Development of lysosome-mimicking vesicles to study the effect of abnormal accumulation of sphingosine on membrane properties
Development of lysosome-mimicking vesicles to study the effect of abnormal accumulation of sphingosine on membrane properties
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DOI：
10.1038/s41598-017-04125-6
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发表时间：
2017-06-21
影响因子：
4.6
通讯作者：
Silva, Liana C.
中科院分区：
文献类型：
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作者：
Carreira, Ana C.;de Almeida, Rodrigo F. M.;Silva, Liana C.
合成系统被广泛用于揭示复杂细胞事件的分子机制。人工膜是用于解决脂质-脂质和脂质-蛋白质相互作用的模型的关键示例。在这项工作中，我们开发了一种新的合成系统，它更类似于溶酶体-溶酶体模拟囊泡（LMV）-显示稳定的跨膜酸性至中性pH梯度。为了评估这种合成系统的优点，我们评估了鞘氨醇（Sph）积累在膜结构和生物物理特性的标准脂质体（无pH梯度）和LMV与脂质组合物调整模拟生理或NPC 1样溶酶体的不同影响。制备分别具有低和高Chol/SM水平的1-棕榈酰基-2-油酰基-sn-甘油基-3-磷酸胆碱（POPC）/鞘磷脂（SM）/胆固醇（Chol）三元混合物。通过荧光光谱、电泳和动态光散射等方法研究了Spha对细胞膜通透性和生物物理性质的影响。结果表明，总的Spha有能力引起囊泡表面电荷的移动，增加膜的有序性，并促进膜通透性的快速增加。这些作用在NPC 1-LMV中增强。结果表明，在病理条件下观察到的这些脂质的溶酶体蓄积可能显著影响溶酶体膜结构和完整性，因此导致细胞功能受损。
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.