Polarity reversal of stable microtubules during neuronal development

Long-distance transport in neurons relies on motor proteins that can move towards either the plus- or minus-end of microtubules. In axons, microtubules uniformly have a plus-end-out orientation, whereas dendrites of vertebrate neurons contain mixed polarity bundles: stable microtubules are typically minus-end-out, and dynamic microtubules are plus-end-out. This organization supports selective transport, yet how this dedicated microtubule organization is established is unclear. Here, we use single-molecule localization microscopy, expansion microscopy and live-cell imaging to examine how the microtubule cytoskeleton is reorganized during neuronal development in cultured rat hippocampal neurons. We find that early neurites contain mixed polarity microtubules, with stable microtubules initially mostly plus-end-out and often connected to centrioles. As neurons mature, these microtubules detach, slide and gradually reorient to become predominantly minus-end-out within the future dendrites. Moreover, prior to axon specification, neurons often have one or two minor neurites with an almost uniformly plus-end-out microtubule network. Our findings show how reorganization of stable microtubules underlies the establishment of the characteristic microtubule network in mature vertebrate neurons.