Dynein and dynactin deficiencies affect the formation and function of the Spitzenkorper and distort hyphal morphogenesis of Neurospora crassa

The impact of mutations affecting microtubule-associated motor proteins on the morphology and cytology of hyphae of Neurospora crassa was studied. Two ropy mutants, ro-1 and ro-3, deficient in dynein and dynactin, respectively, were examined by video-enhanced phase-contrast microscopy and image analysis. In contrast to the regular, hyphoid morphology of wildtype hyphae, the hyphae of the ropy mutants exhibited a great variety of distorted, non-hyphoid morphologies. The ropy hyphae were slow-growing and manifested frequent loss of growth directionality, Cytoplasmic appearance, including organelle distribution and movement, were ostensibly different in the ropy hyphae. The Spitzenkorper (Spk) of wild-type hyphae was readily seen by phase-contrast optics; the Spk of both ro-1 and ro-3 was less prominent and sometimes undetectable. Only the fast-growing ropy hyphae displayed a Spk, and it was smaller and less phase-dark than the wild-type Spk. Growth rate in both wild-type and ropy mutants was directly correlated with the size of the Spk. Spk efficiency, measured in terms of cell area generated per Spk travelled distance, was lower in ropy mutants. Another salient difference between ropy mutants and wild-type hyphae was in Spk trajectory, Whereas the Spk of wild-type hyphae maintained a trajectory close to the cell growth axis, the Spk of ropy hyphae moved much more erratically. Sustained departures in the trajectory Of the ropy Spk produced corresponding distortions in hyphal morphology. A causal correlation between Spk trajectory and cell shape was tested with the Fungus Simulator program. The characteristic morphologies of wild-type or ropy hyphae were reproduced by the Fungus Simulator, whose vesicle supply centre (VSC) was programmed to follow the corresponding Spk trajectories. This is evidence that the Spk controls hyphal morphology by operating as a VSC. These findings on dynein or dynactin deficiency support the notion that the microtubular cytoskeleton plays a major role in the formation and positioning of the Spk, with dramatic consequences on hyphal growth and morphogenesis.