Atlastin-1, the dynamin-like GTPase responsible for spastic paraplegia SPG3A, remodels lipid membranes and may form tubules and vesicles in the endoplasmic reticulum

We examined the effects of wild-type and mutant atlastin-1 on vesicle transport in the endoplasmic reticulum (ER)-Golgi interface and vesicle budding from ER-derived microsomes using the temperature-sensitive reporter vesicular stomatitis virus glycoprotein (VSV-G), and the ability of purified atlastin-1 to form tubules or vesicles from protein-free phosphatidylserine liposomes. A GTPase domain mutation (T162P) altered the cellular distribution of the ER, but none of the mutations studied significantly affected transport from the ER to the Golgi apparatus. The mutations also had no significant effect on the incorporation of VSV-G into vesicles formed from ER microsomes. Atlastin-1, however, was also incorporated into microsome-derived vesicles, suggesting that it might be implicated in vesicle formation. Purified atlastin-1 transformed phosphatidylserine liposomes into branched tubules and polygonal networks of tubules and vesicles, an action inhibited by GDP and the synthetic dynamin inhibitor dynasore. The GTPase mutations T162P and R217C decreased but did not totally prevent this action; the C-terminal transmembrane domain mutation R495W was as active as the wild-type enzyme. Similar effects were observed in human embryonic kidney cells over-expressing mutant atlastin-1. We concluded that atlastin-1, like dynamin, might be implicated in membrane tubulation and vesiculation and participated in the formation as well as the function of the ER.     

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.     

Cotton fibre differentiation: Occurrence and distribution of coated and smooth vesicles during primary and secondary wall formation

Summary Coated vesicles occur in differentiating cotton fibres during primary and secondary wall formation. The coated vesicles are often associated with the plasmalemma, or with membranes at the secreting face of dictyosomes, corresponding positionally to GERL. During secondary wall formation the number of dictyosome-associated coated vesicles seems to be smaller than during primary wall formation. When sections are stained for periodateoxidizable polysaccharides (Thiéry reaction) the membrane of plasmalemma-associated coated vesicles is intensely stained. The membrane of dictyosome-associated coated vesicles is only weakly stained. On the basis of the present evidence it is not possible to clearly decide, whether the staining in plasmalemma-associated coated vesicles is due to obliquely cut membrane or to vesicle contents. The vesicle coat material is not stained. Possible functions of coated vesicles in differentiating cotton fibres are discussed.Vesicles with contents positively stained with the Thiéry reaction are observed only during primary wall formation. The membrane of these vesicles is smooth and seems to bud from the same cisternae, probably GERL, as do the coated vesicles. During secondary wall formation no vesicles containing periodate-oxidizable polysaccharides could be detected, even under conditions that result in a strong, specific reaction in the cellulosic secondary wall. In some instances polysaccharidic material, resembling secondary wall material, has been seen to adhere to the outside of the plasmalemma. These results are consistent with the hypothesis that, in higher plants, at least part of primary wall material may already be synthesized in dictyosome vesicles, whereas cellulose biosynthesis occurs at the cell surface.     

Kinetics of the micelle-to-vesicle transition: aqueous lecithin-bile salt mixtures

Important routes to lipid vesicles (liposomes) are detergent removal techniques, such as dialysis or dilution. Although they are widely applied, there has been only limited understanding about the structural evolution during the formation of vesicles and the parameters that determine their properties. We use time-resolved static and dynamic light scattering to study vesicle formation in aqueous lecithin-bile salt mixtures. The kinetic rates and vesicle sizes are found to strongly depend on total amphiphile concentration and, even more pronounced, on ionic strength. The observed trends contradict equilibrium calculations, but are in agreement with a kinetic model that we present. This model identifies the key kinetic steps during vesicle formation: rapid formation of disk-like intermediate micelles, growth of these metastable micelles, and their closure to form vesicles once line tension dominates bending energy. A comparison of the rates of growth and closure provides a kinetic criterion for the critical size at which disks close and thus for the vesicle size. The model suggests that liposomes are nonequilibrium, kinetically trapped structures of very long lifetime. Their properties are hence controlled by kinetics rather than thermodynamics.     

Identification of Plasmolipin as a Major Constituent of White Matter Clathrin-Coated Vesicles

We have isolated and characterized coated vesicles from bovine white matter and compared them to those isolated from gray matter. The virtual absence of synaptic vesicle antigens in the white matter coated vesicles indicates they are distinct from those found in gray matter and from vesicles derived from synaptic membranes. The white matter coated vesicles also lack compact myelin components, e.g., the myelin proteolipid, galactocerebroside, and sulfatides, as well as the periaxolemmal myelin marker myelin-associated glycoprotein. On the other hand, these vesicles contain 2',3'-cyclic nucleotide phosphohydrolase. The vesicles also contain high levels of plasmolipin, a protein present in myelin and oligodendrocytes. Plasmolipin was found to be four to five times higher in white matter coated vesicles than in gray matter coated vesicles. Based on western blot quantitation, the concentration of plasmolipin in white matter coated vesicles is 3-4% of the vesicle bilayer protein. These studies indicate that a significant proportion of coated vesicles from white matter may be derived from unique membrane domains of the myelin complex or oligodendroglial membrane, which are enriched in plasmolipin.     

Association of the fusion protein NSF with clathrin-coated vesicle membranes.

N-ethylmaleimide-sensitive fusion protein (NSF) is a component of intracellular transport reactions. In order to understand the role of NSF during the fusion of endocytic transport vesicles with the endosome, we have investigated the binding of NSF to purified clathrin-coated vesicle components. First, we have examined whether detergent-solubilized coated vesicle membranes will support formation of NSF-containing 'fusion complexes'. Our results show that these membranes are substantially enriched in components capable of driving formation of these complexes, when compared with membranes from other sources. Secondly, we have analysed coated vesicle preparations for their NSF content. Coated vesicle preparations contain significant amounts of NSF. This was shown to be associated with coated vesicles rather than contaminating membranes by a number of criteria, and was found to be bound in an ATP-independent manner. These findings are discussed in the light of current models for vesicle fusion.     

The role of coated vesicles in recycling of synaptic vesicle membrane

The uptake of extracellular tracers into synaptic nerve terminals has been a phenomenon of persistent interest. Uptake is into synaptic vesicles, hence vesicles spend part of their life in continuity with the plasma membrane, as expected if exocytosis underlies the quantal discharge of neurotransmitters. However, exactly how or when synaptic vesicles acquire extracellular tracers has not been unambiguously determined. Two schools of thought have developed, one holding that vesicles acquire tracers directly via a reversible exo/endocytotic sequence in which they consistently maintain their biochemical identity during their transient continuity with the plasma membrane, the other holding that synaptic vesicles acquire tracers indirectly, via the formation of clathrin-coated vesicles which are spatially and temporally separate from exocytosis and reverse a temporary loss of the vesicles' individual identity upon merger with the plasma membrane. Efforts to distinguish between these two alternatives have generated an interesting diversity of electron microscopic experiments, many of which are reviewed here. However, definitive determination of which view is correct may ultimately require direct visualization of synaptic vesicle turnover in living nerve terminals. To this end, we here review the results of visualizing endocytosis in tissue cultured cells, where light microscopy can provide sufficient resolution to reveal membrane dynamics in living cells. This has allowed visual discrimination of two different types of endocytosis, one clathrin-mediated (coated vesicle formation) and the other actin-mediated (macropinocytosis). Current work is also reviewed which aims at determining experimental methods for inhibiting each type of endocytosis selectively. Hypertonicity and severe cytoplasmic acidification turn out to inhibit coated vesicle formation, while cytochalasin D and mild cytoplasmic acidification selectively inhibit macropinocytosis. Applied to nerves, these various treatments affect synaptic vesicle turnover in a manner that supports the notion that synaptic vesicle membrane recycles via the "indirect" route of coated vesicle formation.     

Induction of mutant dynamin specifically blocks endocytic coated vesicle formation

Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline- inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.     

The role of dynamin and its binding partners in coated pit invagination and scission

Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission.To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits.We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.     

Cytosolic ARFs are required for vesicle formation but not for cell-free intra-Golgi transport: evidence for coated vesicle-independent transport.

We investigated the role of ADP-ribosylation factors (ARFs) in Golgi function using biochemical and morphological cell-free assays. An ARF-free cytosol produced from soluble Chinese hamster ovary (CHO) extracts supports intra-Golgi transport by a mechanism that is biochemically indistinguishable from control transport reactions: ARF-free transport reactions are NSF-dependent, remain sensitive to the donor Golgi-specific inhibitor ilimaquinone, and exhibit kinetics that are identical to that of control reactions containing ARFs. In contrast, ARF-free cytosol does not support the formation of coated vesicles on Golgi cisternae. However, vesicle formation is reconstituted upon the addition of ARF1. These data suggest that neither soluble ARFs nor coated vesicle formation are essential for transport. We conclude that cell-free intra-Golgi transport proceeds via a coated vesicle-independent mechanism regardless of vesicle formation on Golgi cisternae.     

Light scattering characterization of extruded lipid vesicles

By modeling extruded unilamellar lipid vesicles as thin-walled ellipsoidal shells, mathematical analysis provides simple equations which relate the mean elongation and other morphological characteristics of a vesicle population to quantities readily obtained from combined static and dynamic light scattering measurements. For SOPC vesicles extruded through a 100 nm pore-size filter into a 72.9 mm NaCl solution, the inferred elongation ratio (vesicle long axis to short axis) is approximately 3.7±0.6. When these vesicles were dialyzed into hypertonic or hypotonic solutions, this elongation ratio varied from 1 (for spherical liposomes) in strongly hypotonic solutions to greater than 6 in increasingly hypertonic solutions, beyond which abrupt morphological transformations appear. These results are quantitatively consistent with a mechanism of vesicle formation by extrusion and with the expectation that vesicle volumes change to equalize internal and external osmolarity via water flow, subject to the constraint of constant bilayer area. Our analysis also provides simplified equations to assess the effects of vesicle elongation and polydispersity on liposome parameters that are commonly required to characterize vesicle preparations for diverse applications. The implications of this study for routine light scattering characterization of extruded vesicles are discussed. Received: 31 July 1998 / Revised version: 26 October 1998 / Accepted: 5 November 1998     

Scission of COPI and COPII Vesicles Is Independent of GTP Hydrolysis

Intracellular transport and maintenance of the endomembrane system in eukaryotes depends on formation and fusion of vesicular carriers. A seeming discrepancy exists in the literature about the basic mechanism in the scission of transport vesicles that depend on GTP‐binding proteins. Some reports describe that the scission of COP‐coated vesicles is dependent on GTP hydrolysis, whereas others found that GTP hydrolysis is not required. In order to investigate this pivotal mechanism in vesicle formation, we analyzed formation of COPI‐ and COPII‐coated vesicles utilizing semi‐intact cells. The small GTPases Sar1 and Arf1 together with their corresponding coat proteins, the Sec23/24 and Sec13/31 complexes for COPII and coatomer for COPI vesicles were required and sufficient to drive vesicle formation. Both types of vesicles were efficiently generated when GTP hydrolysis was blocked either by utilizing the poorly hydrolyzable GTP analogs GTPγS and GMP‐PNP, or with constitutively active mutants of the small GTPases. Thus, GTP hydrolysis is not required for the formation and release of COP vesicles.     

Effect of alkali cations on freeze-thaw-dependent reconstitution of amino acid transport from Ehrlich ascites cell plasma membrane

Na+-dependent amino acid transport can be reconstituted by gel filtration of disaggregated plasma membrane and asolectin vesicles coupled to a freeze-thaw cycle. The resultant transport activity is markedly affected by the nature of the reconstitution medium. Reconstitution in K+ permits the formation of active liposomes, whereas reconstitution in Na+, Li+, or choline does not. Electron micrographs of K+ liposomes show a wide variation in liposome sizes. Ficoll density gradient fractionation of K+ liposomes shows that the largest vesicles are lipid rich, have the lowest density, and have the highest level of Na+-dependent amino acid transport. Liposomes formed in Na+ have a 34% smaller trapped volume than K+ liposomes and lack a population of large vesicles. A second freeze-thaw in K+ restores activity to Na+ liposomes which now contain large low density active vesicles. Fluorescence measurements of freeze-thaw-induced mixing of vesicle lipids indicates that the absence of large vesicles in Na+ liposomes is due to inhibition by Na+ of lipid vesicle fusion events during freezing and thawing. The large vesicle fraction is enriched in a 125-kDa peptide. It has not yet been established whether this peptide is part of the transport system for neutral amino acids.     

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.     

Role of coated vesicles, microfilaments, and calmodulin in receptor- mediated endocytosis by cultured B lymphoblastoid cells

Cell surface receptor IgM molecules of cultured human lymlphoblastoid cells (WiL2) patch and redistribute into a cap over the Golgi region of the cell after treatment with multivalent anti-IgM antibodies. During and after the redistribution, ligand-receptor clusters are endocytosed into coated pits and coated vesicles. Morphometric analysis of the distribution of ferritin-labeled ligand at EM resolution reveals the following sequence of events in the endocytosis of cell surface IgM: (a) binding of the multivalent ligand in a diffuse cell surface distribution, (b) clustering of the ligand-receptor complexes, (c) recruitment of clathrin coats to the cytoplasmic surface of the cell membrane opposite ligand-receptor clusters, (d) assembly and (e) internalization of coated vesicles, and (f) delivery of label into a large vesicular compartment, presumably partly lysosomal. Most of the labeled ligand enters this pathway. The recruitment of clathrin coats to the membrane opposite ligand-receptor clusters is sensitive to the calmodulin-directed drug Stelazine (trifluoperazine dihydrochloride). In addition, Stelazine inhibits an alternate pathway of endocytosis that does not involve coated vesicle formation. The actin-directed drug dihydrocytochalasin B has no effect on the recruitment of clathrin to the ligand-receptor clusters and the formation of coated pits and little effect on the alternate pathway, but this drug does interfere with subsequent coated vesicle formation and it inhibits capping. Cortical microfilaments that decorate with heavy meromyosin with constant polarity are observed in association with the coated regions of the plasma membrane and with coated vesicles. SDS-polyacrylamide gel electrophoresis analysis of a coated vesicle preparation isolated from WiL2 cells demonstrates that the major polypeptides in the fraction are a 175-kdalton component that comigrates with calf brain clathrin, a 42- kdalton component that comigrates with rabbit muscle actin and a 18.5- kdalton minor component that comigrates with calmodulin as well as 110- , 70-, 55-, 36-, 30-, and 17-kdalton components. These results clarify the pathways of endocytosis in this cell and suggest functional roles for calmodulin, especially in the formation of clathrin-coated pits, and for actin microfilaments in coated vesicle formation and in capping.     

Interactions of peptides with liposomes: pore formation and fusion

Leakage from liposomes induced by several peptides is reviewed and a pore model is described. According to this model peptide molecules become incorporated into the vesicle bilayer and aggregate reversibly or irreversibly within the surface. When a peptide aggregate reaches a critical size, peptide translocation can occur and a pore is formed. With the peptide GALA the pores are stable and persist for at least 10 minutes. The model predicts that for a given lipid/peptide ratio, the extent of leakage should decrease as the vesicle diameter decreases, and for a given amount of peptide bound per vesicle less leakage would be observed at higher temperatures due to the increase in reversibility of surface aggregates of the peptide. Effect of membrane composition on pore formation is reviewed. When cholesterol was included in the liposomes the efficiency of inducation of leakage by the peptide GALA was reduced due to reduced binding and increased reversibility of surface aggregation of the peptide. Phospholipids which contain less ordered acyl-chains and have a slightly wedge-like shape, can better accommodate peptide surface aggregates, and consequently insertion and translocation of the peptide may be less favored. Demonstrations of antagonism between pore formation and fusion are presented. The choice of factors which promote vesicle aggregation, e.g., larger peptides, increased vesicle and peptide concentration results in enhanced vesicle fusion at the expense of formation of intravesicular pores. FTIR studies with HIV-1 fusion peptides indicate that in systems where extensive vesicle fusion occurred the beta conformation of the peptides was predominant, whereas the alpha conformation was exhibited in cases where leakage was the main outcome. Antagonism between leakage and fusion was exhibited by 1-palmitoyl-2-oleoylphosphatidylglycerol vesicles, where the order of addition of peptide (HIV(arg)) or Ca(2+)dictated whether pore formation or vesicle fusion would occur. The current study emphasizes that the addition of Ca(2+), which promotes vesicle aggregation can also reduce peptide translocation in isolated vesicles.     

Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling

The recycling of synaptic vesicles in nerve terminals is thought to involve clathrin-coated vesicles. However, the properties of nerve terminal coated vesicles have not been characterized. Starting from a preparation of purified nerve terminals obtained from rat brain, we isolated clathrin-coated vesicles by a series of differential and density gradient centrifugation steps. The enrichment of coated vesicles during fractionation was monitored by EM. The final fraction consisted of greater than 90% of coated vesicles, with only negligible contamination by synaptic vesicles. Control experiments revealed that the contribution by coated vesicles derived from the axo-dendritic region or from nonneuronal cells is minimal. The membrane composition of nerve terminal-derived coated vesicles was very similar to that of synaptic vesicles, containing the membrane proteins synaptophysin, synaptotagmin, p29, synaptobrevin and the 116-kD subunit of the vacuolar proton pump, in similar stoichiometric ratios. The small GTP-binding protein rab3A was absent, probably reflecting its dissociation from synaptic vesicles during endocytosis. Immunogold EM revealed that virtually all coated vesicles carried synaptic vesicle proteins, demonstrating that the contribution by coated vesicles derived from other membrane traffic pathways is negligible. Coated vesicles isolated from the whole brain exhibited a similar composition, most of them carrying synaptic vesicle proteins. This indicates that in nervous tissue, coated vesicles function predominantly in the synaptic vesicle pathway. Nerve terminal-derived coated vesicles contained AP-2 adaptor complexes, which is in agreement with their plasmalemmal origin. Furthermore, the neuron-specific coat proteins AP 180 and auxilin, as well as the alpha a1 and alpha c1-adaptins, were enriched in this fraction, suggesting a function for these coat proteins in synaptic vesicle recycling.     

Phosphorylation of Brain Synaptic and Coated Vesicle Proteins by Endogenous Ca2+/Calmodulin- and cAMP-Dependent Protein Kinases

Phosphorylation of brain synaptic and coated vesicle proteins was stimulated by Ca2+ and calmodulin. As determined by 5-15% sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE), molecular weights (Mr) of the major phosphorylated proteins were 55,000 and 53,000 in synaptic vesicles and 175,000 and 55,000 in coated vesicles. In synaptic vesicles, phosphorylation was inhibited by affinity-purified antibodies raised against a 30,000 Mr protein doublet endogenous to synaptic and coated vesicles. When this doublet, along with clathrin, was extracted from coated vesicles, phosphorylation did not take place, implying that the protein doublet may be closely associated with Ca2+/calmodulin-dependent protein kinase. Affinity-purified antibodies, raised against clathrin used as a control antibody, failed to inhibit Ca2+/calmodulin-dependent phosphorylation in either synaptic or coated vesicles. Immunoelectron cytochemistry revealed that this protein doublet was present in axon terminal synaptic and coated vesicles. Synaptic vesicles also displayed cAMP-dependent kinase activity; coated vesicles did not. The molecular weights of phosphorylated synaptic vesicle proteins in the presence of Mg2+ and cAMP were: 175,000, 100,000, 80,000, 57,000, 55,000, 53,000, 40,000, and 30,000. Based on the different phosphorylation patterns observed in synaptic and coated vesicles, we propose that brain vesicle protein kinase activities may be involved in the regulation of exocytosis and in retrieval of synaptic membrane in presynaptic axon terminals.     

pH-sensitive liposomes mediate cytoplasmic delivery of encapsulated macromolecules

Negatively charged liposomes are endocytosed by the coated vesicle system and accumulate in acidic intracellular vesicles. Liposomes that become unstable at acidic pH improve cytoplasmic delivery of membrane-impermeant macromolecules such as calcein (CAL) and FITC dextran (18 or 40 kDa). Oleic acid (OA): phosphatidylethanolamine (PE) (3:7 mole ratio) liposomes become permeable to CAL at pH less than 7.0. Control liposomes of phosphatidylserine:PE or OA:phosphatidylcholine are stable at pH 4-8. OA:PE liposomes promote cytoplasmic delivery of encapsulated CAL to CV-1 cells, as evidenced by the emergence of diffuse, cytoplasmic CAL fluorescence. Delivery requires metabolic energy and is partially inhibited by chloroquine or monensin, which raise the pH of intracellular vesicles.     

Energetics of liposomes encapsulating silica nanoparticles

Nanoparticles may be taken up into cells via endocytotic processes whereby the foreign particles are encapsulated in vesicles formed by lipid bilayers. After uptake into these endocytic vesicles, intracellular targeting processes and vesicle fusion might cause transfer of the vesicle cargo into other vesicle types, e.g., early or late endosomes, lysosomes, or others. In addition, nanoparticles might be taken up as single particles or larger agglomerates and the agglomeration state of the particles might change during vesicle processing. In this study, liposomes are regarded as simple models for intracellular vesicles. We compared the energetic balance between two liposomes encapsulating each a single silica nanoparticle and a large liposome containing two silica nanoparticles. Analytical expressions were derived that show how the energy of the system depends on the particle size and the distance between the particles. We found that the electrostatic contributions to the total energy of the system are negligibly small. In contrast, the van der Waals term strongly favors arrangements where the liposome snugly fits around the nanoparticle(s). Thus the two separated small liposomes have a more favorable energy than a larger liposome encapsulating two nanoparticles.