Use of the Vital Stain FM4-64 for Visualizing Membrane Dynamics in Living Fungal Cells

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Allomyces macrogynus is a zoosporic fungus (Chytridiomycota) that exhibits a true alternation of generations between the diploid and haploid thalli. During the diploid stage in the life cycle, sporothalli are produced which are capable of forming asexual zoosporangia containing a multinucleate protoplast bound by a single plasma membrane and cell wall. Though able to remain in a quiescent, coenocytic state for an extended period of time, under certain environmental conditions the cytoplasm of mature zoosporangia is cleaved and gives rise to numerous uninucleate, uniflagellate zoospores within 40 to 50 min. Previous studies of cleavage membrane development m Allomyces zoosporangia have been conducted using light microscopy and transmission electron microscopy (TEM). Despite these investigations, our understanding of cytokinesis in zoosporangia of Allomyces and other zoosporic organisms remains incomplete. This is due, in part, to the difficulty of detecting cleavage elements using standard light microscope optics (i.e., phase contrast, differential interference contrast) and because ultrastructural analysis often provides limited temporal and spatial detail and may suffer from artifacts of sample preparation.