Tubulin acetylation governs organelle remodeling and lysosomal reformation during neuronal differentiation

A functional nervous system depends on neuronal morphology established during differentiation. The microtubule (MT) cytoskeleton supports neuronal differentiation by organizing organelle positioning and facilitating transport. The dynamics and properties of MTs are regulated by a variety of post-translational modifications (PTMs), with many organelle interactions occurring preferentially on modified MTs. Here we find that tubulin acetylation is enriched at specific subcellular locations during differentiation of human induced neurons. We apply a quantitative multispectral imaging pipeline to simultaneously analyze eight membrane-bound organelles and define how tubulin acetylation reshapes organelle architecture and interaction networks during neuronal differentiation. We find that loss of tubulin acetylation broadly alters organelle morphology, spatial distribution, and inter-organelle interactions, with lysosome-organelle interactions most affected. Loss of acetylated MTs leads to enlarged, highly acidified lysosomes, impaired lysosomal fission, and accumulation of autolysosomes, consistent with defective lysosomal reformation. Super-resolution microscopy further reveals that lysosome-endoplasmic reticulum (ER) contacts preferentially associate with acetylated MTs. Together, our data support a model in which tubulin acetylation coordinates lysosome-ER interactions to facilitate lysosome remodeling and turnover. This work establishes tubulin acetylation as a key cytoskeletal regulator that links organelle interactions to organelle homeostasis important for neuronal differentiation.