Supplementary Material for: Rear-Side Localization of the Centrosome in Migrating Neuroblastoma Neuro-2a Cells and Its Roles in Process Elongation

2012-06-01T00:00:00Z (GMT) by Kawabata W. Yonezawa S. Hayashi K.
Axon elongation is usually performed by the migration of growth cones that leave axons. Axon microtubules are generated by enhanced polymerization of tubulin in the growth cones. Some kinds of neurons like cerebellar granule cells, however, generate axons as a result of migration of the cell body leaving axons at the rear. The mechanism to generate microtubules during such growth cone-independent elongation of axons is not well understood. To establish an experimental model to study this mechanism, we cultured neuroblastoma (Neuro-2a) cells on substrates that facilitate cell migration. When cultured on laminin-treated substrate, cells migrated actively and left processes at the rear. We investigated the role of the centrosome in this process formation. The centrosomes were always located at the base of the processes, i.e., at the rear side of the migrating cell body. Close observation of cytoskeletons revealed microtubules limited around the centrosomes, but concentrated at the periphery of the cells or within the processes. Microtubule regrowth experiments showed the ability of the centrosomes to nucleate microtubules. We thus examined the role of microtubule release from the centrosomes, by knocking down the expression of spastin, a microtubule-severing enzyme. Introducing siRNA for spastin into Neuro-2a cells reduced both the migration speed and the length of the processes. Taken together, Neuro-2a cells on laminin proved useful as a model to study the alternative type of axon elongation in which cell migration leaves axons at the rear. This model provided evidence for the involvement of microtubule release from centrosomes in the mechanisms for this type of process elongation.