Daniel S. Friend

Endocytosis of liposomes and intracellular fate of encapsulated molecules: Encounter with a low pH compartment after internalization in coated vesicles

We have compared the intracellular fate of several fluorescent probes and colloidal gold entrapped in negatively charged liposomes. Weakly acidic molecules (carboxyfluorescein) appear in the cytoplasm of CV-1 cells in 30 min; agents that raise lysosomal pH block this process. Highly charged molecules (calcein) and large molecules (FITC-dextran: 18 kd) remain confined to extra-or intracellular vesicles. Thin section electron micrographs show gold-containing liposomes bound to coated pits, in intracellular coated and uncoated vesicles, and in secondary lysosomes, including dense bodies. Free gold was not observed in the cytoplasm. We conclude that negatively charged liposomes are endocytosed and processed intracellularly by the coated vesicle pathway, and acidification of the endocytic vesicle, rather than liposome fusion, permits escape of certain molecules to the cytoplasm.

PHYSICOCHEMICAL CHARACTERIZATION OF LARGE UNILAMELLAR PHOSPHOLIPID-VESICLES PREPARED BY REVERSE-PHASE EVAPORATION

Properties of large unilamellar vesicles (LUV), composed of phosphatidylcholine and prepared by reverse-phase evaporation and subsequent extrusion through Unipore polycarbonate membranes, have been investigated and compared with those of small unilamellar vesicles (SUV) and of multilamellar vesicles (MLV). The unilamellar nature of the LUV is shown by 1H-NMR using Pr3+ as a shift reagent. The gel to liquid-crystalline phase transition of LUV composed of dipalmitoylphosphatidylcholine (DPPC) monitored by differential scanning calorimetry, fluorescence polarization of diphenylhexatriene and 90 degrees light scattering, occurs at a slight lower temperature (40.8 degrees C) than that of MLV (42 degrees C) and is broadened by about 50%. The phase transition of SUV is shifted to considerably lower temperatures (mid-point, 38 degrees C) and extends over a wide temperature range. In LUV a well-defined pretransition is not observed. The permeability of LUV (DPPC) monitored by leakage of carboxyfluorescein, increases sharply at the phase transition temperature, and the extent of release is greater than that from MLV. Leakage from SUV occurs in a wide temperature range. Freeze-fracture electron microscopy of LUV (DPPC) reveals vesicles of 0.1-0.2 micron diameter with mostly smooth fracture faces. At temperatures below the phase transition, the larger vesicles in the population have angled faces, as do extruded MLV. A banded pattern, seen in MLV at temperatures between the pretransition and the main transition, is not observed in the smaller LUV, although the larger vesicles reveal a dimpled appearance.

Morphology and metabolism of intact muscle cells isolated from adult rat heart.

Morphologically intact muscle cells were prepared by perfusing adult rat hearts with a balanced salt medium containing 0.1% collagenase and 0.2% hyaluronidase. Yields of intact cells representing up to 25% of the weight of the heart were obtained. The cells separated along the line of the intercalated discs, with cleavage of desmosomes but with tearing of the plasma membrane in the region of the gap junction, so that when two contiguous cells were parted, the gap junction was retained intact attached to one of the cells. The fine structure of undamaged cells was indistinguishable from that of normal myocardial cells in situ, whereas damaged cells characteristically revealed numerous cytoplasmic vacuoles, clumping of myofilaments, and blebbing of the cell membrane, but morphologically normal mitochondria. The earliest lesion detected was a dilatation of the T (transverse tubular) system. Respiration in the intact cells was linear for 30 to 60 minutes and approximately two to three times the rate observed with heart muscle slices or the arrested isolated perfused heart. Oxygen uptake was stimulated by pyruvate but not by lactate. These observations demonstrate the feasibility of preparing intact isolated cells from adult rat heart and their potential value in histologic, pharmacologic, and metabolic studies.

Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion

We have examined the early events in Ca2+-induced fusion of large (0.2 microns diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1-2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal HII phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca2+, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca2+ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol.

β-Hydroxysterol distribution as determined by freeze-fracture cytochemistry

SummaryFilipin a polyene antibiotic, fluoresces and forms 15–25 nm aggregates when combined with β-hydroxysterols, rendering sterols detectable by fluorescence microscopy and by electron microscopy of thin sections and freeze-fracture replicas. We applied filipin in a glutaraldehyde fixative to tissue-cultured cells ofDrosophila melanogaster larvae, in which sterol concentration can be regulated. Since the number of filipin-sterol aggregates observed in membranes was found to be preportional to the amount of sterol experimentally inserted, utilizing filipin is a valid method for quantifying, as well as for mapping, sterol distribution in biological membranes. Other antibiotics may be similarly used for localizing some species of negatively charged phospholipids.In addition to cytochemical identification of specific lipids, rapid freezing and deep etching of unfixed, non-cryoprotected cells may permit us to examine membrane lipids in different physical states liquid-crystalline and gel. Combining these several techniques has resulted in new data concerning the disposition of lipids during the intimate juxtaposition of membranes preceding fusion. For example, in guinea-pig sperm, foci of closely apposed membranes are bereft of β-hydroxysterols and intramembranous particles. Such regions of membrane sometimes exist in a crystalline state and may be rimmed by negatively charged phospholipids. As previously noted in other areas of cytochemistry, thein situ localization of specific substances provides information unobtainable by morphological or biochemical techniques alone.

Liposomes containing colloidal gold are a useful probe of liposome-cell interactions

A method is described for the preparation of liposomes containing colloidal gold as an electron-dense marker to trace liposome-cell interactions. Since gold sols would precipitate at the high concentrations necessary for loading a large proportion of liposomes, gold sols were formed within preformed liposomes which had encapsulated gold chloride. The optimal conditions for encapsulating the marker were ascertained for liposomes prepared by the method of reverse-phase evaporation. Gold sols formed rapidly at ambient temperature and without organic solvent, and produced homogeneous populations of gold granules inside liposomes. Most vesicles contained the marker, allowing us to determine unambiguously the intracellular fate of liposomes and their contents. The in vitro experiments showed that gold-liposomes were internalized by African green monkey kidney cells in a manner similar to receptor-mediated endocytosis of well-characterized ligands. Preliminary in vivo studies also indicated that liposomes were endocytosed by Kupffer cells via the coated vesicle pathway.

Localization of lactate dehydrogenase-X on the surfaces of mouse spermatozoa

Abstract Enzymatic assays and immunological studies indicate that a large proportion of lactate dehydrogenase X (LDH-X) is present in the fluid phase of spermatozoal preparations. However, immunofluorescence techniques demonstrate that LDH-X can be found on the surfaces of spermatozoa as well, and immunoelectronmicroscopic investigations afford good evidence that the antigen is specifically localized at the site of the plasma membrane overlying the postacrosomal dense lamina.

Light microscopic localization of silver-enhanced liposome-entrapped colloidal gold in mouse tissues

Silver-enhanced liposome-entrapped colloidal gold was developed for light microscopic localization of liposomes. Preparation of colloidal gold entrapped in liposomes was achieved by a modified method of Hong, et al. (1983) Biochim. Biophys. Acta 732, 320-323). In this report, a gold chloride/citrate solution of low pH (3.4) was used to inhibit the formation of gold granules during the liposome preparation. The diameter of most liposomes ranged from 80 to 100 nm. Following liposome preparation, the pH was adjusted to 6, and the temperature increased to 55 degrees C. The majority of the liposomes contained one to three gold particles. Liposomes were injected into mice via tail vein; 24 h later, tissues were collected. Sections were processed for silver enhancement of the gold particles and examined by light microscopy. Silver-enhanced gold particles were clearly observed in both liver and implanted tumor. Localization was confirmed by electron and fluorescence microscopy. Thus, we have shown that silver enhancement of colloidal gold liposomes is a direct and sensitive method for tracing the fate of liposomes in vivo, providing minimal background interference and a good definition of various cell types.

Localization of nucleoside triphosphatase activity to the inner nuclear envelope and associated heterochromatin

Nuclear-envelope nucleoside triphosphatase activity (NTPase), an enzymatic activity thought to participate in RNA transport, was localized in rat liver in situ after brief perfusion with 3% paraformaldehyde. Reaction product was distributed along the nucleoplasmic side of the nuclear envelope (NE) in heterochromatin, was only occasionally found at nuclear pores, and nuclear deposition was selectively blocked by inhibitors of NE NTPase activity. Our results suggest that NTPases, which are active in the NE and which participate in RNA transport, are not specifically associated with nuclear-pore complexes.

Influence of the membrane undercoat on filipin perturbation of the red blood cell membrane

Filipin, a polyene antibiotic, interacts with beta-hydroxy sterols such as cholesterol in most cell membranes, forming bumps and pits that are visible by electron microscopy of freeze-fracture replicas. The markedly reduced perturbability of the red blood cell (RBC) membrane, compared to other cells, has been attributed to the constraining influence of the red cell membrane skeleton, the undercoat composed of spectrin, actin, and protein 4.1. To test the influence of the membrane skeleton on filipin-induced perturbation of the RBC membrane, we studied the interaction of filipin with red cells that were inherently devoid of spectrin and RBC in which spectrin had been crosslinked or denatured. These spectrin-deficient, crosslinked, and denatured cells have a fivefold increase in the number of filipin-induced perturbations as compared to control cells, despite equivalent membrane cholesterol content. These findings confirm that the spectrin-based membrane skeleton strongly influences the organization of the membrane so as to limit perturbation by filipin:cholesterol interaction and that for membranes in which the cholesterol content is known, filipin is a useful probe for testing the avidity of spectrin-based cytoskeletal attachment.