Journal: bioRxiv

Article Title: Cellulose Synthase Complex and Remorin Nanodomains Mediate Stress Resilience Through Cell Wall-Plasma Membrane Attachments

doi: 10.1101/2025.08.01.664786

Figure Lengend Snippet: (A) A 5-d-old Arabidopsis seedling grown on MS media. Box demarcates the root tip. Scale bar: 0.2 cm. (B) Diagram of the longitudinal section of an Arabidopsis root tip in (A). Epidermal cells in the elongation zone are color coded in blue, which represent the regions where images in (C) were taken. (C) 3D rendering of root epidermal cells in 5-d-old seedlings expressing a plasma membrane marker, YFP-LTl6b, under the control condition or treated with 1 M sorbitol for 5 min. Scale bar: 10 µm. (D) Laser scanning confocal images of root epidermal cells from 5-day-old seedlings expressing the plasma membrane marker YFP-LTl6b and co-stained with the cell wall dye calcofluor white. Seedlings were imaged under control conditions or after treatment with 1 M sorbitol for 5 min. XY and YZ projections are shown. The positions where YZ projections were made are indicated by arrowheads. Scale bar: 5 µm. (E) 3D segmentation of Hechtian structure and wall-membrane attachment sites from the dashed box region in (0). Segmented Hechtian structure is shown either in yellow, overlaid on YFP-LTl6b signals, or with depth-based color coding. Wall-membrane attachment sites (indicated by white dashed circles) are visualized by applying an oblique slicer (outlined in red) to the YZ projection. Scale bars: 2 µm. See also Video S1.

Article Snippet: For calcofluor white staining, five-day-old seedlings were stained with 0.1 μM (10 -5 %, w/v) calcofluor white (Santa Cruz Biotechnology, catalog #sc-218504) diluted in MS liquid in the dark for 30 min, washed with MS liquid, treated with fresh MS liquid or 1 M sorbitol for 5 min on a glass slide, and imaged on a Leica TCS SP8 microscope with an HC PL APO 93x/1.3 NA objective.

Techniques: Expressing, Clinical Proteomics, Membrane, Marker, Control, Staining

Journal: bioRxiv

Article Title: Cellulose Synthase Complex and Remorin Nanodomains Mediate Stress Resilience Through Cell Wall-Plasma Membrane Attachments

doi: 10.1101/2025.08.01.664786

Figure Lengend Snippet: (A) Laser scanning confocal images in root epidermal cells of 5-d-old seedlings expressing YFP-REM1.2 treated with OM, 0.28 M, 0.5 M, or 1 M sorbitol for 5 min, respectively. Scale bar: 10 µm. (B) Bar graph showing YFP-REM1.2 nanodomain density at different sorbitol concentrations quantified from (A). n ≥ 21 cells from at least 5 seedlings per treatment. Error bars indicate SD. Different letters indicate significant differences by one-way ANOVA and Tukey’s test. (C) Laser scanning confocal images of root epidermal cells in 5-d-old seedlings expressing YFP-REM1.2 co-stained with calcofluor white under the control condition or treated with 1 M sorbitol for 5 min. XY and YZ projections are shown. The positions where YZ projections were made are indicated by arrowheads. Scale bar: 10 µm. (D) Laser scanning confocal images of root epidermal cells in 5-d-old seedlings expressing YFP-REM1.2 and mCherry-LTl6b under the control condition or treated with 1 M sorbitol for 5 min. XY and YZ projections are shown. The positions where YZ projections were made are indicated by arrowheads. Scale bar: 10 µm. (E) Quantification of Manders overlap coeffiicient in 5-d-old seedlings of YFP-REM1.2/mCherry-LTl6b under the control condition or treated with 1 M sorbitol for 5 min. Error bars indicate SD. n ≥ 9 cells per genotype per treatment. ns, no significance by Student’s t-test. (F) Root growth of wild type, rem1.2 1.3 1.4 triple mutants, and YFP-REM1.2/rem1.2 1.3 1.4 lines 1 day after transfer to either fresh MS media(-) or MS media supplemented with 0.28 M sorbitol (+). n ≥ 27 seedlings per genotype per treatment. Error bars indicate SD. ns, no significance; ***p < 0.001, ****p < 0.0001 by two-way ANOVA. (G) Laser scanning confocal images and manual tracings of plasmolyzed root epidermal cells in 5-d-old seedlings expressing YFP-LTl6b in Col-0 or rem1.2 1.3 1.4 background, respectively. Plasmolysis was induced by 0.5 M sorbitol for 5 min. Each displayed ratio corresponds to the same individual cell. Scale bar: 10 µm. (H) Histogram showing plasmolysis ratio in root epidermal cells of 5-d-old seedlings expressing YFP-LTl6b in Col-0 or rem1.2 1.3 1.4 background, respectively. n ≥ 102 cells from at least 10 seedlings per genotype. ****p < 0.0001 by Student’s t-test.

Article Snippet: For calcofluor white staining, five-day-old seedlings were stained with 0.1 μM (10 -5 %, w/v) calcofluor white (Santa Cruz Biotechnology, catalog #sc-218504) diluted in MS liquid in the dark for 30 min, washed with MS liquid, treated with fresh MS liquid or 1 M sorbitol for 5 min on a glass slide, and imaged on a Leica TCS SP8 microscope with an HC PL APO 93x/1.3 NA objective.

Techniques: Expressing, Staining, Control

Journal: bioRxiv

Article Title: Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus

doi: 10.1101/2024.03.02.582832

Figure Lengend Snippet: (A) Ergosterol biosynthesis pathways in A. fumigatus ( Af, top pathway) and S. cerevisiae ( Sc, bottom pathway). In A. fumigatus , C24-methylation of lanosterol by sterol C24-methyltransferase ( Af Erg6A) is favored over C14-methylation. In yeasts, lanosterol is the favored substrate of sterol C14-demethylase ( Sc Erg11). Both pathways converge with the formation of fecosterol ( 10 ), which is then further processed to ergosterol ( 14 ). (B) Proposed alternative sterol biosynthesis pathway upon inhibition of sterol C14-demethylase (AfCyp51A/B and Sc Erg11). In yeast, lanosterol is converted to 14-methylcholesta-8,24-dien-3β-ol ( 17 ), which in turn is converted to 14-methylergosta-8,24(28)-dien-3β-ol ( 15 ). In A. fumigatus , eburicol ( 2 ) is directly converted to 14-methylergosta-8,24(28)-dien-3β-ol ( 15 ). 14-methylergosta-8,24(28)-dien-3β-ol ( 15 ) is then converted to the 14-methylergosta-8,24(28)-dien-3β,6α-diol ( 16 ) which is considered a “toxic diol”. (A and B) Ergosterol biosynthesis enzymes in A. fumigatus ( Af ) and S. cerevisiae ( Sc ): sterol C24-methyltransferase ( Af Erg6A, Af Erg6B and Sc Erg6), sterol C14-demethylase ( Af Cyp51A/B, Sc Erg11), sterol C14-reductase ( Af Erg24A/B, Sc Erg24), sterol C4-demethylase complex ( Af Erg25A/B, Sc Erg25/26/27), sterol C8-isomerase ( Af Erg2, Sc Erg2), sterol C22-desturase ( Af Erg5, Sc Erg5), sterol C5-desaturase ( Af Erg3A/B/C, Sc Erg3), sterol C24 reductase ( Af Erg4A/B, Sc Erg4). Sterols: lanosterol, ( 2 ) eburicol, ( 3 ) 4,4-dimethylergosta-8,14,24(28)-trien-3β-ol, ( 4 ) 4,4-dimethylergosta-8,24(28)-dien-3β-ol, ( 5 ) 4-methylergosta-8,24(28)-dien-3β-ol, ( 6 ) 4,4-dimethylcholesta-8,14,24-trien-3β-ol, ( 7 ) 4,4-dimethylcholesta-8,24-dien-3β-ol, ( 8 ) 4-methylcholesta-8,24-dien-3β-ol, ( 9 ) zymosterol, ( 10 ) fecosterol, ( 11 ) episterol, ( 12 ) ergosta-7,22,24(28)-trien-3β-ol, ( 13 ) ergosta-5,7,22,24(28)-tetraen-3β-ol, ( 14 ) ergosterol, ( 15 ) 14-methylergosta-8,24(28)-dien-3β-ol, ( 16 ) 14-methylergosta-8,24(28)-dien-3β,6α-diol, and ( 17 ) 14-methylcholesta-8,24-dien-3β-ol. (C) Conidia of A. fumigatus wild type and two Candida species ( C. albicans ATCC14053 and C. glabrata ATCC2950) were inoculated in Sabouraud medium and incubated at 37 °C. After 9.5 h of incubation, the samples were either fixed and stored at 4 °C (control) or, after the medium was supplemented with 3 µg ml - voriconazole (+Vori), further incubated at 37 °C. After 15 h additional incubation, the voriconazole-exposed hyphae and yeasts were also fixed. Samples were then stained with calcofluor white and analyzed with a confocal laser scanning microscope. Depicted are representative images of z-stack projections of optical stacks of the calcofluor white fluorescence covering the entire hyphae in focus. Upper panel, voriconazole-treated hyphae; lower panel, controls. Bars represent 10 μm.

Article Snippet: Calcofluor white (Fluorescent brightener 28; #ICNA0215806705) was obtained from VWR International (Radnor, PA, USA).

Techniques: Methylation, Inhibition, Incubation, Staining, Laser-Scanning Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus

doi: 10.1101/2024.03.02.582832

Figure Lengend Snippet: (A) In a series of 10-fold dilutions derived from a starting suspension of 5 × 10 conidia ml - of wild type (wt), the conditional mutant erg6A tetOn , and the deletion mutant Δ erg6B , aliquots of 3 µl were spotted onto AMM agar plates. AMM was supplemented with 20 μg ml −1 doxycycline when indicated. Agar plates were incubated at 37 °C, representative photos were taken after 28 h. (B) 1.5 × 10 conidia of the indicated strains were spotted on AMM agar supplemented with the indicated amount of doxycycline. The plates were then incubated at 37 °C, representative photos were taken after 40 h. (C and D) Conidia of the erg6A tetOn and cyp51A tetOn Δ cyp51B mutants which express mitochondria-targeted GFP (D) or not (C) were inoculated in Sabouraud medium supplemented with 20 µg mL - doxycycline and incubated at 37 °C. After 6 h, doxycycline was depleted by washing the wells three times with Sabouraud medium without doxycycline. The hyphae were then incubated in Sabouraud medium without doxycycline at 37 °C for an additional 40 h. (C) Hyphae were stained with calcofluor white and analyzed with a confocal microscope. Depicted are representative images of z-stack projections of optical stacks of the calcofluor white fluorescence covering the entire hyphae in focus. The right images show magnifications of the framed sections in the left images. Bars represent 50 μm and are applicable to all images in the respective panel. (D, column graph) At the indicated time points after doxycycline depletion, the viability of the hyphae of the indicated strains expressing mitochondria-targeted GFP was analyzed with time-lapse spinning disc confocal microscopy. The bars indicate the percentage of hyphal compartments with evident mitochondrial dynamics (viable compartments). Data points represent the means of five technical replicates for each timepoint, with an average of approx. 80 analyzed compartments per strain for each timepoint. The error bars indicate standard deviations based on the five technical replicates. (D, lower panel) Representative overlay images of the bright field channel and of z-stack projections of optical stacks of the GFP fluorescence covering the entire hyphae in focus. Depicted are exemplary images of a viable hypha with tubular mitochondrial morphology (also showing mitochondrial dynamics in time-lapse microscopy; left image) and of hyphae with fragmented mitochondria (showing no mitochondrial dynamics in time-lapse microscopy; middle image) or with no or cytosolic GFP signal (right image) which were considered to be dead. Bars represent 50 μm and are applicable to all respective subpanels. (E) 1 × 10 conidia of the indicated strains were spread on Sabouraud agar plates. Agar was supplemented with the indicated amount of doxycycline to achieve a different induction of the conditional promoters. Voriconazole Etest strips were applied. The plates were incubated at 37 °C and representative photos were taken after 42 h.

Article Snippet: Calcofluor white (Fluorescent brightener 28; #ICNA0215806705) was obtained from VWR International (Radnor, PA, USA).

Techniques: Derivative Assay, Suspension, Mutagenesis, Incubation, Staining, Microscopy, Fluorescence, Expressing, Confocal Microscopy, Time-lapse Microscopy

Journal: bioRxiv

Article Title: Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus

doi: 10.1101/2024.03.02.582832

Figure Lengend Snippet: (A) 1.5 × 10 conidia of wild type (wt) or of the conditional squalene epoxidase ( erg9 tetOn ) or of the squalene synthase ( erg1 tetOn ) mutants were spotted on AMM agar supplemented with the indicated amount of doxycycline. The plates were then incubated at 37 °C, representative photos were taken after 40 h. (B) Conidia of the indicated strains were inoculated in Sabouraud medium supplemented with 20 µg ml - doxycycline and incubated at 37 °C. After 6 h, doxycycline was depleted by washing the wells three times with Sabouraud medium without doxycycline. The hyphae were then incubated in Sabouraud medium without doxycycline at 37 °C for an additional 40 h and subsequently stained with calcofluor white and analyzed with a confocal microscope. Depicted are representative images of z-stack projections of optical stacks of the calcofluor white fluorescence covering the entire hyphae in focus. The lower images show magnifications of the framed sections in the upper images. Bars represent 50 μm and are applicable to all images in the respective panel. (C) Conidia of wild-type expressing mitochondria-targeted GFP were inoculated in Sabouraud medium. After 8 h incubation at 37 °C, medium supplemented with 8 µg ml - terbinafine (upper panel) or with 4 µg ml - voriconazole (lower panel). After an additional 16 h incubation at 37 °C, hyphae were analyzed with a fluorescence microscope. Fluorescence signals were analyzed sequentially, first the GFP signal was recorded followed by staining with calcofluor white and recording of the calcofluor white signal. Depicted are representative images of bright-field (left) and z-stack projections of optical stacks of the calcofluor white fluorescence (middle) and GFP fluorescence (right) after deconvolution, that cover the entire hyphae in focus. The bar represents 50 µm and is applicable to all images. (E) 1 × 10 conidia of the indicated strains were spread on Sabouraud agar plates. Agar was supplemented with the indicated amount of doxycycline to achieve a different induction of the conditional promoters. Voriconazole Etest strips were applied. The plates were incubated at 37 °C and representative photos were taken after 42 h.

Article Snippet: Calcofluor white (Fluorescent brightener 28; #ICNA0215806705) was obtained from VWR International (Radnor, PA, USA).

Techniques: Incubation, Staining, Microscopy, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus

doi: 10.1101/2024.03.02.582832

Figure Lengend Snippet: (B) 1 × 10 conidia of wild type (wt) and the indicated strains were spread on Sabouraud agar plates. When indicated, agar was supplemented with 25 μg ml −1 doxycycline. Voriconazole Etest strips were applied. The plates were incubated at 37 °C and representative photos were taken after 42 h. (A) Conidia of wild type and the conditional sterol C5-desaturase mutant ( erg3A tetOn Δ erg3B Δ erg3C ) were inoculated in Sabouraud medium. After 10 h, the samples were either directly analyzed (controls without azole) or the medium was supplemented with 4 µg ml - voriconazole (+Vori). The voriconazole-exposed hyphae were then incubated at 37 °C for another 5 h. The untreated hyphae (upper images per indicated strain) and the voriconazole-treated hyphae (lower images per indicated strain) were stained with calcofluor white and analyzed with a confocal microscope. Depicted are representative images of z-stack projections of optical stacks of the calcofluor white fluorescence covering the entire hyphae in focus. The right panels show magnifications of the framed sections in the left panels. Bars represent 50 μm and are applicable to all images in the respective panel.

Article Snippet: Calcofluor white (Fluorescent brightener 28; #ICNA0215806705) was obtained from VWR International (Radnor, PA, USA).

Techniques: Incubation, Mutagenesis, Staining, Microscopy, Fluorescence