Assessing the use of ellipsoidal microparticles for determining lipid membrane viscosity

The viscosity of lipid membranes sets the timescales of membrane-associated flows and therefore influences the dynamics of a wide range of cellular processes. Techniques to measure membrane viscosity remain sparse, however, and reported measurements to date, even of similar systems, give viscosity values that span orders of magnitude. To address this, we improve a method based on measuring both the rotational and translational diffusion of membrane-anchored microparticles and apply this approach and one based on tracking the motion of phase-separated lipid domains to the same system of phase-separated giant vesicles. We find good agreement between the two methods, with inferred viscosities within a factor of two of each other. Our technique uses ellipsoidal microparticles, and we show that the extraction of physically meaningful viscosity values from their motion requires consideration of their anisotropic shape. The validation of our method on phase-separated membranes makes possible its application to other systems, which we demonstrate by measuring the viscosity of bilayers composed of lipids with different chain lengths ranging from 14 to 20 carbon atoms, revealing a very weak dependence of two-dimensional viscosity on lipid size. The experimental and analysis methods described here should be generally applicable to a variety of membrane systems, both reconstituted and cellular.

Lipid Droplets and Their Autophagic Turnover via the Raft-Like Vacuolar Microdomains

Although once perceived as inert structures that merely serve for lipid storage, lipid droplets (LDs) have proven to be the dynamic organelles that hold many cellular functions. The LDs’ basic structure of a hydrophobic core consisting of neutral lipids and enclosed in a phospholipid monolayer allows for quick lipid accessibility for intracellular energy and membrane production. Whereas formed at the peripheral and perinuclear endoplasmic reticulum, LDs are degraded either in the cytosol by lipolysis or in the vacuoles/lysosomes by autophagy. Autophagy is a regulated breakdown of dysfunctional, damaged, or surplus cellular components. The selective autophagy of LDs is called lipophagy. Here, we review LDs and their degradation by lipophagy in yeast, which proceeds via the micrometer-scale raft-like lipid domains in the vacuolar membrane. These vacuolar microdomains form during nutrient deprivation and facilitate internalization of LDs via the vacuolar membrane invagination and scission. The resultant intra-vacuolar autophagic bodies with LDs inside are broken down by vacuolar lipases and proteases. This type of lipophagy is called microlipophagy as it resembles microautophagy, the type of autophagy when substrates are sequestered right at the surface of a lytic compartment. Yeast microlipophagy via the raft-like vacuolar microdomains is a great model system to study the role of lipid domains in microautophagic pathways.

Wnt3 Is Lipidated at Conserved Cysteine and Serine Residues in Zebrafish Neural Tissue

Wnt proteins are a family of hydrophobic cysteine-rich secreted glycoproteins that regulate a gamut of physiological processes involved in embryonic development and tissue homeostasis. Wnt ligands are post-translationally lipidated in the endoplasmic reticulum (ER), a step essential for its membrane targeting, association with lipid domains, secretion and interaction with receptors. However, at which residue(s) Wnts are lipidated remains an open question. Initially it was proposed that Wnts are lipid-modified at their conserved cysteine and serine residues (C77 and S209 in mWnt3a), and mutations in either residue impedes its secretion and activity. Conversely, some studies suggested that serine is the only lipidated residue in Wnts, and substitution of serine with alanine leads to retention of Wnts in the ER. In this work, we investigate whether in zebrafish neural tissues Wnt3 is lipidated at one or both conserved residues. To this end, we substitute the homologous cysteine and serine residues of zebrafish Wnt3 with alanine (C80A and S212A) and investigate their influence on Wnt3 membrane organization, secretion, interaction and signaling activity. Collectively, our results indicate that Wnt3 is lipid modified at its C80 and S212 residues. Further, we find that lipid addition at either C80 or S212 is sufficient for its secretion and membrane organization, while the lipid modification at S212 is indispensable for receptor interaction and signaling.

Vacuole phase-partitioning boosts mitochondria activity and cell lifespan through an inter-organelle lipid pipeline

Functional linkage between mitochondria and lysosomes is crucial for survival under starvation and lifespan extension. Despite such co-dependency, the supportive pathways connecting mitochondria and lysosomes remain unclear. Here, we identify an inter-organelle lipid trafficking pathway linking yeast vacuole and mitochondria that results in increased mitochondria growth and respiratory activity under glucose starvation. The pathway depends on vacuolar phase-separated, lipid domains, which provide zones for: activation of the vacuolar proton pump; lipid droplet (LD) docking and internalization; and, lipid transfer from vacuole-to-ER-to-mitochondria. Partitioned vacuolar domains form through a specialized type of macro-autophagy, triggered only under acute glucose starvation, that delivers sterol-rich, endosomal-derived lipids to the vacuole. To balance this lipid influx, the vacuole reroutes lipids back to the ER to support both LD biogenesis and mitochondria growth and activity. Energy produced by enhanced mitochondrial activity then feeds back to support the inter-organelle lipid trafficking pathways to ensure survival under nutrient stress.

Quercetin Induces Lipid Domain-Dependent Permeability

In this work, we present our results on quercetin interaction with distinct model membranes exploring the importance of lipid phases, ld, ld/lo and ld+lo+so, to the action of this flavonoid in bilayers and possibly contributing to clarifying some controversial aspects related to quercetin multiple activities. We found out that quercetin is able to increase membrane permeability in a manner dependent on the presence and characteristics of lipid domains. In the presence of sphingomyelin, we found the greatest increase in mean membrane permeability (at least 10 times higher than the other lipid compositions). We also observed the presence of micrometric domains whose shape and size were disturbed by the action of quercetin. The presence of cholesterol increased membrane rigidity. This effect was enhanced with the presence of quercetin, but for chol-sphingomyelin combination, the bilayers became more flaccid at low quercetin/lipid proportions (< 1/5) and moderately rigid at proportions of the 1/1 order. The affinity parameters were higher for the most homogeneous systems and with larger areas and extensions of disordered liquid phase than for those systems of higher heterogeneity.

Quercetin Induces Lipid Domain-Dependent Permeability

In this work, we present our results on quercetin interaction with distinct model membranes exploring the importance of lipid phases, ld, ld/lo and ld+lo+so, to the action of this flavonoid in bilayers and possibly contributing to clarifying some controversial aspects related to quercetin multiple activities. We found out that quercetin is able to increase membrane permeability in a manner dependent on the presence and characteristics of lipid domains. In the presence of sphingomyelin, we found the greatest increase in mean membrane permeability (at least 10 times higher than the other lipid compositions). We also observed the presence of micrometric domains whose shape and size were disturbed by the action of quercetin. The presence of cholesterol increased membrane rigidity. This effect was enhanced with the presence of quercetin, but for chol-sphingomyelin combination, the bilayers became more flaccid at low quercetin/lipid proportions (< 1/5) and moderately rigid at proportions of the 1/1 order. The affinity parameters were higher for the most homogeneous systems and with larger areas and extensions of disordered liquid phase than for those systems of higher heterogeneity.

Quercetin Induces Lipid Domain-Dependent Permeability

In this work, we present our results on quercetin interaction with distinct model membranes exploring the importance of lipid phases, ld, ld/lo and ld+lo+so, to the action of this flavonoid in bilayers and possibly contributing to clarifying some controversial aspects related to quercetin multiple activities. We found out that quercetin is able to increase membrane permeability in a manner dependent on the presence and characteristics of lipid domains. In the presence of sphingomyelin, we found the greatest increase in mean membrane permeability (at least 10 times higher than the other lipid compositions). We also observed the presence of micrometric domains whose shape and size were disturbed by the action of quercetin. The presence of cholesterol increased membrane rigidity. This effect was enhanced with the presence of quercetin, but for chol-sphingomyelin combination, the bilayers became more flaccid at low quercetin/lipid proportions (< 1/5) and moderately rigid at proportions of the 1/1 order. The affinity parameters were higher for the most homogeneous systems and with larger areas and extensions of disordered liquid phase than for those systems of higher heterogeneity.

Insights into the Action Mechanism of the Antimicrobial Peptide Lasioglossin III

Lasioglossin III (LL-III) is a cationic antimicrobial peptide derived from the venom of the eusocial bee Lasioglossum laticeps. LL-III is extremely toxic to both Gram-positive and Gram-negative bacteria, and it exhibits antifungal as well as antitumor activity. Moreover, it shows low hemolytic activity, and it has almost no toxic effects on eukaryotic cells. However, the molecular basis of the LL-III mechanism of action is still unclear. In this study, we characterized by means of calorimetric (DSC) and spectroscopic (CD, fluorescence) techniques its interaction with liposomes composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-rac-phosphoglycerol (POPG) lipids as a model of the negatively charged membrane of pathogens. For comparison, the interaction of LL-III with the uncharged POPC liposomes was also studied. Our data showed that LL-III preferentially interacted with anionic lipids in the POPC/POPG liposomes and induces the formation of lipid domains. Furthermore, the leakage experiments showed that the peptide could permeabilize the membrane. Interestingly, our DSC results showed that the peptide-membrane interaction occurs in a non-disruptive manner, indicating an intracellular targeting mode of action for this peptide. Consistent with this hypothesis, our gel-retardation assay experiments showed that LL-III could interact with plasmid DNA, suggesting a possible intracellular target.