disorganize the microtubules but the addition of colchicine did. The increase in beta-tubulin mRNA expression which was observed after cell culture and after stimulation by dB-cAMP was suppressed by the addition of Con A. Con A did not affect the beta-tubulin mRNA expression when the cells had already been cultured, while colchicine drastically decreased it. Thus, Con A appeared to affect the beta-tubulin mRNA expression in a different manner from colchicine, probably through inhibition of cell movement.     

Presence of glycoconjugates in prolactin granules of male rats

Summary Prolactin granules in the anterior pituitary glands of male rats contain densely stained materials at the periphery of the matrix. These occur in both small spherical and large polymorphic types of granules. The presence of densely stained materials around secretory granules may be a useful criterion for identification of prolactin cells since the dense structure was observed in 95% of these cells after conventional staining by uranyl acetate and lead citrate. The localization of glycoconjugates in the prolactin granules was examined by applying concanavalin A (Con A) on the ultrathin sections. HRP-Con A or ferritinconjugated Con A bound specifically to the densely stained materials in the peripheral region of the prolactin granule matrix, indicating that this densely stained matrix contains glycoconjugates; the significance thereof is discussed with reference to the concentration and packaging of secretory product.     

Ligand induced changes in stability and distribution of acetylcholine receptors on surface membranes of muscle cells

Distribution and stability of acetylcholine receptor (AChR) in cultured muscle cells was studied following the binding to these cells of concanavalin A (Con A) and specific antibodies against the receptor molecules. Con A (10 μg/ml) significantly slowed the rate of receptor degradation. In contrast, the rate of AChR degradation was enhanced 4-fold in the presence of the antibodies while the monovalent antibody fragments (Fab) were without effect. Divalent antibodies induced formation of large clusters on the surface of the muscle cultures within 2 h of incubation at 37°C. Monovalent antibody fragments and Con A had no effect on receptor distribution. It is suggested that receptor aggregation and turnover can be modulated by specific ligands acting at the cell surface.     

Effects of glutathione-oxidizing agents on microtubule assembly and microtubule-dependent surface properties of human neutrophils

In human peripheral blood polymorphonuclear leukocytes and lymphocytes, GSH-oxidizing agents promote the movement of surface-bound concanavalin A (Con A) into caps and inhibit the assembly of microtubules (MT) that is normally induced by Con A binding. Con A capping and inhibition of MT assembly occur when GSH levels in cell suspensions are decreased by 30-70%, and return to GSH to control levels is accompanied by the appearance of cytoplasmic MT and by inhibition of the capping response with Con A. Oxidation of GSH markedly stimulates the hexose monophosphate shunt, and regeneration of GSH occurs rapidly. The data indicate that MT cannot be assembled or maintained in the face of decreased GSH levels. Thus, GSH homeostasis becomes critical during physiological events such as phagocytosis which simultaneously induce the assembly of MT and the production of agents like H2O2 that can oxidize GSH.     

Microtubule organization in lymphoid malignancies.

The abnormal organization of actin-containing microfilaments and vimentin-containing intermediate filaments in neoplastic lymphocytes of T and B cell origin has been described. We investigated microtubules of pathologic cells from 34 lymphoid malignancies, by immunofluorescence microscopy, using monoclonal tubulin antibody. In most cases, apart from two cases of lymphoma, one T cell lymphoma and one B cell lymphoma, interphase leukemia cells, lymphoma cells, and myeloma cells were shown to contain well-organized microtubules which were associated with a microtubule organization center at one end. In the cells of a patient with T cell lymphoma, although microtubules were not visible in the lymphoma cells from lymph nodes, they became visible after 72 hours in culture with concanavalin A (Con A) and interferon alpha. Cap formation was observed with antitubulin monoclonal antibody in the peripheral blood lymphocytes from a chronic lymphocytic leukemia patient, but well-developed microtubules were observed on other occasions in the same patient. There were no obvious structural differences between microtubules in T and B cell lymphoid malignancies, but leukemia cells and lymphoma cells with irregularly shaped nuclei, such as adult T cell leukemia cells and B cell lymphoma cells with cleaved nuclei, had complicated microtubules surrounding their irregular nuclei. In general, after blastogenic stimuli with phytohemagglutinin-P (PHA-P), Con A, and pokeweed mitogen (PWM), the development of the microtubules was proportional to the incorporation of 3H thymidine (3H-TDR). In most cases, after incubation with granulocyte colony-stimulating factor (G-CSF) and interferon alpha, the number of intact cells decreased and the number of degenerated cells increased, but the intact cells had intact microtubules.     

Microtubule and microfilament rearrangements during capping of concanavalin A receptors on cultured ovarian granulosa cells

Thin-section electron microscope analysis of rat and rabbit-cultured granulosa cells treated with concanavalin A (Con A) at 37 degrees C revealed coordinated changes in the cytoplasmic disposition of microfilaments, thick filaments, and microtubules during cap formation and internalization of lectin-receptor complexes. Con A-receptor clustering is accompanied by an accumulation of subplasmalemmal microfilaments which assemble into a loosely woven ring as patches of receptor move centrally on the cell surface. Periodic densities appear in the microfilament ring which becomes reduced in diameter as patches coalesce to form a single central cap. Microtubules and thick filaments emerge associated with the capped membrane. Capping is followed by endocytosis of the con A-receptor complexes. During this process, the microfilament ring is displaced basally into the cytoplasm and endocytic vesicles are transported to the paranuclear Golgi complex along microtubules and thick filaments. Eventually, these vesicles aggregate near the cell center where they are embedded in a dense meshwork of thick filaments. Freeze-fracture analysis of Con A-capped granulosa cells revealed no alteration in the arrangement of peripheral intramembrane particles but large, smooth domains were conspicuous in the capped region of the plasma membrane. The data are discussed with reference to the participation of microtubules and microfilaments in the capping process.     

Binding of microtubules to pituitary secretory granules and secretory granule membranes

Microtubules assembled in vitro were bound to purified porcine pituitary secretory granules and to isolated granule membranes. The interaction between microtubules and whole secretory granules was demonstrated by alteration in the sedimentation properties of the microtubules. Incubation of secretory granules with microtubules resulted in pelleting of microtubules which increased as a function of the number of granules added. Binding was quantitated by measurement of the tubulin remaining in the supernate after centrifugation. The interaction of secretory granules and microtubules was inhibited by nucleoside triphosphates and augmented by adenosine 5'-monophosphate and adenosine. When depolymerized protein from microtubules was incubated with secretory granules, the granules did not appear to bind the soluble tubulin dimer present in these preparations. However, the high molecular weight protein associated with microtubules was adsorbed by secretory granules during the binding process. Incubation of isolated secretory granule membranes with microtubules followed by centrifugation to density equilibrium in a discontinuous sucrose density gradient caused pelleting of the membranes, which otherwise banded higher in the gradient. The visible alteration in membrane sedimentation was confirmed by measurements of the membrane-associated magnesium-ATPase activity and by a shift in radioactivity in iodinated membrane preparations. Our data suggest a role for microtubules in the intracellular movement of secretory granules; this movement is perhaps brought about by dynein-like cross bridges which link the tubulin backbone and granule surface.     

Accelerated calcium ion uptake in murine thymocytes induced by concanavalin A.

The mechanism of enhancement of Ca2+ uptake by the T cell mitogen concanavalin A (Con A) was studied in murine thymocytes. Native Con A enhanced the rate of Ca2+ uptake as much as 9-fold, an increase being observed within five minutes after Con A addition. The effect of Con A was reversed completely by alpha-methyl mannopyranoside (alpha-MM). Increased Ca2+ uptake was observed with increasing concentrations of Con A, between 2 and 400 microgram/ml, indicating that the stimulation of Ca2+ uptake is not restricted to mitogenic lectin concentrations (0.5-2 microgram/ml). Succinyl Con A exhibits only a slight effect in the same concentration ranges as native Con A. Ca2+ uptake, both in the absence and presence of Con A, is strongly dependent on energy metabolism and is carrier mediated. The augmentation of Ca2+ uptake by native Con A is due to an enhanced Vmax. Uptake of the anion, CrO42-, by thymocytes, found to be a non-saturable process, was also enhanced by Con A. The effect of Con A on CrO42- permeability appears to be independent of its effect on Ca2+ uptake.