MICROTUBULE FRAGMENTATION AND PARTITIONING IN THE AXON DURING COLLATERAL BRANCH FORMATION

Axons within the brain branch principally by the formation of collater als rather than by bifurcation of the terminal growth cone (O'Leary an d Terashima, 1988). This same behavior is recapitulated in cultures of embryonic hippocampal neurons (Dotti et al., 1988), rendering them id eal for studies on the cell biological mechanisms underlying collatera l branch formation. In the present study, we focused on changes in the microtubule (MT) array that occur as these axons branch. In particula r, we explored the mechanism by which MT number is locally increased t o accommodate the need for more MTs during collateral branch formation . Serial reconstruction analyses indicate that MT number increases by severalfold and that MT length decreases correspondingly within the pa rent axon in the discrete region giving rise to the branch. These obse rvations strongly suggest that MTs within the parent axon undergo a lo cal fragmentation in this region, and hence raise the possibility that a portion of these new MTs might be destined for transport into the b ranch. To address this latter issue, we used quantitative immunofluore scence to compare the proportion of newly assembled to total MT polyme r in different regions of the axon. As previously reported (Brown et a l., 1992), the region of the axon contiguous with the terminal growth cone is particularly rich in newly assembled polymer. In contrast, the re was no distinguishable difference in the proportion of newly assemb led polymer in the newly formed collateral branches compared to the sh aft region of the parent axon. These results indicate that the MTs wit hin the newly formed collateral branches are on average assembled at t he same time as those within the parent axon, and thus strongly sugges t that the MTs in the collateral branch were assembled in the parent a xon and then translocated into the branch. We conclude on the basis of these observations that collateral branch formation requires a local fragmentation of MTs within the parent axon, followed by the partition ing of a portion of the MT fragments into the branch. These short MTs presumably then resume their movement and elongation down the collater al branch as well as down the parent axon for the steady and orderly i ncrease of both MT arrays.