BIN1 regulates actin-membrane interactions during IRSp53-dependent filopodia formation.

Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation.

Phosphorylation of the F-BAR protein Hof1 drives septin ring splitting in budding yeast.

A double septin ring accompanies cytokinesis in yeasts and mammalian cells. In budding yeast, reorganisation of the septin collar at the bud neck into a dynamic double ring is essential for actomyosin ring constriction and cytokinesis. Septin reorganisation requires the Mitotic Exit Network (MEN), a kinase cascade essential for cytokinesis.

Caveolae and Bin1 form ring-shaped platforms for T-tubule initiation.

Excitation-contraction coupling requires a highly specialized membrane structure, the triad, composed of a plasma membrane invagination, the T-tubule, surrounded by two sarcoplasmic reticulum terminal cisternae. Although the precise mechanisms governing T-tubule biogenesis and triad formation remain largely unknown, studies have shown that caveolae participate in T-tubule formation and mutations of several of their constituents induce muscle weakness and myopathies. Here, we demonstrate that, at the plasma membrane, Bin1 and caveolae composed of caveolin-3 assemble into ring-like structures from which emerge tubes enriched in the dihydropyridine receptor.

The Syp1/FCHo2 protein induces septin filament bundling through its intrinsically disordered domain.

The septin collar of budding yeast is an ordered array of septin filaments that serves a scaffolding function for the cytokinetic machinery at the bud neck and compartmentalizes the membrane between mother and daughter cell. How septin architecture is aided by septin-binding proteins is largely unknown. Syp1 is an endocytic protein that was implicated in the timely recruitment of septins to the newly forming collar through an unknown mechanism.

Structural organization and dynamics of FCHo2 docking on membranes.

Clathrin-mediated endocytosis (CME) is a central trafficking pathway in eukaryotic cells regulated by phosphoinositides. The plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P) plays an instrumental role in driving CME initiation. The F-BAR domain-only protein 1 and 2 complex (FCHo1/2) is among the early proteins that reach the plasma membrane, but the exact mechanisms triggering its recruitment remain elusive.

Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partner.

One challenge in cell biology is to decipher the biophysical mechanisms governing protein enrichment on curved membranes and the resulting membrane deformation. The ERM protein ezrin is abundant and associated with cellular membranes that are flat, positively or negatively curved. Using and cell biology approaches, we assess mechanisms of ezrin's enrichment on curved membranes.

ProLIF - quantitative integrin protein-protein interactions and synergistic membrane effects on proteoliposomes.

Integrin transmembrane receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. Diverse methods have been used to investigate interactions between integrins and intracellular proteins, and predominantly include peptide-based pulldowns and biochemical immuno-isolations from detergent-solubilised cell lysates. However, quantitative methods to probe integrin-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking.

The emerging role of phosphoinositide clustering in intracellular trafficking and signal transduction.

Phosphoinositides are master regulators of multiple cellular processes: from vesicular trafficking to signaling, cytoskeleton dynamics, and cell growth. They are synthesized by the spatiotemporal regulated activity of phosphoinositide-metabolizing enzymes. The recent observation that some protein modules are able to cluster phosphoinositides suggests that alternative or complementary mechanisms might operate to stabilize the different phosphoinositide pools within cellular compartments.

Chiral habit selection on nanostructured epitaxial quartz films.

Understanding the crystallization of enantiomorphically pure systems can be relevant to diverse fields such as the study of the origins of life or the purification of racemates. Here we report on polycrystalline epitaxial thin films of quartz on Si substrates displaying two distinct types of chiral habits that never coexist in the same film. We combine Atomic Force Microscopy (AFM) analysis and computer-assisted crystallographic calculations to make a detailed study of these habits of quartz.

BIN1/M-Amphiphysin2 induces clustering of phosphoinositides to recruit its downstream partner dynamin.

Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its downstream partner dynamin.

Mechanics of proteins with a focus on atomic force microscopy.

The capacity of proteins to function relies on a balance between molecular stability to maintain their folded state and structural flexibility allowing conformational changes related to biological function. Among many others, four different examples can be chosen. The giant protein titin is stretched and can unfold during muscle contraction providing passive elasticity to muscle tissue; myoglobin adsorbs and releases oxygen molecules thank to conformational changes in its structure; the outer membrane protein G (OmpG) is a bacterial porin with a long and flexible loop that modulates gating; and the proton pump bacteriorhodopsin adapts its cytosolic half to allow proton pumping.

Structural and mechanical heterogeneity of the erythrocyte membrane reveals hallmarks of membrane stability.

The erythrocyte membrane, a metabolically regulated active structure that comprises lipid molecules, junctional complexes, and the spectrin network, enables the cell to undergo large passive deformations when passing through the microvascular system. Here we use atomic force microscopy (AFM) imaging and quantitative mechanical mapping at nanometer resolution to correlate structure and mechanics of key components of the erythrocyte membrane, crucial for cell integrity and function. Our data reveal structural and mechanical heterogeneity modulated by the metabolic state at unprecedented nanometer resolution.

Atomic force microscopy: a versatile tool to probe the physical and chemical properties of supported membranes at the nanoscale.

Atomic force microscopy (AFM) was developed in the 1980s following the invention of its precursor, scanning tunneling microscopy (STM), earlier in the decade. Several modes of operation have evolved, demonstrating the extreme versatility of this method for measuring the physicochemical properties of samples at the nanoscopic scale. AFM has proved an invaluable technique for visualizing the topographic characteristics of phospholipid monolayers and bilayers, such as roughness, height or laterally segregated domains.

Direct measurement of the mechanical properties of lipid phases in supported bilayers.

Biological membranes define not only the cell boundaries but any compartment within the cell. To some extent, the functionality of membranes is related to the elastic properties of the lipid bilayer and the mechanical and hydrophobic matching with functional membrane proteins. Supported lipid bilayers (SLBs) are valid biomimetic systems for the study of membrane biophysical properties.

Miscibility behavior and nanostructure of monolayers of the main phospholipids of Escherichia coli inner membrane.

We report a thermodynamic study of the effect of calcium on the mixing properties at the air-water interface of two phospholipids that mimic the inner membrane of Escherichia coli: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. In this study, pure POPE and POPG monolayers and three mixed monolayers, χ(POPE) = 0.25, 0.

Lactose permease lipid selectivity using Förster resonance energy transfer.

The phospholipid composition that surrounds a membrane protein is critical to maintain its structural integrity and, consequently, its functional properties. To understand better this in the present work we have performed FRET measurements between the single tryptophan residue of a lactose permease Escherichia coli mutant (single-W151/C154G LacY) and pyrene-labeled phospholipids (Pyr-PE and Pyr-PG) at 37 degrees C. We have reconstituted this LacY mutant in proteoliposomes formed with heteroacid phospholipids, POPE and POPG, and homoacid phospholipids DOPE and DPPE, resembling the same PE/PG proportion found in the E.

Force spectroscopy study of Langmuir-Blodgett asymmetric bilayers of phosphatidylethanolamine and phosphatidylglycerol.

Phosphatidylethanolamine (PE) and phosphatidylgycerol (PG) are the main components of the inner membrane of Escherichia coli. Mixtures of PE and PG mimicking the proportions found in E. coli have been extensively used to reconstitute transmembrane proteins as lactose permease (LacY) in proteoliposomes because in this environment the protein shows maximal activity.

Preferential insertion of lactose permease in phospholipid domains: AFM observations.

We report the insertion of a transmembrane protein, lactose permease (LacY) from Escherichia coli (E. coli), in supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), in biomimetic molar proportions. We provide evidence of the preferential insertion of LacY in the fluid domains.

Evidence of phosphatidylethanolamine and phosphatidylglycerol presence at the annular region of lactose permease of Escherichia coli.

Biochemical and structural work has revealed the importance of phospholipids in biogenesis, folding and functional modulation of membrane proteins. Therefore, the nature of protein-phospholipid interaction is critical to understand such processes. Here, we have studied the interaction of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) mixtures with the lactose permease (LacY), the sugar/H(+) symporter from Escherichia coli and a well characterized membrane transport protein.

Calcium-induced formation of subdomains in phosphatidylethanolamine-phosphatidylglycerol bilayers: a combined DSC, 31P NMR, and AFM study.

We study the effect of Ca(2+) on the lateral segregation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). Supported lipid bilayers (SLBs) were observed by atomic force microscopy (AFM). Since SLBs are formed from liposomes of POPE:POPG, we examined the effect of calcium on these suspensions by differential scanning calorimetry (DSC) and (31)P nuclear magnetic resonance spectroscopy ((31)P NMR).

Phase changes in supported planar bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine.

We studied the thermal response of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) by comparing the differential scanning calorimetry (DSC) data of liposomes with atomic force microscopy (AFM) observations on supported planar bilayers. Planar bilayers were obtained by using the Langmuir-Blodgett (LB) technique: the first leaflet transferred at 30 mN m(-1) and the second at 25 mN m(-1). The topographic evaluation of supported POPE bilayers above room temperature showed changes between 43.

Atomic force microscopy characterization of supported planar bilayers that mimic the mitochondrial inner membrane.

In this study we examined the properties of supported planar bilayers (SPBs) formed from phospholipid components that comprise the mitochondrial inner membrane. We used 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL). Liposomes of binary POPE:POPC (1:1, mol:mol) and ternary (POPE:POPC:CL (0.

Thermal response of Langmuir-Blodgett films of dipalmitoylphosphatidylcholine studied by atomic force microscopy and force spectroscopy.

The topographic evolution of supported dipalmitoylphosphatidylcholine (DPPC) monolayers with temperature has been followed by atomic force microscopy in liquid environment, revealing the presence of only one phase transition event at approximately 46 degrees C. This finding is a direct experimental proof that the two phase transitions observed in the corresponding bilayers correspond to the individual phase transition of the two leaflets composing the bilayer. The transition temperature and its dependency on the measuring medium (liquid saline solution or air) is discussed in terms of changes in van der Waals, hydration, and hydrophobic/hydrophilic interactions, and it is directly compared with the transition temperatures observed in the related bilayers under the same experimental conditions.

Monitoring pyrene excimers in lactose permease liposomes: revealing the presence of phosphatidylglycerol in proximity to an integral membrane protein.

In this study, we examined the annular lipid composition of the transmembrane protein lactose permease (LacY) from Escherichia coli. LacY was reconstituted into 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (POPE) and 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-3-[Phospho-rac-(1-glycerol)] (POPG) and labeled with 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-Glycero-3-phosphoglycerol (PPDPG) at a 3:0.99:0.