Abstract
Semaphorin3A (Sema3A) guides axonal growth during neuronal network development. Accumulating evidence indicates that Sema3A-induced growth cone collapse and repulsion involve endocytic membrane trafficking in the growth cone. It is now possible to visualize endocytic processes in living cells using total internal reflection fluorescence microscopy (TIRFM), a powerful tool for imaging dynamic subcellular events at the plasma membrane. In this chapter, we describe a method for TIRFM observation and analysis of clathrin-mediated endocytosis in growth cones of chicken dorsal root ganglion neurons that receive an extracellular concentration gradient of Sema3A in a culture medium.
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References
Luo Y, Raible D, Raper JA (1993) Collapsin: a protein in brain that induces the collapse and paralysis of neuronal growth cones. Cell 75:217–227
Fan J, Mansfield SG, Redmond T et al (1993) The organization of F-actin and microtubules in growth cones exposed to a brain-derived collapsing factor. J Cell Biol 121:867–878
Fan J, Raper JA (1995) Localized collapsing cues can steer growth cones without inducing their full collapse. Neuron 14:263–274
Song H, Ming G, He Z et al (1998) Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides. Science 281:1515–1518
Campbell DS, Regan AG, Lopez JS et al (2001) Semaphorin 3A elicits stage-dependent collapse, turning, and branching in Xenopus retinal growth cones. J Neurosci 21:8538–8547
Tojima T, Itofusa R, Kamiguchi H (2010) Asymmetric clathrin-mediated endocytosis drives repulsive growth cone guidance. Neuron 66:370–377
Fournier AE, Nakamura F, Kawamoto S et al (2000) Semaphorin3A enhances endocytosis at sites of receptor-F-actin colocalization during growth cone collapse. J Cell Biol 149:411–422
Jurney WM, Gallo G, Letourneau PC et al (2002) Rac1-mediated endocytosis during ephrin-A2- and semaphorin 3A-induced growth cone collapse. J Neurosci 22:6019–6028
Piper M, Salih S, Weinl C et al (2005) Endocytosis-dependent desensitization and protein synthesis-dependent resensitization in retinal growth cone adaptation. Nat Neurosci 8:179–186
Kabayama H, Nakamura T, Takeuchi M et al (2009) Ca2+ induces macropinocytosis via F-actin depolymerization during growth cone collapse. Mol Cell Neurosci 40:27–38
Carcea I, Ma'ayan A, Mesias R et al (2010) Flotillin-mediated endocytic events dictate cell type-specific responses to semaphorin 3A. J Neurosci 30:15317–15329
Toomre D, Manstein DJ (2001) Lighting up the cell surface with evanescent wave microscopy. Trends Cell Biol 11:298–303
Millis BA (2012) Evanescent-wave field imaging: an introduction to total internal reflection fluorescence microscopy. Methods Mol Biol 823:295–309
Toomre D (2012) Cellular imaging using total internal reflection fluorescence microscopy: theory and instrumentation. Cold Spring Harb Protoc 2012:414–424
Merrifield CJ, Feldman ME, Wan L et al (2002) Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits. Nat Cell Biol 4:691–698
Merrifield CJ, Perrais D, Zenisek D (2005) Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell 121:593–606
Rappoport JZ, Simon SM (2003) Real-time analysis of clathrin-mediated endocytosis during cell migration. J Cell Sci 116:847–855
Rappoport JZ, Simon SM, Benmerah A (2004) Understanding living clathrin-coated pits. Traffic 5:327–337
Gaidarov I, Santini F, Warren RA et al (1999) Spatial control of coated-pit dynamics in living cells. Nat Cell Biol 1:1–7
Shaner NC, Campbell RE, Steinbach PA et al (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572
Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88:49–92
Kamiguchi H, Yoshihara F (2001) The role of endocytic L1 trafficking in polarized adhesion and migration of nerve growth cones. J Neurosci 21:9194–9203
He Y, Baas PW (2003) Growing and working with peripheral neurons. Methods Cell Biol 71:17–35
Toomre D (2012) Generating live cell data using total internal reflection fluorescence microscopy. Cold Spring Harb Protoc 2012:439–446
Toomre D (2012) Alignment and calibration of total internal reflection fluorescence microscopy systems. Cold Spring Harb Protoc 2012:504–509
Zheng JQ, Felder M, Connor JA et al (1994) Turning of nerve growth cones induced by neurotransmitters. Nature 368:140–144
Acknowledgments
We are grateful to H. Akiyama and A.T. Guy for helpful comments on this manuscript. We also thank J.H. Keen and R.Y. Tsien for providing plasmid constructs, and RIKEN BSI Research Resources Center for DNA sequencing. This work was partially funded by the Japan Science and Technology Agency PRESTO program (T.T.) and Grants-in-Aid for Scientific Research on Innovative Areas (23110005, T.T.), Scientific Research (C) (24500393, T.T.), and Young Scientists (B) (22700353, T.T.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Itofusa, R., Tojima, T., Kamiguchi, H. (2017). Visualization of Clathrin-Mediated Endocytosis During Semaphorin-Guided Axonal Growth. In: Terman, J. (eds) Semaphorin Signaling. Methods in Molecular Biology, vol 1493. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6448-2_21
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DOI: https://doi.org/10.1007/978-1-4939-6448-2_21
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