Abstract
Rotary shadow electron microscopy (EM) of growth cone cytoskeletons provides a high-resolution method for detecting both global and macromolecular changes in cytoskeletal organization or structure. This approach can be used to study responses to repulsive guidance factors such as semaphorin 3A. Here I describe the procedures used to prepare cultured neurons for rotary-shadow EM, allowing detailed comparisons of cytoskeletal structure.
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References
Fan J, Mansfield SG, Redman T, Phillip R, Gordon-Weeks PR, Raper JA (1993) The organization of F-actin and microtubules in growth cones exposed to a brain-derived collapsing factor. J Cell Biol 121:867–878
Kapfhammer JP, Xu H, Raper JA (2007) The detection and quantification of growth cone collapsing activities. Nat Protoc 2(8):2005–2011, doi:nprot.2007.295 [pii]10.1038/nprot.2007.295
Rauch P, Heine P, Goettgens B, Kas JA (2013) Different modes of growth cone collapse in NG 108-15 cells. Eur Biophys J 42(8):591–605. doi:10.1007/s00249-013-0907-z
Marx A, Godinez WJ, Tsimashchuk V, Bankhead P, Rohr K, Engel U (2013) Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules. Mol Biol Cell 24(10):1544–1558, doi:mbc.E12-08-0573 [pii]10.1091/mbc.E12-08-0573
Brown JA, Bridgman PC (2009) Disruption of the cytoskeleton during Semaphorin 3A induced growth cone collapse correlates with differences in actin organization and associated binding proteins. Dev Neurobiol 69(10):633–646. doi:10.1002/dneu.20732
Gallo G (2006) RhoA-kinase coordinates F-actin organization and myosin II activity during semaphorin-3A-induced axon retraction. J Cell Sci 119(16):3413–3423, doi:119/16/3413 [pii]10.1242/jcs.03084
Gallo G, Letourneau PC (2004) Regulation of growth cone actin filaments by guidance cues. J Neurobiol 58(1):92–102
Bridgman PC (2002) Growth cones contain myosin II bipolar filament arrays. Cell Motil Cytoskeleton 52(2):91–96. doi:10.1002/cm.10038
Lewis AK, Bridgman PC (1992) Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity. J Cell Biol 119(5):1219–1243
Svitkina TM, Borisy GG (1999) Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia. J Cell Biol 145(5):1009–1026
Bridgman PC, Dailey ME (1989) The organization of myosin and actin in rapid frozen nerve growth cones. J Cell Biol 108(1):95–109
Schliwa M, van Blerkom J (1981) Structural interaction of cytoskeletal components. J Cell Biol 90(1):222–235
Bridgman PC, Lewis AK, Victor JC (1993) Comparison of the ability of freeze etch and freeze substitution to preserve actin filament structure. Microsc Res Tech 24(5):385–394. doi:10.1002/jemt.1070240504
Hirokawa N, Heuser JE (1981) Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells. J Cell Biol 91(2 Pt 1):399–409
Dailey ME, Bridgman PC (1991) Structure and organization of membrane organelles along distal microtubule segments in growth cones. J Neurosci Res 30(1):242–258. doi:10.1002/jnr.490300125
Letourneau PC (1983) Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos. J Cell Biol 97(4):963–973
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This work was supported by NIH grant; contract grant number: NS026150.
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Bridgman, P.C. (2017). Using Rotary Shadow Electron Microscopy to Characterize Semaphorin-Mediated Growth Cone Collapse. 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_13
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DOI: https://doi.org/10.1007/978-1-4939-6448-2_13
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