Spatially patterned cytoskeletal organization shapes astrocyte branch complexity

Astrocytes, the most abundant cells in the brain, extend elaborate branches that enable diverse functions, from synapse maintenance to blood–brain–barrier integrity. The cytoskeletal basis of this architecture has remained unclear, since traditional culturing methods produce minimal branching. Using immunopanning and serum-free conditions, we generated primary rodent astrocytes with in–vivo–like morphology and surveyed their cytoskeleton using confocal microscopy and cryogenic electron tomography. We show that astrocyte microtubules are oriented primarily plus–ends–out. Proximally, microtubules appear stabilized by post-translational modifications (PTMs) and microtubule inner proteins (MIPs). Distal regions lack stabilizing microtubule PTMs, and are enriched in intermediate filament (IF) GFAP. Additionally, diverse actin microstructures, including reticular webbing, extend astrocyte boundaries beyond the microtubule—IF framework. Our results uncover fundamental principles of astrocyte cytoskeletal organization that underlie their intricate branching.