Fluorescent lipids: functional parts of fusogenic liposomes and tools for cell membrane labeling and visualization

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.     

Near-field scanning optical microscopy: a tool for nanometric exploration of biological membranes

Near-field scanning optical microscopy (NSOM) is an emerging optical technique that enables simultaneous high-resolution fluorescence and topography measurements. Here we discuss selected applications of NSOM to biological systems that help illustrate the utility of its high spatial resolution and simultaneous collection of both fluorescence and topography. For the biological sciences, these attributes seem particularly well suited for addressing ongoing issues in membrane organization, such as those regarding lipid rafts, and protein–protein interactions. Here we highlight a few NSOM measurements on model membranes, isolated biological membranes, and cultured cells that help illustrate some of these capabilities. We finish by highlighting nontraditional applications of NSOM that take advantage of the small probe to create nanometric sensors or new modes of imaging.     

Interaction of nucleic acids with the glycocalyx

Mammalian cells resist the uptake of nucleic acids. The lipid bilayer of the plasma membrane presents one barrier. Here, we report on a second physicochemical barrier for uptake. To create a sensitive probe for nucleic acid-cell interactions, we synthesized fluorescent conjugates in which lipids are linked to DNA oligonucleotides. We found that these conjugates incorporate readily into the plasma membrane but are not retained there. Expulsion of lipid-oligonucleotide conjugates from the plasma membrane increases with oligonucleotide length. Conversely, the incorporation of conjugates increases markedly in cells that lack the major anionic components of the glycocalyx, sialic acid and glycosaminoglycans, and in cells that had incorporated highly cationic lipids into their plasma membrane. We conclude that anionic oligosaccharides provide a formidable barrier to the uptake of nucleic acids by mammalian cells. This conclusion has implications for genomic stability, as well as the delivery of genes and siRNAs into mammalian cells.     

CHARMM‐GUI Membrane Builder toward realistic biological membrane simulations

CHARMM‐GUI Membrane Builder, http://www.charmm‐gui.org/input/membrane , is a web‐based user interface designed to interactively build all‐atom protein/membrane or membrane‐only systems for molecular dynamics simulations through an automated optimized process. In this work, we describe the new features and major improvements in Membrane Builder that allow users to robustly build realistic biological membrane systems, including (1) addition of new lipid types, such as phosphoinositides, cardiolipin (CL), sphingolipids, bacterial lipids, and ergosterol, yielding more than 180 lipid types, (2) enhanced building procedure for lipid packing around protein, (3) reliable algorithm to detect lipid tail penetration to ring structures and protein surface, (4) distance‐based algorithm for faster initial ion displacement, (5) CHARMM inputs for P₂₁ image transformation, and (6) NAMD equilibration and production inputs. The robustness of these new features is illustrated by building and simulating a membrane model of the polar and septal regions of E. coli membrane, which contains five lipid types: CL lipids with two types of acyl chains and phosphatidylethanolamine lipids with three types of acyl chains. It is our hope that CHARMM‐GUI Membrane Builder becomes a useful tool for simulation studies to better understand the structure and dynamics of proteins and lipids in realistic biological membrane environments. © 2014 Wiley Periodicals, Inc.     

Practical Labeling Methodology for Choline‐Derived Lipids and Applications in Live Cell Fluorescence Imaging

Lipids of the plasma membrane participate in a variety of biological processes, and methods to probe their function and cellular location are essential to understanding biochemical mechanisms. Previous reports have established that phosphocholine‐containing lipids can be labeled by alkyne groups through metabolic incorporation. Herein, we have tested alkyne, azide and ketone‐containing derivatives of choline as metabolic labels of choline‐containing lipids in cells. We also show that 17‐octadecynoic acid can be used as a complementary metabolic label for lipid acyl chains. We provide methods for the synthesis of cyanine‐based dyes that are reactive with alkyne, azide and ketone metabolic labels. Using an improved method for fluorophore conjugation to azide or alkyne‐modified lipids by Cu(I)‐catalyzed azide‐alkyne cycloaddition (CuAAC), we apply this methodology in cells. Lipid‐labeled cell membranes were then interrogated using flow cytometry and fluorescence microscopy. Furthermore, we explored the utility of this labeling strategy for use in live cell experiments. We demonstrate measurements of lipid dynamics (lateral mobility) by fluorescence photobleaching recovery (FPR). In addition, we show that adhesion of cells to specific surfaces can be accomplished by chemically linking membrane lipids to a functionalized surface. The strategies described provide robust methods for introducing bioorthogonal labels into native lipids.     

The Remarkable Effect of Halogen Substitution on the Membrane Transport of Fluorescent Molecules in Living Cells

Small‐molecule‐based fluorescent probes have become important tools in biology for sensing and imaging applications. However, the biological applications of many of the fluorescent molecules are hampered by low cellular uptake and high toxicity. In this paper, we show for the first time that the introduction of halogen atoms enhances the cellular uptake of fluorescent molecules and the nature of halogen atoms plays a crucial role in the plasma membrane transport in mammalian cells. The remarkably higher uptake of iodinated compounds compared to that of their chloro or bromo analogues suggests that the strong halogen bonding ability of iodine atoms may play an important role in the membrane transport. This study provides a novel strategy for the transport of fluorescent molecules across the plasma membrane in living cells.     

Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light‐Activated Guide RNA

We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5′‐protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA‐target hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off‐to‐on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.     

Mechanical Trap Surface‐Enhanced Raman Spectroscopy for Three‐Dimensional Surface Molecular Imaging of Single Live Cells

Reported is a new shell‐based spectroscopic platform, named mechanical trap surface‐enhanced Raman spectroscopy (MTSERS), for simultaneous capture, profiling, and 3D microscopic mapping of the intrinsic molecular signatures on the membrane of single live cells. By leveraging the functionalization of the inner surfaces of the MTs with plasmonic gold nanostars, and conformal contact of the cell membrane, MTSERS permits excellent signal enhancement, reliably detects molecular signatures, and allows non‐perturbative, multiplex 3D surface imaging of analytes, such as lipids and proteins on the surface of single cells. The demonstrated ability underscores the potential of MTSERS to perform 3D spectroscopic microimaging and to furnish biologically interpretable, quantitative, and dynamic molecular maps in live cell populations.     

A Remarkably Simple Class of Imidazolium‐Based Lipids and Their Biological Properties

A series of imidazolium salts bearing two alkyl chains in the backbone of the imidazolium core were synthesized, resembling the structure of lipids. Their antibacterial activity and cytotoxicity were evaluated using Gram‐positive and Gram‐negative bacteria and eukaryotic cell lines including tumor cells. It is shown that the length of alkyl chains in the backbone is vital for the antibiofilm activities of these lipid‐mimicking components. In addition to their biological activity, their surface activity and their membrane interactions are shown by film balance and quartz crystal microbalance (QCM) measurements. The structure–activity relationship indicates that the distinctive chemical structure contributes considerably to the biological activities of this novel class of lipids.     

Spectral Imaging to Measure Heterogeneity in Membrane Lipid Packing

Physicochemical properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the molecular order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane‐embedded polarity‐sensitive dyes, whose emission spectrum is dependent on the molecular order of the immediate membrane environment. Here, we report on an improved determination of such spectral shifts in the emission spectrum of the polarity‐sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved analysis, which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity‐sensitive dyes C‐Laurdan or Di‐4‐ANEPPDHQ, we were able to image—with high accuracy—minute differences in the lipid packing of model and cellular membranes.     

Interaction of hexadecylbetainate chloride with biological relevant lipids

The present work investigates the interaction of hexadecylbetainate chloride (C(16)BC), a glycine betaine-based ester with palmitoyl-oleoyl-phosphatidylcholine (POPC), sphingomyelin (SM), and cholesterol (CHOL), three biological relevant lipids present in the outer leaflet of the mammalian plasma membrane. The binding affinity and the mixing behavior between the lipids and C(16)BC are discussed based on experimental (isothermal titration calorimetry (ITC) and Langmuir film balance) and molecular modeling studies. The results show that the interaction between C(16)BC and each lipid is thermodynamically favorable and does not affect the integrity of the lipid vesicles. The primary adsorption of C(16)BC into the lipid film is mainly governed by a hydrophobic effect. Once C(16)BC is inserted in the lipid film, the polar component of the interaction energy between C(16)BC and the lipid becomes predominant. Presence of CHOL increases the affinity of C(16)BC for membrane. This result can be explained by the optimal matching between C(16)BC and CHOL within the film rather by a change of membrane fluidity due to the presence of CHOL. The interaction between C(16)BC and SM is also favorable and gives rise to highly stable monolayers probably due to hydrogen bonds between their hydrophilic groups. The interaction of C(16)BC with POPC is less favorable but does not destabilize the mixed monolayer from a thermodynamic point of view. Interestingly, for all the monolayers investigated, the exclusion surface pressures are above the presumed lateral pressure of the plasma membranes suggesting that C(16)BC would be able to penetrate into mammalian plasma membranes in vivo. These results may serve as a useful basis in understanding the interaction of C(16)BC with real membranes.     

The effect of phenyltin chlorides on osmotically induced erythrocyte haemolysis

The toxicity of many amphiphilic compounds may result from their effect on the lipid phase of biological membranes. Upon incorporation such compounds may change the properties of membranes in general and in particular alter the organization of membrane lipids. These changes should affect, among other things, the mechanical properties of membranes. We selected two amphiphilic compounds, diphenyltin dichloride (Ph₂SnCl₂) and triphenyltin chloride (Ph₃SnCl), which are known to be located at different regions of the lipid bilayer and to be toxic. As a model biological membrane the erythrocyte plasma membrane was used. Analysis of the haemolysis kinetics showed differences between the effect of the compound studied on mechanical properties at so‐called non‐lytic concentrations. Diphenyltin dichloride showed a limited effect on erythrocyte haemolysis, whereas triphenyltin chloride affected all the parameters measured (extent of initial haemolysis, extent of final haemolysis and membrane mechanical strength). We correlated these effects with the location of the investigated compounds in liposomes. The presented data show that triphenyltin chloride reduces the erythrocyte plasma membrane mechanical strength and increases the extent of haemolysis under osmotic stress conditions. Copyright © 2005 John Wiley & Sons, Ltd.     

Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry‐Based Approach

Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three‐dimensional structures. As lipids are dynamic by nature, their “structure” does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid‐raft‐related molecules, lipid–protein interactions, and membrane‐active natural products, we discuss current perspectives on membrane structural biology.     

Boron‐Containing Probes for Non‐optical High‐Resolution Imaging of Biological Samples

Boron has been employed in materials science as a marker for imaging specific structures by electron energy loss spectroscopy (EELS) or secondary ion mass spectrometry (SIMS). It has a strong potential in biological analyses as well; however, the specific coupling of a sufficient number of boron atoms to a biological structure has proven challenging. Herein, we synthesize tags containing closo‐1,2‐dicarbadodecaborane, coupled to soluble peptides, which were integrated in specific proteins by click chemistry in mammalian cells and were also coupled to nanobodies for use in immunocytochemistry experiments. The tags were fully functional in biological samples, as demonstrated by nanoSIMS imaging of cell cultures. The boron signal revealed the protein of interest, while other SIMS channels were used for imaging different positive ions, such as the cellular metal ions. This allows, for the first time, the simultaneous imaging of such ions with a protein of interest and will enable new biological applications in the SIMS field.     

Hydrophobic/superhydrophobic oxidized metal surfaces showing negligible contact angle hysteresis

The blood coagulation system relies on lipid membrane constituents to act as regulators of the coagulation process upon vascular trauma, and in particular the 2D configuration of the lipid membranes is known to efficiently catalyze enzymatic activity of blood coagulation factors. This work demonstrates a new application of a recently developed methodology to study blood coagulation at lipid membrane interfaces with the use of imaging technology. Lipid membranes with varied net charges were formed on silica supports by systematically using different combinations of lipids where neutral phosphocholine (PC) lipids were mixed with phospholipids having either positively charged ethylphosphocholine (EPC), or negatively charged phosphatidylserine (PS) headgroups. Coagulation imaging demonstrated that negatively charged SiO(2) and membrane surfaces exposing PS (obtained from liposomes containing 30% of PS) had coagulation times which were significantly shorter than those for plain PC membranes and EPC exposing membrane surfaces (obtained from liposomes containing 30% of EPC). Coagulation times decreased non-linearly with increasing negative surface charge for lipid membranes. A threshold value for shorter coagulation times was observed below a PS content of ～6%. We conclude that the lipid membranes on solid support studied with the imaging setup as presented in this study offers a flexible and non-expensive solution for coagulation studies at biological membranes. It will be interesting to extend the present study towards examining coagulation on more complex lipid-based model systems.     

Bilayer lipid membrane formation on a chemically modified S-layer lattice

The present paper describes the generation of a biomimetic model lipid membrane on bacterial surface (S-)layer which covered the entire surface of various sensors. The S-layer lattice allows one to be independent from the underlying solid material and provides a biological surface and anchoring structure for lipid membranes. S-layer proteins were chemically modified via binding of two amine-terminated phospholipids. Subsequently, a bimolecular lipid membrane anchored to the previously generated viscoelastic lipid monolayer was generated by the rapid solvent exchange technique. Characterization of the intermediate (monolayer) and final membrane structures (bilayer) was performed by imaging, surface-sensitive, and electrochemical techniques. This bilayer lipid membrane generated on an S-layer lattice revealed a thickness of ～6 nm and constitutes a stable supported model membrane system with highly isolating properties showing a membrane resistance of 8.5 MΩ × cm(2).     

Biological Investigations of (+)‐Danicalipin A Enabled Through Synthesis

A total synthesis of the chlorosulfolipid (+)‐danicalipin A has been accomplished in 12 steps and 4.4 % overall yield. The efficient and scalable synthesis enabled in‐depth investigations of the lipid’s biological properties, in particular cytotoxicity towards various mammalian cell lines. Furthermore, the ability of (+)‐danicalipin A to increase the uptake of fluorophores into bacteria and mammalian cells was demonstrated, indicating it may enhance membrane permeability. By comparing (+)‐danicalipin A with racemic 1,14‐docosane disulfate, and the diol precursor of (+)‐danicalipin A, we have shown that both chlorine and sulfate functionalities are necessary for biological activity.     

Electro-optical BLM chips enabling dynamic imaging of ordered lipid domains

Studies of lipid rafts, ordered microdomains of sphingolipids and cholesterol within cell membranes, are essential in probing the relationships between membrane organization and cellular function. While in vitro studies of lipid phase separation are commonly performed using spherical vesicles as model membranes, the utility of these models is limited by a number of factors. Here we present a microfluidic device that supports simultaneous electrical measurements and confocal imaging of on-chip bilayer lipid membranes (BLMs), enabling real-time multi-domain imaging of membrane organization. The chips further support closed microfluidic access to both sides of the membrane, allowing the membrane boundary conditions to be rapidly changed and providing a mechanism for dynamically adjusting membrane curvature through application of a transmembrane pressure gradient. Here we demonstrate the platform through the study of dynamic generation and dissolution of ordered lipid domains as membrane components are transported to and from the supporting annulus containing solvated lipids and cholesterol.     

Design of quantum dot-conjugated lipids for long-term, high-speed tracking experiments on cell surfaces

The current study reports the facile design of quantum dot (QD)-conjugated lipids and their application to high-speed tracking experiments on cell surfaces. CdSe/ZnS core/shell QDs with two types of hydrophilic coatings, 2-(2-aminoethoxy)ethanol (AEE) and a 60:40 molar mixture of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine and 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol-2000], are conjugated to sulfhydryl lipids via maleimide reactive groups on the QD surface. Prior to lipid conjugation, the colloidal stability of both types of coated QDs in aqueous solution is confirmed using fluorescence correlation spectroscopy. A sensitive assay based on single lipid tracking experiments on a planar solid-supported phospholipid bilayer is presented that establishes conditions of monovalent conjugation of QDs to lipids. The QD-lipids are then employed as single-molecule tracking probes in plasma membranes of several cell types. Initial tracking experiments at a frame rate of 30 frames/s corroborate that QD-lipids diffuse like dye-labeled lipids in the plasma membrane of COS-7, HEK-293, 3T3, and NRK cells, thus confirming monovalent labeling. Finally, QD-lipids are applied for the first time to high-speed single-molecule imaging by tracking their lateral mobility in the plasma membrane of NRK fibroblasts with up to 1000 frames/s. Our high-speed tracking data, which are in excellent agreement with previous tracking experiments that used larger (40 nm) Au labels, not only push the time resolution in long-time, continuous fluorescence-based single-molecule tracking but also show that highly photostable, photoluminescent nanoprobes of 10 nm size can be employed (AEE-coated QDs). These probes are also attractive because, unlike Au nanoparticles, they facilitate complex multicolor experiments.     

Pseudo‐Membrane Jackets: Two‐Dimensional Coordination Polymers Achieving Visible Phase Separation in Cell Membrane

Cell membranes contain lateral systems that consist of various lipid compositions and actin cytoskeleton, providing two‐dimensional (2D) platforms for chemical reactions. However, such complex 2D environments have not yet been used as a synthetic platform for artificial 2D nanomaterials. Herein, we demonstrate the direct synthesis of 2D coordination polymers (CPs) at the liquid‐cell interface of the plasma membrane of living cells. The coordination‐driven self‐assembly of networking metal complex lipids produces cyanide‐bridged CP layers with metal ions, enabling “pseudo‐membrane jackets” that produce long‐lived micro‐domains with a size of 1–5 μm. The resultant artificial and visible phase separation systems remain stable even in the absence of actin skeletons in cells. Moreover, we show the cell application of the jackets by demonstrating the enhancement of cellular calcium response to ATP.