title: Imaging the Polarized Sorting of Proteins from the Golgi Complex in Live Neurons.
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.
authors.affiliation: Cell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Ginny G
authors.initials: GG
authors.lastname: Farías
authors.affiliation: Cell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Dylan J
authors.initials: DJ
authors.lastname: Britt
authors.affiliation: Cell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 35A Convent Drive, Room 2F226, MSC 3758, Bethesda, MD, 20892, USA. bonifacinoj@helix.nih.gov.
authors.forename: Juan S
authors.initials: JS
authors.lastname: Bonifacino
mesh_terms: D000818:Animals; D001369:Axons / Q000378:metabolism*; D003712:Dendrites / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051381:Rats
chemical_list: D009419:Nerve Tissue Proteins
keywords: Axon initial segment; Hippocampal neurons; Kymograph analysis; Live-cell imaging; Neuron dissection; Neuron transfection; Neuronal culture; Polarized trafficking; RUSH system; Trafficking from the Golgi complex