An improved method for affinity probe localization in whole cells of filamentous fungi.
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.
Bourett
T M
TM
DuPont Agricultural Products, Wilmington, Delaware, 19880-0402, USA.
Czymmek
K J
KJ
Howard
R J
RJ
D000345:Affinity Labels; D003599:Cytoskeleton / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005615:Freezing; D005658:Fungi / Q000648:ultrastructure*; D007075:Immunoglobulin M; D037102:Lectins; D008689:Methacrylates; D018613:Microscopy, Confocal; D008870:Microtubules / Q000648:ultrastructure; D015388:Organelles / Q000648:ultrastructure; D016610:Tissue Embedding / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*
D000345:Affinity Labels; D007075:Immunoglobulin M; D037102:Lectins; D008689:Methacrylates