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Stimulation of monocytes with immunomodulating agents can harness the immune system to treat a long range of diseases, including cancers, infections and autoimmune diseases. To this end we aimed to develop a monocyte-targeting delivery platform based on cationic liposomes, which can be utilized to deliver immunomodulators and thus induce monocyte-mediated immune responses while avoiding off-target side-effects. The cationic liposome design is based on functionalizing the liposomal membrane with a cholesterol-anchored tri-arginine peptide (TriArg). We demonstrate that TriArg liposomes can target monocytes with high specificity in both human and murine blood and that this targeting is dependent on the content of TriArg in the liposomal membrane. In addition, we show that the mechanism of selective monocyte targeting involves the CD14 co-receptor, and selectivity is compromised when the TriArg content is increased, resulting in complement-mediated off-target uptake in granulocytes. The presented mechanistic findings of uptake by peripheral blood leukocytes may guide the design of future drug delivery systems utilized for immunotherapy. STATEMENT OF SIGNIFICANCE: Monocytes are attractive targets for immunotherapies of cancers, infections and autoimmune diseases. Specific delivery of immunostimulatory drugs to monocytes is typically achieved using ligand-targeted drug delivery systems, but a simpler approach is to target monocytes using cationic liposomes. To achieve this, however, a deep understanding of the mechanisms governing the interactions of cationic liposomes with monocytes and other leukocytes is required. We here investigate these interactions using liposomes incorporating a cationic arginine-rich lipopeptide. We demonstrate that monocyte targeting can be achieved by fine-tuning the lipopeptide content in the liposomes. Additionally, we reveal that the CD14 receptor is involved in the targeting process, whereas the complement system is not. These mechanistic findings are critical for future design of monocyte-targeting liposomal therapies.

Biomimetic high-density lipoproteins (b-HDL) have in the past two decades been applied for various drug delivery applications. As b-HDL inherently have relatively long circulation half-life and high tumor accumulation, this has inspired researchers to use b-HDL to selectively deliver drugs to tumors. PEGylation of the b-HDL has been pursued to increase the circulation half-life and therapeutic efficacy even further. The b-HDL consist of lipids stabilized by a protein/peptide scaffold, and while PEGylation of the scaffold has been shown to greatly increase the circulation half-life of the scaffold, the effect of PEGylation of the lipids is much less significant. Still, it remains to be evaluated how the biological fate, including cellular uptake, biodistribution, and circulation half-life, of the b-HDL lipids is affected by PEGylation of the b-HDL scaffold. We studied this with apolipoprotein A-I (apoA-I)-based b-HDL and mono-PEGylated b-HDL (PEG b-HDL) both in vitro and in vivo. We found that PEGylation of the b-HDL scaffold only seemed to have minimal effect on the biological fate of the lipids. Both b-HDL and PEG b-HDL overall shared similar biological fates, which includes cellular uptake through the scavenger receptor class B type 1 (SR-BI) and relatively high tumor accumulation. This highlights that b-HDL are dynamic particles, and the biological fates of the b-HDL components (lipids and scaffold) can differ. A phenomenon that may also apply for other multicomponent nanoparticles.

Liposomes are the most used drug delivery vehicle and their therapeutic function is closely linked to their lipid composition. Since most liposome characterization is done using bulk techniques, providing only ensemble averages, the lipid composition of all liposomes within the same formulation are typically assumed to be identical. Here we image individual liposomes using confocal microscopy to quantify that liposomal drug delivery formulations, including multiple component mixtures mimicking Doxil, display more than 10-fold variation in their relative lipid composition. Since liposome function is tightly regulated by the physicochemical properties bestowed by the lipid composition, such significant variations could render only a fraction of liposomes therapeutically active. Additionally, we quantified how this degree of compositional inhomogeneity was modulated by liposome preparation method, the saturation state of the membrane lipid, and whether anti-fouling polyethylene glycol (PEG) conjugated lipids were added to the initial lipid mix or inserted after liposome formation. We believe the insights into the factors governing the degree of inhomogeneity offers the possibility for producing more uniform liposomal drug delivery systems, potentially increasing their therapeutic efficacy.

Biological membranes have distinct geometries that confer specific functions. However, the molecular mechanisms underlying the phenomenological geometry/function correlations remain elusive. We studied the effect of membrane geometry on the localization of membrane-bound proteins. Quantitative comparative experiments between the two most abundant cellular membrane geometries, spherical and cylindrical, revealed that geometry regulates the spatial segregation of proteins. The measured geometry-driven segregation reached 50-fold for membranes of the same mean curvature, demonstrating a crucial and hitherto unaccounted contribution by Gaussian curvature. Molecular-field theory calculations elucidated the underlying physical and molecular mechanisms. Our results reveal that distinct membrane geometries have specific physicochemical properties and thus establish a ubiquitous mechanistic foundation for unravelling the conserved correlations between biological function and membrane polymorphism., Cellular organelles display highly conserved morphologies, e.g., cylindrical (tubes) or spherical (vesicles), and here we show that their Gaussian curvature differences can regulate protein recruitment.

Targeting nanocarrier drug delivery systems, that deliver drug payloads to the site of disease action, are frequently viewed as the future of nanocarrier based therapies but have struggled to breakthrough to the clinic in comparison to non-targeting counterparts. Using unilamellar liposomes as model nanocarriers, we show that cell targeting strategy (electrostatic, ligand and antigen) influences both the intracellular fate of the liposomes and the corresponding efficacy of the loaded drug, doxorubicin, in endothelial cells. We show that increased liposome uptake by cells does not translate to improved efficacy in this scenario but that liposome intracellular trafficking, particularly distribution between recycling endosomes and lysosomes, influences in vitro efficacy. Choosing targeting strategies that promote desired nanocarrier intracellular trafficking may be a viable strategy to enhance the in vivo efficacy of drug delivery systems.

Membrane-active peptides (MAPs) have several potential therapeutic uses, including as antimicrobial drugs. Many traditional methods used to evaluate the membrane interactions of MAPs have limited applicability. Low-throughput methods, such as microscopy, provides detailed information but often relies on fluorophore-labeled MAPs, and high-throughput assays, such as the calcein release assay, cannot assess the mechanism behind the disruption of vesicular-based lipid membranes. Here we present a flow cytometric assay that provides detailed information about the peptide-lipid membrane interactions on single artificial lipid vesicles while being high-throughput (1000–2000 vesicles/s) and based on label-free MAPs. We synthesized and investigated six MAPs with different modes of action to evaluate the versatility of the assay. The assay is based on the flow cytometric readouts from artificial lipid vesicles, including the fluorescence from membrane-anchored and core-encapsulated fluorophores, and the vesicle concentration. From these parameters, we were able to distinguish between MAPs that induce vesicle solubilization, permeation (pores/membrane distortion), and aggregation or fusion. Our flow cytometry findings have been verified by traditional methods, including the calcein release assay, dynamic light scattering, and fluorescence microscopy on giant unilamellar vesicles. We envision that the presented flow cytometric assay can be used for various types of peptide-lipid membrane studies, e.g. to identify new antibiotics. Moreover, the assay can easily be expanded to derive additional valuable information.

Flow cytometry (FCM) is a high-throughput fluorescence-based technique for multiparameter analysis of individual particles, including cells and nanoparticles. Currently, however, FCM does in many cases not permit proper counting of fluorophore-tagged markers on individual particles, due to a lack of tools for translating FCM output intensities into accurate numbers of fluorophores. This lack hinders derivation of detailed biologic information and comparison of data between experiments with FCM. To address this technological void, the authors here use DNA nanotechnology to design and construct barrel-shaped DNA-origami nanobeads for fluorescence/antigen quantification in FCM. Each bead contains a specific number of calibrator fluorophores and a fluorescent trigger domain with an alternative fluorophore for proper detection in FCM. Using electron microscopy, single-particle fluorescence microscopy, and FCM, the design of each particle is verified. To validate that the DNA bead-based FCM calibration enabled the authors to determine the number of antigens on a biological particle, the uniform and well-characterized murine leukemia virus (MLV) is studied. 48 ± 11 envelope surface protein (Env) trimers per MLV is obtained, which is consistent with reported numbers that relied on low-throughput imaging. Thus, the authors’ DNA-beads should accelerate quantitative studies of the biology of individual particles with FCM.

Within nanomedicine, liposomes are investigated for their ability to deliver drug cargoes specifically into subcellular compartments of target cells. Such studies are often based on flow cytometry or microscopy, where researchers rely on fluorescently labeled lipids (FLLs) incorporated into the liposomal membrane to determine the localization of the liposomes within cells. These studies assume that the FLLs stay embedded in the liposomal membrane throughout the duration of the experiment. Here, we used size exclusion chromatography (SEC) to investigate the validity of this assumption by quantitatively determining the propensity of various widely used FLLs to dissociate from liposomes during incubation in human plasma. For certain commonly used off-the-shelf FLLs, up to 75% of the dye dissociated from the liposomes, while others dissociated less than 10%. To investigate the implications of this finding, we measured the peripheral blood leukocyte uptake of liposomes formulated with different FLLs using flow cytometry, and observed a significant difference in uptake correlating with the FLL's dissociation tendencies. Consequently, the choice of FLL can dramatically influence the conclusions drawn from liposome uptake and localization studies due to uptake of dissociated FLLs. The varying dissociation propensities for the FLLs were not reflected when incubating in buffer, showing that non-biological environments are unsuitable to mimic liposomal stability in a drug delivery context. Overall, our findings suggest that it is crucial for researchers to evaluate the stability of their FLL-labeled liposomes in biological environments, and the simplicity of the SEC assay put forward here makes it very applicable for the purpose.

Proteins anchored to membranes through covalently linked fatty acids and/or isoprenoid groups play crucial roles in all forms of life. Sorting and trafficking of lipidated proteins has traditionally been discussed in the context of partitioning to membrane domains of different lipid composition. We recently showed that membrane shape/curvature can in itself mediate the recruitment of lipidated proteins. However, exactly how membrane curvature and composition synergize remains largely unexplored. Here we investigated how three critical structural parameters of lipids, namely acyl chain saturation, headgroup size, and acyl chain length, modulate the capacity of membrane curvature to recruit lipidated proteins. As a model system we used the lipidated minimal membrane anchor of the GTPase, N-Ras (tN-Ras). Our data revealed complex synergistic effects, whereby tN-Ras binding was higher on planar DOPC than POPC membranes, but inversely higher on curved POPC than DOPC membranes. This variation in the binding to both planar and curved membranes leads to a net increase in the recruitment by membrane curvature of tN-Ras when reducing the acyl chain saturation state. Additionally, we found increased recruitment by membrane curvature of tN-Ras when substituting PC for PE, and when decreasing acyl chain length from 14 to 12 carbons (DMPC versus DLPC). However, these variations in recruitment ability had different origins, with the headgroup size primarily influencing tN-Ras binding to planar membranes whereas the change in acyl chain length primarily affected binding to curved membranes. Molecular field theory calculations recapitulated these findings and revealed lateral pressure as an underlying biophysical mechanism dictating how curvature and composition synergize to modulate recruitment of lipidated proteins. Our findings suggest that the different compositions of cellular compartments could modulate the potency of membrane curvature to recruit lipidated proteins and thereby synergistically regulate the trafficking and sorting of lipidated proteins.

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

A key prerequisite for image-based research on nanocarriers for drug delivery is that the recorded fluorescence can be accurately assigned to originate from the nanocarrier in question. For liposomal nanocarriers, fluorescent labeling is typically achieved by labeling a minority of the lipid species making up the liposome. Early work determined that lipid species can transfer between membrane components within a solution, nevertheless the fluorescently labeled lipids (FLLs) of drug delivery liposomes are intrinsically assumed to stay associated with the liposome, even when placed in a biological environment. To efficiently test this assumption, routine methods capable of investigating the dissociation of FLLs from liposomes should be implemented. Here we present two experimental methodologies able to quantitatively characterize the degree of FLL dissociation from liposomes when subjected to human blood plasma, mimicking the biological environment experienced by the carrier when travelling in the human body. Routine implementation of such methodologies could facilitate the appropriate selection of FLLs displaying low liposome dissociation, hereby facilitating more reliable liposomal uptake and trafficking studies.

The use of high-throughput flow cytometry to characterize nanoparticles has received increased interest in recent years. However, to fully realize the potential of flow cytometry for the characterization of nanometer-sized objects, suitable calibrators for size estimation must be developed and the sensitivity of conventional flow cytometers has to be advanced. Based on the scattered signal, silica and plastic beads have often been used as flow cytometric size calibrators to evaluate the size of extracellular vesicles and artificial vesicles (liposomes). However, several studies have shown that these beads are unable to accurately correlate scatter intensity to vesicle size. In this work, we present a novel method to estimate the size of individual liposomes in flow cytometry based on liposomal size calibrators prepared by fluorescence-activated cell sorting (FACS), here coined fluorescence-activated nanoparticle sorting (FANS). These calibration liposomes exhibit sizes, structures, and refractive indexes identical to the particles being studied and thus can serve as unique calibrators. First, a sample of polydisperse fluorophore-labeled unilamellar liposomes was prepared and analyzed by flow cytometry. Next, different fractions of the polydisperse liposomes were FANS-sorted according to their fluorescence intensity. Thereafter, we employed nanoparticle tracking analysis (NTA) to evaluate the liposome sizes of the FANS-sorted liposome fractions. Finally, we correlated the flow cytometric readouts (side scatter and fluorescence intensity) of the FANS-sorted liposome fractions with their corresponding size obtained by NTA. This procedure enabled us to translate the liposome fluorescence intensity to the liposome size in nanometers for all detected individual liposomes. We validated the size distribution of our polydisperse liposome sample obtained from flow cytometry in combination with our FANS-calibrators against standard methods for sizing nanoparticles, including NTA and cryo-transmission electron microscopy. This work also highlights the limitation of using the flow cytometric side scattering readout to determine the size of small (30-300 nm) artificial vesicles. © 2019 International Society for Advancement of Cytometry.

Summary BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila , disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis.

Protein recruitment to biological membranes is motivated by either highly selective recognition of specific target membrane components or non-specific attraction to general physical properties of the membrane, such as charge, lipid heterogeneity, and curvature. Here we discuss the interaction between lipid-anchored proteins and lipid membranes from a comprehensive examination of how features of the membrane and its lipid constituents, including lipid head-group size, composition, heterogeneity, membrane thickness, degree of unsaturation, and membrane geometry, effect the adsorption ability of the proteins. Of key importance is the strong interconnection among these compositional and morphological elements in mediating the binding of peripheral membrane proteins. As a model protein, we use the dual lipidated (palmitoyl and farnesyl) anchoring motif of the signaling GTPase N-Ras (tN-Ras). We find marked augmentation in tN-Ras adsorption with increasing degree of membrane curvature—a trend that is tightly regulated by the bilayer characteristics mentioned above. Experimental results are fully reproduced by a molecular level theoretical model of the systems under study. Of note, the theory suggests an explicit dependence on the lateral pressure profile of the membrane's hydrophobic region to be the mechanism and cause of variation in protein density with membrane curvature and composition. Relief in the lateral pressure of the bilayer's outer leaf, upon its expansion induced by increasing curvature, reduces the work requirement for lipid-anchor insertion into the membrane. Furthermore, the inherent pressure profile of the hydrophobic channel, at a given curvature, is unique with regard to membrane composition, which allows for fine-tuning of lipidated-protein density.

Membrane shape or geometrical curvature emerged recently as a potent regulator of membrane recruitment during protein trafficking and sorting. Cellular membranes display distinct curvature geometries e.g. spherical (trafficking- and synaptic vesicles) or cylindrical (tubes in the ER and Golgi), however quantitative studies of protein recruitment by membrane curvature typically focus on a single geometry. Thus the biological implications of different curvature geometries remain largely unexplored.We recently used our single liposome assay to show that the N-Ras lipid anchor (tN-Ras) is recruited by spherical membrane curvature. Here we report the development of a novel membrane tube assay, allowing us to quantitatively compare the recruitment of tN-Ras by spherical and cylindrical membrane curvature. Furthermore we expand the study to include representatives of the four most common families of membrane-binding domains (MBDs); the lipid anchor of N-Ras (tN-Ras), the C2AB-domain of Synaptotagmin1 (Syt), AnnexinB12 (Anx) and Amphiphysin NBAR (Amph). Our data revealed an increased recruitment of all four MBDs by spherical as compared to cylindrical curvature. Molecular field theory calculations attributed this trend to the greater perturbation of lipid packing parameters by spherically curved membranes. Importantly, the four MBDs displayed markedly distinct abilities to discriminate the two curvature geometries e.g. tN-Ras had a moderate 2-fold discrimination while Amph a remarkable absolute preference for spherical membranes. This demonstrated that discrimination of curvature geometry can be modulated and likely adapted to specific biological functions. Our results reveal membrane curvature geometry as a novel regulator of protein recruitment during trafficking and sorting for a plethora of membrane-binding proteins, and thus suggest a novel functional role to the diversity of conserved organelle morphologies.

A confocal fluorescence microscopy-based assay was used for studying the influence of the preparation route on the supramolecular organization of lipids in a vesicular system. In this work, vesicles composed of cholesterol and CTAB (1/1 mol %) or cholesterol and DOPC (2/8 mol %) and incorporating two membrane dyes were prepared by either a compressed fluid (CF)-based method (DELOS-susp) or a conventional film hydration procedure. They were subsequently immobilized and imaged individually using a confocal fluorescence microscope. Two integrated fluorescence intensities, I(dye1) and I(dye2), were assigned to each tracked vesicle, and their ratio, I(dye1)/I(dye2), was used for quantifying the degree of membrane inhomogeneity between individual vesicles within each sample. A distribution of I(dye1)/I(dye2) values was obtained for all the studied vesicular systems, indicating intrasample heterogeneity. The degree of inhomogeneity (DI) was similar for Chol/DOPC vesicles prepared by both procedures. In contrast, DI was more than double for the hydration method compared to the CF-based method in the case of Chol/CTAB vesicles, which can suffer from lipid demixing during film formation. These findings reveal a more homogeneous vesicle formation path by CFs, which warranted good homogeneity of the vesicular system, independently of the lipid mixture used.

A 'chemical biology of cellular membranes' must capture the way that mesoscale perturbations tune the biochemical properties of constituent lipid and protein molecules and vice versa. Whereas the classical paradigm focuses on chemical composition, dynamic modulation of the physical shape or curvature of a membrane is emerging as a complementary and synergistic modus operandi for regulating cellular membrane biology.

In vivo studies have reported preferential partitioning of Ras GTPases into ordered lipid-protein membrane domains, a process believed to regulate both cellular signaling and protein trafficking.1 However studies in vitro have failed to quantify a preferential partitioning of full length Ras proteins into the liquid ordered phase2,3 and thus a biophysically validated mechanism for in vivo sorting of Ras is still missing. We recently showed that lipidated proteins localize to highly curved membranes in vitro.4 Here we study both in vitro and in vivo whether recruitment by membrane curvature can sort full length lipid-anchored Ras.We employ a single vesicle fluorescence based assay to quantify in vitro the sorting by membrane curvature of full-length Ras proteins. We demonstrate a more than 50 fold increase in protein density on membranes of high curvature as compared to the density on flat membranes. To test for recruitment by membrane curvature in vivo we utilize hypo-osmotic swelling of cells, which flattens curved membrane regions. By measuring the local protein density using FRET,5 we detect a significant reduction in the clustering of Ras and other lipidated proteins upon membrane flattening. This demonstrates that recruitment by membrane curvature can sort Ras and potentially other lipidated proteins in cellular membranes. Furthermore sorting by membrane curvature constitutes the first biophysical sorting mechanism for Ras validated by both in vitro and in vivo measurements.1 Hancock, J. F. Nat. Rev. Mol. Cell Biol.4 (2003).2 Johnson, S. A. et al.Biochim. Biophys. Acta - Biomembranes1798 (2010).3 Nicolini, C. et al.J. Am. Chem. Soc.128 (2006).4 Hatzakis, N. S. et al.Nat. Chem. Biol.5 (2009).5 Kohnke, M. et al.Chem. Biol.19 (2012).

Liposomes are one of the most extensively used model systems for studying the physical and chemical properties of membranes and the majority of these studies have implicitly assumed that all liposomes in an ensemble are identical. However recent measurements performed at the level of single liposomes revealed the existence of intrinsic intra-sample heterogeneities that were otherwise averaged out in ensemble experiments.By employing fluorescently labeled lipids and measuring at the single liposome level we examined the compositional inhomogeneity between individual liposomes within an ensemble. In a recent publication we demonstrated an up to ten-fold variation in the relative lipid composition of individual liposomes with diameters between 50 nm and 15 μm.1 This observation is made for both a variety of different lipid-labels and compositions, which suggest that compositional inhomogeneity is a general phenomenon present in liposome systems. As a result, bulk measurement of physical and chemical properties that depend on bilayer composition, e.g. phase-transition temperature, will produce results representing the ensemble average, which will be a convolution of the properties arising from many different bilayer compositions of the individual liposomes. Furthermore, we saw that the choice of liposome preparation method greatly influences the degree of compositional inhomogeneity. This could be particularly important when using liposomes as drug delivery carriers where the monodispersity of, and control over, bilayer properties is instrumental.Since the physicochemical properties of liposomes are directly linked to their composition a direct consequence of compositional inhomogeneities is a polydispersity in the properties of the individual liposomes in an ensemble.(1) Larsen, J.; Hatzakis, N. S.; Stamou, D. J. Am. Chem. Soc. 2011, 133, 10685-10687.

Liposomes, or vesicles, have been studied extensively both as models of biological membranes and as drug delivery vehicles. Typically it is assumed that all liposomes within the same preparation are identical. Here by employing pairs of fluorescently labeled lipids we demonstrated an up to 10-fold variation in the relative lipid composition of individual liposomes with diameters between 50 nm and 15 μm. Since the physicochemical properties of liposomes are directly linked to their composition, a direct consequence of compositional inhomogeneities is a polydispersity in the properties of the individual liposomes in an ensemble.

A critical feature of neurotransmission is that only a subset of vesicles react at a given Ca++ pulse. To achieve such a fractional response the synapse utilises a number of complex spatio-temporal and biochemical mechanisms that are still under debate but collectively appear to create distinct vesicle pools. We currently ignore whether differentiation of seemingly identical vesicles exists also for membrane trafficking vesicles.We recently established that vesicles within an ensemble display large compositional inhomogeneities(1). Annexins, as well as other Ca2+-dependent membrane binding proteins, are known to bind specific phospholipid components in cellular membranes(2). Here we investigated how the unique capability of Ca2+-regulated membrane-binding proteins to bind certain lipids enables them to discriminate between single vesicles based on their lipid compositional heterogeneity. We employed human Annexin A5 (AnxA5), which is widely used as a model system for the membrane binding properties of the Annexin superfamily(2), and our previously described single vesicle assay(3, 4). We demonstrate that heterogeneities in the lipid composition of individual vesicles within a population, in conjunction with Ca2+ concentration, regulate the fraction of vesicles recruiting Annexins with digital precision. This is a recurrent phenomenon observed in multiple vesicle samples and for different Ca2+ sensing proteins. Thus, we anticipate that the cell utilize this feature to translate analog Ca2+ influxes into a selective digital targeting of vesicle sub-populations within the ensemble for specific functions.1. J. Am. Chem. Soc. 133: 10685-10687.2. Nat Rev Mol Cell Biol. 6: 449-461.3. The EMBO Journal. 28: 3303-3314.4. Nat. Chem. Biol. 5: 835-841.

Lipids and several specialized proteins are thought to be able to sense the curvature of membranes (MC). Here we used quantitative fluorescence microscopy to measure curvature-selective binding of amphipathic motifs on single liposomes 50-700 nm in diameter. Our results revealed that sensing is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity. We proposed a model based on curvature-induced defects in lipid packing that related these findings to lipid sorting and accurately predicted the existence of a new ubiquitous class of curvature sensors: membrane-anchored proteins. The fact that unrelated structural motifs such as alpha-helices and alkyl chains sense MC led us to propose that MC sensing is a generic property of curved membranes rather than a property of the anchoring molecules. We therefore anticipate that MC will promote the redistribution of proteins that are anchored in membranes through other types of hydrophobic moieties.

Sorting and trafficking of membrane-anchored Ras GTPases is critical for signaling and is believed to rely on their preferential portioning in ordered lipid-protein membrane domains (1). However studies in vitro have failed to quantify the preferential partitioning of full length Ras proteins into the liquid ordered phase(2), indicating that a physical principle underlying sorting of Ras is missing. We recently showed that lipidated proteins localize to highly curved membranes in vitro(3, 4). Here we provide a mechanistic insight on how membrane curvature can drive N-Ras sorting.Combining the results of our in vitro assays, measurements on single vesicles, with in vivo studies, hypo-osmotic swelling of cells that flattens curved membrane regions, revealed that : a) N-Ras is preferentially recruited in areas of high membrane curvature and b) membrane curvature is the enabling factor underlying the selective partitioning of NRas in ordered domains. The combined readout of mean field theory calculations and coarse grain simulations provided a mechanistic insight on preferential partitioning in highly curved areas, via the changes in lateral pressure of the outer monolayer when curving an ordered versus a disordered membrane. In addition to providing the first biophysical sorting mechanism for Ras validated by both in vitro and in vivo measurements, our data indicate that membrane curvature may act as a generic cue underlying trafficking and sorting of multiple lipidated proteins.References1. J. F. Hancock, Nat. Rev. Mol. Cell Biol. 4, 373 (2003).2. S. A. Johnson Biochim. Biophys. Acta - Biomembranes 1798, 1427 (2010).3. N. S. Hatzakis et al., Nat. Chem. Biol. 5, 835 (2009).4. V. K. Bhatia, et al Semin. Cell Dev. Biol. 21, 381 (2010).

Ras proteins are small GTPases that are post-translationally modified by the attachment of lipid moieties (1). This modification is essential for the correct trafficking and sorting of Ras proteins through the vesicular pathway from the Golgi to the plasma membrane (2).Traditionally the sorting of Ras is primarily discussed in the context of membrane domains in flat membranes, neglecting the influence of membrane shape. Recently we have demonstrated, utilizing our single liposome curvature (SLiC) assay that the minimal anchoring motif of N-Ras (tN-Ras) up-concentrates in areas of high membrane curvature (not published), suggesting that curvature might act as a cue for the spatial localization of Ras proteins.In the SLiC assay, curvature-sensing molecules are added from aqueous solution to vesicles of different curvatures, but the vesicles are not in diffusive contact (3, 4). In vivo Ras proteins are anchored to membranes and laterally sorts between curved and planar membranes, which are in diffusive contact. To study the curvature-sensing ability of tN-Ras in a setup mimicking the in vivo scenario we developed a membrane tube based assay in which the tubes are in diffusive contact with a lipid bilayer.Membrane tubes are formed by heating a confined lipid bilayer (5). The tubes eventually adsorb to the flat membrane, which enable imaging by confocal fluorescence microscopy. After addition of tN-Ras we observed a preferential sorting into curved tubes rather than the flat bilayer. This observation further implies a pivoting role of membrane shape as a regulator of Ras-protein localization.1. Prior & Hancock, Semin Cell Dev Biol 23:145(2012).2. Choy et al., Cell 98:69(1999).3. Kunding et al., Biophysical Journal 95:1176(2008).4. Hatzakis et al., Nat Chem Biol 5:835(2009).5. Weirich & Fygenson, Poster Abstract 2731, Biophys. Soc. 2011.