Spatially patterned cytoskeletal organization shapes astrocyte branch complexity

Abstract

Astrocytes, one of the most abundant cell types 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 complex, hierarchically branched morphology and surveyed their cytoskeleton using confocal microscopy and cryogenic electron tomography. We show that microtubules in the primary branches of immunopanned astrocytes are oriented primarily plus-end-out. Proximally, microtubules appear stabilized by post-translational modifications (PTMs) and microtubule inner proteins. Distal regions lack stabilizing microtubule PTMs, and are enriched in intermediate filament GFAP. Additionally, diverse actin microstructures, including reticular webbing, extend astrocyte boundaries beyond the microtubule-GFAP framework. Finally, pharmacological disruption of actin polymerization alters primary branch number and length, providing functional support for the interplay of cytoskeletal classes in defining astrocyte branching. Together, our results uncover spatial principles of astrocyte cytoskeletal organization that support complex branching morphology.