Live-cell imaging of microtubule dynamics in hyphae of Neurospora crassa

Due to the large number of microtubules in the wild-type strain, total internal reflection fluorescence (TIRF) microscopy was used to study cortical microtubule dynamics in leading hyphae of Neurospora crassa expressing beta-tubulin-GFP. Detection of plus-end dynamics of individual microtubule was much improved with this approach compared to the other commonly used methods such as confocal and widefield fluorescence microscopy. In order to address the roles of motor proteins in microtubule dynamics, microtubule-motor mutant strains, deltankin and ro-1 were examined. Unlike the wild-type strain, there were fewer microtubules in these hyphal cells; therefore, imaging was done using widefield fluorescence microscopy. We have shown that polymerization and depolymerization rates as well as hyphal extension rates were reduced by one half relative to those of wild type. Therefore, we believe that the hyphal extension rates are dependent upon the dynamic characteristics of microtubules, which are then regulated by microtubule motors in N. crassa.