Abstract
Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Autophagosomes / metabolism*
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Autophagosomes / ultrastructure
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Azides / chemistry*
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Biological Transport
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Carbocyanines / metabolism
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Choline / analogs & derivatives*
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Click Chemistry / methods
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Endoplasmic Reticulum / metabolism*
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Endoplasmic Reticulum / ultrastructure
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Fluorescent Dyes / metabolism
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Golgi Apparatus / metabolism
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Golgi Apparatus / ultrastructure
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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HeLa Cells
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Humans
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Lysosomes / metabolism
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Lysosomes / ultrastructure
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Mitochondria / metabolism
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Mitochondria / ultrastructure
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Molecular Imaging / methods
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Phosphatidylcholines / chemistry
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Phosphatidylcholines / metabolism*
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Red Fluorescent Protein
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Rhodamine 123 / metabolism
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Staining and Labeling / methods*
Substances
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Alexa Fluor 647
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Azides
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Carbocyanines
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Fluorescent Dyes
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Luminescent Proteins
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Phosphatidylcholines
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Green Fluorescent Proteins
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Rhodamine 123
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Choline