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, respectivel y, were examined by video-enhanced phase-contrast microscopy and image anal ysis. In contrast to the regular, hyphoid morphology of wildtype hyphae, th e hyphae of the ropy mutants exhibited a great variety of distorted, non-hy phoid morphologies. The ropy hyphae were slow-growing and manifested freque nt loss of growth directionality, Cytoplasmic appearance, including organel le distribution and movement, were ostensibly different in the ropy hyphae. The Spitzenkorper (Spk) of wild-type hyphae was readily seen by phase-cont rast 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 wa s smaller and less phase-dark than the wild-type Spk. Growth rate in both w ild-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 ro py mutants and wild-type hyphae was in Spk trajectory, Whereas the Spk of w ild-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 mo rphology. A causal correlation between Spk trajectory and cell shape was te sted with the Fungus Simulator program. The characteristic morphologies of wild-type or ropy hyphae were reproduced by the Fungus Simulator, whose ves icle supply centre (VSC) was programmed to follow the corresponding Spk tra jectories. This is evidence that the Spk controls hyphal morphology by oper ating as a VSC. These findings on dynein or dynactin deficiency support the notion that the microtubular cytoskeleton plays a major role in the format ion and positioning of the Spk, with dramatic consequences on hyphal growth and morphogenesis.