Cytochalasin B binding and actin content of endoplasmic reticulum subfractions isolated from L-929 cells

Heavy rough (HR) endoplasmic reticulum (ER) membranes and a dense fraction of light rough (LR) membranes (LR I) of L-929 cells bind H-cytochalasin B extremely poorly in comparison to smooth (S) membranes and a fraction of LR membranes of low density (LR II). The LR and S subfractions of ER are apparently heterogeneous membrane populations with respect to cytochalasin B binding. The separation of proteins in HR and LR subfractions by electrophoresis followed by immunoblotting with monoclonal antibodies against actin showed that actin was not present in the former membranes while there were large amounts in the LR subfraction. It is concluded that membranes in the LR II fraction of ER are associated with actin-containing microfilaments of the cytoskeleton, but that HR membranes are not.     

Compartmentation of the rough endoplasmic reticulum

It has become evident during recent years that a wide variety of proteins are synthesized on membrane-bound polysomes, very many of which are not ultimately secreted from the cell. The majority of proteins appear to go through some form of post-translational modification before the final appearance of an 'active' product, and in some cases the polypeptide chain may be modified before the completed protein molecule is released from the ribosome. This then raises the question concerning the possibility of the organization of the rough endoplasmic reticulum into individual domains, or compartments, each of which may have the responsibility of performing definite and well defined functions. During recent years the behaviour of two subfractions of the rough endoplasmic reticulum in a variety of cell types and under a variety of conditions has been studied in order to gain insight into a possible compartmentation of this organelle. Throughout the studies disruption of cells has been performed by nitrogen cavitation. This technique was chosen in order to provide conditions of homogenization which were extremely reproducible since shearing forces, mechanical damage and the effects of local heating were eliminated. Endoplasmic reticulum (ER) membranes isolated from the post-mitochondrial supernatant have been separated into subfractions by centrifugation on discontinuous sucrose gradients. By virtue of their high density imparted by the association of ribosomes, rough ER (RER) membranes penetrate 1.4 M sucrose accumulating above either 2.0 M sucrose (light rough -LR membranes) or a cushion of 2.3 M sucrose (heavy rough -HR membranes). Smooth (S) membranes, which are virtually devoid of ribosomes, collect above 1.4 M sucrose. The HR, LR and S subfractions in MPC-11 cells differ in a number of respects: RNA/protein and RNA/phospholipid ratios, polysome profiles and marker enzymes. When cells were homogenized in buffer containing 25 mM KCl then all three ER subfractions were observed, however, when the buffer contained 100 mM KCl then only the LR and S subfractions were observed in gradients, radioactivity equivalent to that in the HR fraction was not recovered in the other two subfractions. Four times as many light chain immunoglobulin polypeptides were found associated with polysomes of HR membranes compared to LR membranes. The nuclear associated ER (NER), though very active in protein synthesis, was only 20% as active in the synthesis of light chain as the combined LR/HR fraction. Studies with MPC-11 cells showed that the relative amounts of the three ER subfractions were related to the phase of the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

The importance of salt concentration during nitrogen cavitation of MPC-11 cells for the isolation of heavy rough endoplasmic reticulum membranes

When MPC-11 cells are disrupted by nitrogen cavitation in the presence of buffer containing 25-40 mM KCl then endoplasmic reticulum membranes can be separated into three subfractions by sucrose density gradient centrifugation: heavy rough (HR), light rough (LR) and smooth (S) membranes. An increase in the salt concentration of the buffer to 50 mM or above results in the occurrence of only the LR and S membranes in gradients. However, when cells equilibrated at high pressure in the bomb in 100 mM KCl buffer were expelled into a diluting buffer such that the final buffer concentration was reduced to 25 mM KCl upon cell disruption, then appreciable amounts of HR membranes are observed in sucrose gradients. The results would suggest that salt concentrations above 25-40 mM KCl stabilize the interaction between HR membranes and the cytoskeleton to such a degree that these membranes are pelleted at low speed together with the nuclei. The yields of LR and S membranes are apparently not affected to any significant degree by altered salt concentration.     

Ultrastructure and polysome content of the microsomal subfractions of mouse plasmacytoma cells

Summary The endoplasmic reticulum (ER) of MPC-11 cells released as vesicles upon cell disruption by nitrogen cavitation was separated from the bulk of mitochondria, lysosomes and plasma membranes by a low speed centrifugation. The ER membranes were fractionated on discontinuous sucrose gradients into heavy rough (HR), light rough (LR) and smooth (S) membranes. The morphology of subcellular fractions was studied by electron microscopy and the ER membranes were shown to be virtually free of contaminating organelles. The S fraction was easily distinguishable because of the lack of ribosomes but there were no apparent morphological differences between the HR and LR fractions. Of total activity in the microsomal subfractions, 70% of the UDPase and 67% of the 5′-nucleotidase activity was associated with the S fraction. Polysomes were present in the HR, LR and nuclear-associated ER fractions but not in the S fraction. The HR and LR fractions did not appear to be contaminated to any great extent with free polysomes. RNA/protein and RNA/phospholipid ratios of the HR fraction were higher than those of the LR fraction, indicating a greater density of ribosomes in the former fraction. These ratios were much lower in the S fraction reflecting the low ribosome content.     

Correlation of the effect of feeding upon the cell cycle distribution profiles of MPC-11 cells with the relative appearance of [3H]-choline labeled material in microsomal subfractions and other cell fractions

Summary Cultures of mouse plasmacytoma cells (MPC-11) grown within the range 6–23 × 10⁵ cells/ml showed considerable variation in cell cycle distribution profiles and also differences with regard to relative amounts of microsomal subfractions. The variability of appearance of heavy rough (HR) and light rough (LR) microsomal subfractions was not merely due to differences in nutritional state of the culture. Cultures containing a high S/G₂ + M cell cycle distribution ratio showed a high content of HR microsomal membranes; as the S/G₂ + M ratio decreased, so too decreased the amount of HR material whilst the amount of LR microsomal membranes increased. The results indicate that there is a direct correlation between phase of cell cycle and both amount and relative distribution of rough microsomal membranes, the smooth fraction (S), however, remains relatively unchanged.     

Reversible inhibition of secretion in root cap cells of cress after treatment with cytochalasin B: Support for the membrane flow concept

After treatment of cress roots with cytochalasin B (cytB) (25 μg/ml. 5.2 × 10^?5 M) for 4 h, marked structural changes are observed in the peripheral secretory calyptra cells. Deposits of slime outside the plasma membrane are smaller than in cells of untreated roots, whereas secretory vesicles accumulate within the treated cells. Dictyosomes are no longer present and the number of cisternae of rough endoplasmic reticulum surrounding the nucleus is increased at least three-fold. After an 8 h leaching of the drug, the structure of the secretory cells changes again. Accumulation of secretory vesicles no longer takes place, slime is deposited outside the plasma membrane and the number of ER cisternae surrounding the nucleus decreases. On the other hand, dictyosomes are now present. However, they are different from those in the hypertrophied stage of cells exhibiting high secretory activity, but are similar to those of an early developmental stage found at the beginning of the secretion process. This indicates that the dictyosomes are rebuilt during the leaching procedure. The results show that ER membranes accumulate near the nuclear envelope. They also indicate that bulk membrane material is transferred from the RER to the plasma membrane via dictyosome membranes and secretory vesicles, i.e. that membrane flow occurs in secretory cells of higher plants.     

Light rough endoplasmic reticulum membranes re-appear before heavy rough membranes after feeding starved MPC-11 cells

In 48 hr starved MPC-11 cells smooth (S) endoplasmic reticulum (ER) membranes accounted for more than 80% of the total ER membrane fraction. After 2 1/2 hr feeding the amount of S membranes was reduced to below 55% while light rough (LR) membranes had increased from about 16% of the total to 42%. The percentage of heavy rough (HR) membranes only showed a minor increase during the incubation period. The results show that following an accumulation of S membranes in starved cells the LR membranes 're-appear' more rapidly than the HR membranes. This difference in behaviour provides further support to the concept that LR and HR membranes represent distinct domains of the ER system.     

Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal membrane origins

HEK293 cell detergent-resistant membranes (DRMs) isolated by the standard homogenization protocol employing a Teflon pestle homogenizer yielded a prominent opaque band at approximately 16% sucrose upon density gradient ultracentrifugation. In contrast, cell disruption using a ground glass tissue homogenizer generated three distinct DRM populations migrating at approximately 10%, 14%, and 20% sucrose, named DRM subfractions A, B, and C, respectively. Separation of the DRM subfractions by mechanical disruption suggested that they are physically associated within the cellular environment, but can be dissociated by shear forces generated during vigorous homogenization. All three DRM subfractions possessed cholesterol and ganglioside G_M1, but differed in protein composition. Subfraction A was enriched in flotillin-1 and contained little caveolin-1. In contrast, subfractions B and C were enriched in caveolin-1. Subfraction C contained several mitochondrial membrane proteins, including mitofilin and porins. Only subfraction B appeared to contain significant amounts of plasma membrane-associated proteins, as revealed by cell surface labeling studies. A similar distribution of DRM subfractions, as assessed by separation of flotillin-1 and caveolin-1 immunoreactivities, was observed in CHO cells, in 3T3-L1 adipocytes, and in HEK293 cells lysed in detergent-free carbonate. Teflon pestle homogenization of HEK293 cells in the presence of the actin-disrupting agent latrunculin B generated DRM subfractions A–C. The microtubule-disrupting agent vinblastine did not facilitate DRM subfraction separation, and DRMs prepared from fibroblasts of vimentin-null mice were present as a single major band on sucrose gradients, unless pre-treated with latrunculin B. These results suggest that the DRM subfractions are interconnected by the actin cytoskeleton, and not by microtubes or vimentin intermediate filaments. The subfractions described may prove useful in studying discrete protein populations associated with detergent-resistant membranes, and their potential interactions in cell signaling.     

Effect of membrane and cytoskeleton modification on the heat response of BP-8 murine sarcoma cells

Summary Mammalian cells subjected to hyperthermia in the presence of glycerol exhibit greatly enhanced resistance to thermal death. The mechanisms responsible for this effect remain unknown, but it has been suggested that glycerol may act by stabilizing cell membranes or by preventing heat induced disruption of cytoskeletal networks. To test these hypotheses, BP-8 murine sarcoma cells were treated with various combinations of glycerol, procaine, colcemid, and cytochalasin B, followed by 1 hour in vitro heating at temperatures ranging from 37°C to 45.5°C. After heating, the tumor cells were inoculated intraperitoneally into mice and cell survival was evaluated in vivo with the¹²⁵I-iododeoxyuridine prelabeling assay. Addition of 5% glycerol to the incubation medium caused a pronounced increase in the heat resistance of BP-8 cells. Exposure to colcemid (microtubule disrupting agent) or cytochalasin B (micro-filament disrupting agent) did not influence the thermal response of control cells, nor did it counteract the protective effects of glycerol. This suggests that the ability of heat to dissociate cytoskeletal networks may not be an important factor in cellular heat death. In contrast, treatment with the membrane modulator procaine induced significant thermal sensitization in control cells, and caused a complete reversal of the protective action of glycerol. These findings are consistent with the hypothesis that membranes play an important role in the genesis of cellular heat death.Abbreviations ¹²⁵IUdR iodine-125 labeled iododeoxyuridine - EBSS Earle's balanced salt solution This work was supported by Grant CA 21673 from the National Cancer Institute, NIH.     

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. II. Rat liver microsomal subfractions contain equimolar amounts of ribophorins and ribosomes

Ribophorins I and II, two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum (ER) are thought to be part of the translocation apparatus for proteins made on membrane bound polysomes. To study the stoichiometry between ribophorins and membrane-bound ribosomes we have determined the RNA and ribophorin content in rat liver microsomes or in microsomal subfractions of different density (i.e., ribosome content). The specificity of antibodies against the ribophorins was demonstrated by Western blot analysis of rat liver rough microsomes separated by 2-dimensional gel electrophoresis. The ribophorin content of microsomal subfractions was determined by indirect immunoprecipitation and for ribophorin I by a radioimmune assay. In the latter assay a molar ratio of ribophorin I/ribosomes approaching one was calculated for total microsomes as well as in the gradient subfractions. We therefore suggest that ribophorins mediate the binding of ribosomes to endoplasmic reticulum membranes or play a role in co-translational process which depend on this binding, such as the insertion of nascent polypeptides into the membrane or their transfer into the cisternal lumen.     

Identification of the d-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[³H]Cytochalasin B binding and its competitive inhibition by d-glucose have been used to identify the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [³H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for d-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the d-glucose-inhibitable site. If 280 nM (40 000 μunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of d-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

Distribution of microsomal triglyceride transfer protein within sub-endoplasmic reticulum regions in human hepatoma cells

Very low-density lipoprotein (VLDL) particles are formed in the endoplasmic reticulum (ER) through the association of lipids with apolipoprotein B (apoB). Microsomal triglyceride transfer protein (MTP), which transfers lipid molecules to nascent apoB, is essential for VLDL formation in ER. However, little is known of the distribution and interaction of MTP with apoB within ER. In this study, distribution patterns of apoB and MTP large subunit (lMTP) within ER were examined. Microsomes prepared from HuH-7 cells, a human hepatoma cell line, were further fractionated into rough ER (RER)-enriched subfractions (ER-I fraction) and smooth ER (SER)-enriched subfractions (ER-II fraction) by iodixanol density-gradient ultracentrifugation. ApoB was evenly distributed in the ER-I and the ER-II fractions, while 1.5 times more lMTP molecules were present in the ER-I fraction than in the ER-II fraction. lMTP and apoB were coprecipitated both in the ER-I and in the ER-II fractions by immunoprecipitation whenever anti-apoB or an anti-lMTP antibodies were used. ApoB-containing lipoprotein particles showed a lower density in the ER-II fraction than those in the ER-I fraction. From these results, it is suggested that MTP can function in both rough and smooth regions of ER in human hepatoma cells.     

Characterization of plasma membrane domains of mouse EL4 lymphoma cells obtained by affinity chromatography on concanavalin A-Sepharose

Purified plasma membranes of mouse EL₄ lymphoma cells were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (MF1) eluted freely from the affinity column, the second (MF2) adhered specifically to Con A-Sepharose. Both membrane subfractions proved to be of plasma membrane origin, as evidenced by the following criteria. (i) The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. (ii) When isolated plasma membranes were labelled with tritiated NaBH₄, both subfractions exhibited identical specific radioactivities. (iii) After enzymatic radioiodination of the cells, the total content of labelled proteins was very similar in isolated plasma membranes and in both subfractions. (iv) Some plasma membrane marker enzymes exhibited nearly identical specific activities in plasma membranes, MF1 or MF2 including γ-glutamyl transpeptidase, 5′-nucleotidase and Mg²⁺-ATPase. Both subfractions exhibited characteristic differences. Thus the specific activities of (Na⁺ + K⁺)-ATPase, Ca²⁺-ATPase and lysophosphatidylcholine acyltransferase were several-fold enriched in MF2 compared to MF1. SDS-polyacrylamide gel electrophoresis revealed a different polypeptide composition of the two subfractions. Polypeptides of apparent molecular mass of 116, 95, 42, 39, 30 and 28 kDa were highly enriched in MF2, whereas MF1 contained another set of proteins, of apparent molecular mass of 70, 55 and 24 kDa. The phospholipid fatty acid composition of the subfractions proved to be different, as well, MF2 contained more saturated fatty acids than MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of the mouse EL₄ lymphoma cells, containing a set of polypeptides, among others membrane bound enzymes, embedded in a different phospholipid milieu.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

The use of lanthanum and cytochalasin B to study calcium effects on skeletal muscle differentiation in vitro

A dual effect of external Ca²⁺ on creatine kinase (CPK) accumulation during myogenesis has recently been demonstrated (Morris and Cole, '79). Ca²⁺ inhibits muscle-specific CPK accumulation at intermediate (50–100 μ) concentrations compared with both lower (no added Ca²⁺) and higher (2–3 μ) concentrations. Myoblast fusion, however, requires high Ca²⁺ and is inhibited at both low and intermediate Ca²⁺ levels. These effects are now investigated further by studying the effects of lanthanum ion (La³⁺), which interferes with Ca²⁺-binding to membranes and Ca²⁺-transport, and cytochalasin B, which affects the cell membrane and prevents cell fusion without inhibiting CPK accumulation. The results show that low concentrations (10–100 μ) of La³⁺ inhibit the appearance of the muscle-specific (MM) CPK isoenzyme during myogenesis without significantly affecting cell fusion or intracellular cyclic AMP levels. Three further observations are consistent with the existence of myotube-specific membrane-binding sites for Ca²⁺, which are involved in the stimulation of CPK accumulation on increasing external Ca²⁺ from intermediate to high concentrations. (1) CPK levels are not affected by La³⁺ at 0–50 μ external Ca²⁺. (2) CPK levels in cytochalasin B treated myoblasts are hardly affected by La³⁺ at any Ca²⁺ concentration. (3) In cytochalasin B treated cultures, CPK levels are not increased by raising external Ca²⁺ from intermediate to high levels. In contrast, the stimulation of CPK accumulation on decreasing external Ca²⁺ from intermediate to very low concentrations is not affected by either La³⁺ or cytochalasin B. Some alternative interpretations of the data are also considered, including direct disruption of a membrane Ca²⁺-binding site by cytochalasin B.     

Cytochalasin B inhibits phosphoinositide hydrolysis in rat hippocampal slices

The effect of cytochalasin B on phosphoinositide (PI) hydrolysis was examined in rat hippocampal slices. Pretreatment of the slices with cytochalasin B caused a significant decrease in PI hydrolysis elicited by carbachol, norepinephrine, or by high K⁺. This effect was cytochalasin B dose- and time-dependent and was not mimicked by cytochalasin D, vinblastine, colchicine, or phloretin. In contrast, in [³H]inositol-prelabeled hippocampal membranes, cytochalasin B did not affect PI hydrolysis elicited by GTPS and GTPS plus carbachol. Similar result was obtained using the membranes prepared from the slices pretreated with cytochalasin B. The inhibitory effect of cytochalasin B on the carbachol-response was observed in SK-N-SH human neuroblastoma cells, but not in cultured rat astrocytes. These results indicate that cytochalasin B inhibits PI hydrolysis in neuron-specific manner and that its action may be an indirect cellular mechanism other than interaction with cytoskeleton elements.     

Protein retention in yeast rough endoplasmic reticulum: expression and assembly of human ribophorin I

The RER retains a specific subset of ER proteins, many of which have been shown to participate in the translocation of nascent secretory and membrane proteins. The mechanism of retention of RER specific membrane proteins is unknown. To study this phenomenon in yeast, where no RER- specific membrane proteins have yet been identified, we expressed the human RER-specific protein, ribophorin I. In all mammalian cell types examined, ribophorin I has been shown to be restricted to the membrane of the RER. Here we ascertain that yeast cells correctly target, assemble, and retain ribophorin I in their RER. Floatation experiments demonstrated that human ribophorin I, expressed in yeast, was membrane associated. Carbonate (pH = 11) washing and Triton X-114 cloud-point precipitations of yeast microsomes indicated that ribophorin I was integrated into the membrane bilayer. Both chromatography on Con A and digestion with endoglycosidase H were used to prove that ribophorin I was glycosylated once, consistent with its expression in mammalian cells. Proteolysis of microsomal membranes and subsequent immunoblotting showed ribophorin I to have assumed the correct transmembrane topology. Sucrose gradient centrifugation studies found ribophorin I to be included only in fractions containing rough membranes and excluded from smooth ones that, on the basis of the distribution of BiP, included smooth ER. Ribosome removal from rough membranes and subsequent isopycnic centrifugation resulted in a shift in the buoyant density of the ribophorin I-containing membranes. Furthermore, the rough and density-shifted fractions were the exclusive location of protein translocation activity. Based on these studies we conclude that sequestration of membrane proteins to rough domains of ER probably occurs in a like manner in yeast and mammalian cells.     

The isolation of endoplasmic reticulum from barley aleurone layers

Techniques for the isolation and purification of endoplasmic reticulum (ER) from aleurone layers of barley (Hordeum vulgare L.) were assessed. Neither differential centrifugation nor density gradient centrifugation of a homogenate separate the ER or other organelles of this tissue from the lipidcontaining spherosomes. Isopycnic sucrose gradient centrifugation of organelles first purified by molecular sieve chromatography on Sepharose 4B, however, results in separation of the organelles based on their differing buoyant densities. Manipulation of the magnesium concentration of the isolation media and density-gradient solutions affords isolation of ER at a density of 1.13–1.14 g cc^-1 and 1.17–1.18 g cc^-1. Electron microscopy shows that the membranes sedimenting at 1.13–1.14 g cc^-1 are devoid of ribosomes and are characteristic of smooth ER, while those sedimenting at 1.17–1.18 g cc^-1 are studded with ribosomes and have the features of rough ER. Endoplasmic reticulum isolated by isopycnic density gradient centrifugation can be further purified by rate-zonal centrifugation.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid - Trizma tris(hydroxymethyl)aminomethane     

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.     

Auxin-binding Sites of Maize Coleoptiles Are Localized on Membranes of the Endoplasmic Reticulum

Sites in maize (Zea mays L.) coleoptile homogenates that reversibly bind naphthalene-1-acetic acid with high affinity and may represent receptor sites for auxins are located primarily on cellular membranes that show the enzymic and buoyant density characteristics of membranes of the rough endoplasmic reticulum. The sites remain attached to the endoplasmic reticulum (ER) membranes after the ribosomes have been stripped off them. Binding sites for naphthylphthalamic acid, an inhibitor of auxin transport, are located on membranes different from those that carry the naphthalene-1-acetic-acid (NAA)-binding sites, and which are probably plasma membrane. The two kinds of binding sites can be largely separated by appropriate density gradient centrifugation. The results raise the possibility that primary auxin action occurs at ER membranes and could represent facilitation of the transfer of hydrogen ions and nascent secretory protein into the ER lumen followed by secretory transport of these products to the cell exterior via the Golgi system.