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Updated: Sep 25, 2025
Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography
Published on: February 12, 2014
Cryo-EM Visualization of Neuronal Particles Inside Microtubules
Sylvie Gory-Fauré1, Julie Delaroche1, Camille Cuveillier1
1Univ. Grenoble Alpes, Inserm U1216, CNRS, Grenoble Institut Neurosciences, Grenoble, France.
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.
Insights
Neuronal microtubules contain inner proteins (MIPs) crucial for their stability. This study details cryo-electron microscopy methods to visualize these MIPs within microtubules from neuronal cultures and in vitro assays.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Neuronal microtubules possess intraluminal particles, termed MIPs (microtubule inner proteins).
- MIPs are hypothesized to contribute to the remarkable stability of neuronal microtubules.
Purpose of the Study:
- To describe a cryo-electron microscopy (cryo-EM) based assay for visualizing neuronal MIPs within microtubules.
- To present protocols for preparing MIPs-containing microtubules for visualization.
Main Methods:
- Development of a cryo-EM assay for visualizing MIPs within microtubules.
- Two preparation protocols: in vitro microtubule polymerization and extraction from cultured mouse hippocampal neurons.
Main Results:
- Successful visualization of microtubules containing neuronal MIPs using the described cryo-EM assay.
- Demonstration of two distinct methods for obtaining samples suitable for MIP visualization.
Conclusions:
- The presented cryo-EM assay provides a method for studying neuronal MIPs.
- The protocols enable the preparation of MIPs-containing microtubules for further structural and functional investigations.
