Investigation of microtubule assembly and organization accompanying tension-induced neurite initiation

Pulling on the margin of embryonic chick sensory neurons induces neurite formation de novo. We find that these neurites contain microtubules within minutes after the application of tension and apparently normal microtubule arrays within 10-20 min. We wished to determine whether these microtubules reflected existing microtubules that were reorganized, e.g. pulled into the neurite by the applied forces, or whether they reflected primarily new assembly of tubulin. We investigated tension-induced neurite initiation in the presence of 4 nM vinblastine, a concentration that poisons net microtubule assembly but does not depolymerize extant polymers, thus separating new assembly from movements of existing microtubules. We find that vinblastine seriously compromises the ability of chick sensory neurons to initiate neurites in response to tension. The few poisoned neurites that did form were abnormal in several respects. In contrast to unpoisoned cells, poisoned neurites were prone to stretching and breaking while pulling, as though they lacked normal structural support. Indeed, poisoned neurites possessed only short microtubule fragments. We conclude that the microtubule array seen in tension-induced neurites reflects primarily new microtubule assembly, rather than existing microtubules that were reorganized to invade the neurite. This implies that tension applied to unpoisoned chick sensory neurons rapidly stimulates new microtubule assembly concomitant with neurite initiation. Examination of the tension-induced microtubules shows that both their spatial pattern and their acetylation are similar to that reported for normal growth cone-mediated neurites.