Abstract
The study of polarized protein trafficking in live neurons is critical for understanding neuronal structure and function. Given the complex anatomy of neurons and the numerous trafficking pathways that are active in them, however, visualization of specific vesicle populations leaving the Golgi complex presents unique challenges. Indeed, several approaches used in non-polarized cells, and even in polarized epithelial cells, have been less successful in neurons. Here, we describe an adaptation of the recently developed Retention Using Selective Hooks (RUSH) system (Boncompain et al., Nat Methods 9:493–498, 2012), previously used in non-polarized cells, to analyze the polarized sorting of proteins from the Golgi complex to dendrites and axons in live neurons. The RUSH system involves the retention of a fluorescently tagged cargo protein fused to the streptavidin-binding peptide (SBP) in the endoplasmic reticulum (ER) through the expression of an ER-hook protein fused to streptavidin. Upon d-biotin addition, the cargo protein is released and its traffic to dendrites and axons can be analyzed in live neurons.
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Acknowledgement
This work was funded by the Intramural Program of NICHD, NIH (ZIA HD001607).
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Farías, G.G., Britt, D.J., Bonifacino, J.S. (2016). Imaging the Polarized Sorting of Proteins from the Golgi Complex in Live Neurons. In: Brown, W. (eds) The Golgi Complex. Methods in Molecular Biology, vol 1496. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6463-5_2
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DOI: https://doi.org/10.1007/978-1-4939-6463-5_2
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