Optical manipulation reveals strong attracting forces at membrane contact sites between endoplasmic reticulum and chloroplasts

Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209-220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.     

Role of membrane organization and membrane domains in endocytic lipid trafficking

Lipid compositions vary greatly among organelles, and specific sorting mechanisms are required to establish and maintain these distinct compositions. In this review, we discuss how the biophysical properties of the membrane bilayer and the chemistry of individual lipid molecules play a role in the intracellular trafficking of the lipids themselves, as well as influencing the trafficking of transmembrane proteins. The large diversity of lipid head groups and acyl chains lead to a variety of weak interactions, such as ionic and hydrogen bonding at the lipid/water interfacial region, hydrophobic interactions, and van-der-Waals interactions based on packing density. In simple model bilayers, these weak interactions can lead to large-scale phase separations, but in more complex mixtures, which mimic cell membranes, such phase separations are not observed. Nevertheless, there is growing evidence that domains (i.e., localized regions with non-random lipid compositions) exist in biological membranes, and it is likely that the formation of these domains are based on interactions similar to those that lead to phase separations in model systems. Sorting of lipids appears to be based in part on the inclusion or exclusion of certain types of lipids in vesicles or tubules as they bud from membrane organelles.     

Design of supported membranes tethered via metal-affinity ligand-receptor pairs

Model lipid layers are very promising in investigating the complex network of recognition, transport and signaling processes at membranes. We have developed a novel and generic approach to create supported lipid membranes tethered by metal-affinity binding. By self-assembly we have generated various interfaces that display histidine sequences (6xHis) via polymer spacers. These histidine-functionalized interfaces are designed to allow specific docking and fusion of vesicles containing metal-chelating lipids. By means of surface plasmon resonance and atomic force microscopy we analyzed the formation and subsequently the structure of these solid-supported membranes. Although the affinity constant of single ligand-receptor pairs is only in the micromolar range, very stable immobilization of these membranes was observed. This behavior can be explained by multivalent interactions resembling many features of cell adhesion. The process is highly specific, because vesicle docking and bilayer formation are strictly dependent on the presence of metal-affinity ligand-receptor pairs. The surface accessibility and geometry of these tethered membranes were probed by binding of histidine-tagged polypeptides. The supported membranes show adsorption kinetics and values similar to planar supported monolayers. Using various combinations of metal-chelating and histidine-tagged lipids or thiols these metal-affinity-tethered membranes should make a great impact on probing and eventually understanding the dynamic dialog of reconstituted membrane proteins.     

Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides

The indiscriminate use of conventional antibiotics is leading to an increase in the number of resistant bacterial strains, motivating the search for new compounds to overcome this challenging problem. Antimicrobial peptides, acting only in the lipid phase of membranes without requiring specific membrane receptors as do conventional antibiotics, have shown great potential as possible substituents of these drugs. These peptides are in general rich in basic and hydrophobic residues forming an amphipathic structure when in contact with membranes. The outer leaflet of the prokaryotic cell membrane is rich in anionic lipids, while the surface of the eukaryotic cell is zwitterionic. Due to their positive net charge, many of these peptides are selective to the prokaryotic membrane. Notwithstanding this preference for anionic membranes, some of them can also act on neutral ones, hampering their therapeutic use. In addition to the electrostatic interaction driving peptide adsorption by the membrane, the ability of the peptide to perturb lipid packing is of paramount importance in their capacity to induce cell lysis, which is strongly dependent on electrostatic and hydrophobic interactions. In the present research, we revised the adsorption of antimicrobial peptides by model membranes as well as the perturbation that they induce in lipid packing. In particular, we focused on some peptides that have simultaneously acidic and basic residues. The net charges of these peptides are modulated by pH changes and the lipid composition of model membranes. We discuss the experimental approaches used to explore these aspects of lipid membranes using lipid vesicles and lipid monolayer as model membranes.     

Sequence and functional similarities between pro-apoptotic Bid and plant lipid transfer proteins

Pro-apoptotic proteins of the Bcl-2 family are known to act on mitochondria and facilitate the release of cytochrome c, but the biochemical mechanism of this action is unknown. Association with mitochondrial membranes is likely to be important in determining the capacity of releasing cytochrome c. The present work provides new evidence suggesting that some pro-apoptotic proteins like Bid have an intrinsic capacity of binding and exchanging membrane lipids. Detailed analysis indicates a significant sequence similarity between a subset of Bcl-2 family proteins including Bid and Nix and plant lipid transfer proteins. The similar structural signatures could be related to common interactions with membrane lipids. Indeed, isolated Bid shows a lipid transfer activity that is even higher than that of plant lipid transfer proteins. To investigate the possible relevance of these structure-function correlations to the apoptotic action of Bid, cell free assays were established with isolated mitochondria, recombinant Bid and a variety of exogenous lipids. Micromolar concentrations of lysolipids such as lysophosphatidylcholine were found to change the association of Bid with mitochondria and also stimulate the release of cytochrome c promoted by Bid. The changes in mitochondrial association and cytochrome c release were enhanced by the presence of liposomes of lipid composition similar to that of mitochondrial membranes. Thus, a mixture of liposomes, mitochondria and key lysolipids could reproduce the conditions enabling Bid to transfer lipids between donor and acceptor membranes, and also change its reversible association with mitochondria. Bid was also found to enhance the incorporation of a fluorescent lysolipid, but not of a related fatty acid, into mitochondria. On the basis of the results presented here, it is hypothesised that Bid action may depend upon its capacity of exchanging lipids and lysolipids with mitochondrial membranes. The hypothesis is discussed in relation to current models for the integrated action of pro-apoptotic proteins of the Bcl-2 family.     

Calcium-Lipid Interactions Observed with Isotope-Edited Infrared Spectroscopy

Calcium ions bind to lipid membranes containing anionic lipids; however, characterizing the specific ion-lipid interactions in multicomponent membranes has remained challenging because it requires nonperturbative lipid-specific probes. Here, using a combination of isotope-edited infrared spectroscopy and molecular dynamics simulations, we characterize the effects of a physiologically relevant (2 mM) Ca²⁺ concentration on zwitterionic phosphatidylcholine and anionic phosphatidylserine lipids in mixed lipid membranes. We show that Ca²⁺ alters hydrogen bonding between water and lipid headgroups by forming a coordination complex involving the lipid headgroups and water. These interactions distort interfacial water orientations and prevent hydrogen bonding with lipid ester carbonyls. We demonstrate, experimentally, that these effects are more pronounced for the anionic phosphatidylserine lipids than for zwitterionic phosphatidylcholine lipids in the same membrane.     

Thermosensing via transmembrane protein–lipid interactions

Cell membranes are composed of a lipid bilayer containing proteins that cross and/or interact with lipids on either side of the two leaflets. The basic structure of cell membranes is this bilayer, composed of two opposing lipid monolayers with fascinating properties designed to perform all the functions the cell requires. To coordinate these functions, lipid composition of cellular membranes is tailored to suit their specialized tasks. In this review, we describe the general mechanisms of membrane–protein interactions and relate them to some of the molecular strategies organisms use to adjust the membrane lipid composition in response to a decrease in environmental temperature. While the activities of all biomolecules are altered as a function of temperature, the thermosensors we focus on here are molecules whose temperature sensitivity appears to be linked to changes in the biophysical properties of membrane lipids. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Membrane perturbation and the mechanism of lipid-mediated transfer of DNA into cells

Mixtures of cationic lipids and unsaturated phosphatidylethanolamine are used extensively for the intracellular delivery of plasmids and antisense oligodeoxynucleotides (ODN) in vitro. However, the mechanism by which cytoplasmic delivery of these large molecules is achieved remains unclear. The common hypothesis is that phosphatidylethanolamine promotes fusion of lipid/DNA particles with endosomal membranes, but this is inconsistent with several reports that have failed to correlate the fusogenic activity of a wide variety of lipid/DNA particles, measured by lipid mixing techniques, with their transfection activity. To address this issue further we have conducted a detailed analysis of the lipid mixing and DNA transfer activity of two, physically similar but functionally different, lipid/DNA particles composed of equimolar dioleyldimethylammonium chloride (DODAC) and dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC). In combination with DODAC both phospholipids form almost identical lipid/DNA particles, they are endocytosed by cells to the same extent and each undergoes equivalent lipid mixing with cell membranes after uptake. Despite this, DNA transfer is 10- to 100-fold more extensive for lipid/DNA particles containing DOPE. We conclude that lipid mixing between lipid-based delivery systems and endosomal membranes must occur for DNA transfer to occur. However, the potency of different lipid/DNA particles correlates better with the ability of the exogenous lipid to disrupt membrane integrity.     

Biomimetic membrane systems to study cellular organization

During many cellular processes such as cell division, polarization and motility, the plasma membrane does not only represent a passive physical barrier, but also provides a highly dynamic platform for the interplay between lipids, membrane binding proteins and cytoskeletal elements. Even though many regulators of these interactions are known, their mutual interdependence appears to be highly complex and difficult to study in a living cell. Over the past few years, in vitro studies on membrane–cytoskeleton interactions using biomimetic membranes turned out to be extremely helpful to get better mechanistic insight into the dynamics of these processes. In this review, we discuss some of the recent developments using in vitro assays to dissect the role of the players involved: lipids in the membrane, proteins binding to membranes and proteins binding to membrane proteins. We also summarize advantages and disadvantages of supported lipid bilayers as model membrane.     

Sequence and functional similarities between pro-apoptotic Bid and plant lipid transfer proteins

Pro-apoptotic proteins of the Bcl-2 family are known to act on mitochondria and facilitate the release of cytochrome c, but the biochemical mechanism of this action is unknown. Association with mitochondrial membranes is likely to be important in determining the capacity of releasing cytochrome c. The present work provides new evidence suggesting that some pro-apoptotic proteins like Bid have an intrinsic capacity of binding and exchanging membrane lipids. Detailed analysis indicates a significant sequence similarity between a subset of Bcl-2 family proteins including Bid and Nix and plant lipid transfer proteins. The similar structural signatures could be related to common interactions with membrane lipids. Indeed, isolated Bid shows a lipid transfer activity that is even higher than that of plant lipid transfer proteins. To investigate the possible relevance of these structure-function correlations to the apoptotic action of Bid, cell free assays were established with isolated mitochondria, recombinant Bid and a variety of exogenous lipids. Micromolar concentrations of lysolipids such as lysophosphatidylcholine were found to change the association of Bid with mitochondria and also stimulate the release of cytochrome c promoted by Bid. The changes in mitochondrial association and cytochrome c release were enhanced by the presence of liposomes of lipid composition similar to that of mitochondrial membranes. Thus, a mixture of liposomes, mitochondria and key lysolipids could reproduce the conditions enabling Bid to transfer lipids between donor and acceptor membranes, and also change its reversible association with mitochondria. Bid was also found to enhance the incorporation of a fluorescent lysolipid, but not of a related fatty acid, into mitochondria. On the basis of the results presented here, it is hypothesised that Bid action may depend upon its capacity of exchanging lipids and lysolipids with mitochondrial membranes. The hypothesis is discussed in relation to current models for the integrated action of pro-apoptotic proteins of the Bcl-2 family.     

Diversity and versatility of lipid-protein interactions revealed by molecular genetic approaches

The diversity in structures and physical properties of lipids provides a wide variety of possible interactions with proteins that affect their assembly, organization, and function either at the surface of or within membranes. Because lipids have no catalytic activity, it has been challenging to define many of their precise functions in vivo in molecular terms. Those processes responsive to lipids are attuned to the native lipid environment for optimal function, but evidence that lipids with similar properties or even detergents can sometimes partially replace the natural lipid environment has led to uncertainty as to the requirement for specific lipids. The development of strains of microorganisms in which membrane lipid composition can be genetically manipulated in viable cells has provided a set of reagents to probe lipid functions. These mutants have uncovered previously unrecognized roles for lipids and provided in vivo verification for putative functions described in vitro. In this review, we summarize how these reagent strains have provided new insight into the function of lipids. The role of specific lipids in membrane protein folding and topological organization is reviewed. The evidence is summarized for the involvement of anionic lipid-enriched domains in the organization of amphitropic proteins on the membrane surface into molecular machines involved in DNA replication and cell division.     

The role of the phosphoinositides at the Golgi complex

Eukaryotic cells are organized into a complex system of subcompartments, each with its distinct protein and lipid composition. A continuous flux of membranes crosses these compartments, and in some cases direct connections exist between the different organelles. It is thus surprising that they can maintain their individual identities. Small GTPases and the phosphoinositides have emerged as the key regulators in the maintenance of the identity of the Golgi complex. This property is due to their ability to act either alone or, more often, in combination, as cues directing and controlling the recruitment of proteins that possess phosphoinositide-binding domains. Among these many proteins there are the lipid transfer proteins, which can transfer ceramide, oxysterol, cholesterol and possibly glucosylceramide. By regulating these lipid transfer proteins in this way, this binomial combination of the small GTPases and the phosphoinositides acquires a further important role: control of the synthesis and/or distribution of other important integral constituents of cell organelles, such as the sphingolipids and cholesterol. This role is particularly relevant at the level of the Golgi complex, a key organelle in the biosynthesis, transport and sorting of both lipids and proteins that is located at the intersection of the secretory and endocytic pathways.     

Synthesis and biosynthetic trafficking of membrane lipids

Eukaryotic cells can synthesize thousands of different lipid molecules that are incorporated into their membranes. This involves the activity of hundreds of enzymes with the task of creating lipid diversity. In addition, there are several, typically redundant, mechanisms to transport lipids from their site of synthesis to other cellular membranes. Biosynthetic lipid transport helps to ensure that each cellular compartment will have its characteristic lipid composition that supports the functions of the associated proteins. In this article, we provide an overview of the biosynthesis of the major lipid constituents of cell membranes, that is, glycerophospholipids, sphingolipids, and sterols, and discuss the mechanisms by which these newly synthesized lipids are delivered to their target membranes.     

Lipid domains in bacterial membranes and the action of antimicrobial agents

There has been increasing interest in recent years in describing the lateral organization of membranes and the formation of membrane domains. Much of the focus in this area has been on the formation of cholesterol-rich domains in mammalian membranes. However, it is likely that there are domains in all biological membranes. One of the challenges has been to define the chemical composition, lifetime and size of these domains. There is evidence that bacteria have domains that are enriched in cardiolipin. In addition, the formation of lipid domains can be induced in bacteria by clustering negatively charged lipids with polycationic substances. Many antimicrobial compounds have multiple positive charges. Such polycationic compounds can sequester anionic lipids to induce lipid phase separation. The molecular interactions among lipids and their lateral packing density will be different in a domain from its environment. This will lead to phase boundary defects that will lower the permeability barrier between the cell and its surroundings. The formation of these clusters of anionic lipids may also alter the stability or composition of existing membrane domains that may affect bacterial function. Interestingly many antimicrobial agents are polycationic and therefore likely have some effect in promoting lipid phase segregation between anionic and zwitterionic lipids. However, this mechanism is expected to be most important for substances with sequential positive charges contained within a flexible molecule that can adapt to the arrangement of charged groups on the surface of the bacterial cell. When this mechanism is dominant it can allow the prediction of the bacterial species that will be most affected by the agent as a consequence of the nature of the lipid composition of the bacterial membrane.     

Lipid domains in bacterial membranes and the action of antimicrobial agents

There has been increasing interest in recent years in describing the lateral organization of membranes and the formation of membrane domains. Much of the focus in this area has been on the formation of cholesterol-rich domains in mammalian membranes. However, it is likely that there are domains in all biological membranes. One of the challenges has been to define the chemical composition, lifetime and size of these domains. There is evidence that bacteria have domains that are enriched in cardiolipin. In addition, the formation of lipid domains can be induced in bacteria by clustering negatively charged lipids with polycationic substances. Many antimicrobial compounds have multiple positive charges. Such polycationic compounds can sequester anionic lipids to induce lipid phase separation. The molecular interactions among lipids and their lateral packing density will be different in a domain from its environment. This will lead to phase boundary defects that will lower the permeability barrier between the cell and its surroundings. The formation of these clusters of anionic lipids may also alter the stability or composition of existing membrane domains that may affect bacterial function. Interestingly many antimicrobial agents are polycationic and therefore likely have some effect in promoting lipid phase segregation between anionic and zwitterionic lipids. However, this mechanism is expected to be most important for substances with sequential positive charges contained within a flexible molecule that can adapt to the arrangement of charged groups on the surface of the bacterial cell. When this mechanism is dominant it can allow the prediction of the bacterial species that will be most affected by the agent as a consequence of the nature of the lipid composition of the bacterial membrane.     

Lipid synthesis and transport are coupled to regulate membrane lipid dynamics in the endoplasmic reticulum

Structural lipids are mostly synthesized in the endoplasmic reticulum (ER), from which they are actively transported to the membranes of other organelles. Lipids can leave the ER through vesicular trafficking or non-vesicular lipid transfer and, curiously, both processes can be regulated either by the transported lipid cargos themselves or by different secondary lipid species. For most structural lipids, transport out of the ER membrane is a key regulatory component controlling their synthesis. Distribution of the lipids between the two leaflets of the ER bilayer or between the ER and other membranes is also critical for maintaining the unique membrane properties of each cellular organelle. How cells integrate these processes within the ER depends on fine spatial segregation of the molecular components and intricate metabolic channeling, both of which we are only beginning to understand. This review will summarize some of these complex processes and attempt to identify the organizing principles that start to emerge. This article is part of a Special Issue entitled Endoplasmic reticulum platforms for lipid dynamics edited by Shamshad Cockcroft and Christopher Stefan.     

SHIP164 is a chorein motif lipid transfer protein that controls endosome–Golgi membrane traffic

Cellular membranes differ in protein and lipid composition as well as in the protein–lipid ratio. Thus, progression of membranous organelles along traffic routes requires mechanisms to control bilayer lipid chemistry and their abundance relative to proteins. The recent structural and functional characterization of VPS13-family proteins has suggested a mechanism through which lipids can be transferred in bulk from one membrane to another at membrane contact sites, and thus independently of vesicular traffic. Here, we show that SHIP164 (UHRF1BP1L) shares structural and lipid transfer properties with these proteins and is localized on a subpopulation of vesicle clusters in the early endocytic pathway whose membrane cargo includes the cation-independent mannose-6-phosphate receptor (MPR). Loss of SHIP164 disrupts retrograde traffic of these organelles to the Golgi complex. Our findings raise the possibility that bulk transfer of lipids to endocytic membranes may play a role in their traffic.     

Membrane lipid therapy: Modulation of the cell membrane composition and structure as a molecular base for drug discovery and new disease treatment

Nowadays we understand cell membranes not as a simple double lipid layer but as a collection of complex and dynamic protein–lipid structures and microdomains that serve as functional platforms for interacting signaling lipids and proteins. Membrane lipids and lipid structures participate directly as messengers or regulators of signal transduction. In addition, protein–lipid interactions participate in the localization of signaling protein partners to specific membrane microdomains. Thus, lipid alterations change cell signaling that are associated with a variety of diseases including cancer, obesity, neurodegenerative disorders, cardiovascular pathologies, etc. This article reviews the newly emerging field of membrane lipid therapy which involves the pharmacological regulation of membrane lipid composition and structure for the treatment of diseases. Membrane lipid therapy proposes the use of new molecules specifically designed to modify membrane lipid structures and microdomains as pharmaceutical disease-modifying agents by reversing the malfunction or altering the expression of disease-specific protein or lipid signal cascades. Here, we provide an in-depth analysis of this emerging field, especially its molecular bases and its relevance to the development of innovative therapeutic approaches.     

Protein–lipid interactions studied with designed transmembrane peptides: role of hydrophobic matching and interfacial anchoring (Review)

Biological membranes are characterized by a heterogeneous composition, which is not only manifested in the wide variety of their components, but also in aspects like the lateral organization, topology, and conformation of proteins and lipids. In bringing about the correct membrane structure, protein–lipid interactions can be expected to play a prominent role. The extent of hydrophobic matching between transmembrane protein segments and lipids potentially constitutes a versatile director of membrane organization, because a tendency to avoid hydrophobic mismatch could result in compensating adaptations such as tilt of the transmembrane segment or segregation into distinct domains. Also, interfacial interactions between lipid headgroups and the aromatic and charged residues that typically flank transmembrane domains may act as an organizing element. In this review, we discuss the numerous model studies that have systematically explored the influence of hydrophobic matching and interfacial anchoring on membrane structure. Designed peptides consisting of a polyleucine or polyleucine/alanine hydrophobic stretch, which is flanked on both sides by tryptophan or lysine residues, reflect the general layout of transmembrane protein segments. It is shown for phosphatidylcholine bilayers and for other model membranes that these peptides adapt a transmembrane topology without extensive peptide or lipid adaptations under conditions of hydrophobic matching, but that significant rearrangements can result from hydrophobic mismatch. Moreover, these effects depend on the nature of the flanking residues, implying a modulation of the mismatch response by interfacial interactions of the flanking residues. The implications of these model studies for the organization of biomembranes are discussed in the context of recent experiments with more complex systems.     

Is the protein/lipid hydrophobic matching principle relevant to membrane organization and functions?

Biological membranes are complex and well-organized multimolecular assemblies composed of a wide variety of protein and lipid molecular species. If such a diversity in protein and lipid polar headgroup structures may easily be related to a large panel of functions, the wide dispersion in acyl chain length and structure which the lipids display is more difficult to understand. It is not required for maintaining bilayer assembly and fluidity. Direct information on the lateral distribution of these various molecular species, on their potential specificity for interaction between themselves and with proteins and on the functional implications of these interactions is also still lacking. Because hydrophobic interactions play a major role in stabilizing membrane structures, we suggest considering the problem from the point of view of the matching of the hydrophobic surface of proteins by the acyl chains of the lipids. After a brief introduction to the hydrophobic matching principle, we will present experimental results which demonstrate the predictive power of the current theories and then, we will introduce the new and important concept of protein/lipid sorting in membranes. Finally, we will show how the hydrophobic matching condition may play a key role in the membrane organization and function.