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First published online 4 November 2008
doi: 10.1242/jcs.033852

Journal of Cell Science 121, 3878-3889 (2008)
Published by The Company of Biologists 2008
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Growth-speed-correlated localization of exocyst and polarisome components in growth zones of Ashbya gossypii hyphal tips

Michael Köhli1, Virginie Galati1, Kamila Boudier1, Robert W. Roberson2 and Peter Philippsen1,*

1 Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland
2 School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA


Figure 1
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  		Fig. 1. Light and electron microscopy of A. gossypii hyphae. (A) Slow- and fast-growing hyphae. The square brackets indicate the section of the hyphae that grew for 7.5 minutes. (B) Hyphal diameters measured 5 µm behind the tip (y-axis) plotted against growth speed (x-axis). Mycelium from the border of a 3-day-old A. gossypii colony was inoculated on thin layer of AFM agar on microscopy slides at room temperature. Elongation speeds were determined as described in the Materials and Methods. (C) Surface expansion rate (SER, y-axis) plotted against growth speed (x-axis). The SER was determined as the product of hyphal circumference and growth speed. The circumference was calculated from the hyphal diameter assuming a circular hyphal profile. The broken line indicates the surface expansion rate of a (theoretical) hypha with a fixed diameter of 3.8 µm. (D) Spitzenkörper (white arrowhead) visible on AFM containing 19% gelatin. The numbers indicate time in seconds. The hyphae were covered with a cover slide and allowed to recover for 2 hours prior to imaging. Scale bars in A,D: 5 µm. (E) Transmission electron microscopy micrograph of 60 nm section of a cryofixed A. gossypii hyphal tip. The inset shows a higher magnification of the region enclosed by the rectangle. The white arrowheads depict small vesicles and the asterisk indicates a large vesicle. Scale bars: 400 nm. (F) The diameters of 140 tip-based vesicles were measured and plotted on the y-axis. The vesicle with the lowest diameter marks the left end of the x-axis, the vesicle with the biggest diameter indicates the right end.

 

Figure 2
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  		Fig. 2. The localization of exocyst components depends on growth speed. (A) AgExo70-GFP in hyphal tips (central planes of z-series). The white numbers indicate growth speed in µm/minute. (B) Growth speed and AgExo70-GFP localization. The dimensions of the AgExo70-GFP signals (y) were measured as shown in the sketch and plotted against growth speed in µm/minute (v). Micrographs for the data-points labeled `a', `b' and `c' in A. (C) AgSEC3-YFP expressing hypha. Growth speed is indicated in µm/minute. (D) Simplified speed/localization plot. Growth speeds are given in µm/minute on the y-axis. The two different localization patterns are shown on the x-axis. Growth speeds of hyphae that displayed a cortical AgSec3-YFP localization are shown on the left half of the plot, growth speeds of hyphae with a spherical AgSec3-YFP localization are shown on the right half. Hyphae that displayed a crescent-like AgSec3-YFP localization were not considered in this analysis. Scale bars: 5 µm.

 

Figure 3
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  		Fig. 3. Exocyst components localize to the A. gossypii Spitzenkörper. (A) FM4-64-stained hyphal tips. The arrowhead indicates an FM4-64-stained Spitzenkörper that is observed in some hyphae. (B) Correlation between growth speed and FM4-64-stained Spitzenkörper. Growth speeds of hyphae without accumulation of internal FM4-64 fluorescence in the hyphal tip are plotted on the left side of the graph; growth speeds of hyphae with a Spitzenkörper are plotted on the right side. (C) FM4-64 staining (red in the overlay) of an AgEXO70-GFP and an AgSEC3-YFP hypha (green in the overlay). Overlaps appear yellow. Scale bars: 5 µm.

 

Figure 4
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  		Fig. 4. Cell polarity factors localize to the cortex tip. DIC and fluorescence images of GFP-AgCDC42, AgCDC24-GFP and AgBEM1-GFP hyphae stained with FM4-64. Scale bar: 5 µm.

 

Figure 5
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  		Fig. 5. Size variations of cortical AgExo70-GFP caps. (A) A z-series of AgEXO70-GFP hypha that grew faster than 1.50 µm/minute was subjected to blind deconvolution. Maximum projections of central planes are shown. Red in the false-colored image indicates high GFP-fluorescence intensity, blue and purple indicate low intensity. Eighty-seven percent of the hyphae showed an accumulation of AgExo70-GFP at the cortex (arrowhead, n=31). (B) The sizes of the cortical AgExo70-GFP areas were estimated assuming that the arc-shaped fluorescent zone visible in the central plane represents the central section of a spherical cap. These values (y-axis) were plotted against growth speed. (C) Frames from time-lapse Movie 1 of an AgEXO70-GFP hypha. The broken lines indicate the hyphal tip, the arrowheads indicate the border of the vesicle fusion zone. Scale bars for A,C: 5 µm. (D) The area of the vesicle fusion zone was estimated for every frame of Movie 1 and plotted against time. The red line results from plotting the mean vesicle fusion area for every 4-second interval. The kymograph shows GFP fluorescence enclosed by the red rectangle in D over time. The advancing tip front is indicated with a broken line. The constant slope of this line indicates a constant growth speed. Scale bars: 2 µm. (E) Model for the size variations of cortical AgExo70-GFP caps.

 

Figure 6
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  		Fig. 6. The polarisome components AgSpa2 and AgPea2 and the formin AgBni1 localize to the Spitzenkörper at high growth speeds. (A) Radial growth of polarisome deletion strains. Mycelium of wt, Agspa2{Delta}, Agpea2{Delta} and Agbud6{Delta} was inoculated on AFM agar, spores of a heterokaryonic Agbni1{Delta} strain were spotted on selective medium and incubated for 6 days at 30°C. Scale bar: 2 cm. (B) DIC and fluorescence images of GFP-AgBNI1, AgSPA2-GFP, AgPEA2-YFP and GFP-AgBUD6 hyphae. The white numbers indicate growth speed in µm/minute. (C) Correlation between localization and growth speed of the polarisome components. Growth speeds are plotted on the y-axis in µm/minute, the localization of the GFP- or YFP-tagged proteins are shown on the x-axis. (D) Overlay of fluorescently labeled polarisome components (green) and FM4-64 staining (red). (E) Stacks of GFP-AgBni1-expressing hypha that grew faster than 1.50 µm/minute were subjected to blind deconvolution. Maximum projections of central planes are shown. Seventy-one displayed a core-like localization of GFP-AgBni1 (black arrowhead, n=31). The gray arrowhead indicates a zone of reduced GFP-AgBni1 fluorescence. (F) Deconvoluted fluorescence images of AgSPA2-GFP hyphae. Fifty-two percent of fast hyphae displayed an enrichment of AgSpa2-GFP in a core region in the Spitzenkörper (top row, black arrowhead). Thirty-four percent of the hyphae displayed a uniform Spitzenkörper localization (bottom row, n=29). (G) GFP-AgBni1 in polarisome deletion strains. The hyphae grew with speeds of between 0.9 and 1.1 µm/minute. (H) Fluorescence ratios between the tip and the cytoplasm. Maximal GFP or YFP fluorescence was determined in two circular areas of 5 µm diameter, the centers of which were located in the tip and in the cytoplasm 10 µm away from the tip. The average ratios tip/cytoplasm were plotted. More than 15 hyphae with a growth speed between 0.75 and 1.3 µm/minute were assessed per strain. Error bars indicate s.e.m. Scale bars for B,D-G: 5 µm.

 

Figure 7
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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.

 

Figure 8
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  		Fig. 8. A model for Spitzenkörper formation in A. gossypii. (A) A slow-growing hyphal tip is outlined, localization of the proteins listed below the sketch are shown in red. (B) Localization of the same proteins as in A in fast growing hyphae. (C) A model for Spitzenkörper formation. The relative amount of vesicle transport is symbolized by the width of the gray arrows. (D) Actin patches representing sites of endocytosis in slow and fast hyphae. In slow hyphae the tip front lacks actin patches (Knechtle et al., 2003Go). An extended tip zone lacks actin patches in fast hyphae (Fig. 7E).

 

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© The Company of Biologists Ltd 2008