Immunolocalization of tubulin and actin in thick-sectioned fungal hyphae after freeze-substitution fixation and methacrylate de-embedment

Immunolocalizations in whole mounts of walled cells require an empirically derived, species-specific permeabilization strategy. Particularly when cell walls are recalcitrant to enzymatic digestion, as with many fungi, permeabilization can degrade structure and reduce or destroy antigenicity. To avoid these potential problems, hyphae were freeze-substituted, embedded in a methacrylate resin, thick-sectioned and de-embedded prior to immunolocalization. The cytoskeletal proteins actin and β-tubulin were labelled via indirect immunofluorescence in hyphal tip cells of Magnaporthe grisea , and Trichoderma viride . Well-defined punctate actin plaques were observed concentrated at the subapical cell periphery in both species, especially near the hypha–substratum interface. In addition, a Spitzenkörper core and less intense cytoplasmic binding were observed. Immunoelectron microscopy confirmed localization of actin in the Spitzenkörper core and filasomes. β-Tubulin was immunolocalized as both linear arrays and punctate dots via epifluorescence microscopy. Analysis of 450–550-nm-thick serial sections contributed to improved resolution by the reduction of out-of-focus blur. Overall, the freeze substitution methacrylate de-embedment technique provided superior preservation, serial section analysis, and improved antibody penetration and resolution of these cytoskeletal proteins. The technique will be a valuable tool for use with a variety of reporter molecules in cells of fungi.     

Microinjecting FM4-64 validates it as a marker of the endocytic pathway in plants

The amphiphilic dye FM4–64 is used to investigate endocytosis and vesicle trafficking in living eukaryotic cells. The standing hypothesis is that it is inserted into the outer leaflet of the plasma membrane and, from there, is passed on to intracellular membrane compartments by endocytosis. We tested this hypothesis by microinjecting FM4–64 into the cytoplasm and vacuole of Nicotiana tabacum BY-2 suspension culture cells and Tradescantia virginiana stamen hair cells. We found that the dye did not label any membranes when injected into the cytoplasm, but clearly labelled the tonoplast when injected directly into the vacuole. However, because the dye is pH-sensitive, the fluorescence intensity between the plasma membrane and tonoplast varied. We conclude that FM4–64 is a specific marker for the endocytic pathway. Nevertheless, little is known about the molecular interactions of FM4–64 with these particular phospholipid membrane leaflets. We, therefore, appeal for biochemical research to determine which membrane lipids FM4–64 interacts with.     

Confocal microscopy as a tool for the study of the intranuclear topography of chromosomes

A scanning confocal microscope was used to investigate the spatial positions of specific regions within blood cell nuclei. These centromeric regions were fluorescently labelled by in-situ hybridization to suspended nuclei with a centromere-1-specific DNA probe. The 3-D image data sets, obtained by optical sectioning of the cells, were used to determine the spatial position of the centromeric regions in the nuclei by means of specially developed software. The centromeres were found to be localized near the nuclear boundary. This spatial pattern was tested against a random distribution model by means of the Kolmogorov—Smirnov test. The difference between the two patterns was at a P < 0?01 significance level.