Abstract
The advent of photoactivatable tools has revolutionized imaging of dynamic cellular processes. One such application is to visualize axonal transport—an intricate and dynamic process by which proteins and other macromolecules are conveyed from their sites of synthesis in the cell bodies to their destinations within axons and synapses. High-quality dynamic imaging of axonal transport using photoactivatable vectors can now be routinely performed using epifluorescence microscopes and CCD cameras that are standard in most laboratories, yet this is largely underutilized. Here we describe detailed protocols for imaging cargoes moving in fast and slow axonal transport in axons of cultured hippocampal neurons.
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References
Droz B, Leblond CP (1962) Migration of proteins along the axons of the sciatic nerve. Science 137(3535):1047–1048
Black MM, Lasek RJ (1980) Slow components of axonal transport: two cytoskeletal networks. J Cell Biol 86(2):616–623
Brown A (2003) Axonal transport of membranous and nonmembranous cargoes: a unified perspective. J Cell Biol 160(6):817–821. doi: 10.1083/jcb.200212017, jcb.200212017 [pii]
Goldstein AY, Wang X, Schwarz TL (2008) Axonal transport and the delivery of pre-synaptic components. Curr Opin Neurobiol 18(5):495–503. doi:10.1016/j.conb.2008.10.003, S0959-4388(08)00130-X [pii]
Roy S, Zhang B, Lee VM, Trojanowski JQ (2005) Axonal transport defects: a common theme in neurodegenerative diseases. Acta Neuropathol 109(1):5–13. doi:10.1007/s00401-004-0952-x
Tang Y, Scott DA, Das U, Edland SD, Radomski K, Koo EH, Roy S (2012) Early and selective impairments in axonal transport kinetics of synaptic cargoes induced by soluble amyloid beta-protein oligomers. Traffic 13(5):681–693. doi:10.1111/j.1600-0854.2012.01340.x
Gauthier-Kemper A, Weissmann C, Golovyashkina N, Sebo-Lemke Z, Drewes G, Gerke V, Heinisch JJ, Brandt R (2011) The frontotemporal dementia mutation R406W blocks tau’s interaction with the membrane in an annexin A2-dependent manner. J Cell Biol 192(4):647–661. doi:10.1083/jcb.201007161, jcb.201007161 [pii]
Roy S, Yang G, Tang Y, Scott DA (2011) A simple photoactivation and image analysis module for visualizing and analyzing axonal transport with high temporal resolution. Nat Protoc 7(1):62–68. doi:10.1038/nprot.2011.428, nprot.2011.428 [pii]
Scott DA, Das U, Tang Y, Roy S (2011) Mechanistic logic underlying the axonal transport of cytosolic proteins. Neuron 70(3):441–454. doi:10.1016/j.neuron.2011.03.022, S0896-6273(11)00295-9 [pii]
Tang Y, Das U, Scott DA, Roy S (2012) The slow axonal transport of alpha-synuclein—mechanistic commonalities amongst diverse cytosolic cargoes. Cytoskeleton (Hoboken) 69(7):506–513. doi:10.1002/cm.21019
Trivedi N, Jung P, Brown A (2007) Neurofilaments switch between distinct mobile and stationary states during their transport along axons. J Neurosci 27(3):507–516. doi:10.1523/JNEUROSCI.4227-06.2007, 27/3/507 [pii]
Weissmann C, Reyher HJ, Gauthier A, Steinhoff HJ, Junge W, Brandt R (2009) Microtubule binding and trapping at the tip of neurites regulate tau motion in living neurons. Traffic 10(11):1655–1668. doi:10.1111/j.1600-0854.2009.00977.x, TRA977 [pii]
Taylor NJ, Wang L, Brown A (2012) Neurofilaments are flexible polymers that often fold and unfold, but they move in a fully extended configuration. Cytoskeleton (Hoboken) 69(7): 535–544. doi:10.1002/cm.21039
Fath T, Ke YD, Gunning P, Gotz J, Ittner LM (2009) Primary support cultures of hippocampal and substantia nigra neurons. Nat Protoc 4(1):78–85. doi:10.1038/nprot.2008.199, nprot.2008.199 [pii]
Roy S, Winton MJ, Black MM, Trojanowski JQ, Lee VM (2007) Rapid and intermittent cotransport of slow component-b proteins. J Neurosci 27(12):3131–3138
Acknowledgments
Past and ongoing work on slow axonal transport in the Roy lab is supported by grants from the NIH (R01NS075233), the March of Dimes (Basil O’ Connor), and start-up funds from UCSD to SR. The authors thank the many researchers who have generously shared constructs with us.
Conflict of interest: The authors declare no conflict of interest.
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Ganguly, A., Roy, S. (2014). Using Photoactivatable GFP to Track Axonal Transport Kinetics. In: Cambridge, S. (eds) Photoswitching Proteins. Methods in Molecular Biology, vol 1148. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0470-9_13
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DOI: https://doi.org/10.1007/978-1-4939-0470-9_13
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