Neurite varicosities are highly specialized compartments that are involved in neurotransmitter/ neuromodulator release and provide a physiological platform for neural functions. However, it remains unclear how microtubule organization contributes to the form of varicosity. Here, we examine the three-dimensional structure of microtubules in varicosities of a differentiated PC12 neural cell line using ultra-high voltage electron microscope tomography. Three-dimensional imaging showed that a part of the varicosities contained an accumulation of organelles that were separated from parallel microtubule arrays. Further detailed analysis using serial sections and whole-mount tomography revealed microtubules running in a spindle shape of swelling in some other types of varicosities. These electron tomographic results showed that the structural diversity and heterogeneity of microtubule organization supported the form of varicosities, suggesting that a different distribution pattern of microtubules in varicosities is crucial to the regulation of varicosities development.
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Aletta JM, Greene LA (1988) Growth cone configuration and advance: a time-lapse study using video-enhanced differential interference contrast microscopy. J Neurosci 8:1425–1435
Braunfeld MB, Koster AJ, Sedat JW, Agard DA (1994) Cryo automated electron tomography: towards high-resolution reconstructions of plastic-embedded structures. J Microsc 174:75–84
Brown CL, Maier KC, Stauber T, Ginkel LM, Wordeman L, Vernos I, Schroer TA (2005) Kinesin-2 is a motor for late endosomes and lysosomes. Traffic 6:1114–1124
Dent EW, Callaway JL, Szebenyi G, Baas PW, Kalil K (1999) Reorganization and movement of microtubules in axonal growth cones and developing interstitial branches. J Neurosci 19:8894–8908
Ferrari-Toninelli G, Bonini SA, Bettinsoli P, Uberti D, Memo M (2008) Microtubule stabilizing effect of notch activation in primary cortical neurons. Neuroscience 154:946–952
Ferrari-Toninelli G, Bonini SA, Uberti D, Napolitano F, Stante M, Santoro F, Minopoli G, Zambrano N, Russo T, Memo M (2009) Notch activation induces neurite remodeling and functional modifications in SH-SY5Y neuronal cells. Dev Neurobiol 69:378–339
Jacobs JR, Stevens JK (1986) Experimental modification of PC12 neurite shape with the microtubule-depolymerizing drug Nocodazole: a serial electron microscopic study of neurite shape control. J Cell Biol 103:907–915
Kolkova K, Novitskaya V, Pedersen N, Berezin V, Bock E (2000) Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway. J Neurosci 20:2238–2246
Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76
Lee CW, Peng HB (2008) The function of mitochondria in presynaptic development at the neuromuscular junction. Mol Biol Cell 19:150–158
Luther PK, Lawrence MC, Crowther RA (1988) A method for monitoring the collapse of plastic sections as a function of electron dose. Ultramicroscopy 24:7–18
Makihara M, Watanabe T, Usukura E, Kaibuchi K, Narita A, Tanaka N, Usukura J (2016) A new approach for the direct visualization of the membrane cytoskeleton in cryo-electron microscopy: a comparative study with freeze-etching electron microscopy. Microscopy (Oxf) 65:488–498
Nechipurenko IV, Broihier HT (2012) FoxO limits microtubule stability and is itself negatively regulated by microtubule disruption. J Cell Biol 196:345–362
Nishida T, Yoshimura R, Endo Y (2013) Three-dimensional distribution of TrkA neurotrophin receptors in neurite varicosities of differentiated PC12 cells treated with NGF determined by immunoelectron tomography. Cell Tissue Res 351:1–13
Perlson E, Hendricks AG, Lazarus JE, Ben-Yaakov K, Gradus T, Tokito M, Holzbaur EL (2013) Dynein interacts with the neural cell adhesion molecule (NCAM180) to tether dynamic microtubules and maintain synaptic density in cortical neurons. J Biol Chem 288:27812–27824
Roos J, Hummel T, Ng N, Klämbt C, Davis GW (2000) Drosophila Futsch regulates synaptic microtubule organization and is necessary for synaptic growth. Neuron 26:371–382
Takei K, Chan TA, Wang FS, Deng H, Rutishauser U, Jay DG (1999) The neural cell adhesion molecules L1 and NCAM-180 act in different steps of neurite outgrowth. J Neurosci 19:9469–9479
This work was supported by the “Nanotechnology Platform” (project No. 12024046) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
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Three-dimensional models of cytoskeletons and membrane organelles in the neurite varicosity. Microtubules, neurofilaments, clathrin-coated vesicles, endosome and mitochondria are colored green, purple, blue, yellow and white, respectively. Scale bar, 500 nm. (related to Fig. 2 and S1) (AVI 12881 kb)
Three-dimensional models of microtubules and membrane organelles in a small cytoplasmic expansion at the neurite shaft. Microtubules, clathrin-coated vesicles, endosome and plasma membrane are in green, blue, yellow and white respectively. A part of the plasma membrane of varicosity and swollen areas are colored red and purple, respectively. Scale bar, 500 nm. (related to Fig. 2 and S2) (AVI 12282 kb)
Three-dimensional models of microtubules and mitochondrion in a beaded varicosity that was reconstructed from two serial tomograms. Microtubules, spherical structures of microtubule tip and mitochondrion are in green, yellow and purple, respectively. Scale bar, 500 nm. (related to Fig. 3 and S3) (AVI 9363 kb)
Three-dimensional models of microtubule distribution in a globular varicosity branch that was reconstructed from four serial tomograms. Microtubules, looped microtubule, spherical structures of microtubule tip, plasma membrane and clathrin-coated vesicles are colored green, red, yellow, white and blue, respectively. Scale bar, 500 nm. (related to Fig. 3 and S4) (AVI 12484 kb)
Three-dimensional models of microtubules in a swelled neurite branching site that was reconstructed from two serial tomograms. Microtubules, spherical structures of microtubule tip, looped microtubule, two kinds of large circular turn microtubules and mitochondria are colored green, yellow, red, blue, yellow and white, respectively. Scale bar, 500 nm. (related to Fig. 4 and S5) (AVI 10112 kb)
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Nishida, T., Yoshimura, R. & Endo, Y. Three-dimensional fine structure of the organization of microtubules in neurite varicosities by ultra-high voltage electron microscope tomography. Cell Tissue Res 369, 467–476 (2017). https://doi.org/10.1007/s00441-017-2645-5
- Electron tomography
- Ultra-high voltage electron microscope