Abstract
Neuronal microtubules have long been known to contain intraluminal particles, called MIPs (microtubule inner proteins), most likely involved in the extreme stability of microtubules in neurons. This chapter describes a cryo-electron microscopy-based assay to visualize microtubules containing neuronal MIPs. We present two protocols to prepare MIPs-containing microtubules, using either in vitro microtubule polymerization assays or extraction of microtubules from mouse hippocampal neurons in culture.
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Acknowledgments
We thank Lisa De Macedo and Nicolas Chaumontel for technical help in protein purification and culture of neurons. We thank Naïs Martin for help in Fig. 1 scheme drawing. This work was supported by INSERM, CNRS and Grenoble Alpes University, and by grants from the French Research National Agency (2017-CE11-0026 MAMAs). This work used the EM facilities at the Grenoble Instruct-ERIC Center (ISBG; UMS 3518 CNRS CEA-UGA-EMBL) with support from the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) within the Grenoble Partnership for Structural Biology. The IBS Electron Microscope facility is supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA. We thank the IBS facility members for their help.
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Gory-Fauré, S., Delaroche, J., Cuveillier, C., Delphin, C., Arnal, I. (2022). Cryo-EM Visualization of Neuronal Particles Inside Microtubules. In: Inaba, H. (eds) Microtubules. Methods in Molecular Biology, vol 2430. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1983-4_24
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DOI: https://doi.org/10.1007/978-1-0716-1983-4_24
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