Maria J. Sarmento

Reorganization of lipid domain distribution in giant unilamellar vesicles upon immobilization with different membrane tethers.

Characterization of phase coexistence in biologically relevant lipid mixtures is often carried out through confocal microscopy of giant unilamellar lipid vesicles (GUVs), loaded with fluorescent membrane probes. This last analysis is generally limited to the vesicle hemisphere further away from the coverslip, in order to avoid artifacts induced by the interaction with the solid surface, and immobilization of vesicles is in many cases required in order to carry out intensity, lifetime or single-molecule based microscopy. This is generally achieved through the use of membrane tethers adhering to a coverslip surface. Here, we aimed to determine whether GUV immobilization through membrane tethers induces changes in lipid domain distribution within liposomes displaying coexistence of lipid lamellar phases. Confocal imaging and a Förster resonance energy transfer (FRET) methodology showed that biotinylated phospholipids present significantly different membrane phase partition behavior upon protein binding, depending on the presence or absence of a linker between the lipid headgroup and the biotinyl moiety. Membrane phases enriched in a membrane tether displayed in some cases a dramatically increased affinity for the immobilization surface, effectively driving sorting of lipid domains to the adherent membrane area, and in some cases complete sequestering of a lipid phase to the interaction surface was observed. On the light of these results, we conclude that tethering of lipid membranes to protein surfaces has the potential to drastically reorganize the distribution of lipid domains, and this reorganization is solely dictated by the partition properties of the protein-tether complex.

Ca(2+) induces PI(4,5)P2 clusters on lipid bilayers at physiological PI(4,5)P2 and Ca(2+) concentrations.

Calcium has been shown to induce clustering of PI(4,5)P2 at high and non-physiological concentrations of both the divalent ion and the phosphatidylinositol, or on supported lipid monolayers. In lipid bilayers at physiological conditions, clusters are not detected through microscopic techniques. Here, we aimed to determine through spectroscopic methodologies if calcium plays a role in PI(4,5)P2 lateral distribution on lipid bilayers under physiological conditions. Using several different approaches which included information on fluorescence quantum yield, polarization, spectra and diffusion properties of a fluorescent derivative of PI(4,5)P2 (TopFluor(TF)-PI(4,5)P2), we show that Ca(2+) promotes PI(4,5)P2 clustering in lipid bilayers at physiological concentrations of both Ca(2+) and PI(4,5)P2. Fluorescence depolarization data of TF-PI(4,5)P2 in the presence of calcium suggests that under physiological concentrations of PI(4,5)P2 and calcium, the average cluster size comprises ~15 PI(4,5)P2 molecules. The presence of Ca(2+)-induced PI(4,5)P2 clusters is supported by FCS data. Additionally, calcium mediated PI(4,5)P2 clustering was more pronounced in liquid ordered (lo) membranes, and the PI(4,5)P2-Ca(2+) clusters presented an increased affinity for lo domains. In this way, PI(4,5)P2 could function as a lipid calcium sensor and the increased efficiency of calcium-mediated PI(4,5)P2 clustering on lo domains might provide targeted nucleation sites for PI(4,5)P2 clusters upon calcium stimulus.

Liquid-liquid extraction of a recombinant protein, cytochrome b5, with aqueous two-phase systems of polyethylene glycol and potassium phosphate salts

The partitioning of cytochrome b5 in aqueous two-phase systems of polyethylene glycol (PEG) and potassium phosphate salts was investigated. Cytochrome b5 partitioning is enhanced with decreasing polymer molecular mass and with increasing tieline length and pH. The effect of cytochrome b5 mutation, by substitution of the glutamic acid at positions 56 and 92 of the polypeptide chain by a lysine, on protein partitioning was also studied. Partitioning of cytochrome b5 mutants shows the same dependence on tieline length and pH, following the order cytochrome b5 > mutant 56 > mutant 92.

Role of calcium in membrane interactions by PI(4,5)P2-binding proteins

Ca²⁺ and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] are key agents in membrane-associated signalling events. Their temporal and spatial regulation is crucial for activation or recruitment of proteins in the plasma membrane. In fact, the interaction of several signalling proteins with PI(4,5)P₂ has been shown to be tightly regulated and dependent on the presence of Ca²⁺, with co-operative binding in some cases. In these proteins, PI(4,5)P₂ and Ca²⁺ binding typically occurs at different binding sites. In addition, several PI(4,5)P₂-binding proteins are known targets of calmodulin (CaM), which, depending on the presence of calcium, can compete with PI(4,5)P₂ for protein interaction, translating Ca²⁺ transient microdomains into variations of PI(4,5)P₂ lateral organization in time and space. The present review highlights different examples of calcium-dependent PI(4,5)P₂-binding proteins and discusses the possible impact of this dual regulation on fine-tuning of protein activity by triggering target membrane binding in the presence of subtle changes in the levels of calcium or PI(4,5)P₂.

Liquid-liquid extraction of a recombinant protein, cytochrome b5, from an impure extract using aqueous two-phase systems

Abstract The recovery and purification of a recombinant protein, cytochrome b5, from an impure extract of Escherichia coli disrupted cells was carried out in one step using a liquid–liquid extraction process of aqueous two-phase systems of polyethylene glycol (PEG) and potassium phosphate salts. With this separation process it was possible in one single step to remove the cell debris, that precipitate at interface of the system, and to obtain relatively high recovery yields, nearly 67%, of the target protein in the salt-rich phase, with purification factors up to 6.

Accurate quantification of inter-domain partition coefficients in GUVs exhibiting lipid phase coexistence

Giant unilamellar vesicles (GUVs) with phase coexistence allow for the recovery of inter-domain partition coefficients (Kp) of fluorescent molecules through comparison of fluorescence intensities in each phase. This method has been extensively used to gather qualitative information regarding the preference of both lipid analogues and other fluorescent molecules for insertion into ordered lipid membrane phases, which is often used as a predictor for incorporation in ordered plasma membrane domains. Methods aiming to recover quantitative information on partition properties from GUV imaging fail to correct for brightness and area per lipid differences, skewing recovered values. Additionally, photoselection effects occurring in the presence of linearly polarized excitation are generally neglected in these calculations. Here, we describe a new methodology for correction of fluorescence imaging data obtained from GUVs to recover accurate partition coefficients, which accounts for changes in the probes quantum yield, area per lipid differences and photoselection effects. This general methodology is used to quantify liquid ordered/liquid disordered lipid phase partition coefficients of several commonly used fluorescent analogues of phospholipids. Importantly, several sources of error in spectroscopic measurements of Kp, such as incorporation of a fluorescent probe in domain boundaries, clustering in water or in the membrane, or formation of three coexisting phases, are either irrelevant for quantification of inter-domain Kps through the method presented here or are readily recognized through imaging with GUVs.

Membrane Order Is a Key Regulator of Divalent Cation-Induced Clustering of PI(3,5)P2 and PI(4,5)P2

Although the evidence for the presence of functionally important nanosized phosphorylated phosphoinositide (PIP)-rich domains within cellular membranes has accumulated, very limited information is available regarding the structural determinants for compartmentalization of these phospholipids. Here, we used a combination of fluorescence spectroscopy and microscopy techniques to characterize differences in divalent cation-induced clustering of PI(4,5)P2 and PI(3,5)P2. Through these methodologies we were able to detect differences in divalent cation-induced clustering efficiency and cluster size. Ca2+-induced PI(4,5)P2 clusters are shown to be significantly larger than the ones observed for PI(3,5)P2. Clustering of PI(4,5)P2 is also detected at physiological concentrations of Mg2+, suggesting that in cellular membranes, these molecules are constitutively driven to clustering by the high intracellular concentration of divalent cations. Importantly, it is shown that lipid membrane order is a key factor in the regulation of clustering for both PIP isoforms, with a major impact on cluster sizes. Clustered PI(4,5)P2 and PI(3,5)P2 are observed to present considerably higher affinity for more ordered lipid phases than the monomeric species or than PI(4)P, possibly reflecting a more general tendency of clustered lipids for insertion into ordered domains. These results support a model for the description of the lateral organization of PIPs in cellular membranes, where both divalent cation interaction and membrane order are key modulators defining the lateral organization of these lipids.

High Affinity Immobilization of Giant Unilamellar Vesicles (GUVs) Induces Redistribution of Lipid Domains

Fluorescence microscopy imaging of giant unilamellar vesicles (GUVs) is a popular tool for the study of lipid phase separation. Studies with GUVs frequently require immobilization of the vesicles, and several methods are available for that effect. One of the most common methods of immobilization requires the inclusion of a very small fraction of biotin labeled lipids in the lipid mixture. Following vesicle formation, the sample is added to avidin coated imaging chambers and the interaction between biotin and avidin ensures that GUVs remain fully immobile during data acquisition. Here, we analyze the effect of biotinylated lipids with different structures on the distribution of lipid domains within the GUVs. We show that the different biotinylated lipids present differential affinities for liquid disordered, liquid ordered and gel lipid phases, and as a consequence the distribution of lipid domains inside GUVs was dramatically affected. Additionally, even in the absence of immobilization, interaction of the GUVs with the coverslip solid surface induced changes in lipid domain distribution. A very common methodology in GUV imaging is the acquisition of data only from the top hemisphere. Taking our results into account, it is clear that this creates a bias relatively to the total composition of the vesicles. Thus, in order to correctly quantify lipid phase fractions in GUVs, it is absolutely essential to acquire data from the entire vesicle.