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Updated: Feb 7, 2026
Directly Measuring Forces Within Reconstituted Active Microtubule Bundles
Published on: May 10, 2022
A genetically encoded microtubule bundler for causal dissection of microtubule bundling in cells
Soei Watari1,2, Takumi Chinen3, Yuto Kunitatsu4
1Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori 680-8552, Japan.
Abstract:
Microtubule (MT) bundling is a conserved organizational feature of the cytoskeleton that accompanies MT stabilization. MT bundling is suggested to engage with diverse cellular processes, including mitosis, migration, and axon morphogenesis. Although microtubule-associated proteins are known to induce MT bundling, whether bundling itself is sufficient to alter MT properties and cellular behavior has remained difficult to address due to the lack of tools that selectively manipulate MT bundling in living cells. Here, we describe the development of a genetically encoded, protein-based "MT-Bundler" by coupling an MT-binding motif to a biologically inert oligomerization scaffold, enabling direct and tunable crosslinking of intracellular MTs. The expression of MT-Bundler consisting of MAP4 and Azami-Green not only drove robust MT bundling but also conferred marked resistance to depolymerization and elevated MT acetylation. Functionally, enforced MT bundling disrupts cell division and migration and suppresses neurite and axon outgrowth. To confirm the causal relationship behind these findings, we further engineered MT-Bundlers to make them chemically and optically inducible to permit rapid, reversible, and spatiotemporally precise control of MT bundling. Acute induction of MT bundling triggers a rapid increase in MT acetylation, implying bundling as an upstream organizational cue that promotes luminal access of the acetyltransferase ATAT1. Notably, MT stabilization persists even in the absence of acetylation, demonstrating that bundling itself is sufficient to mechanically stabilize MTs. Together, these results identify MT bundling as a primary determinant of MT stability and modification, establishing MT-Bundlers as a versatile tool to dissect the mechanistic basis of MT bundling in living cells.
Insights
Microtubule bundling, a key cytoskeletal organization, stabilizes microtubules. New protein tools called MT-Bundlers demonstrate that bundling itself drives microtubule stability and modification, impacting cell functions.
Area of Science:
- Cell Biology
- Cytoskeletal Dynamics
- Molecular Biology
Background:
- Microtubule (MT) bundling is a fundamental cytoskeletal feature linked to MT stabilization and various cellular processes like mitosis and migration.
- Existing methods using microtubule-associated proteins lack specificity for studying bundling's direct effects.
- A gap exists in understanding if MT bundling alone can alter MT properties and cellular behavior.
Purpose of the Study:
- To develop a novel tool for selectively manipulating MT bundling in living cells.
- To investigate the direct impact of MT bundling on MT stability, modification, and cellular functions.
- To establish a causal link between MT bundling and its downstream effects.
Main Methods:
- Development of a genetically encoded, protein-based "MT-Bundler" by fusing an MT-binding motif to an oligomerization scaffold.
- Expression of MT-Bundlers (e.g., MAP4 and Azami-Green) to induce and control intracellular MT crosslinking.
- Engineering of chemically and optically inducible MT-Bundlers for precise, reversible spatiotemporal control.
Main Results:
- Engineered MT-Bundlers successfully induced robust MT bundling, increased resistance to depolymerization, and elevated MT acetylation.
- Enforced MT bundling disrupted cell division, migration, and suppressed neurite/axon outgrowth.
- Acute MT bundling induction rapidly increased MT acetylation, suggesting bundling as an upstream cue for ATAT1 access.
- MT stabilization was observed independently of acetylation, confirming bundling's mechanical stabilization role.
Conclusions:
- MT bundling is a primary determinant of MT stability and modification.
- MT-Bundlers provide a versatile tool to dissect the mechanistic basis of MT bundling in living cells.
- Bundling itself is sufficient to mechanically stabilize microtubules, independent of post-translational modifications like acetylation.
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