Endogenous βⅢ-tubulin FRAP reveals microtubule dynamics in living developing neurons

Microtubules support neuronal morphology, intracellular transport, and neurite growth, but how microtubule turnover is regulated across neuronal compartments remains incompletely understood. To examine microtubule dynamics under physiological expression conditions, we expressed EGFP-tagged βIII-tubulin from the endogenous Tubb3 locus in cultured rat hippocampal neurons and measured tubulin turnover using fluorescence recovery after photobleaching (FRAP). FRAP analysis revealed pronounced spatial differences in microtubule dynamics at 9 days in vitro (DIV), with the highest turnover observed in growth cones and substantially slower turnover in dendrites, axons, and the axon initial segment (AIS). Microtubule turnover further decreased between 9 and 17 DIV in dendrites and axons, indicating progressive stabilization during neuronal maturation, whereas turnover in the AIS remained largely unchanged. Analysis of EB3 comet dynamics suggested that microtubule polymerization contributes substantially to tubulin turnover. In addition, manipulation of the dendritic microtubule-associated protein MAP-2 altered tubulin FRAP, indicating that MAP-2 contributes to the regulation of neuronal microtubule dynamics. Together, these results demonstrate that microtubule turnover is spatially and developmentally regulated in neurons.