Macromolecular Crowding Tailors the Microtubule Cytoskeleton Through Tubulin Modifications and Microtubule-Associated Proteins

Cells remodel their cytoskeletal networks to adapt to their environment. Here, we analyze the mechanisms utilized by the cell to tailor its microtubule landscape in response to changes in osmolarity that alter macromolecular crowding. By integrating live cell imaging, ex vivo enzymatic assays, and in vitro reconstitution, we probe the impact of acute perturbations in cytoplasmic density on microtubule-associated proteins (MAPs) and tubulin posttranslational modifications (PTMs), unraveling the molecular underpinnings of cellular adaptation via the microtubule cytoskeleton. We find that cells respond to fluctuations in cytoplasmic density by modulating microtubule acetylation, detyrosination, or MAP7 association, without differentially affecting polyglutamylation, tyrosination, or MAP4 association. These MAP-PTM combinations alter intracellular cargo transport, enabling the cell to respond to osmotic challenges. We further dissect the molecular mechanisms governing tubulin PTM specification, and find that MAP7 promotes acetylation by biasing the conformation of the microtubule lattice, and directly inhibits detyrosination. Acetylation and detyrosination can therefore be decoupled and utilized for distinct cellular purposes. Our data reveal that the MAP code dictates the tubulin code, resulting in remodeling of the microtubule cytoskeleton and alteration of intracellular transport as an integrated mechanism of cellular adaptation.