Abstract
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.
Highlights
Membrane lipids undoubtedly appear to contribute to almost every step in the regulated secretory pathway from the biogenesis of secretory granules to the exocytosis process.[1]
Our results demonstrate that number of the molecules released during exocytosis and the dynamics of exocytosis event from chromaffin single cells are altered by injection of the lipids into the individual cells; the in situ supplementation of PC and LPC decrease the number of released catecholamines, whereas PE shows the opposite effect
A constant applied potential between the working and reference electrodes leads to oxidation of electro-active molecules at the working electrode. Such events are observed in the form of current transients, which appear as spikes in a plot of current versus time (Fig. 1A and B). Integrating these spikes gives the total charge transferred (Q), which is related to the number of neurotransmitter molecules released (N) according to Faraday's law: N 1⁄4 Q/nF, where n is the number of electrons exchanged in the oxidation reaction (2e for catecholamines) and F is Faraday's constant (96 485 C molÀ1)
Summary
Membrane lipids undoubtedly appear to contribute to almost every step in the regulated secretory pathway from the biogenesis of secretory granules to the exocytosis process.[1]. The effect of cytoplasmic membrane engineering, lipid changes in the inner lea et of the cell membrane and the outer lea et of the vesicle membrane, on exocytosis and vesicular content have been examined by combining these methods with a nano-injection method to deliver different phospholipids possessing different intrinsic geometries including cylindrical PC, conical PE, and inverseconical lysophosphatidylcholine (LPC) into the single chromaffin cells using a nanopipette.
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