Effect of vinblastine sulfate, colchicine and lumicolchicine on membrane organization of normal and transformed cells

Differences in the distribution of plasma membrane intramembranous particles (PMP) have been demonstrated in normal and transformed fibroblasts using freeze fracture and electron microscopy. Transformed 3T3 cells contain randomly distributed PMP and contact-inhibited 3T3 cells have aggregated PMP when frozen in medium, glycerol, sucrose, or following stabilization in 1 % formaldehyde. To define some of the mechanisms controlling the organization of PMP in this system we have examined the effects of microtubule disruptive drugs including vinblastine sulfate and colchicine on SV3T3 cells. These drugs were observed to induce a dose- and time-dependent aggregation of PMP at concentrations between 10⁻⁹ and 10⁻⁵ M. These results suggest that modulation of PMP distribution in these cells may be influenced by an interaction of microtubules with plasma membrane components. However, the observation that lumicolchicine, a derivative of colchicine which does not disrupt microtubules, also promotes PMP aggregation, suggests that these drugs may also have a primary effect on the plasma membrane in addition to the disruption of microtubules. This is supported by the observation that reduced temperature (4 °C) which is known to disrupt microtubules fails to induce PMP aggregation in SV3T3 cells, suggesting the hypothesis that changes in the interaction of plasma membrane or plasma membrane associated constituents may control the distribution of PMP in this cell system.     

Effects of retinyl acetate on surface morphology and intramembrane particle distribution in the plasma membrane of 10T1/2 cells.

Scanning electron microscopy and freeze fracture electron microscopy were used to characterize membrane ultrastructural differences between parental, C3H/10T1/2, and carcinogen-initiated, INIT C3H/10T1/2, cells and treatments with retinyl acetate. The intramembranous particle distribution on the E-face was detected and quantitated by the methods of automated image analysis to obtain statistically meaningful numerical characteristics of intramembranous particle size and density. Subtle differences were found when no differences were apparent by light microscopy or by scanning electron microscopy. Initial retinyl acetate treatment caused a significant increase of the intramembranous particle size in parental cells. Intramembranous particle density increased for retinyl acetate treatment in parental and INIT cells and in INIT cells previously maintained but withheld from retinyl acetate. Intramembranous particle distribution analysis includes the interparticle distance of nearest neighbors and the randomness of the distribution by the differential density distribution function, which compares the observed sample to Poisson modified for particle size. These measures show that the three cell groups that have been treated with retinyl acetate have a more even distribution of intramembranous particles than was found for untreated parental cells. The relationship between the freeze fracture morphology and the biological responses to retinyl acetate treatment is discussed.     

Influence of cell cycle and cell movement on the distribution of intramembranous particles in contact-inhibited and transformed cells

Freeze fracture ultrastructure studies have shown that contact inhibited 3T3 cells contain aggregated intramembranous particles (IMP) while transformed 3T3 cells have randomly distributed IMP. The results of this study show that the aggregation of IMP in 3T3 cells is primarily related to the degree of cell contact and not significantly affected by inhibition of cell movement. Cell cycle studies do, however, show a transient disaggregation of IMP during the mitotic phase of the cell cycle. These observations are interpreted to suggest that changes in membrane structure which occur during mitosis or following cell-to-cell contact may be associated with changes in membrane fluidity and the activity of membrane enzymes that appear to be critical for control of cell growth and cell division.     

CRYOPROTECTANT-INDUCED REDISTRIBUTION OF INTRAMEMBRANOUS PARTICLES IN MOUSE LYMPHOCYTES

This study demonstrates, by freeze fracture, clustering of intramembranous particles caused by cryoprotectant treatment of intact unfixed mouse lymphoid cells. Both T and B cells react in a similar fashion, while similar clustering of particles is not observed in some other cell types. The intramembranous particles can be aggregated by incubating unfixed cells in glycerol or dimethylsulfoxide (DMSO) before freezing. The aggregation phenomenon is dependent on the length of time of exposure and the concentration of the cryoprotectants. Further, the cells remain viable and the cryoprotectant-induced clustering is completely reversible. Prefixation of glycerol-treated cells with glutaraldehyde prevents the formation of these particle clusters, and unfixed nonglycerinated cells show no clusters. Thin sections of cells exposed to glycerol or DMSO without previous fixation exhibit bizarre membrane alterations and numerous other degenerative changes. These observations stress the importance of prefixation of lymphoid cells before exposure to glycerol or DMSO, as well as indicate that the membrane characteristics of certain cell types may be probed by glycerol treatment of unfixed cells.     

Intramembranous particles are clustered on microvillus membrane vesicles

Many intramembranous particles in pig jejunal microvillus membranes cluster during cell disruption and membrane vesiculation with the MgCl2 aggregation technique (Hauser, H., Howell, K., Dawson, R.M.C. and Bowyer, D.E. (1980) Biochim. Biophys. Acta 602, 567-577). Isolated brush borders and purified microvillus membrane vesicles were jet-frozen and examined by freeze-fracture electron microscopy. From 30 to 60% of purified vesicles exhibited no intramembranous particles on their fracture face and 22-39% exhibited clustered or aggregated intramembranous particles. Only 6-15% of the vesicles exhibited the random distribution of intramembranous particles that is characteristic of intact enterocytes. Aggregation was not reversed after dialysis to remove divalent cations. Prior freezing of tissue or vesicles (-70 degrees C) gave the same results as fresh unfrozen material. Heterogeneity of microvillus vesicles may occur among the vesicles generated from a single microvillus.     

Freeze-fracture evidence of gel-phase lipid in membranes of senescing cowpea cotyledons

The structural details of membrane organization in germinating and senescing cotyledons of cowpea (Vigna unguiculata (L.) Walp.) were studied by thin section and freeze-fracture electron microscopy. Germination- and senescence-related changes in the ultrastructure of parenchymal cells of cowpea cotyledons, as detected in thin sections, closely resemble those described for other leguminous seeds. Additionally, electron-dense deposits associated with the membranes, particularly the plasmalemma and endoplasmic reticulum, were seen to increase with advancing senescence. Freeze-fracture electron microscopy demonstrated that the membranes of cotyledons of 2-d-old seedings appear to be normal, with evenly dispersed intramembranous particles. However by 4 d, small areas or domains of the plasmalemma were free of intramembranous particles. These particle-free areas increased in both size and number as senescence progressed. We interpret these particle-free areas to be structural evidence for lateral phase separations of the membrane lipids into microdomains of gel-phase lipid from which intrinsic membrane proteins are excluded. Our results support wide-angle X-ray diffraction studies which have demonstrated the presence of gel-phase lipids in senescing bean cotyledons.Abbreviations EF exoplasmic fracture - ER endoplasmic reticulum - ESR electron-spin resonance - IMP(s) intramembranous particle(s) - PF protoplasmic fracture     

Effect of colchicine, vinblastine, and cytochalasin B on cell surface anionic sites of Tritrichomonas foetus

Drugs that interact with microtubules (colchicine and vinblastine) and microfilaments (cytochalasin B) partially inhibited cell growth and motility of Tritrichomonas foetus. Parasites incubated with these substances became rounded and cell division was blocked. Neither colchicine nor vinblastine disrupted the microtubules that form the peltar-axostylar system. Any one of these drugs interfered with the net negative surface charge of T. foetus as evaluated by determination of the cellular electrophoretic mobility (EPM). The decrease in the EPM of cytochalasin B-treated cells was caused by dimethylsulfoxide, which was used as solvent. Untreated cells as well as cytochalasin B-treated cells showed a uniform distribution of anionic sites on the plasma membrane as seen with cationized ferritin particles. In cells treated with colchicine or vinblastine the anionic sites were distributed in patches. These results are discussed in terms of participation of labile cytoplasmic microtubules and microfilaments in the control of the distribution of anionic site-containing macromolecules located on the cell surface of T. foetus.     

Properties of particle movement in the plasma membrane of 3T3 mouse fibroblasts

The effects of cytochalasin B, vinblastine and temperature on particle movement in the plasma membrane of several 3T3 mouse fibroblast lines were investigated. Preincubation of normal 3T3 cells for 24 h in 5–10 μg/ml cytochalasin B had no effect on the mean square relative displacement of marker particles in the membrane (motion constant), but preincubation for 4 h in 40 μg/ml vinblastine reduced the value of this constant by 70%. A 10 °C decrease in temperature decreased the motion constant in normal cells (Q₁₀ = 4) more than in virus-transformed 3T3 cells (Q₁₀ = 1.8). Interpreting the motion constant of the particles as an expression of the viscosity of the membrane material, values of 3 poise for normal 3T3 cells and 6 poise for the transformed cells are obtained for 37 °C and pH = 7.4.A method is suggested to quantitate aggregation of particles on the surface of cells. When this method is applied to gold particles on 3T3 cells, disaggregation of particles is seen to behave as an unordered process, whereas aggregation appears to express cellular control. This consideration and the effect of vinblastine indicate that the interpretation of particle movement as Brownian movement in a viscous membrane material does not cover all phenomena observed.The membrane movement of the flat revertant SVF1 101 [1] was investigated. This cell line occupies an intermediary position between normal 3T3 mouse fibroblasts and the polyoma and SV40 transformed 3T3 cell lines as judged by growth properties. Its membrane movement was found to occupy an intermediary position between the membrane movements of these cell lines, too.     

Density and size distributions of the intramembranous particles in the cytoplasmic membrane of human peripheral blood lymphocytes. II. Ultrastructural differences between T and B cells

Separated T and B lymphocytes from human peripheral blood were studied using the freeze-fracture technique. Quantitative analysis performed on density and size of intramembranous particles (IMPs) present on both fracture faces of the plasma membrane has revealed remarkable differences between cells belonging to the two main lymphocyte populations. In particular: (a) both fracture faces of the cytoplasmic membrane of B lymphocytes exhibit larger particles than T lymphocytes; (b) the mean densities, on both protoplasmic (PF) and external (EF) fracture faces, in B lymphocytes are lower than in T lymphocytes; (c) in B cells the partition ratio of particles between PF and EF is reversed with respect to T cells; (d) on both fracture faces of B lymphocytes, the IMP densities present a normal distribution while on T cells, density values show bimodal distributions indicating the existence of two cell subsets differing in particle density.     

Freeze-fracture planes of methanogen membranes correlate with the content of tetraether lipids.

Methanospirillum hungatei GP1 contained 50% of its ether core lipids (polar lipids less head groups) as tetraether lipids, and its plasma membrane failed to fracture along its hydrophobic domain during freeze-etching. The membrane of Methanosaeta ("Methanothrix") concilii did not contain tetraether lipids and easily fractured to reveal typical intramembranous particles. Methanococcus jannaschii grown at 50 degrees C contained 20% tetraether core lipids, which increased to 45% when cells were grown at 70 degrees C. The frequency of membrane fracture was reduced as the membrane-spanning tetraether lipids approached 45%. As the tetraether lipid content increased, and while fracture was still possible, the particle density in the membrane increased; these added particles could be tetraether lipid complexes torn from the opposing membrane face. The diether membrane (no tetraether lipid) of Methanococcus voltae easily fractured, and the intramembranous particle density was low. Protein-free liposomes containing tetraether core lipids (ca. 45%) also did not fracture, whereas those made up exclusively of diether lipids did split, indicating that tetraether lipids add considerable vertical stability to the membrane. At tetraether lipid concentrations below 45%, liposome bilayers fractured to reveal small intramembranous particles which we interpret to be tetraether lipid complexes.     

Effects of amphotericin B and its methyl ester on plasma membranes of Candida albicans and erythrocytes as examined by freeze-fracture electron microscopy

Freeze-fracture electron microscopy of the plasma membranes of Candida albicans yeast cells and red blood cells treated with amphotericin methyl ester and amphotericin B showed that amphotericin B (50 micrograms ml-1) caused extreme aggregation of intramembranous particles on the protoplasmic fracture face of the C. albicans membrane, and a marked reduction of the density of intramembranous particles. On the other hand, the rearrangement of intramembranous particles induced by amphotericin methyl ester (50 micrograms ml-1) produced elevations of the particle-free membrane domains toward the outside of the cells, so that the particles were aggregated in linear furrows surrounding these elevations on the protoplasmic fracture face, and the corresponding ridges on the exoplasmic fracture face. The density of intramembranous particles was greatly reduced on the protoplasmic fracture face. Both polyenes produced only small changes in the erythrocyte membranes at the same concentration. These results suggest that amphotericin methyl ester affects the ergosterol-containing membranes more than amphotericin B, and that ergosterol has a higher sensitivity for these two polyene antibiotics than cholesterol.     

Cold-induced ultrastructural changes in bull and boar sperm plasma membranes

The effect of low temperatures on the ultrastructure of the plasma membrane of bull and boar spermatozoa was investigated. Cold-induced changes in the organization of sperm plasma membrane components were demonstrated by the use of fast-freezing combined with freeze-fracture electron microscopy. This preparation technique ensures fixation without artifacts. At 38 degrees C bull and boar spermatozoa exhibited a random distribution of intramembranous particles over the plasma membrane of both head and tail. Exposure to 0 degree C resulted in redistribution of the intramembranous particles: on the head and principal piece of bull spermatozoa and on the principal piece of boar spermatozoa, particle-free areas were observed, whereas on the boar sperm head, particle aggregates were present. The original particle distribution was restored upon rewarming of bull and boar spermatozoa to 38 degrees C, as well as after freezing and thawing of bull spermatozoa. Dilution of bull and boar semen into Tris-dilution buffer and Beltsville Thaw Solution-dilution buffer, respectively, could not prevent cold-induced redistribution of intramembranous particles. The observed particle reorganization upon cooling was interpreted as the result of lateral phase separation in the plasma membrane. Species-dependent differences in cold-induced ultrastructural changes were considered to be determined by lipid composition and asymmetry of the plasma membrane, and might be related to differences in cold resistance between species.     

Effect of Colchicine,Vinblastine, and Cytochalasin B on Cell Surface Anionic Sites of Tritrichomonas foetus1

ABSTRACT. Drugs that interact with microtubules (colchicine and vinblastine) and microfilaments (cytochalasin B) partially inhibited cell growth and motility of Tritrichomonas foetus. Parasites incubated with these substances became rounded and cell division was blocked. Neither colchicine nor vinblastine disrupted the microtubules that form the peltar-axostylar system. Any one of these drugs interfered with the net negative surface charge of T. foetus as evaluated by determination of the cellular electrophoretic mobility (EPM). The decrease in the EPM of cytochalasin B-treated cells was caused by dimethylsulfoxide, which was used as solvent. Untreated cells as well as cytochalasin B-treated cells showed a uniform distribution of anionic sites on the plasma membrane as seen with cationized ferritin particles. In cells treated with colchicine or vinblastine the anionic sites were distributed in patches. These results are discussed in terms of participation of labile cytoplasmic microtubules and microfilaments in the control of the distribution of anionic sitecontaining macromolecules located on the cell surface of T. foetus.     

Polarization of endocytosis and receptor topography on cultured macrophages

In the 1774.2 macrophage cell line, microtubule disassembly by colchicine causes the polarization of membrane functions ane structure. Colchicine-treated cells develop a bulge or protuberance that is bordered by microvillous membrane. The protuberance is the site of concanavalin A cap formation. The fluid pinocytosis of horseradish peroxidase and of fluorescein- and rhodamine-conjugated high molecular- weight dextrans, the adsorptive pinocytosis of concanavalin A, and the concentration and phagocytosis at 37 degrees C of a range of phagocytic particles (IgG- and complement-opsonized erythrocytes, complement- opsonized zymosan, latex shpres, albumin-stabilized oil droplets) are all similarly restricted to the protuberance. A reduction in the rate of dextran pinocytosis, determined by fluorimetry, and reductions in phagocytic rates for oil emulsion and IgG-opsonized erythrocytes accompany the polarization of endocytic activity in colchicine-trated 1774.2 macrophages. Membrane receptors for phagocytic particles are not confined to the protuberance but rather may display their own unique topographical asymmetry. The inherent topography of receptors was inferred from particle distribution under conditions that limit particle-receptor redistribution (after labeling at 4 degrees C or a very brief incubation at 37 degrees C). Under these restrictive conditions, latex binding sites were detected over the whole membrane whereas receptors for IgG-opsonized erythrocytes, aggregated IgG, complement-opsonized erythrocytes, and complement-opsonized zymosan were excluded from the protuberance. Thus, functional (endocytosis) and structural (inherent receptor distribution) analyses of membrane topography define different patterns of asymmetry in protuberant cells. The asymmetry induced in 1774.2 macrophages by colchicine is highly analogous to the functional and structural polarity of epithelial cells. Exploration of this analogy may provide insight into the development of polarized epithelia and, more generally, into mechanisms by which specialized areas of membrane are established.     

Asymmetric mass distribution of (Na+ + K+)-ATPase in membranes studied by freeze-fracture-etch electron microscopy

SDS-purified porcine kidney (Na+ + K+)-ATPase was studied by thin-section and freeze-etch electron microscopy. Freeze-fracturing of resealed membrane fragments shows no difference in the distribution of intramembranous particles of approx. 9.0 nm in diameter between convex and concave fracture faces. However, two types of convex face are found: FA, which shows a rather smooth background with many intramembranous particles, and FB, which shows a textured background with very few or no intramembranous particles. Etching the fractured samples further reveals that FA faces are covered with many intramembranous particles, while the etched external faces (EA) are either irregularly granulated or reveal many particles half the size of intramembranous particles. FB faces are covered with distinct pits of 9 nm or larger. The etched external surfaces (EB) are covered with many particles of intramembranous particle size. These results suggest that there are two vesicle orientations in our resealed purified membrane preparation: right-side-out, as in vivo, and inside-out. The majority of the protein mass is distributed only on one side of the membranes. Right-side-out resealed membrane vesicles after fracturing and etching show particulated FA convex fracture faces and irregularly granulated or smooth etched EA surfaces, indicating that the FA face is the protoplasmic fracture face and that the majority of the protein mass of the (Na+ + K+)-ATPase is located on the cytoplasmic half of the membrane.     

A freeze-fracture study of afferent and efferent synapses of hair cells in the sensory epithelium of the organ of Corti in the guinea pig

Summary Afferent and efferent synapses of hair cells in the organ of Corti of the guinea pig have been examined in freeze-fracture replicas.Afferent synapse In the inner hair cells, intramembranous particles 10 nm in diameter are aggregated on the ridge on the P-face of the presynaptic membrane directly beneath the synaptic rod. In the outer hair cells, in which the synaptic rod is located in the presynaptic cytoplasm underneath the presynaptic membrane, small aggregations of intramembranous particles 10 nm in diameter can be found on the P-face of the presynaptic membrane corresponding to the site of the presynaptic dense projection. Intramembranous particles 10 nm in diameter are also densely aggregated on the P-face of the postsynaptic membrane of the outer hair cells.Efferent synapse of the outer hair cells Large intramembranous particles 13 nm in diameter are distributed in clusters composed of four to ten particles on the P-face of the presynaptic membrane. In the P-face of the postsynaptic membrane, disc-like aggregations of intramembranous particles 9 nm in diameter are found. The subsynaptic cistern covers the cytoplasmic surface of the postsynaptic membrane of the efferent synapse; it may cover more than one postsynaptic membrane when several efferent synapses are in close proximity to one another.     

EFFECTS OF COLCHICINE, CYTOCHALASIN B, AND 2-DEOXYGLUCOSE ON THE TOPOGRAPHICAL ORGANIZATION OF SURFACE-BOUND CONCANAVALIN A IN NORMAL AND TRANSFORMED FIBROBLASTS

The distribution of surface-bound concanavalin A on the membranes of 3T3, and simian virus 40-transformed 3T3 cultured mouse fibroblasts was examined using a shadow-cast replica technique with a hemocyanin marker. When cells were prefixed in paraformaldehyde, the binding site distribution was always random on both cell types. On the other hand, labeling of transformed cells with concanavalin A (Con A) and hemocyanin at 37°C resulted in the organization of Con A binding sites (CABS) into clusters (primary organization) which were not present on the pseudopodia and other peripheral areas of the membrane (secondary organization). Treatment of transformed cells with colchicine, cytochalasin B, or 2-deoxyglucose did not alter the inherent random distribution of binding sites as determined by fixation before labeling. However, these drugs produced marked changes in the secondary (but not the primary) organization of CABS on transformed cells labeled at 37°C. Colchicine treatment resulted in the formation of a caplike aggregation of binding site clusters near the center of the cell, whereas cytochalasin B and 2-deoxyglucose led to the formation of patches of CABS over the entire membrane, eliminating the inward displacement of patches observed on untreated cells. The distribution of bound Con A on normal cells (3T3) at 37°C was always random, in both control and drug-treated preparations. Pretreatment of cells with Con A enhanced the effect of colchicine on cell morphology, but inhibited the morphological effects of cytochalasin B. The mechanisms that determine receptor movement and disposition are discussed.     

Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro

Freeze-fracture and freeze-etch techniques have been employed to study the supramolecular structure of isolated spinach chloroplast membranes and to monitor structural changes associated with in vitro unstacking and restacking of these membranes. High-resolution particle size histograms prepared from the four fracture faces of normal chloroplast membranes reveal the presence of four distinct categories of intramembranous particles that are nonrandomly distributed between grana and stroma membranes. The large surface particles show a one to one relationship with the EF-face particles. Since the distribution of these particles between grana and stroma membranes coincides with the distribution of photosystem II (PS II) activity, it is argued that they could be structural equivalents of PS II complexes. An interpretative model depicting the structural relationship between all categories of particles is presented. Experimental unstacking of chloroplast membranes in low-salt medium for at least 45 min leads to a reorganization of the lamellae and to a concomitant intermixing of the different categories of membrane particles by means of translational movements in the plane of the membrane. In vitro restacking of such experimentally unstacked chloroplast membranes can be achieved by adding 2-20 mM MgCl2 or 100-200 mM NaCl to the membrane suspension. Membranes allowed to restack for at least 1 h at room temperature demonstrate a resegregation of the EF-face particles into the newly formed stacked membrane regions to yield a pattern and a size distribution nearly indistinguishable from the normally stacked controls. Restacking occurs in two steps: a rapid adhesion of adjoining stromal membrane surfaces with little particle movement, and a slower diffusion of additional large intramembranous particles into the stacked regions where they become trapped. Chlorophyll a:chlorophyll b ratios of membrane fraction obtained from normal, unstacked, and restacked membranes show that the particle movements are paralleled by movements of pigment molecules. The directed and reversible movements of membrane particles in isolated chloroplasts are compared with those reported for particles of plasma membranes.     

Lateral phase separations in Escherichia coli membranes

Membranes from unsaturated fatty acid auxotrophs of Escherichia coli were studied by spin labeling and freeze-fracturing. From measurements of the partition of the spin label TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) between the aqueous phase and fluid lipids in isolated membranes, temperatures, corresponding to the onset and completion of a lateral phase separation of the membrane phospholipids were determined. By freeze-fracture electron microscopy a change in the distribution of particle in the membrane was observed around the temperature of the onset of the lateral phase separation. When cells were frozen from above that temperature a netlike distribution of particles in the plasma membrane was observed for unfixed preparations. When frozen after fixing with glutaraldehyde the particle distribution was random. In membranes of cells frozen with or without fixing from a temperature below the onset of the phase separation, the particles were aggregated and large areas void of particles were present. This behavior can be understood in terms of the freezing rate with the aid of phase diagrams.     

Adenovirus adsorption and sterol redistribution in KB cell plasma membrane

Filipin was used as a chemical probe for localization of sterols in freeze-fractured plasma membrane of KB cells. After adenovirus particle adsorption, marked changes occurred in the number and planar distribution of sterols and of intramembranous particles (IMPs). Filipin-sterol complexes became more abundant and both sterols and IMPs aggregated in a network pattern. It was suggested that redistribution of sterols and rearrangement of IMPs were interconnected phenomena, which represented an early cellular response to adenovirus attachment.