Development of lysosome-mimicking vesicles to study the effect of abnormal accumulation of sphingosine on membrane properties

Abstract

Synthetic systems are widely used to unveil the molecular mechanisms of complex cellular events. Artificial membranes are key examples of models employed to address lipid-lipid and lipid-protein interactions. In this work, we developed a new synthetic system that more closely resembles the lysosome – the lysosome-mimicking vesicles (LMVs) – displaying stable acid-to-neutral pH gradient across the membrane. To evaluate the advantages of this synthetic system, we assessed the distinct effects of sphingosine (Sph) accumulation in membrane structure and biophysical properties of standard liposomes (no pH gradient) and in LMVs with lipid composition tuned to mimic physiological- or NPC1-like lysosomes. Ternary 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/Sphingomyelin (SM)/Cholesterol (Chol) mixtures with, respectively, low and high Chol/SM levels were prepared. The effect of Sph on membrane permeability and biophysical properties was evaluated by fluorescence spectroscopy, electrophoretic and dynamic light scattering. The results showed that overall Sph has the ability to cause a shift in vesicle surface charge, increase membrane order and promote a rapid increase in membrane permeability. These effects are enhanced in NPC1- LMVs. The results suggest that lysosomal accumulation of these lipids, as observed under pathological conditions, might significantly affect lysosomal membrane structure and integrity, and therefore contribute to the impairment of cell function.

Mammalian sphingoid bases: Biophysical, physiological and pathological properties

“…Several studies have addressed the effect of SO on bilayer membranes and its ability to change membrane biophysical properties, by promoting changes in fluidity [92][93][94]97,[100][101][102], domain formation [101,103] or membrane permeability [94,102,104,105] (Fig. 4).…”

“…The impact of SO on membrane structure, thermotropic behavior and lipid phase separation, can have consequences on membrane permeability, both in model and cell membranes [94,102,104,105]. Changes in membrane permeability might derive from i) structural defects created by SO as a consequence of lipid phase separation into rigid and fluid lamellar phases [94,105]; ii) the formation of small short-lived channels that allow the passage of small molecules and ions [104]; and iii) non-lamellar structures formed upon interaction of SO with negatively charged lipids [102]. Additionally, it was shown that SO-induced membrane permeability depends on pH and membrane lipid composition: SO promotes a rapid increase in membrane permeability in conditions mimicking the lysosome pH environment, i.e., with internal acidic and external neutral pH.…”

“…Additionally, it was shown that SO-induced membrane permeability depends on pH and membrane lipid composition: SO promotes a rapid increase in membrane permeability in conditions mimicking the lysosome pH environment, i.e., with internal acidic and external neutral pH. Moreover, high levels of Chol and SM, as observed in NPC1, enhance SO-induced membrane permeability in those lysosomal-mimicking vesicles [105], suggesting that SO might impair membrane structure under pathological conditions (…”

“…4). Indeed, synthetic membranes enriched in SO have impaired stability under acidic conditions and enhanced propensity to form larger aggregates, likely due to SO-induced changes in membrane surface charge [105]. A defective NPC1 function not only affects the storage of lipids, but also reduces the levels of lysosomal Ca 2+ and causes unique endocytic transport and fusion defects [73].…”

The effect of H3O+ on the membrane morphology and hydrogen bonding of a phospholipid bilayer

“…In contrast, the effect of pH on the structure of lipid bilayers has been less examined leading to a poor understanding of this effect at a molecular level. Yet pHinduced changes in membranes play a critical role in many biological processes including the stability and integrity of the lysosomal membranes (Carreira et al 2017), the formation of cholesterol-enriched domains (Redfern and Gericke 2005) and drug partitioning (Krämer et al 1998;Schaper et al 2001). Our understanding of pH on biological membranes is also important to comprehending systemic health issues such as inflammation (Kellum et al 2004), tumour growth (Gerweck and Seetharaman 1996;Kato et al 2013), ischaemic stroke (Huang and McNamara 2004) and epileptic seizures (Ziemann et al 2008).…”