Verplicht vrouwen in de top? Een onderzoek naar het beleidseffect van de Nederlandse inspanningsverplichting van evenwichtige verdeling tussen mannen en vrouwen op de top van grote beursgenoteerde ondernemingen in Nederland

<p>A) Phase contrast images of tip-branching in wild-type (top row) and <i>ΔAgrho2</i> strains. The images show 3 time-points of tip-branching events taken from time-lapse movies. The time-points in minutes are indicated in the top right corner of each image. The growth speed of the hyphae directly prior to tip-branching was determined from the time-lapse movie and is presented on the right side of the figure. Scale bar, 20 µm. B) Actin stained with rhodamine-phalloidin of hyphae from wild-type and <i>ΔAgrho2</i> strains. Scale bar, 5 µm.</p

The Small GTP-Binding Proteins <i>Ag</i>Rho2 and <i>Ag</i>Rho5 Regulate Tip-Branching, Maintenance of the Growth Axis and Actin-Ring-Integrity in the Filamentous Fungus <i>Ashbya gossypii</i>

<div><p>GTPases of the Rho family are important molecular switches that regulate many basic cellular processes. The function of the Rho2 and Rho5 proteins from <i>Saccharomyces cerevisiae</i> and of their homologs in other species is poorly understood. Here, we report on the analysis of the <i>Ag</i>Rho2 and <i>Ag</i>Rho5 proteins of the filamentous fungus <i>Ashbya gossypii</i>. In contrast to <i>S. cerevisiae</i> mutants of both encoding genes displayed a strong morphological phenotype. The <i>Agrho2</i> mutants showed defects in tip-branching, while <i>Agrho5</i> mutants had a significantly decreased growth rate and failed to maintain their growth axis. In addition, the <i>Agrho5</i> mutants had highly defective actin rings at septation sites. We also found that a deletion mutant of a putative GDP-GTP-exchange factor (GEF) that was homologous to a Rac-GEF from other species phenocopied the <i>Agrho5</i> mutant, suggesting that both proteins act in the same pathway, but the <i>Ag</i>Rho5 protein has acquired functions that are fulfilled by Rac-proteins in other species.</p></div

Actin instability at the hyphal tip of a <i>Δ</i><i>Agrho5</i> mutant.

<p>DIC and fluorescence images of a hypha of a <i>ΔAgrho5</i> strain carrying an actin-binding domain fused to GFP. The growth of the strain was followed by time-lapse microscopy for 150 minutes. Images were taken every 5 minutes. For each time-point, pixel intensities were measured over the first 2 µm of the hypha. The difference between the pixel intensities of the upper half of pixels versus the lower half of the hypha is plotted for each time-point in the graph below the images. In addition, for each time-point, the corresponding hyphal tip is shown in heat map coloring, with cold colors representing low-pixel intensities and warm colors represent high-pixel intensities. The scale bar represents 10 µm.</p

Growth of <i>Agrho2</i> mutant strains.

<p>A) Measurement of the radial growth speed on solid medium at 30° for the wild type, <i>Agrho2</i> deletion and a strain carrying a GTP-locked allele of <i>Agrho2</i>. Shown are the arithmetic mean and standard deviation (n> = 3). The diameter of the initial inoculum was subtracted from each time-point, such that all measurements began at 0 mm. B) Example images of mycelia from the measurements in A) that display the difference in diameter taken at day 6 of the measurements. C) Measurement of the radial growth speed on solid medium at 30° for the wild type and <i>Agrho2</i> overexpression. Shown are the arithmetic mean and standard deviation (n = 2). The diameter of the initial inoculum was subtracted from each time-point, such that all measurements began at 0 mm. D) Example images of mycelia from the measurements in C) that display the difference in diameter taken at day 6 of the measurements.</p

Localization of mCherry-<i>Ag</i>Rho5.

<p>A) DIC and fluorescence image of a <i>ΔAgrho5</i> strain expressing a fusion of mCherry to <i>Ag</i>Rho5 from a vector. The scale bar represents 5 µm. B) Mycelia of wild-type, <i>ΔAgrho5</i> and the <i>ΔAgrho5</i> strain expressing the mCherry-<i>AgRHO5</i> construct. C) DIC images of single hyphae and tip-branching hyphae of a <i>ΔAgrho5</i> strain carrying expressing wild-type <i>AgRHO5</i> from a plasmid. D) Mycelia of wild-type, <i>ΔAgrho5</i> and the <i>ΔAgrho5</i> strain expressing wild-type <i>AgRHO5</i> from a plasmid. The scale bar is 1 cm.</p

Phentoype of a <i>ΔAgrho2/ΔAgrho5</i> double deletion.

<p>A) Mycelia of wild-type, <i>ΔAgrho2, ΔAgrho5</i> and a <i>ΔAgrho2/ΔAgrho5</i> double deletion. The scale bar represents 1 cm. B) Phase contrast images of <i>ΔAgrho2/ΔAgrho5</i> double deletion hyphae. The scale bar is 10 µm. The images were produce with a 40× magnification directly from agar plates and therefore display a rather uneven background.</p

<i>A. gossypii</i> strains used in this study.

<p><i>A. gossypii</i> strains used in this study.</p

Microscopic observation of <i>Agrho5</i> and <i>Agdck1</i> mutant strains.

<p>A) DIC images of polarity defects observed in germlings with a single, small germ tube. The angle given for each strain is the average deviation of the germ tube from a 90° angle that was measured from the middle axis of the spore needle. The scale bar represents 10 µm. B) DIC images of polarity defects observed in germlings with two germ tubes. Scale bar, 10 µm. C) DIC images of polarity defects observed in mature hyphae. Scale bar, 5 µm.</p

Growth of <i>Agrho5</i> and <i>Agdck1</i> mutant strains.

<p>A) Measurement of radial growth speed on solid medium at 30° measured for wild type as well as <i>Agrho5</i> and <i>Agdck1</i> deletions. Shown are the arithmetic means and standard deviations (n> = 3). The diameter of the initial inoculum was subtracted from each time-point, such that all measurements began at 0 mm. B) Example images of mycelia from the measurements on the left displaying the difference in diameter taken at day 6 of the measurements. C) Sensitivity of wild type <i>Agrho5</i> and <i>Agdck1</i> deletions against Latrunculin A. Spores in a concentration that results in a lawn were plated on full medium agar in cell culture six well plates. Five microliters of 10 mM Latrunculin A dissolves in DMSO or DMSO as a control were spotted on sterile filter discs and placed in the middle of each well. The plates were incubated for 3 days at 30°C.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p