A genetically encoded microtubule bundler for causal dissection of microtubule bundling in cells.

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.