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The fungal cell wall, though phylogenetically variable, acts universally as a potent barrier to probing intracellular structures. Thus, the use of high-molecular-weight probes such as antibodies and lectins has proven a formidable challenge. We have devised a preparative method for use with various affinity probes that can be applied to a broad spectrum of filamentous fungal species and used for imaging whole cells. In this study, confocal imaging of whole-mount fungal hyphae after freeze substitution, methacrylate embedment/de-embedment, and infiltration with affinity probes has yielded remarkably improved renderings of the three-dimensional distribution of both microtubules (using antibodies against both alpha- and beta-tubulin) and concanavalin A binding sites. Using this protocol we have been able to document: (1) the three-dimensional distribution of microtubules in all regions of hyphae, (2) the presence of apparent foci for cytoplasmic microtubules, (3) persistent cytoplasmic microtubules during mitosis, and (4) a three-dimensional view of many compartments of the endomembrane system including Golgi-equivalent organelles and apical vesicles. The last result represents the first direct confirmation of apical vesicles comprising the Spitzenkörper., (Copyright 1998 Academic Press.)

Whilst the function and organization of the secretory machinery in eukaryotic cells exhibit basic similarities, the compartmentation of the endomembrane system can show significant differences between the fungal, plant and animal kingdoms. The use of the antibiotic brefeldin A (BFA) as an inhibitor of secretion in both animal and yeast cells has resulted in a remarkable advance in our understanding of the modes of action of vesicle shuttles between the endoplasmic reticulum and Golgi apparatus and within the Golgi apparatus itself. It is now apparent that application of the drug to filamentous fungi and plants will also help unravel the workings of the secretory system in these organisms. In this paper we review recent progress in our laboratories on elucidating the effects of BFA on the morphology of the Golgi apparatus and compare these with recently published data on fungal and plant cells. Variation in the response to BFA are reported, which may not all be attributed to differences in drug concentration and time of treatment. These may reflect differences in cellular sensitivity or multiple sites of action of the drug, and the existence of a specific molecular target for BFA is questioned.