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Intracellular injection of phospholipids directly alters exocytosis and the fraction of chemical release in chromaffin cells as measured by nano-electrochemistry
Chemical Science ( IF 7.6 ) Pub Date : 2020-10-06 , DOI: 10.1039/d0sc03683h Mohaddeseh Aref 1 , Elias Ranjbari 1 , Armaghan Romiani 1 , Andrew G Ewing 1
Chemical Science ( IF 7.6 ) Pub Date : 2020-10-06 , DOI: 10.1039/d0sc03683h Mohaddeseh Aref 1 , Elias Ranjbari 1 , Armaghan Romiani 1 , Andrew G Ewing 1
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Using a nano-injection method, we introduced phospholipids having different intrinsic geometries into single secretory cells and used single cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC) with nanotip electrodes to monitor the effects of intracellular incubation on the exocytosis process and vesicular storage. Combining tools, this work provides new information to understand the impact of intracellular membrane lipid engineering on exocytotic release, vesicular content and fraction of chemical release. We also assessed the effect of membrane lipid alteration on catecholamine storage of isolated vesicles by implementing another amperometric technique, vesicle impact electrochemical cytometry (VIEC), outside the cell. Exocytosis analysis reveals that the intracellular nano-injection of phosphatidylcholine and lysophosphatidylcholine decreases the number of released catecholamines, whereas phosphatidylethanolamine shows the opposite effect. These observations support the emerging hypothesis that lipid curvature results in membrane remodeling through secretory pathways, and also provide new evidence for a critical role of the lipid localization in modulating the release process. Interestingly, the IVIEC data imply that total vesicular content is also affected by in situ supplementation of the cells with some lipids, while, the corresponding VIEC results show that the neurotransmitter content in isolated vesicles is not affected by altering the vesicle membrane lipids. This suggests that the intervention of phospholipids inside the cell has its effect on the cellular machinery for vesicle release rather than vesicle structure, and leads to the somewhat surprising conclusion that modulating release has a direct effect on vesicle structure, which is likely due to the vesicles opening and closing again during exocytosis. These findings could lead to a novel regulatory mechanism for the exocytotic or synaptic strength based on lipid heterogeneity across the cell membrane.
中文翻译：
通过纳米电化学测量，细胞内注射磷脂直接改变嗜铬细胞中的胞吐作用和化学物质释放分数
利用纳米注射方法，我们将具有不同固有几何形状的磷脂引入单个分泌细胞中，并使用单细胞电流分析法（SCA）和带有纳米尖端电极的细胞内囊泡冲击电化学细胞术（IVIEC）来监测细胞内孵育对胞吐过程的影响，并囊泡储存。结合工具，这项工作提供了新的信息来了解细胞内膜脂质工程对胞吐释放、囊泡含量和化学释放分数的影响。我们还通过在细胞外实施另一种安培技术，囊泡冲击电化学细胞术（VIEC），评估了膜脂改变对分离囊泡儿茶酚胺储存的影响。胞吐作用分析表明，细胞内纳米注射磷脂酰胆碱和溶血磷脂酰胆碱减少了儿茶酚胺的释放数量，而磷脂酰乙醇胺则显示出相反的效果。这些观察结果支持了脂质曲率通过分泌途径导致膜重塑的新假设，并且还为脂质定位在调节释放过程中的关键作用提供了新的证据。有趣的是，IVIEC 数据表明，向细胞原位补充一些脂质也会影响总囊泡含量，而相应的 VIEC 结果表明，分离囊泡中的神经递质含量不受改变囊泡膜脂质的影响。 这表明细胞内磷脂的干预对囊泡释放的细胞机制产生影响，而不是对囊泡结构产生影响，并得出了一个有点令人惊讶的结论，即调节释放对囊泡结构有直接影响，这可能是由于囊泡在胞吐作用期间再次打开和关闭。这些发现可能会导致基于细胞膜上脂质异质性的胞吐或突触强度的新调节机制。
更新日期：2020-10-14
中文翻译：
通过纳米电化学测量，细胞内注射磷脂直接改变嗜铬细胞中的胞吐作用和化学物质释放分数
利用纳米注射方法，我们将具有不同固有几何形状的磷脂引入单个分泌细胞中，并使用单细胞电流分析法（SCA）和带有纳米尖端电极的细胞内囊泡冲击电化学细胞术（IVIEC）来监测细胞内孵育对胞吐过程的影响，并囊泡储存。结合工具，这项工作提供了新的信息来了解细胞内膜脂质工程对胞吐释放、囊泡含量和化学释放分数的影响。我们还通过在细胞外实施另一种安培技术，囊泡冲击电化学细胞术（VIEC），评估了膜脂改变对分离囊泡儿茶酚胺储存的影响。胞吐作用分析表明，细胞内纳米注射磷脂酰胆碱和溶血磷脂酰胆碱减少了儿茶酚胺的释放数量，而磷脂酰乙醇胺则显示出相反的效果。这些观察结果支持了脂质曲率通过分泌途径导致膜重塑的新假设，并且还为脂质定位在调节释放过程中的关键作用提供了新的证据。有趣的是，IVIEC 数据表明，向细胞原位补充一些脂质也会影响总囊泡含量，而相应的 VIEC 结果表明，分离囊泡中的神经递质含量不受改变囊泡膜脂质的影响。 这表明细胞内磷脂的干预对囊泡释放的细胞机制产生影响，而不是对囊泡结构产生影响，并得出了一个有点令人惊讶的结论，即调节释放对囊泡结构有直接影响，这可能是由于囊泡在胞吐作用期间再次打开和关闭。这些发现可能会导致基于细胞膜上脂质异质性的胞吐或突触强度的新调节机制。
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