Microtubule Severing at Crossover Sites by Katanin Generates Ordered Cortical Microtubule Arrays in Arabidopsis

The noncentrosomal cortical microtubules (CMTs) of land plants form highly ordered parallel arrays that mediate cell morphogenesis by orienting cellulose deposition. Since new CMTs initiate from dispersed cortical sites at random orientations, parallel array organization is hypothesized to require selective pruning of CMTs that are not in the dominant orientation. Severing of CMTs at crossover sites is proposed to be a potential pruning mechanism; however, the parameters and molecular mechanisms underlying this activity are unknown. Here, using live-cell imaging, we show that severing preferentially targets the overlying CMTs at crossover sites and leads to their depolymerization about 85% of the time. In addition, the probability of severing has a sigmoidal relationship to the crossover dwell time, indicating a strong bias for longer-lived crossover sites to be severed. We found that severing at CMT crossover sites was completely abolished in the Arabidopsis katanin mutant. Consistent with this finding, GFP-tagged katanin driven by its native promoter localizes at sites of CMT crossover prior to severing. Furthermore, array recovery experiments showed that CMTs fail to become ordered in the katanin mutant. We conclude that katanin is solely responsible for severing at CMT crossover sites and that this activity is essential to generate ordered CMT arrays.