Patrick Drücker

Lipid segregation and membrane budding induced by the peripheral membrane binding protein annexin A2.

Background: Annexin A2 is a phospholipid binding protein implicated in endo- and exocytosis. Results: Annexin A2 induces microdomain formation and inward vesicle budding in giant unilamellar vesicles. Conclusion: Specific lipid clustering and vesicle budding is triggered by annexin A2. Significance: Membrane binding by the cytosolic protein annexin A2 can induce the formation of microdomains rich in cholesterol and phosphatidylinositol 4,5-bisphosphate. The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P2 and other acidic phospholipids in a Ca2+-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P2 clustering are also capable of forming interconnections between PI(4,5)P2-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P2 and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P2-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.

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.

Active release of pneumolysin prepores and pores by mammalian cells undergoing a Streptococcus pneumoniae attack

BACKGROUND Streptococcus pneumoniae is a potent human pathogen. Its pore-forming exotoxin pneumolysin is instrumental for breaching the hosts epithelial barrier and for the incapacitation of the immune system. METHODS AND RESULTS Using a combination of life imaging and cryo-electron microscopy we show that pneumolysin, released by cultured bacteria, is capable of permeabilizing the plasmalemma of host cells. However, such permeabilization does not lead to cell lysis since pneumolysin is actively removed by the host cells. The process of pore elimination starts with the formation of pore-bearing plasmalemmal nanotubes and proceeds by the shedding of pores that are embedded in the membrane of released microvesicles. Pneumolysin prepores are likewise removed. The protein composition of the toxin-induced microvesicles, assessed by mass spectrometry, is suggestive of a Ca(2+)-triggered mechanism encompassing the proteins of the annexin family and members of the endosomal sorting complex required for transport (ESCRT) complex. CONCLUSIONS S. pneumoniae releases sufficient amounts of pneumolysin to perforate the plasmalemma of host cells, however, the immediate cell lysis, which is frequently reported as a result of treatment with purified and artificially concentrated toxin, appears to be an unlikely event in vivo since the toxin pores are efficiently eliminated by microvesicle shedding. Therefore the dysregulation of cellular homeostasis occurring as a result of transient pore formation/elimination should be held responsible for the damaging toxin action. GENERAL SIGNIFICANCE We have achieved a comprehensive view of a general plasma membrane repair mechanism after injury by a major bacterial toxin.

Imidazolium Salts Mimicking the Structure of Natural Lipids Exploit Remarkable Properties Forming Lamellar Phases and Giant Vesicles.

Tailor-made ionic liquids based on imidazolium salts have recently attracted a large amount of attention because of their extraordinary properties and versatile functionality. An intriguing ability to interact with and stabilize membranes has already been reported for 1,3-dialkylimidazolium compounds. We now reveal further insights into the field by investigating 1,3-dimethyl-4,5-dialkylimidazolium (Cn-IMe·HI, n = 7, 11, 15) and 1,3-dibenzyl-4,5-dialkylimidazolium (Cn-IBn·HBr, n = 7, 11, 15) salts. Diverse alkyl chain lengths and headgroups differing in their steric demand were employed for the membrane interface interaction with bilayer membranes imitating the cellular plasma membrane. Membrane hydration properties and domain fluidization were analyzed by fluorescent bilayer probes in direct comparison to established model membranes in a buffered aqueous environment, which resembles the salt content and pH of the cytosol of living cells. Membrane binding and insertion was analyzed via a quartz crystal microbalance and confocal laser scanning microscopy. We show that short-chain 4,5-dialkylimidazolium salts with a bulky headgroup were able to disintegrate membranes. Long-chain imidazolium salts form bilayer membrane vesicles spontaneously and autonomously without the addition of other lipids. These 4,5-dialkylimidazolium salts are highly eligible for further biochemical engineering and drug delivery.

Formation and characterization of supported lipid bilayers containing phosphatidylinositol-4,5-bisphosphate and cholesterol as functional surfaces.

Solid-supported lipid bilayers (SLBs) mimicking a biological membrane are commonly used to investigate lipid-lipid or lipid-protein interactions. Simple binary or ternary lipid systems are well established, whereas more complex model membranes containing biologically important signaling lipids such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and cholesterol have not been extensively described yet. Here we report the generation of such bilayers and their relevant biophysical properties and in particular the accessibility of PI(4,5)P2 for protein binding. Ternary mixtures of POPC with 20% cholesterol and either 3 or 5 mol % dioleoyl-phosphatidylinositol-4,5-bisphosphate were probed by employing the quartz crystal microbalance and atomic force microscopy. We show that these mixtures form homogeneous solid-supported bilayers that exhibit no intrinsic phase separation and are characterized by long-term stability (>8 h). Bilayers were formed in a pH-dependent manner and were characterized by the accessibility of PI(4,5)P2 on the SLB surface as shown by the interaction with the PI(4,5)P2 binding domain of the cortical membrane-cytoskeleton linker protein ezrin. A time-dependent reduction of PI(4,5)P2 levels in the upper leaflet of SLBs was observed, which could be effectively inhibited by the incorporation of a negatively charged lipid such as phosphatidylserine. Furthermore, quartz crystal microbalance measurements revealed that cholesterol affects bilayer adsorption to the solid support.

Importance of phospholipid bilayer integrity in the analysis of protein–lipid interactions

The integrity of supported phospholipid bilayer membranes is of crucial importance for the investigation of lipid-protein interactions. Therefore we recorded the formation of supported membranes on SiO2 and mica by quartz crystal microbalance and controlled the integrity by atomic force microscopy. This study aims to analyze how membrane defects affect protein-lipid interactions. The experiments focused on a lipid mixture of POPC/DOPC/Chol/POPS/PI(4,5)P2 (37:20:20:20:3) and the binding of the peripheral membrane associated protein annexin A2. We found that formation of a continuous undisturbed bilayer is an indispensable precondition for a reliable determination and quantification of lipid-protein-interactions. If membrane defects were present, protein adsorption causes membrane disruption and lipid detachment on a support thus leading to false determination of binding constants. Our results obtained for PI(4,5)P2 and cholesterol containing supported membranes yield new knowledge to construct functional surfaces that may cover nanoporous substrates, form free standing membranes or may be used for lab-on-a-chip applications.

Host-Derived Microvesicles Carrying Bacterial Pore-Forming Toxins Deliver Signals to Macrophages: A Novel Mechanism of Shaping Immune Responses

Bacterial infectious diseases are a leading cause of death. Pore-forming toxins (PFTs) are important virulence factors of Gram-positive pathogens, which disrupt the plasma membrane of host cells and can lead to cell death. Yet, host defense and cell membrane repair mechanisms have been identified: i.e., PFTs can be eliminated from membranes as microvesicles, thus limiting the extent of cell damage. Released into an inflammatory environment, these host-derived PFTs-carrying microvesicles encounter innate immune cells as first-line defenders. This study investigated the impact of microvesicle- or liposome-sequestered PFTs on human macrophage polarization in vitro. We show that microvesicle-sequestered PFTs are phagocytosed by macrophages and induce their polarization into a novel CD14+MHCIIlowCD86low phenotype. Macrophages polarized in this way exhibit an enhanced response to Gram-positive bacterial ligands and a blunted response to Gram-negative ligands. Liposomes, which were recently shown to sequester PFTs and so protect mice from lethal bacterial infections, show the same effect on macrophage polarization in analogy to host-derived microvesicles. This novel type of polarized macrophage exhibits an enhanced response to Gram-positive bacterial ligands. The specific recognition of their cargo might be of advantage in the efficiency of targeted bacterial clearance.

Pneumolysin-damaged cells benefit from non-homogeneous toxin binding to cholesterol-rich membrane domains

Nucleated cells eliminate lesions induced by bacterial pore-forming toxins, such as pneumolysin via shedding patches of damaged plasmalemma into the extracellular milieu. Recently, we have shown that the majority of shed pneumolysin is present in the form of inactive pre-pores. This finding is surprising considering that shedding is triggered by Ca2+-influx following membrane perforation and therefore is expected to positively discriminate for active pores versus inactive pre-pores. Here we provide evidence for the existence of plasmalemmal domains that are able to attract pneumolysin at high local concentrations. Within such a domain an immediate plasmalemmal perforation induced by a small number of pneumolysin pores would be capable of triggering the elimination of a large number of not yet active pre-pores/monomers and thus pre-empt more frequent and perilous perforation events. Our findings provide further insights into the functioning of the cellular repair machinery which benefits from an inhomogeneous plasmalemmal distribution of pneumolysin.