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Updated: Jul 6, 2026
Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues
Published on: November 17, 2023
Mass spectrometric analysis of microtubule co-sedimented proteins from rat brain
Tatsuhiko Sakamoto1, Akiyoshi Uezu, Shinya Kawauchi
1Department of Molecular Pharmacology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, 1-1-1 Honjo, Kumamoto 860-8556, Japan.
Abstract:
Microtubules (MTs) play crucial roles in a variety of cell functions, such as mitosis, vesicle transport and cell motility. MTs also compose specialized structures, such as centrosomes, spindles and cilia. However, molecular mechanisms of these MT-based functions and structures are not fully understood. Here, we analyzed MT co-sedimented proteins from rat brain by tandem mass spectrometry (MS) upon ion exchange column chromatography. We identified a total of 391 proteins. These proteins were grouped into 12 categories: 57 MT cytoskeletal proteins, including MT-associated proteins (MAPs) and motor proteins; 66 other cytoskeletal proteins; 4 centrosomal proteins; 10 chaperons; 5 Golgi proteins; 7 mitochondrial proteins; 62 nucleic acid-binding proteins; 14 nuclear proteins; 13 ribosomal proteins; 28 vesicle transport proteins; 83 proteins with diverse function and/or localization; and 42 uncharacterized proteins. Of these uncharacterized proteins, six proteins were expressed in cultured cells, resulting in the identification of three novel components of centrosomes and cilia. Our present method is not specific for MAPs, but is useful for identifying low abundant novel MAPs and components of MT-based structures. Our analysis provides an extensive list of potential candidates for future study of the molecular mechanisms of MT-based functions and structures.
Insights
Researchers identified 391 proteins associated with microtubules (MTs) in rat brains, including novel components of cellular structures like centrosomes and cilia. This study advances understanding of MT-based functions and cellular organization.
Area of Science:
- Cell Biology
- Proteomics
- Cytoskeletal Dynamics
Background:
- Microtubules (MTs) are essential for vital cellular processes including mitosis, intracellular transport, and motility.
- The precise molecular mechanisms governing MT-based functions and structures remain incompletely understood.
- Identifying novel proteins interacting with MTs is crucial for elucidating these mechanisms.
Purpose of the Study:
- To comprehensively identify proteins that co-sediment with microtubules from rat brain.
- To characterize the functional and localization diversity of identified MT-associated proteins.
- To discover novel components of MT-based cellular structures.
Main Methods:
- Proteins co-sedimented with microtubules from rat brain were analyzed using tandem mass spectrometry (MS).
- Ion exchange column chromatography was employed for protein separation prior to MS analysis.
- Six uncharacterized proteins were expressed in cultured cells to validate their localization.
Main Results:
- A total of 391 proteins were identified and categorized into 12 functional groups.
- Key categories included MT cytoskeletal proteins (57), other cytoskeletal proteins (66), and nucleic acid-binding proteins (62).
- Three novel components of centrosomes and cilia were identified among the expressed uncharacterized proteins.
Conclusions:
- The applied method effectively identifies novel, low-abundance microtubule-associated proteins (MAPs) and components of MT-based structures.
- This extensive protein list provides valuable candidates for future research into MT functions.
- The findings significantly contribute to understanding the molecular underpinnings of microtubule-based cellular organization and dynamics.
