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First published online 14 September 2004
doi: 10.1242/jcs.01377

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Apical localization of actin patches and vacuolar dynamics in Ashbya gossypii depend on the WASP homolog Wal1p

Junior Research Group: Growth-control of Fungal Pathogens, Hans-Knöll Institute for Natural Products Research and Department of Microbiology, Friedrich-Schiller University, Hans-Knöll Strasse 2, 07745 Jena, Germany

View larger version (77K): [in a new window]   Fig. 1. Growth assay of wild-type and mutant strains. (A) Wild-type and wal1 mutant were grown on YPD plates at the indicated temperatures for 5 days before photography. Optimal growth conditions are at 30°C. Loss of WAL1 results in slow growth and temperature sensitivity (no growth) at elevated temperatures. (B) This temperature-sensitive phenotype can be complemented by introduction of a plasmid bearing the complete WAL1 gene (pWAL1cc) at 37°C. Complementation with plasmid-borne WAL1 at lower temperatures (30°C) results in increased growth of the mutant. However, owing to lack of selective pressure to maintain the plasmid growth rate is slower than in the wild type.

View larger version (51K): [in a new window]   Fig. 2. Comparison of actin cytoskeletal organization in the wild type and in the wal1 mutant. Representative fluorescent images are shown of hyphae stained with rhodamine-phalloidin of the wild type (A,B) and the wal1 mutant (C,D). Small brackets mark the concentration of cortical actin patches in the hyphal tip of the wild type (A,B). Larger brackets point to the subapical region, where patches accumulate in wal1 hyphae (C,D). Scale bar, 10 µm.

View larger version (75K): [in a new window]   Fig. 3. Loss of WAL1 leads to increased isotropic growth and defects in septation. (A) Wild-type hyphae showed accumulation of chitin at septal sites (arrowheads) and more intensely stained hyphal apices (brackets). (B,C) The wal1 hyphae were stained uniformly and rarely displayed chitin-rich septa. Notice the swellings of wal1 hyphae and the short distance between consecutive dichotomous tip branches. Staining was done using calcofluor. Scale bar, 20 µm.

View larger version (85K): [in a new window]   Fig. 4. In vivo time-lapse analysis of the early stages of growth in the wild type and the wal1 mutant. Spores were germinated in liquid YPD or YPD supplemented with G418 for selection of the wal1 mutant for 8 hours at 30°C and then transferred to microwell-containing slides for further observation. Representative frames from Movies 1 and 2 (see supplementary material) are shown at the indicated time points (hours:minutes). Arrowheads point to hyphal tips that ceased growth and asterisks mark positions where contact inhibition occurred. Scale bars, 40 µm (A), 20 µm (B).

View larger version (145K): [in a new window]   Fig. 5. In vivo time-lapse analysis of growth of adult stages of wild-type hyphae. Mycelium from exponentially grown liquid YPD cultures was transferred to slides and growth was monitored. Images represent frames from Movie 3 (see supplementary material) at the corresponding time points (hours:minutes). Septal sites are marked by asterisks. Cytoplasmic flow as visible by the movement of large vacuoles is indicated by arrows. Arrowheads mark the same vacuole at different time points. Septation alters the direction of flow and flow was also reversed in one hypha. Scale bar, 40 µm.

View larger version (157K): [in a new window]   Fig. 6. In vivo time-lapse analysis of growth of adult stages of wal1 hyphae. Mycelium from exponentially grown liquid YPD cultures was transferred to slides and growth was monitored. Images represent frames from Movie 4 (see supplementary material) at the corresponding time points (hours:minutes). Boxes indicate three regions processed for Fig. 7 and Movie 5 (see supplementary material). Scale bar, 40 µm.

View larger version (102K): [in a new window]   Fig. 7. Growth defect of subapical regions of wal1 hyphae. Three rows represent boxed region in Fig. 6. Arrowheads point to subapical regions that result in aberrant swellings. The asterisk marks a hyphal tip that burst during the time-lapse imaging.

View larger version (72K): [in a new window]   Fig. 8. Organelle distribution in A. gossypii wild-type and mutant strains. Vacuolar morphology of the wild type (A,E) and the wal1 (B,F), cla4 (C,G) and bem2 (D,H) mutants was analysed at hyphal tip regions containing early endosomes (A-D) and in older hyphal segments that contained larger vacuoles (E-H) using the vacuolar dye FM4-64. Images are from Movies 6-9 (see supplementary material). Notice the tip localization and the presence of tubular structures of endosomes in the wild type and the cla4 and bem2 mutant strains, whereas wal1 hyphal tips did not contain endocytic vesicles. Also, larger vacuoles of the wild type and the bem2 mutant strain were highly motile over the imaged period, whereas wal1 and cla4 vacuoles appeared immobile. Mitochondria in wild-type (I) and wal1 (J) hyphae were stained with DIOC6 (see Movie 12 in supplementary material).