To determine how microtubule (MT) nucleation and nuclear migration are controlled in multinucleated hyphae we deleted genes encoding MTOC subunits and AgStu2. The novel phenotypes we observed in these mutants compared with analogous deletions in budding yeast allowed us to assign functions to the two types of cMTs that we observe in A. gossypii.
In the multinucleate fungus Ashbya gossypii, cytoplasmic microtubules (cMTs) emerge from the spindle pole body outer plaque (OP) in perpendicular and tangential directions. To elucidate the role of cMTs in forward/backward movements (oscillations) and bypassing of nuclei, we constructed mutants potentially affecting cMT nucleation or stability. Hyphae lacking the OP components AgSpc72, AgNud1, AgCnm67, or the microtubule-stabilizing factor AgStu2 grew like wild- type but showed substantial alterations in the number, length, and/or nucleation sites of cMTs. These mutants differently influenced nuclear oscillation and bypassing. In Agspc72Δ, only long cMTs were observed, which emanate tangentially from reduced OPs; nuclei mainly moved with the cytoplasmic stream but some performed rapid bypassing. Agnud1Δ and Agcnm67Δ lack OPs; short and long cMTs emerged from the spindle pole body bridge/half-bridge structures, explaining nuclear oscillation and bypassing in these mutants. In Agstu2Δ only very short cMTs emanated from structurally intact OPs; all nuclei moved with the cytoplasmic stream. Therefore, long tangential cMTs promote nuclear bypassing and short cMTs are important for nuclear oscillation. Our electron microscopy ultrastructural analysis also indicated that assembly of the OP occurs in a stepwise manner, starting with AgCnm67, followed by AgNud1 and lastly AgSpc72.