Dimitrios Stamou

How curved membranes recruit amphipathic helices and protein anchoring motifs

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.

Amphipathic motifs in BAR domains are essential for membrane curvature sensing

BAR (Bin/Amphiphysin/Rvs) domains and amphipathic α‐helices (AHs) are believed to be sensors of membrane curvature thus facilitating the assembly of protein complexes on curved membranes. Here, we used quantitative fluorescence microscopy to compare the binding of both motifs on single nanosized liposomes of different diameters and therefore membrane curvature. Characterization of members of the three BAR domain families showed surprisingly that the crescent‐shaped BAR dimer with its positively charged concave face is not able to sense membrane curvature. Mutagenesis on BAR domains showed that membrane curvature sensing critically depends on the N‐terminal AH and furthermore that BAR domains sense membrane curvature through hydrophobic insertion in lipid packing defects and not through electrostatics. Consequently, amphipathic motifs, such as AHs, that are often associated with BAR domains emerge as an important means for a protein to sense membrane curvature. Measurements on single liposomes allowed us to document heterogeneous binding behaviour within the ensemble and quantify the influence of liposome polydispersity on bulk membrane curvature sensing experiments. The latter results suggest that bulk liposome‐binding experiments should be interpreted with great caution.

Membrane Curvature Induction and Tubulation Are Common Features of Synucleins and Apolipoproteins

Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, β-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.

An Integrated Self-Assembled Nanofluidic System for Controlled Biological Chemistries†

Reference EPFL-ARTICLE-148631doi:10.1002/anie.200801606View record in Web of Science Record created on 2010-04-30, modified on 2017-05-12

Heat Profiling of Three-Dimensionally Optically Trapped Gold Nanoparticles using Vesicle Cargo Release

Irradiated metallic nanoparticles hold great promise as heat transducers in photothermal applications such as drug delivery assays or photothermal therapy. We quantify the temperature increase of individual gold nanoparticles trapped in three dimensions near lipid vesicles exhibiting temperature sensitive permeability. The surface temperature can increase by hundreds of degrees Celsius even at moderate laser powers. Also, there are significant differences of the heat profiles in two-dimensional and three-dimensional trapping assays.

Surface-based lipidvesicle reactor systems: fabrication and applications

Over the last ten years there has been a strong (bio)technological drive for the development of miniaturised reaction systems, motivated mainly by the need to reduce sample consumption and parallelise. Self-assembled soft-matter containers have naturally evolved to handle small volumes and could provide viable fluidic solutions especially in niche areas where ultra-miniaturisation, biocompatibility or cost are of critical importance. Here we focus on nanocontainers that are made of lipids and are immobilised on surfaces. We will highlight the most prominent contributions to date on the fabrication and the applications of surface-based vesicle systems as miniaturised reactors. Emphasis will be put on single-vesicle experiments.

A Fluorescence-Based Technique to Construct Size Distributions from Single-Object Measurements: Application to the Extrusion of Lipid Vesicles

We report a novel approach to quantitatively determine complete size distributions of surface-bound objects using fluorescence microscopy. We measure the integrated intensity of single particles and relate it to their size by taking into account the object geometry and the illumination profile of the microscope, here a confocal laser scanning microscope. Polydisperse (as well as monodisperse) size distributions containing objects both below and above the optical resolution of the microscope are recorded and analyzed. The data is collected online within minutes, which allows the user to correlate the size of an object with the response from any given fluorescence-based biochemical assay. We measured the mean diameter of extruded fluorescently labeled lipid vesicles using the proposed method, dynamic light scattering, and cryogenic transmission electron microscopy. The three techniques were in excellent agreement, measuring the same values within 7-9%. Furthermore we demonstrated here, for the first time that we know of, the ability to determine the full size distribution of polydisperse samples of nonextruded lipid vesicles. Knowledge of the vesicle size distribution before and after extrusion allowed us to propose an empirical model to account for the effect of extrusion on the complete size distribution of vesicle samples.

Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules.

Sorting nexins (SNXs) are regulators of endosomal sorting. For the SNX‐BAR subgroup, a Bin/Amphiphysin/Rvs (BAR) domain is vital for formation/stabilization of tubular subdomains that mediate cargo recycling. Here, by analysing the in vitro membrane remodelling properties of all 12 human SNX‐BARs, we report that some, but not all, can elicit the formation of tubules with diameters that resemble sorting tubules observed in cells. We reveal that SNX‐BARs display a restricted pattern of BAR domain‐mediated dimerization, and by resolving a 2.8 Å structure of a SNX1‐BAR domain homodimer, establish that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR‐dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX‐BARs, and the formation of high order SNX‐BAR oligomers through selective ‘tip–loop’ interactions. Overall, the restricted and selective nature of these interactions provide a molecular explanation for how distinct SNX‐BAR‐decorated tubules are nucleated from the same endosomal vacuole, as observed in living cells. Our data provide insight into the molecular mechanism that generates and organizes the tubular endosomal network.

BAR domains, amphipathic helices and membrane-anchored proteins use the same mechanism to sense membrane curvature

The internal membranes of eukaryotic cells are all twists and bends characterized by high curvature. During recent years it has become clear that specific proteins sustain these curvatures while others simply recognize membrane shape and use it as “molecular information” to organize cellular processes in space and time. Here we discuss this new important recognition process termed membrane curvature sensing (MCS). First, we review a new fluorescence‐based experimental method that allows characterization of MCS using measurements on single vesicles and compare it to sensing assays that use bulk/ensemble liposome samples of different mean diameter. Next, we describe two different MCS protein motifs (amphipathic helices and BAR domains) and suggest that in both cases curvature sensitive membrane binding results from asymmetric insertion of hydrophobic amino acids in the lipid membrane. This mechanism can be extended to include the insertion of alkyl chain in the lipid membrane and consequently palmitoylated and myristoylated proteins are predicted to display similar curvature sensitive binding. Surprisingly, in all the aforementioned cases, MCS is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity as assumed so far. Finally, we integrate these new insights into the debate about which motifs are involved in sensing versus induction of membrane curvature and what role MCS proteins may play in biology.

Membrane Curvature Sensing by Amphipathic Helices A SINGLE LIPOSOME STUDY USING α-SYNUCLEIN AND ANNEXIN B12

Background: Amphipathic helices preferentially bind highly curved lipid membranes, providing a method of protein sorting. Results: Curvature sensing requires the insertion of hydrophobic residues and is modulated by electrostatic interactions. Conclusion: The relative strength of hydrophobic and electrostatic membrane interactions determines whether helix-containing proteins sense curvature. Significance: Sensing cannot be described through simple physicochemical properties but depends on the total sum of membrane interactions. Preferential binding of proteins on curved membranes (membrane curvature sensing) is increasingly emerging as a general mechanism whereby cells may effect protein localization and trafficking. Here we use a novel single liposome fluorescence microscopy assay to examine a common sensing motif, the amphipathic helix (AH), and provide quantitative measures describing and distinguishing membrane binding and sensing behavior. By studying two AH-containing proteins, α-synuclein and annexin B12, as well as a range of AH peptide mutants, we reveal that both the hydrophobic and hydrophilic faces of the helix greatly influence binding and sensing. Although increased hydrophobic and electrostatic interactions with the membrane both lead to greater densities of bound protein, the former yields membrane curvature-sensitive binding, whereas the latter is not curvature-dependent. However, the relative contributions of both components determine the sensing of AHs. In contrast, charge density in the lipid membrane seems important primarily in attracting AHs to the membrane but does not significantly influence sensing. These observations were made possible by the ability of our assay to distinguish within our samples liposomes with and without bound protein as well as the density of bound protein. Our findings suggest that the description of membrane curvature-sensing requires consideration of several factors such as short and long range electrostatic interactions, hydrogen bonding, and the volume and structure of inserted hydrophobic residues.