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Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography
Published on: February 12, 2014
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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.
Methods in Molecular Biology (Clifton, N.J.)
|April 27, 2022
Summary
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.
