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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. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation., (© 2024. The Author(s).)

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. However, the effectors of MEN in this process are unknown. Here we identify the F-BAR protein Hof1 as a critical target of MEN in septin remodelling. Phospho-mimicking HOF1 mutant alleles overcome the inability of MEN mutants to undergo septin reorganisation by decreasing Hof1 binding to septins and facilitating its translocation to the actomyosin ring. Hof1-mediated septin rearrangement requires its F-BAR domain, suggesting that it may involve a local membrane remodelling that leads to septin reorganisation. In vitro Hof1 can induce the formation of intertwined septin bundles, while a phosphomimetic Hof1 protein has impaired septin-bundling activity. Altogether, our data indicate that Hof1 modulates septin architecture in distinct ways depending on its phosphorylation status., (© 2024. The Author(s).)

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. Bin1 expression lead to the formation of both rings and tubes and we show that Bin1 forms scaffolds on which caveolae accumulate to form the initial T-tubule. Cav3 deficiency caused by either gene silencing or pathogenic mutations results in defective ring formation and perturbed Bin1-mediated tubulation that may explain defective T-tubule organization in mature muscles. Our results uncover new pathophysiological mechanisms that may prove relevant to myopathies caused by Cav3 or Bin1 dysfunction., Competing Interests: EL, JL, MB, GM, AB, CL, AM, AC, PA, JV, NR, MB, VM, IM, BC, LP, SV No competing interests declared, (© 2023, Lemerle et al.)

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. Using advanced microscopy and in vitro reconstitution assays, we show that Syp1 is able to align laterally and tightly pack septin filaments, thereby forming flat bundles or sheets. This property is shared by the Syp1 mammalian counterpart FCHo2, thus emphasizing conserved protein functions. Interestingly, the septin-bundling activity of Syp1 resides mainly in its intrinsically disordered region. Our data uncover the mechanism through which Syp1 promotes septin collar assembly and offer another example of functional diversity of unstructured protein domains., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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 2 ) 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. Here, we show the molecular dynamics of FCHo2 self-assembly on membranes by combining minimal reconstituted in vitro and cellular systems. Our results indicate that PI(4,5)P 2 domains assist FCHo2 docking at specific membrane regions, where it self-assembles into ring-like-shaped protein patches. We show that the binding of FCHo2 on cellular membranes promotes PI(4,5)P 2 clustering at the boundary of cargo receptors and that this accumulation enhances clathrin assembly. Thus, our results provide a mechanistic framework that could explain the recruitment of early PI(4,5)P 2 -interacting proteins at endocytic sites., Competing Interests: FE, IC, DS, CA, RR, VB, AC, TL, JV, SV, AC, LP No competing interests declared, (© 2022, El Alaoui et al.)

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 in vitro and cell biology approaches, we assess mechanisms of ezrin's enrichment on curved membranes. We evidence that wild-type ezrin (ezrinWT) and its phosphomimetic mutant T567D (ezrinTD) do not deform membranes but self-assemble anti-parallelly, zipping adjacent membranes. EzrinTD's specific conformation reduces intermolecular interactions, allows binding to actin filaments, which reduces membrane tethering, and promotes ezrin binding to positively-curved membranes. While neither ezrinTD nor ezrinWT senses negative curvature alone, we demonstrate that interacting with curvature-sensing I-BAR-domain proteins facilitates ezrin enrichment in negatively-curved membrane protrusions. Overall, our work demonstrates that ezrin can tether membranes, or be targeted to curved membranes, depending on conformations and interactions with actin and curvature-sensing binding partners., Competing Interests: FT, AB, HB, JM, YS, MT, LP, SM, EL, EC No competing interests declared, PL, PB Reviewing editor, eLife, (© 2018, Tsai et al.)

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. Here, we describe 'protein-liposome interactions by flow cytometry' (denoted ProLIF), a technique to reconstitute recombinant integrin transmembrane domains (TMDs) and cytoplasmic tail (CT) fragments in liposomes as individual subunits or as αβ heterodimers and, via flow cytometry, allow rapid and quantitative measurement of protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. Moreover, the effect of membrane composition, such as PI(4,5)P 2 incorporation, on protein recruitment to the integrin CTs can be analysed. ProLIF requires no specific instrumentation and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.This article has associated First Person interviews with the first authors of the paper (see doi: 10.1242/jcs.223644 and doi: 10.1242/jcs.223719)., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

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. Herein, we discuss the different known and potential molecular players that are prone to engage phosphoinositide clustering and elaborate on how such a mechanism might take part in the regulation of intracellular trafficking and signal transduction.

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. By estimating the surface energies of the observed crystallites we argue that the films are enantiomorphically pure and we briefly outline a possible mechanism to explain the habit and chiral selection in this system.

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. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.

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. ATP-depletion, reducing skeletal junction phosphorylation in RBC cells, leads to membrane stiffening. Analysis of ghosts and shear-force opened erythrocytes show that, in the absence of cytosolic kinases, spectrin phosphorylation results in membrane stiffening at the extracellular face and a reduced junction remodeling in response to loading forces. Topography and mechanical mapping of single components at the cytoplasmic face reveal that, surprisingly, spectrin phosphorylation by ATP softens individual filaments. Our findings suggest that, besides the mechanical signature of each component, the RBC membrane mechanics is regulated by the metabolic state and the assembly of its structural elements.

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. All these conformational changes triggered either by chemical or by physical cues, require mechanical flexibility or elasticity of certain protein domains. While the methods to determine protein structure, X-ray crystallography above all, have been dramatically improved over the last decades, the number of tools that directly measure the mechanical flexibility of proteins and protein domains is still limited. In this tutorial, after a brief introduction to protein structure, we present some of the available techniques to estimate protein flexibility, then focusing on atomic force microscopy (AFM). We describe the principles of the technique and its various imaging and force spectroscopy modes of operation that allow probing the elasticity of proteins, protein domains and their surrounding environment.

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. Implemented modes such as phase imaging have also provided criteria for discriminating the viscoelastic properties of different supported lipid bilayer (SLB) regions. In this review, we focus on the AFM force spectroscopy (FS) mode, which enables determination of the nanomechanical properties of membrane models. The interpretation of force curves is presented, together with newly emerging techniques that provide complementary information on physicochemical properties that may contribute to our understanding of the structure and function of biomembranes. Since AFM is an imaging technique, some basic indications on how real-time AFM imaging is evolving are also presented at the end of this paper., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

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, 0.5, and 0.75, were analyzed. We show that for χ(POPE) = 0.75, the values of the Gibbs energy of mixing were negative, which implies attractive interactions. We used atomic force microscopy to study the structural properties of Langmuir-Blodgett monolayers that were transferred onto mica substrate at lateral surface pressures of 25 and 30 mN m(-1). The topographic images of pure POPE and POPG monolayers exhibited two domains of differing size and morphology, showing a step height difference within the range expected for liquid-condensed and liquid-expanded phases. The images captured for χ(POPE) = 0.25 were featureless, and for χ(POPE) = 0.5 small microdomains were observed. The composition that mimics quantitatively the proportions found in the inner membrane of E. coli , χ(POPE) = 0.75, showed large liquid condensed domains in the liquid expanded phase. The extension of each domain was quantitatively analyzed. Because calcium is used in the formation of supported bilayers of negatively charged phospholipids, the possible influence of the nanostructure of the apical on the distal monolayer is discussed.

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. coli inner membrane (3:1, mol/mol). A theoretical model has been fitted to the experimental data. In the POPE/POPG system, quantitative model calculations show accordance with the experimental values that requires an annular region composed of approximately approximately 90 mol% PE. The experimental FRET efficiencies for the gel/fluid phase-separated DOPE/POPG system indicate a higher presence of PG in the annular region, from which it can be concluded that LacY shows clear preference for the fluid phase. Similar conclusions are obtained from analysis of excimer-to-monomer (E/M) pyrene ratios. To test the effects of this on cardiolipin (CL) on the annular region, myristoyl-CL and oleoyl-CL were incorporated in the biomimetic POPE/POPG matrix. The experimental FRET efficiency values, slightly larger for Pyr-PE than for Pyr-PG, suggest that CL displaces POPE and, more extensively, POPG from the annular region of LacY. Model fitting indicates that CL enrichment in the annular layer is, in fact, solely produced by replacing PG and that myristoyl-CL is not able to displace PE in the same way that oleoyl-CL does. One of the conclusions of this work is the fact that LacY inserts preferentially in fluid phases of membranes., (2010 Elsevier B.V. All rights reserved.)

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. Analysis of the self-assembled protein arrangements showed that LacY: (i) is inserted as a monomer within fluid domains of SLBs of POPE:POPG (3:1, mol/mol), (ii) has a diameter of approx. 7.8nm; and (iii) keeps an area of phospholipids surrounding the protein that is compatible with shells of phospholipids., (2009 Elsevier B.V. All rights reserved.)

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. Hence, the study of the physicochemical properties of this phospholipid matrix becomes of potential interest. In previous studies, we used atomic force microscopy (AFM) and force spectroscopy (FS) to study the topographic and nanomechanical properties of supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and of POPE and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). The study reported here was extended for completeness to asymmetric SLBs obtained by the Langmuir-Blodgett (LB) method. Thus, we prepared SLBs with the proximal leaflet extracted at 30 mN x m(-1) and the distal leaflet extracted at 25 mN x m(-1). We prepared SLBs with both leaflets with same composition (POPG/POPG), and also with the proximal leaflet of POPE and the distal leaflet of POPG or POPE:POPG (3:1, mol/mol). The topography of the SLBs acquired in liquid was compared with the topography of the monolayers acquired in air. Breakthrough (F(y)) and adhesion forces (F(adh)) of SLBs were extracted from force curves. The values obtained are discussed in terms of the possible involvement of the nanomechanical properties of the SLBs in membrane protein insertion. The results provide means for the observation that insertion of LacY in POPE:POPG (3:1, mol/mol) occurs preferentially in the fluid phase, which is the phase with the lower F(y) and the higher F(adh).

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. FRET measurements between single-W151/C154G LacY reconstituted in a lipid mixture composed of POPE and POPG at different molar ratios and pyrene-labeled PE or PG revealed a different phospholipid distribution between the annular region of LacY and the bulk lipid phase. Results also showed that both PE and PG can be part of the annular region, being PE the predominant when the PE:PG molar ratio mimics the membrane of E. coli. Furthermore, changes in the thermotropic behavior of phospholipids located in this annular region confirm that the interaction between LacY and PE is stronger than that of LacY and PG. Since PE is a proton donor, the results obtained here are discussed in the context of the transport mechanism of LacY., (Copyright 2009 Elsevier B.V. All rights reserved.)

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). AFM images revealed the existence of two separated phases, the higher showing a region with protruding subdomains. Force spectroscopy (FS) was applied to clarify the nature of each phase. The values of breakthrough force (F(y)), adhesion force (F(adh)), and height extracted from the force curves were assigned to the corresponding gel (L(beta)) and fluid (L(alpha)) phase. The endotherms obtained by DSC suggest that, in the presence of Ca(2+), phase separation already exists in the suspensions of POPE:POPG used to form SLBs. Due to the temperature changes applied during preparation of SLBs a (31)P NMR study was performed to assess the lamellar nature of the samples before spreading them onto mica. With in situ AFM experiments we showed that the binding of Ca(2+) to POPG-enriched domains only induces the formation of subdomains in the L(beta) phase.

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 43.8 and 59.8 degrees C. These observations are discussed in relation to the main roughness (Ra) variations and are interpreted as the result of the lamellar liquid crystalline (Lalpha) to inverted hexagonal (HII) phase transition. High-magnification images obtained at 45 degrees C revealed intermediate structures in the transformation. Force spectroscopy (FS) was subsequently applied to gain further structural and nanomechanical insight into the POPE planar bilayers as a function of temperature. These measurements show that the threshold force (Fy), which is the maximum force, that the sample can withstand before breaking, increases from 1.91+/-0.11 nN at 21 degrees C up to 3.08+/-0.17 nN at 43.8 degrees C. This behavior is interpreted as a consequence of the formation of intermediate structures or stalks in the transition from the L alpha to H II phase.

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 3:0.99:0.01 molar ratio. Pyrene excimer formation was monitored by exciting a single tryptophan mutant of the protein (T320W). The results suggest that POPG remains segregated in the vicinity of the protein, most likely forming part of the annular composition. The possible involvement of POPG in hydrogen binding with the protein, as well as the molecular mechanism of LacY, is also discussed in the context of the proteomic network theory.

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 (0.5:0.3:0.2, mol:mol:mol) composition were used in the formation of SPBs on mica. The characterization of the SPBs was carried out below (4 degrees C) and above (24 and 37 degrees C) the phase transition temperature (Tm) of the mixtures in solution. We observed: (i) that the thickness of the bilayers, calculated from a cross-sectional analysis, decreased as the visualization temperature increased; (ii) the existence of laterally segregated domains that respond to temperature in SPBs of POPE:POPC:CL; (iii) a decrease in height and an increase in roughness (Ra) of SPBs after cytochrome c (cyt c) injection at room temperature. To obtain further insight into the nature of the interaction between cyt c and the bilayers, the competition between 8-anilino-1-naphthalene sulfonate (ANS) and the protein for the same binding sites in liposomes was monitored by fluorescence. The results confirm the existence of preferential interaction of cyt c with CL containing liposomes. Taking these results and those of previous papers published by the group, we discuss the preferential adsorption of cyt c in CL domains. This provides support for the relevance of these phospholipids as a proton trap in the oxidative phosphorylation process that occurs in the energy transducing membranes., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

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. Force spectroscopy allows us to probe the nanomechanical properties of such monolayers as a function of temperature. These measurements show that the force needed to puncture the monolayers is highly dependent on the temperature and on the phospholipid phase, ranging from 120+/-4 pN at room temperature (liquid condensed phase) to 49+/-2 pN at 65 degrees C (liquid expanded phase), which represents a two orders-of-magnitude decrease respective to the forces needed to puncture DPPC bilayers. The topographic study of the monolayers in air around the transition temperature revealed the presence of boundary domains in the monolayer surface forming 120 degrees angles between them, thus suggesting that the cooling process from the liquid-expanded to the liquid-condensed phase follows a nucleation and growth mechanism.

Two cases of amyloidosis of the bladder are reported: 1) a 45 year old man with haematuria. Cystoscopic examination reveals several tumours in the wall of the bladder. Transuretral resection was performed and histopathological examination revealed primary amyloidosis. Systemic amyloidosis was excluded. He was treated with oral colchicine with success. 2) a 71 year old male with diagnosis of rheumatoid arthritis and systemic amyloidosis visited our hospital with massive haematuria. Transuretral resection was unsuccessful and was necessary surgical ligation of hypogastric arteries.