A live cell imaging-based assay for tracking particle uptake by clathrin-mediated endocytosis.

A popular strategy for therapeutic delivery to cells and tissues is to encapsulate therapeutics inside particles that cells internalize via endocytosis. The efficacy of particle uptake by endocytosis is often studied in bulk using flow cytometry and Western blot analysis and confirmed using confocal microscopy. However, these techniques do not reveal the detailed dynamics of particle internalization and how the inherent heterogeneity of many types of particles may impact their endocytic uptake. Toward addressing these gaps, here we present a live-cell imaging-based method that utilizes total internal reflection fluorescence microscopy to track the uptake of a large ensemble of individual particles in parallel, as they interact with the cellular endocytic machinery. To analyze the resulting data, we employ an open-source tracking algorithm in combination with custom data filters. This analysis reveals the dynamic interactions between particles and endocytic structures, which determine the probability of particle uptake. In particular, our approach can be used to examine how variations in the physical properties of particles (size, targeting, rigidity), as well as heterogeneity within the particle population, impact endocytic uptake. These data impact the design of particles toward more selective and efficient delivery of therapeutics to cells.

Stable DOPG/Glycyrrhizin Vesicles with a Wide Range of Mixing Ratios: Structure and Stability as Seen by Scattering Experiments and Cryo-TEM.

Phosphatidylglycerols represent a large share of the lipids in the plasmamembrane of procaryotes. Therefore, this study investigates the role of charged lipids in the plasma membrane with respect to the interaction of the antiviral saponin glycyrrhizin with such membranes. Glycyrrhizin is a natural triterpenic-based surfactant found in licorice. Vesicles made of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1'-glycerol) (DOPG)/glycyrrhizin are characterized by small-angle scattering with neutrons and X-rays (SANS and SAXS). Small-angle scattering data are first evaluated by the model-independent modified Kratky-Porod method and afterwards fitted by a model describing the shape of small unilamellar vesicles (SUV) with an internal head-tail contrast. Complete miscibility of DOPG and glycyrrhizin was revealed even at a ratio of lipid:saponin of 1:1. Additional information about the chain-chain correlation distance of the lipid/saponin mixtures in the SUV structures is obtained from wide-angle X-ray scattering (WAXS).

Altering the intra-liver distribution of phospholipid-free small unilamellar vesicles using temperature-dependent size-tunability.

We demonstrated that phospholipid-free small unilamellar vesicles (PFSUVs) composed of TWEEN 80 and cholesterol (25/75, mol%) could be fabricated using a staggered herringbone micromixer with precise controlling of their mean size between 54 nm and 147 nm. Increasing the temperature or decreasing the flow rate led to an increase in the resulting particle diameter. In zebrafish embryos, 120-nm PFSUVs showed 3-fold higher macrophage clearance compared to the 60-nm particles, which exhibited prolonged blood circulation. In mice, the 60-nm particles showed dominant accumulation in the liver hepatocytes (66% hepatocytes positive), while the 120-nm particles were delivered equally to the liver and spleen macrophages. Accordingly, in a murine model of acetaminophen-induced hepatotoxicity the 60-nm particles loaded with chlorpromazine reduced the serum alanine aminotransferase level and liver necrosis 2- to 4-fold more efficiently than their 120-nm counterparts and the free drug, respectively. This work showed that the intra-liver distribution of PFSUVs was largely determined by the size. Most other nanoparticles published to date are predominantly cleared by the liver Kupffer cells. The 60-nm PFSUVs, on the other hand, focused the delivery to the hepatocytes with significant advantages for the therapy of liver diseases.

E46K-like α-synuclein mutants increase lipid interactions and disrupt membrane selectivity.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders, and both genetic and histopathological evidence have implicated the ubiquitous presynaptic protein α-synuclein (αSyn) in its pathogenesis. Recent work has investigated how disrupting αSyn's interaction with membranes triggers trafficking defects, cellular stress, and apoptosis. Special interest has been devoted to a series of mutants exacerbating the effects of the E46K mutation (associated with autosomal dominant PD) through homologous Glu-to-Lys substitutions in αSyn's N-terminal region (i.e. E35K and E61K). Such E46K-like mutants have been shown to cause dopaminergic neuron loss and severe but L-DOPA-responsive motor defects in mouse overexpression models, presenting enormous translational potential for PD and other "synucleinopathies." In this work, using a variety of biophysical techniques, we characterize the molecular pathology of E46K-like αSyn mutants by studying their structure and membrane-binding and remodeling abilities. We find that, although a slight increase in the mutants' avidity for synaptic vesicle-like membranes can be detected, most of their deleterious effects are connected to their complete disruption of αSyn's curvature selectivity. Indiscriminate binding can shift αSyn's subcellular localization away from its physiological interactants at the synaptic bouton toward trafficking vesicles and organelles, as observed in E46K-like cellular and murine models, as well as in human pathology. In conclusion, our findings suggest that a loss of curvature selectivity, rather than increased membrane affinity, could be the critical dyshomeostasis in synucleinopathies.

Analyzing Protein-Phosphoinositide Interactions with Liposome Flotation Assays.

Liposome flotation assays are a convenient tool to study protein-phosphoinositide interactions. Working with liposomes resembles physiological conditions more than protein-lipid overlay assays, which makes this method less prone to detect false positive interactions. However, liposome lipid composition must be well-considered in order to prevent nonspecific binding of the protein through electrostatic interactions with negatively charged lipids like phosphatidylserine. In this protocol we use the PROPPIN Hsv2 (homologous with swollen vacuole phenotype 2) as an example to demonstrate the influence of liposome lipid composition on binding and show how phosphoinositide binding specificities of a protein can be characterized with this method.

Investigating the interactions of the 18kDa translocator protein and its ligand PK11195 in planar lipid bilayers.

The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug-membrane interactions may contribute to the apparent affinity of TSPO ligands.

IFABP portal region insertion during membrane interaction depends on phospholipid composition.

Intestinal fatty acid-binding protein (IFABP) is highly expressed in the intestinal epithelium and it belongs to the family of soluble lipid binding proteins. These proteins are thought to participate in most aspects of the biology of lipids, regulating its availability for specific metabolic pathways, targeting and vectorial trafficking of lipids to specific subcellular compartments. The present study is based on the ability of IFABP to interact with phospholipid membranes, and we characterized its immersion into the bilayer's hydrophobic central region occupied by the acyl-chains. We constructed a series of Trp-mutants of IFABP to selectively probe the interaction of different regions of the protein, particularly the elements forming the portal domain that is proposed to regulate the exit and entry of ligands to/from the binding cavity. We employed several fluorescent techniques based on selective quenching induced by soluble or membrane confined agents. The results indicate that the portal region of IFABP penetrates deeply into the phospholipid bilayer, especially when CL-containing vesicles are employed. The orientation of the protein and the degree of penetration were highly dependent on the lipid composition, the superficial net charge and the ionic strength of the medium. These results may be relevant to understand the mechanism of ligand transfer and the specificity responsible for the unique functions of each member of the FABP family.

Rubber particle proteins, HbREF and HbSRPP, show different interactions with model membranes.

The biomembrane surrounding rubber particles from the hevea latex is well known for its content of numerous allergen proteins. HbREF (Hevb1) and HbSRPP (Hevb3) are major components, linked on rubber particles, and they have been shown to be involved in rubber synthesis or quality (mass regulation), but their exact function is still to be determined. In this study we highlighted the different modes of interactions of both recombinant proteins with various membrane models (lipid monolayers, liposomes or supported bilayers, and multilamellar vesicles) to mimic the latex particle membrane. We combined various biophysical methods (polarization-modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS)/ellipsometry, attenuated-total reflectance Fourier-transform infrared (ATR-FTIR), solid-state nuclear magnetic resonance (NMR), plasmon waveguide resonance (PWR), fluorescence spectroscopy) to elucidate their interactions. Small rubber particle protein (SRPP) shows less affinity than rubber elongation factor (REF) for the membranes but displays a kind of "covering" effect on the lipid headgroups without disturbing the membrane integrity. Its structure is conserved in the presence of lipids. Contrarily, REF demonstrates higher membrane affinity with changes in its aggregation properties, the amyloid nature of REF, which we previously reported, is not favored in the presence of lipids. REF binds and inserts into membranes. The membrane integrity is highly perturbed, and we suspect that REF is even able to remove lipids from the membrane leading to the formation of mixed micelles. These two homologous proteins show affinity to all membrane models tested but neatly differ in their interacting features. This could imply differential roles on the surface of rubber particles.

Macrophage mitochondrial damage from StAR transport of 7-hydroperoxycholesterol: implications for oxidative stress-impaired reverse cholesterol transport.

StAR family proteins in vascular macrophages participate in reverse cholesterol transport (RCT). We hypothesize that under pathophysiological oxidative stress, StARs will transport not only cholesterol to macrophage mitochondria, but also pro-oxidant cholesterol hydroperoxides (7-OOHs), thereby impairing early-stage RCT. Upon stimulation with dibutyryl-cAMP, RAW264.7 macrophages exhibited a strong time-dependent induction of mitochondrial StarD1 and plasma membrane ABCA1, which exports cholesterol. 7α-OOH uptake by stimulated RAW cell mitochondria (like cholesterol uptake) was strongly reduced by StarD1 knockdown, consistent with StarD1 involvement. Upon uptake by mitochondria, 7α-OOH (but not redox-inactive 7α-OH) triggered lipid peroxidation and membrane depolarization while reducing ABCA1 upregulation. These findings provide strong initial support for our hypothesis.

The roles of C-terminal helices of human apolipoprotein A-I in formation of high-density lipoprotein particles.

Apolipoprotein A-I (apoA-I) accepts cholesterol and phospholipids from ATP-binding cassette transporter A1 (ABCA1)-expressing cells to form high-density lipoprotein (HDL). Human apoA-I has two tertiary structural domains and the C-terminal domain (approximately amino acids 190-243) plays a key role in lipid binding. Although the high lipid affinity region of the C-terminal domain of apoA-I (residues 223-243) is essential for the HDL formation, the function of low lipid affinity region (residues 191-220) remains unclear. To evaluate the role of residues 191-220, we analyzed the structure, lipid binding properties, and HDL formation activity of Δ191-220 apoA-I, in comparison to wild-type and Δ223-243 apoA-I. Although deletion of residues 191-220 has a slight effect on the tertiary structure of apoA-I, the Δ191-220 variant showed intermediate behavior between wild-type and Δ223-243 regarding the formation of hydrophobic sites and lipid interaction through the C-terminal domain. Physicochemical analysis demonstrated that defective lipid binding of Δ191-220 apoA-I is due to the decreased ability to form α-helix structure which provides the energetic source for lipid binding. In addition, the ability to form HDL particles in vitro and induce cholesterol efflux from ABCA1-expressing cells of Δ191-220 apoA-I was also intermediate between wild-type and Δ223-243 apoA-I. These results suggest that despite possessing low lipid affinity, residues 191-220 play a role in enhancing the ability of apoA-I to bind to and solubilize lipids by forming α-helix upon lipid interaction. Our results demonstrate that the combination of low lipid affinity region and high lipid affinity region of apoA-I is required for efficient ABCA1-dependent HDL formation.

Aggregates of nisin with various bactoprenol-containing cell wall precursors differ in size and membrane permeation capacity.

Many lantibiotics use the membrane bound cell wall precursor Lipid II as a specific target for killing Gram-positive bacteria. Binding of Lipid II usually impedes cell wall biosynthesis, however, some elongated lantibiotics such as nisin, use Lipid II also as a docking molecule for pore formation in bacterial membranes. Although the unique nisin pore formation can be analyzed in Lipid II-doped vesicles, mechanistic details remain elusive. We used optical sectioning microscopy to directly visualize the interaction of fluorescently labeled nisin with membranes of giant unilamellar vesicles containing Lipid II and its various bactoprenol precursors. We quantitatively analyzed the binding and permeation capacity of nisin when applied at nanomolar concentrations. Specific interactions with Lipid I, Lipid II and bactoprenol-diphosphate (C55-PP), but not bactoprenol-phosphate (C55-P), resulted in the formation of large molecular aggregates. For Lipid II, we demonstrated the presence of both nisin and Lipid II in these aggregates. Membrane permeation induced by nisin was observed in the presence of Lipid I and Lipid II, but not in the presence of C55-PP. Notably, the size of the C55-PP-nisin aggregates was significantly smaller than that of the aggregates formed with Lipid I and Lipid II. We conclude that the membrane permeation capacity of nisin is determined by the size of the bactoprenol-containing aggregates in the membrane. Notably, transmitted light images indicated that the formation of large aggregates led to a pinch-off of small vesicles, a mechanism, which probably limits the growth of aggregates and induces membrane leakage.

Double-tailed lipid modification as a promising candidate for oligonucleotide delivery in mammalian cells.

BACKGROUND: The potential use of nucleic acids as therapeutic drugs has triggered the quest for oligonucleotide conjugates with enhanced cellular permeability. To this end, the biophysical aspects of previously reported potential lipid oligodeoxyribonucleotide conjugates were studied including its membrane-binding properties and cellular uptake. METHODS: These conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography. Their ability to insert into lipid model membrane systems was evaluated by Langmuir balance and confocal microscopy followed by the study of the internalization of a lipid oligodeoxyribonucleotide conjugate bearing a double-tail lipid modification (C28) into different cell lines by confocal microscopy and flow cytometry. This compound was also compared with other lipid containing conjugates and with the classical lipoplex formulation using Transfectin as transfection reagent. RESULTS: This double-tail lipid modification showed better incorporation into both lipid model membranes and cell systems. Indeed, this lipid conjugation was capable of inserting the oligodeoxyribonucleotide into both liquid-disordered and liquid-ordered domains of model lipid bilayer systems and produced an enhancement of oligodeoxyribonucleotide uptake in cells, even better than the effect caused by lipoplexes. In addition, in ß2 integrin (CR3) expressing cells this receptor was directly involved in the enhanced internalization of this compound. CONCLUSIONS: All these features confirm that the dual lipid modification (C28) is an excellent modification for enhancing nucleic acid delivery without altering their binding properties. GENERAL SIGNIFICANCE: Compared to the commercial lipoplex approach, oligodeoxyribonucleotide conjugation with C28 dual lipid modification seems to be promising to improve oligonucleotide delivery in mammalian cells.

Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods.

We characterized phosphoinositide binding of the S. cerevisiae PROPPIN Hsv2 qualitatively with density flotation assays and quantitatively through isothermal titration calorimetry (ITC) measurements using liposomes. We discuss the design of these experiments and show with liposome flotation assays that Hsv2 binds with high specificity to both PtdIns3P and PtdIns(3,5)P 2. We propose liposome flotation assays as a more accurate alternative to the commonly used PIP strips for the characterization of phosphoinositide-binding specificities of proteins. We further quantitatively characterized PtdIns3P binding of Hsv2 with ITC measurements and determined a dissociation constant of 0.67 µM and a stoichiometry of 2:1 for PtdIns3P binding to Hsv2. PtdIns3P is crucial for the biogenesis of autophagosomes and their precursors. Besides the PROPPINs there are other PtdIns3P binding proteins with a link to autophagy, which includes the FYVE-domain containing proteins ZFYVE1/DFCP1 and WDFY3/ALFY and the PX-domain containing proteins Atg20 and Snx4/Atg24. The methods described could be useful tools for the characterization of these and other phosphoinositide-binding proteins.

Experimental aspects of colloidal interactions in mixed systems of liposome and inorganic nanoparticle and their applications.

In the past few years, growing attention has been devoted to the study of the interactions taking place in mixed systems of phospholipid membranes (for instance in the form of vesicles) and hard nanoparticles (NPs). In this context liposomes (vesicles) may serve as versatile carriers or as a model system for biological membranes. Research on these systems has led to the observation of novel hybrid structures whose morphology strongly depends on the charge, composition and size of the interacting colloidal species as well as on the nature (pH, ionic strength) of their dispersing medium. A central role is played by the phase behaviour of phospholipid bilayers which have a tremendous influence on the liposome properties. Another central aspect is the incorporation of nanoparticles into vesicles, which is intimately linked to the conditions required for transporting a nanoparticle through a membrane. Herein, we review recent progress made on the investigations of the interactions in liposome/nanoparticle systems focusing on the particularly interesting structures that are formed in these hybrid systems as well as their potential applications.

Supported membrane formation, characterization, functionalization, and patterning for application in biological science and technology.

Supported membranes, formed as a single continuous lipid bilayer on a solid substrate, such as silica, have been used extensively as a model for protein-protein and cell-cell interaction, to study the molecular interactions at interfaces and the heterogeneities of plasma membranes. The advantages of a supported membrane system include the ability to control membrane composition and the compatibility it has with various surface-sensitive microscopic and spectroscopic techniques. Recent advances in micro- and nanotechnology have greatly extended the use of supported membranes to address key questions in cell biology. Although supported membranes can be easily made by vesicle fusion, the samples need careful preparation for this process to be efficient. The protocols in this unit comprehensively describe procedures to prepare, functionalize, and characterize supported membranes. Curr. Protoc. Chem. Biol. 2:235-269 © 2010 by John Wiley & Sons, Inc.