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Fig. 7. Disruption of the microtubule and the actin cytoskeleton in fast-growing hyphae. Hyphal growth after addition of (A) 30 µg/ml nocodazole or (B) 400 µM latrunculin A. The drugs were pipetted onto the border of a 2-day-old mycelium on AFM agar. The numbers indicate the time after drug addition in minutes. Scale bars: 20 µm. (C) Growth speed of hyphae treated with 15 µg/ml nocodazole. Micrographs show anti-tubulin staining (aTub) of nocodazole- or DMSO-treated hyphae prior to the growth speed assessments. The 20-hour-old mycelia were treated with nocodazole for 2 minutes and transferred to solid medium containing nocodazole. Three movies were acquired per condition and the speed of 10 hyphae was measured per movie. Error bars=s.e.m. (D) Immunofluorescence staining of microtubules (aTub) in AgEXO70-GFP. Mycelia were grown and treated as above. Actin staining of AgEXO70-GFP (E) and AgSPA2-GFP (F) grown for 20 hours in liquid AFM. The faint actin cables (arrowhead) are visible only in a subapical region of an overexposed micrograph owing to the clustered actin patches in the tip. (G) Latrunclin A treatment of AgEXO70-GFP. Samples were fixed prior to and 15, 30, 60 and 180 seconds after drug addition and stained with Alexa568-phalloidin. Micrographs of three time points are shown. (H) Quantification of characteristic AgExo70-GFP localization patterns observed upon latrunculin A-treatment. Forty to sixty hyphae were analyzed for the latrunclin A-treated samples and more than 20 for the DMSO controls. Schemes of the different localization patterns are shown below the graphs. The different categories do not add up to 100% as a few hyphae that displayed crescent-like or aberrant localization were not included. (I) Images of AgSPA2-GFP hyphae treated with latrunculin A as described in F. (J) Quantification of AgSpa2-GFP localization in latrunculin A-treated samples. Scale bars for C-G,I: 5 µm.
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