Quantitative image mean squared displacement (iMSD) analysis of the dynamics of Aquaporin 2 within the membrane of live cells.

Nanodomains are a biological membrane phenomenon which have a large impact on various cellular processes. They are often analysed by looking at the lateral dynamics of membrane lipids or proteins. The localization of the plasma membrane protein aquaporin-2 in nanodomains has so far been unknown. In this study, we use total internal reflection fluorescence microscopy to image Madin-Darby Canine Kidney (MDCK) cells expressing aquaporin-2 tagged with mEos 3.2. Then, image mean squared displacement (iMSD) approach was used to analyse the diffusion of aquaporin-2, revealing that aquaporin-2 is confined within membrane nanodomains. Using iMSD analysis, we found that the addition of the drug forskolin increases the diffusion of aquaporin-2 within the confined domains, which is in line with previous studies. Finally, we observed an increase in the size of the membrane domains and the extent of trapping of aquaporin-2 after stimulation with forskolin.

Angiotensin II Treatment Induces Reorganization and Changes in the Lateral Dynamics of Angiotensin II Type 1 Receptor in the Plasma Membrane Elucidated by Photoactivated Localization Microscopy Combined with Image Spatial Correlation Analysis.

The mechanisms by which angiotensin II type 1 receptor is distributed and the diffusional pattern in the plasma membrane (PM) remain unclear, despite their crucial role in cardiovascular homeostasis. In this work, we obtained quantitative information of angiotensin II type 1 receptor (AT1R) lateral dynamics as well as changes in the diffusion properties after stimulation with ligands in living cells using photoactivated localization microscopy (PALM) combined with image spatial-temporal correlation analysis. To study the organization of the receptor at the nanoscale, expansion microscopy (ExM) combined with PALM was performed. This study revealed that AT1R lateral diffusion increased after binding to angiotensin II (Ang II) and the receptor diffusion was transiently confined in the PM. In addition, ExM revealed that AT1R formed nanoclusters at the PM and the cluster size significantly decreased after Ang II treatment. Taking these results together suggest that Ang II binding and activation cause reorganization and changes in the dynamics of AT1R at the PM.

What can we learn about amphiphile-membrane interaction from model lipid membranes?

Surface-active amphiphiles find applications in a wide range of areas of industry such as agrochemicals, personal care, and pharmaceuticals. In many of these applications, interaction with cell membranes is a key factor for achieving their purpose. How do amphiphiles interact with lipid membranes? What are their bases for membrane specificity? Which biophysical properties of membranes are susceptible to modulation by amphiphilic membrane-effectors? What aspects of this interaction are important for performing their function? In our work on membrane biophysics over the years, questions like these have arisen and we now share some of our findings and discuss them in this review. This topic was approached focusing on the membrane properties and their alterations rather than on the amphiphile structure requirements for their interaction. Here, we do not aim to provide a comprehensive list of the modes of action of amphiphiles of biological interest but to help in understanding them.

Special Issue: Dynamics and Nano-Organization in Plasma Membranes.

Cell membranes develop extraordinarily complex lipids and proteins geared to perform functions required by cells [...].

Self-assembled nanostructures of L-ascorbic acid alkyl esters support monomeric amphotericin B.

HYPOTHESIS: Amphotericin B (AmB) is a highly effective antimicrobial, with broad antimycotic and antiparasitic effect. However, AmB poor water-solubilisation and aggregation tendency limits its use for topical applications. We studied the capacity of nanostructures formed by alkyl esters of L-ascorbic acid (ASCn) to solubilise AmB and tested the relationship between the prevalence of the monomeric form of AmB and its effectiveness as antimicrobial agent. EXPERIMENTS: We developed self-assembled nanostructures formed by the commercial compound, palmitoyl ascorbic acid, as well as the shorter chained myristoyl and lauroyl ascorbic acid. AmB loaded ASCn nanostructures were studied by a combination of spectroscopic techniques, together with particle analysis, differential scanning calorimetry, microbiological tests, and Langmuir monolayer visualisation. FINDINGS: We found no direct relation between the antimicrobial capacity and the prevalence of the monomeric form of the drug. However, the later was related to chemical stability and colloidal robustness. Nanostructures formed by ASC16 in its anionic state provide an appropriate environment for AmB in its monomeric form, maintaining its antimicrobial capacity. Langmuir film visualisation supports spectrophotometric evidence, indicating that ASC16 allows the in-plane solubilisation of AmB. Coagels formed by ASC16 appear as promising for carrying AmB for dermal delivery.

Miltefosine inhibits the membrane remodeling caused by phospholipase action by changing membrane physical properties.

Miltefosine (hexadecylphosphocholine or HePC) is an alkylphosphocholine approved for the treatment of visceral and cutaneous Leishmaniasis. HePC exerts its effect by interacting with lipid membranes and affecting membrane-dependent processes. The molecular geometry of HePC suggests that the pharmacological function of HePC is to alter membrane curvature. As a model system, we studied the enzyme production in model membranes of diacylglycerol (DAG) or ceramide (CER), lipids involved in cell signaling which alter the structure of membranes. Here, we studied the effect of HePC on changes in phospholipase activity and on the effect that the lipid products have on the curvature and fusogenicity of membranes where they accumulate. Our results indicate that HePC inhibits the long-time restructuring of membranes, characteristic of the DAG and CER enzyme formation processes. In addition, the drug also reduces the fusogenicity of phospholipase-derived products. We postulate that the effect of HePC is due to a non-specific geometric compensation of HePC to the inverted cone-shape of DAG and CER products, acting as a relaxation agent of membrane curvature stress. These data are important for understanding the mechanism of action by which HePC regulates the lipid metabolism and signal transduction pathways in which these enzymes are involved.

l-Ascorbic acid alkyl esters action on stratum corneum model membranes: An insight into the mechanism for enhanced skin permeation.

L-ascorbic acid alkyl esters (ASCn) are lipophilic forms of vitamin C, which act as skin permeation enhancers. We investigated the physical changes induced by incorporating ASCn into stratum corneum (SC) lipid membranes and correlated this with the mechanism proposed in the literature for skin permeation enhancement phenomena. We used lipid monolayers to explore the 2D structure and elasticity of the lipid-enhancer systems. As a comparison, the classic permeation enhancer, oleic acid (OA) and the non-enhancer analogue stearic acid (SA) were analysed. The incorporation of ASCn or OA into SC membranes resulted in more liquid-like films, with a dose-dependent lowering of the compressibility modulus. Brewster angle microscopy (BAM) evidenced partial miscibility of the enhancer with SC lipid components, stabilising the liquid-expanded phase. At the nanoscale, AFM showed that SC lipids form heterogeneous membranes, which underwent structural alterations after incorporating ASCn and fatty acids, such as SA and OA. The lower, cholesterol-enriched phase appears to concentrate the enhancers, whilst the higher ceramide-enriched phase concentrated the non-enhancer SA. Our results and previously reported pieces of evidence indicate a strong pattern in which the rheological properties of SC lipid films are determinant for skin permeation phenomena.

Psychosine remodels model lipid membranes at neutral pH.

ß-Galactosylsphingosine or psychosine (PSY) is a single chain sphingolipid with a cationic group, which is degraded in the lysosome lumen by ß-galactosylceramidase during sphingolipid biosynthesis. A deficiency of this enzyme activity results in Krabbe's disease and PSY accumulation. This favors its escape to extralysosomal spaces, with its pH changing from acidic to neutral. We studied the interaction of PSY with model lipid membranes in neutral conditions, using phospholipid vesicles and monolayers as classical model systems, as well as a complex lipid mixture that mimics the lipid composition of myelin. At pH 7.4, when PSY is mainly neutral, it showed high surface activity, self-organizing into large structures, probably lamellar in nature, with a CMC of 38 ±â€¯3 µM. When integrated into phospholipid membranes, PSY showed preferential partition into disordered phases, shifting phase equilibrium. The presence of PSY reduces the compactness of the membrane, making it more easily compressible. It also induces lipid domain disruption in vesicles composed of the main myelin lipids. The surface electrostatics of lipid membranes was altered by PSY in a complex manner. A shift to positive zeta potential values evidenced its presence in the vesicles. Furthermore, the increase of surface potential and surface water structuring observed may be a consequence of its location at the interface of the positively charged layer. As Krabbe's disease is a demyelinating process, PSY alteration of the membrane phase state, lateral lipid distribution and surface electrostatics appears important to the understanding of myelin destabilization at the supramolecular level.

Crossregulation between the insertion of Hexadecylphosphocholine (miltefosine) into lipid membranes and their rheology and lateral structure.

Hexadecylphosphocholine (HePC, miltefosine) is an alkylphospholipid used clinically for the topical treatment of cancer and against leishmaniasis. The mechanism of action of HePC, not yet elucidated, involves its insertion into the plasma membrane, affecting lipid homeostasis. It has also been proposed that HePC directly affects lipid raft stability and function in cell membranes. The present work deals with two main questions in the understanding of the action of HePC: the bases for membrane selectivity and as a membrane perturbator agent. We explored the interaction of HePC with lipid monolayers and bilayer vesicles, combining monolayer penetration experiments, Brewster angle microscopy and differential scanning calorimetry. Several membrane compositions were tested to explore different rheological conditions, phase states and lateral structures. Additionally, the kinetics between the soluble and the membrane form of HePC was explored. Our results showed an increase in elasticity induced by HePC incorporation in all the membranes studied. Differential incorporation was found for membranes in different phase states, supporting a preferential partitioning and a higher dynamic kinetics of HePC incorporation into fluid membranes in comparison with condensed or liquid-ordered ones. This effect resulted in phase equilibrium displacement in phospholipids and membranes containing liquid-ordered domains. The presence of cholesterol or ergosterol induced a fast incorporation and slow desorption of HePC from sterol-containing monolayers, favoring a long residence period within the membrane. This contributes to a better understanding of the HePC regulation of membrane-mediated events and lipid homeostasis.