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SummaryConfocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4-64, FM1-43 or TMA-DPH, three of the most commonly used membrane-selective dyes reported as markers of endocytosis. All three dyes were rapidly internalized within hyphae. FM4-64 was found best for imaging the dynamic changes in size, morphology and position of the apical vesicle cluster within growing hyphal tips because of its staining pattern, greater photostability and low cytotoxicity. FM4-64 was taken up into both the apical and subapical compartments of living hyphae in a time-dependent manner. The pattern of stain distribution was broadly similar in a range of fungal species tested (Aspergillus nidulans, Botrytis cinerea, Magnaporthe grisea, Neurospora crassa, Phycomyces blakesleeanus, Puccinia graminis, Rhizoctonia solani, Sclerotinia sclerotiorum and Trichoderma viride). With time, FM4-64 was internalized from the plasma membrane appearing in structures corresponding to putative endosomes, the apical vesicle cluster, the vacuolar membrane and mitochondria. These observations are consistent with dye internalization by endocytosis. A speculative model of the vesicle trafficking network within growing hyphae is presented.

Methylation of cytosines silences transposable elements and selected cellular genes in mammals, plants, and some fungi. Recent findings have revealed mechanistic connections between DNA methylation and features of specialized condensed chromatin, "heterochromatin." In Neurospora crassa, DNA methylation depends on trimethylation of Lys9 in histone H3 by DIM-5. Heterochromatin protein HP1 binds methylated Lys9 in vitro. We therefore investigated the possibility that a Neurospora HP1 homolog reads the methyl-Lys9 mark to signal DNA methylation. We identified an HP1 homolog and showed that it is essential for DNA methylation, is localized to heterochromatic foci, and that this localization is dependent on the catalytic activity of DIM-5. We conclude that HP1 serves as an adaptor between methylated H3 Lys9 and the DNA methylation machinery. Unlike mutants that lack DNA methyltransferase, mutants with defects in the HP1 gene hpo exhibit severe growth defects, suggesting that HP1 is required for processes besides DNA methylation.

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