Filipin labelling and intramembrane particles on the membranes of early and later autophagic vacuoles in Ehrlich ascites cells

Cholesterol and intramembrane particle distribution on autophagic vacuole membranes was studied in Ehrlich ascites cells using filipin labelling and freeze-fracture electron microscopy. Unsaturated fatty acids were stained using imidazole-buffered osmium tetroxide. Autophagocytosis was induced with vinblastine, and early autophagic vacuoles were accumulated by lowering the ATP level in the cells with iodoacetate. Filipin labelling was observed in the limiting membranes of later, apparently hydrolase-containing autophagic vacuoles, whereas the most newly-formed, double-membrane limited vacuoles were not labelled. The limiting membranes of late, residual body-type vacuoles either showed patchy filipin-induced deformation or were completely smooth. Imidazole-buffered osmium tetroxide stained the membranes of newly-formed or developing autophagic vacuoles partly or entirely. The membranes of older vacuoles stained more weakly. Intramembrane particle density on the P-face of the outer limiting membranes of newly-formed autophagic vacuoles was similar to that on endoplasmic reticulum, and the density seemed to increase slightly later on. The size of the P-face particles increased when the vacuoles became older. The limiting membranes of late, residual body-type vacuoles were almost smooth. The inner limiting membranes and the membranes inside the autophagic were always almost particle-free. In conclusion, the amount of cholesterol, unsaturated fatty acids and protein in autophagic vacuole membranes changes during vacuole maturation.     

Intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver

Summary Morphologically detectable protein (intramembrane particles) and cholesterol (filipin labelling) in the membranes of autophagic vacuoles and lysosomes were studied in mouse hepatocytes using thin-section and freeze-fracture electron microscopy. Both isolated autophagic vacuoles and lysosomes, and intact tissue blocks were used due to the facts (i) that lysosomes are difficult to recognize in freeze-fracture replicas of intact hepatocytes, and (i) that filipin penetration into the tissue blocks is unsatisfactory. Intramembrane particle density was low in the membranes of early autophagic vacuoles (defined as round-shaped vacuoles in which an inner membrane parallel with the outer limiting membrane was clearly visible). The lysosomal membranes contained considerably more intramembrane particles. Particle-rich lysosomes or other vesicles were observed to fuse with the early autophagic vacuoles. The membranes of nascent autophagic vacuoles with morphologically intact contents were usually not labelled by filipin, whereas the membranes of all other autophagic vacuoles and lysosomes were heavily labelled. The increased cholesterol in the membranes of slightly older autophagic vacuoles is presumably derived from cholesterol-rich lysosomes or other vesicles fusing with the vacuoles and from the degrading organelles inside the autophagic vacuoles.     

Maturation of autophagic vacuoles in Mammalian cells

The autophagic process was first described in mammalian cells several decades ago. After their formation as double-membraned vacuoles containing cytoplasmic material, autophagic vacuoles or autophagosomes undergo a stepwise maturation including fusion with both endosomal and lysosomal vesicles. However, the molecular mechanisms regulating these fusion steps have begun to emerge only recently. The list of newly discovered molecules that regulate the maturation of autophagosomes to degradative autolysosomes includes the AAA ATPase SKD1, the small GTP binding protein Rab7, and possibly also the Alzheimer-linked presenilin 1. This review combines previous data on the endo/lysosomal fusion steps during autophagic vacuole maturation with recent findings on the molecules regulating these fusion steps. Interestingly, autophagic vacuole maturation appears to be blocked in certain human diseases including neuronal ceroid lipofuscinosis and Danon disease. This suggests that autophagy has important housekeeping or protective functions because a block in autophagic maturation causes a disease.     

Morphological changes in Ehrlich ascites tumor cells during the cell fusion reaction with HVJ (Sendai virus): II. Cluster formation of intramembrane particles in the early stage of cell fusion

The morphological events in the cell membrane of Ehrlich ascites tumor (EAT) cells associated with cell fusion caused by HVJ were investigated with freeze-fracture technique. When cell fusion was carried out at 37 °C, the EATC fusion was too rapid to allow identification of the sequential steps of membrane fusion and no deleterious changes in the plasma membrane could be detected. However, on lowering the incubation temperature from 37 to 28 °C, the process of cell fusion was slower and there was a distinct alteration in the plasma membrane. On incubation of cell aggregates with HVJ at 28 °C, the fusion reaction proceeded very slowly. On incubation for 10 min, fusion was initiated in a few cells, but most of the cells remained agglutinated with their cell membranes close to those of neighboring cells and often in direct contact in small localized regions. When cells in this stage were chilled and fixed at 4 °C, large clusters of intramembrane particles (IMPs) were seen all over the P face. On further incubation of the cells at 37 °C, cell fusion proceeded rapidly and the IMPs became randomly redistributed, indicating that clustering is a reversible phenomenon occurring in the early stage of cell fusion. This clustering was temperature-dependent. It was seen in cell fixations at 4 °C, but not at 28 °C without chilling, and it was prevented by inhibitors of cell fusion, such as cytochalasin D (CD) or glucose at high concentration. These findings suggest that certain structural changes in the plasma membrane that may induce thermotropic aggregation of IMP are required to initiate cell fusion.     

Low temperature-induced displacement of cholesterol and intramembrane particles in nuclear membranes of mouse leukemia cells

Chilling of GRSL mouse leukemia cells to 0 °C induces a rearrangement of intramembrane particles and filipin-cholesterol complexes in the membranes of the outer nuclear envelope and of the endoplasmic reticulum, but not in the plasma membrane. Both rearrangements are reversible, and are inhibited by prior fixation with glutaraldehyde. After chilling, filipin-cholesterol complexes are concentrated as rings around areas that are concomittantly cleared of intramembrane particles. Aldehyde fixation inhibits displacement of cholesterol in those areas that contain intramembrane particles, but not in the regions that are devoid of intramembrane particles.     

Synchronization of replicons in Ehrlich ascites cells

Ehrlich ascites cells, in which replication units at the beginning of the S phase started and grew synchronously, were obtained by the following protocol: (1) selection of G1 cells by zonal centrifugation, (2) hypoxia for 12 h, (3) reaeration, (4) addition of cycloheximide (30 microM) within the first minute after reoxygenation. Studies on the effectiveness of the different steps revealed: (i) G1 cells reoxygenated after 12 h of hypoxia traverse two succeeding cell cycles highly synchronously. This was shown by monitoring the thymidine incorporation rate, the thymidine pulse-labeling index, and the mitotic index. (ii) Cycloheximide, like hypoxia, suppresses replicon initiation in Ehrlich ascites cells without interfering with DNA chain growth and DNA maturation. The reversibility of the suppression is less complete than in the case of hypoxia. This was shown by DNA fiber autoradiography and by analyzing the length distribution of pulse- or pulse/pulse-chase-labeled daughter DNA in alkaline sucrose gradients. The alkaline sedimentation patterns of daughter-strand DNA, pulse labeled immediately after the cycloheximide addition at the end of the elaborated protocol and 1 and 2 h later, indicated synchronous initiation and growth of a homogeneous population of DNA molecules to replicon-sized lengths.     

Lectin staining patterns of plasma membranes of daunorubicin and vincristine resistant Ehrlich ascites tumour cells

The plasma membrane is considered to play a major role in the development of resistance to anthracycline and vinca alkaloid drugs (pleiotropic resistance). Previous studies have reported an increase in plasma membrane carbohydrates in pleiotropic resistant cells compared with wild-type cells. The present study has utilized a panel of 11 lectins and the streptavidin-biotin histochemical technique in order to compare plasma membrane carbohydrates from wild-type Ehrlich ascites tumour cells with cells from daunorubicin and vincristine resistant sublines. While the lectins ConA, LCA, PSA, PNA after neuraminidase and WGA stained plasma membranes of daunorubicin-resistant cells to a significantly greater degree than those of wild-type cells, no difference was apparent between vincristine-resistant and wild-type cells. PWM and WGA after neuraminidase pretreatment showed similar staining of the wild-type and both resistant sublines, while SBA with and without neuraminidase pretreatment, HPA, DBA, LTA and UEA I demonstrated either very weak or negative reactions with all sublines. We conclude that the observed increase in plasma membrane carbohydrate found in anthracycline-resistant cells is possibly due to drug action during acquisition and maintainance of resistance, and, though conceivably of importance in the development of resistance towards anthracyclines, is without significance for the pleiotropic resistance phenotype itself.     

Vital staining of autophagic vacuoles in differentiating cells of Dictyostelium discoideum

Abstract. Vital staining of differentiating slime mold cells of Dictyostelium discoideum was studied, with reference to autophagic vacuoles they contain. By microscopically comparing within individual cells neutral-red staining granules with acid phosphatase positive granules, we identified vitally stained granules as autophagic vacuoles. At the early stages of differentiation, although the majority of cells were strongly acid phosphatase positive and there was little difference in the number of autophagic vacuoles between prestalk and prespore cells, only the former (about 25% of the total) were strongly vitally stained. It was thus concluded that autophagic vacuoles of prestalk cells are intensely stained with neutral-red while those of prespore cells are only weakly stained. Strong vital staining of prestalk cells was bleached by lysosomotropic agents such as NH₄Cl, methylamine, and chloroquine which are known to increase intra-lysosomal pH. This suggests that autophagic vacuoles of prestalk cells are strongly stained because of their acidity.     

Uptake of amino acids in reconstituted vesicles derived from plasma membranes of Ehrlich ascites cells.

To obtain a clearer concept of the mechanism of organic solute transport in mammalian cells, we have attempted to reconstitute a functional transport system for amino acids from plasma membranes of Ehrlich ascites cells. Purified plasma membranes were dissolved in 2% Na cholate--4 M urea, a mixture which brought over 85% of the membrane proteins into solution. After centrifugation of the solubilized material for 2 hrs at 100,000 x g, the supernatant was dialyzed in the cold for 20 hrs with additional lipid. The reformed vesicles were tested for the ability to transport amino acids. The preliminary results obtained show that the uptake of alpha-aminoisobutyric acid can be inhibited by L-methionine and much less by L-leucine as would be predicted from the known properties of alpha-aminoisobutyric transport in the intact cells. In addition, it has been possible to show accelerated efflux of intravesicular phenylalanine when phenylalanine is added to the trans side (medium side). The data are consistent with the conclusion that there is carrier mediated transport in the reconstituted vesicles.     

Glycolysis and glutaminolysis in perifused Ehrlich ascites tumour cells

A perifusion system was designed in order to study glucose and glutamine metabolism by freshly harvested Ehrlich ascites tumour cells in steady state conditions. Cells were perifused in the presence of 5 mM glucose, 0.5 mM glutamine or 5 mM glucose and 0.5 mM glutamine. The results in steady state reveal that both substrates glucose and glutamine are continuously wasted by tumour cells, excreting two moles of lactate per mol of glucose and one mol of glutamate and ammonia per mol of glutamine consumed into the medium. Glutamine consumption in the presence of glucose was higher than with glutamine alone.