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We show that pyruvate decarboxylase (PDC) 8- to 10-nm-diameter filaments, first described in vegetative cells of Neurospora crassa, are ubiquitously present in filamentous fungi. The cellular arrangement of these structures was examined over the entire sexual cycle of the ascomycetes N. crassa, N. tetraesperma, Podospora anserina, and Sordaria macrospora. PDC-filaments were found associated with the cortical microtubule array of asci and ascospores and absent from the vicinity of spindles and spindle pole bodies. Nocodazole-induced depolymerization of the cortical microtubules results in the loss of PDC-filaments. Moreover, a S. macrospora mutant defective in cortical MT distribution shows abnormal PDC organization. Neurospora asci generated on various metabolic conditions, which modify the presence and relative abundance of PDC-filaments in vegetative cells, have identical patterns of subcellular distribution of these structures. A N. crassa mutant (snowflake) that accumulates giant bundles of PDC-filaments during vegetative growth, shows normal distribution of the filaments during ascogenesis. Thus, the regulation conditioning the presence and supramolecular assembly of PDC-filaments in Neurospora differs between vegetative and sexual cells. Taken together, these results suggest that PDC in filamentous fungi may perform two functions, intervening as an enzyme in vegetative metabolism and as a structural protein associated with the cytoskeleton during sexual development., (Copyright 1999 Academic Press.)

The Podospora anserina cro1 gene was identified as a gene required for sexual sporulation. Crosses homozygous for the cro1-1 mutation yield fruiting bodies which produce few asci due to the formation of giant plurinucleate cells instead of dikaryotic cells after fertilization. This defect does not impair karyogamy, but meioses of the resultant polyploid nuclei are most often abortive. Cytological studies suggest that the primary defect of the mutant is its inability to form septa between the daughter nuclei after each mitosis, a step specific for normal dikaryotic cell divisions. The cro1-1 mutant would thus be unable to leave the syncytial vegetative state while abiding by the meiotic programme. cro1-1 also shows defects in ascospore germination and growth rate. GFP-tagging of the CRO1 protein reveals that it is a cytosolic protein mainly expressed at the beginning of the dikaryotic stage and at the time of ascospore maturation. The CRO1 protein exhibits significant similarity to the SHE4 protein, which is required for asymmetric mating-type switching in budding yeast cells. Thus, a gene involved in asymmetric cell divisions in a unicellular organism plays a key role at the transition between the syncytial (vegetative) state and the cellular (sexual) state in a filamentous fungus.

In heterothallic filamentous ascomycetes, two nuclei of opposite mating type must recognize one another in a plurinucleate cell to form a pair prior to karyogamy. In pseudohomothallic species, two nuclei of opposite mating type must also pair after meiosis to form a binucleate spore. We have examined the cytoskeletal involvement in nuclear pairings by immunofluorescence and drug disruption, using heterothallic and pseudohomothallic species, as well as species without defined mating type (homothallic). Nuclei of species with defined mating type have spindle pole bodies which react with chromatin stains; those of homothallic species do not. The reactivity is seen only in interphase, not during nuclear divisions; thus, the DNA concerned is nuclear and not organellar. From light and immunofluorescence microscopy, the DNA is located at the nuclear face of the spindle pole body (SPB). We suggest that the DNA-SPB association may be involved in the recognition of self and nonself between nuclei of opposite mating types. Nuclei which cooperate in cell formation during ascus development or sporulation are placed in close proximity by the arrangement of spindles during the division preceding cell formation; after division, each nuclear pair remains linked by intertwined microtubule asters. Nuclear pairs must migrate before binucleate spore formation. Drug disruptions established that actin-myosin interaction was the most important cytoskeletal factor in normal spore production. The ascomycete SPB shows unexpected flexibility in form and location during development. Prior to sporulation the outer plaque shows extensive modification in size and orientation. The modified portion detaches from the nucleus and acts as a cortical microtubule organizing center, while the rest of the spindle pole body remains at the nucleus.